Patrick O. Brown

All Publications

• Quantitative proteomic analysis reveals concurrent RNA-protein interactions and identifies new RNA-binding proteins in Saccharomyces cerevisiae GENOME RESEARCH Klass, D. M., Scheibe, M., Butter, F., Hogan, G. J., Mann, M., Brown, P. O. 2013; 23 (6): 1028-1038

  Abstract

  A growing body of evidence supports the existence of an extensive network of RNA-binding proteins (RBPs) whose combinatorial binding affects the post-transcriptional fate of every mRNA in the cell-yet we still do not have a complete understanding of which proteins bind to mRNA, which of these bind concurrently, and when and where in the cell they bind. We describe here a method to identify the proteins that bind to RNA concurrently with an RBP of interest, using quantitative mass spectrometry combined with RNase treatment of affinity-purified RNA-protein complexes. We applied this method to the known RBPs Pab1, Nab2, and Puf3. Our method significantly enriched for known RBPs and is a clear improvement upon previous approaches in yeast. Our data reveal that some reported protein-protein interactions may instead reflect simultaneous binding to shared RNA targets. We also discovered more than 100 candidate RBPs, and we independently confirmed that 77% (23/30) bind directly to RNA. The previously recognized functions of the confirmed novel RBPs were remarkably diverse, and we mapped the RNA-binding region of one of these proteins, the transcriptional coactivator Mbf1, to a region distinct from its DNA-binding domain. Our results also provided new insights into the roles of Nab2 and Puf3 in post-transcriptional regulation by identifying other RBPs that bind simultaneously to the same mRNAs. While existing methods can identify sets of RBPs that interact with common RNA targets, our approach can determine which of those interactions are concurrent-a crucial distinction for understanding post-transcriptional regulation.

  View details for DOI 10.1101/gr.153031.112

  View details for Web of Science ID 000319803700012

  View details for PubMedID 23636942

• An interview with Patrick O Brown on the origins and future of open access BMC BIOLOGY Brown, P. O. 2013; 11

  View details for DOI 10.1186/1741-7007-11-33

  View details for Web of Science ID 000317692700002

  View details for PubMedID 23587113

• Improved discovery of molecular interactions in genome-scale data with adaptive model-based normalization. PloS one Salzman, J., Klass, D. M., Brown, P. O. 2013; 8 (1)

  Abstract

  High throughput molecular-interaction studies using immunoprecipitations (IP) or affinity purifications are powerful and widely used in biology research. One of many important applications of this method is to identify the set of RNAs that interact with a particular RNA-binding protein (RBP). Here, the unique statistical challenge presented is to delineate a specific set of RNAs that are enriched in one sample relative to another, typically a specific IP compared to a non-specific control to model background. The choice of normalization procedure critically impacts the number of RNAs that will be identified as interacting with an RBP at a given significance threshold - yet existing normalization methods make assumptions that are often fundamentally inaccurate when applied to IP enrichment data.In this paper, we present a new normalization methodology that is specifically designed for identifying enriched RNA or DNA sequences in an IP. The normalization (called adaptive or AD normalization) uses a basic model of the IP experiment and is not a variant of mean, quantile, or other methodology previously proposed. The approach is evaluated statistically and tested with simulated and empirical data.The adaptive (AD) normalization method results in a greatly increased range in the number of enriched RNAs identified, fewer false positives, and overall better concordance with independent biological evidence, for the RBPs we analyzed, compared to median normalization. The approach is also applicable to the study of pairwise RNA, DNA and protein interactions such as the analysis of transcription factors via chromatin immunoprecipitation (ChIP) or any other experiments where samples from two conditions, one of which contains an enriched subset of the other, are studied.

  View details for DOI 10.1371/journal.pone.0053930

  View details for PubMedID 23349766

• The Yeast Rab GTPase Ypt1 Modulates Unfolded Protein Response Dynamics by Regulating the Stability of HAC1 RNA PLOS GENETICS Tsvetanova, N. G., Riordan, D. P., Brown, P. O. 2012; 8 (7)

  Abstract

  The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins that specifically interact with HAC1 RNA. Protein microarray experiments revealed that HAC1 interacts specifically with small ras GTPases of the Ypt family. We characterized the interaction of HAC1 RNA with one of these proteins, the yeast Rab1 homolog Ypt1. We found that Ypt1 protein specifically associated in vivo with unspliced HAC1 RNA. This association was disrupted by conditions that impaired protein folding in the ER and induced the UPR. Also, the Ypt1-HAC1 interaction depended on IRE1 and ADA5, the two genes critical for UPR activation. Decreasing expression of the Ypt1 protein resulted in a reduced rate of HAC1 RNA decay, leading to significantly increased levels of both unspliced and spliced HAC1 RNA, and delayed attenuation of the UPR, when ER stress was relieved. Our findings establish that Ypt1 contributes to regulation of UPR signaling dynamics by promoting the decay of HAC1 RNA, suggesting a potential regulatory mechanism for linking vesicle trafficking to the UPR and ER homeostasis.

  View details for DOI 10.1371/journal.pgen.1002862

  View details for Web of Science ID 000306840400056

  View details for PubMedID 22844259

• Extensive Gene-Specific Translational Reprogramming in a Model of B Cell Differentiation and Abl-Dependent Transformation PLOS ONE Bates, J. G., Salzman, J., May, D., Garcia, P. B., Hogan, G. J., McIntosh, M., Schlissel, M. S., Brown, P. O. 2012; 7 (5)

  Abstract

  To what extent might the regulation of translation contribute to differentiation programs, or to the molecular pathogenesis of cancer? Pre-B cells transformed with the viral oncogene v-Abl are suspended in an immortalized, cycling state that mimics leukemias with a BCR-ABL1 translocation, such as Chronic Myelogenous Leukemia (CML) and Acute Lymphoblastic Leukemia (ALL). Inhibition of the oncogenic Abl kinase with imatinib reverses transformation, allowing progression to the next stage of B cell development. We employed a genome-wide polysome profiling assay called Gradient Encoding to investigate the extent and potential contribution of translational regulation to transformation and differentiation in v-Abl-transformed pre-B cells. Over half of the significantly translationally regulated genes did not change significantly at the level of mRNA abundance, revealing biology that might have been missed by measuring changes in transcript abundance alone. We found extensive, gene-specific changes in translation affecting genes with known roles in B cell signaling and differentiation, cancerous transformation, and cytoskeletal reorganization potentially affecting adhesion. These results highlight a major role for gene-specific translational regulation in remodeling the gene expression program in differentiation and malignant transformation.

  View details for DOI 10.1371/journal.pone.0037108

  View details for Web of Science ID 000305338500030

  View details for PubMedID 22693568

• Widespread mRNA Association with Cytoskeletal Motor Proteins and Identification and Dynamics of Myosin-Associated mRNAs in S. cerevisiae PLOS ONE Casolari, J. M., Thompson, M. A., Salzman, J., Champion, L. M., Moerner, W. E., Brown, P. O. 2012; 7 (2)

  Abstract

  Programmed mRNA localization to specific subcellular compartments for localized translation is a fundamental mechanism of post-transcriptional regulation that affects many, and possibly all, mRNAs in eukaryotes. We describe here a systematic approach to identify the RNA cargoes associated with the cytoskeletal motor proteins of Saccharomyces cerevisiae in combination with live-cell 3D super-localization microscopy of endogenously tagged mRNAs. Our analysis identified widespread association of mRNAs with cytoskeletal motor proteins, including association of Myo3 with mRNAs encoding key regulators of actin branching and endocytosis such as WASP and WIP. Using conventional fluorescence microscopy and expression of MS2-tagged mRNAs from endogenous loci, we observed a strong bias for actin patch nucleator mRNAs to localize to the cell cortex and the actin patch in a Myo3- and F-actin dependent manner. Use of a double-helix point spread function (DH-PSF) microscope allowed super-localization measurements of single mRNPs at a spatial precision of 25 nm in x and y and 50 nm in z in live cells with 50 ms exposure times, allowing quantitative profiling of mRNP dynamics. The actin patch mRNA exhibited distinct and characteristic diffusion coefficients when compared to a control mRNA. In addition, disruption of F-actin significantly expanded the 3D confinement radius of an actin patch nucleator mRNA, providing a quantitative assessment of the contribution of the actin cytoskeleton to mRNP dynamic localization. Our results provide evidence for specific association of mRNAs with cytoskeletal motor proteins in yeast, suggest that different mRNPs have distinct and characteristic dynamics, and lend insight into the mechanism of actin patch nucleator mRNA localization to actin patches.

  View details for DOI 10.1371/journal.pone.0031912

  View details for Web of Science ID 000302796200110

  View details for PubMedID 22359641

  View details for PubMedCentralID PMC3281097

• Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types PLOS ONE Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. 2012; 7 (2)

  Abstract

  Most human pre-mRNAs are spliced into linear molecules that retain the exon order defined by the genomic sequence. By deep sequencing of RNA from a variety of normal and malignant human cells, we found RNA transcripts from many human genes in which the exons were arranged in a non-canonical order. Statistical estimates and biochemical assays provided strong evidence that a substantial fraction of the spliced transcripts from hundreds of genes are circular RNAs. Our results suggest that a non-canonical mode of RNA splicing, resulting in a circular RNA isoform, is a general feature of the gene expression program in human cells.

  View details for DOI 10.1371/journal.pone.0030733

  View details for Web of Science ID 000301977500016

  View details for PubMedID 22319583

  View details for PubMedCentralID PMC3270023

• Interferon and Biologic Signatures in Dermatomyositis Skin: Specificity and Heterogeneity across Diseases PLOS ONE Wong, D., Kea, B., Pesich, R., Higgs, B. W., Zhu, W., Brown, P., Yao, Y., Fiorentino, D. 2012; 7 (1)

  Abstract

  Dermatomyositis (DM) is an autoimmune disease that mainly affects the skin, muscle, and lung. The pathogenesis of skin inflammation in DM is not well understood.We analyzed genome-wide expression data in DM skin and compared them to those from healthy controls. We observed a robust upregulation of interferon (IFN)-inducible genes in DM skin, as well as several other gene modules pertaining to inflammation, complement activation, and epidermal activation and differentiation. The interferon (IFN)-inducible genes within the DM signature were present not only in DM and lupus, but also cutaneous herpes simplex-2 infection and to a lesser degree, psoriasis. This IFN signature was absent or weakly present in atopic dermatitis, allergic contact dermatitis, acne vulgaris, systemic sclerosis, and localized scleroderma/morphea. We observed that the IFN signature in DM skin appears to be more closely related to type I than type II IFN based on in vitro IFN stimulation expression signatures. However, quantitation of IFN mRNAs in DM skin shows that the majority of known type I IFNs, as well as IFN g, are overexpressed in DM skin. In addition, both IFN-beta and IFN-gamma (but not other type I IFN) transcript levels were highly correlated with the degree of the in vivo IFN transcriptional response in DM skin.As in the blood and muscle, DM skin is characterized by an overwhelming presence of an IFN signature, although it is difficult to conclusively define this response as type I or type II. Understanding the significance of the IFN signature in this wide array of inflammatory diseases will be furthered by identification of the nature of the cells that both produce and respond to IFN, as well as which IFN subtype is biologically active in each diseased tissue.

  View details for DOI 10.1371/journal.pone.0029161

  View details for Web of Science ID 000301123400030

  View details for PubMedID 22235269

  View details for PubMedCentralID PMC3250414

• ESRRA-C11orf20 Is a Recurrent Gene Fusion in Serous Ovarian Carcinoma PLOS BIOLOGY Salzman, J., Marinelli, R. J., Wang, P. L., Green, A. E., Nielsen, J. S., Nelson, B. H., Drescher, C. W., Brown, P. O. 2011; 9 (9)

  Abstract

  Every year, ovarian cancer kills approximately 14,000 women in the United States and more than 140,000 women worldwide. Most of these deaths are caused by tumors of the serous histological type, which is rarely diagnosed before it has disseminated. By deep paired-end sequencing of mRNA from serous ovarian cancers, followed by deep sequencing of the corresponding genomic region, we identified a recurrent fusion transcript. The fusion transcript joins the 5' exons of ESRRA, encoding a ligand-independent member of the nuclear-hormone receptor superfamily, to the 3' exons of C11orf20, a conserved but uncharacterized gene located immediately upstream of ESRRA in the reference genome. To estimate the prevalence of the fusion, we tested 67 cases of serous ovarian cancer by RT-PCR and sequencing and confirmed its presence in 10 of these. Targeted resequencing of the corresponding genomic region from two fusion-positive tumor samples identified a nearly clonal chromosomal rearrangement positioning ESRRA upstream of C11orf20 in one tumor, and evidence of local copy number variation in the ESRRA locus in the second tumor. We hypothesize that the recurrent novel fusion transcript may play a role in pathogenesis of a substantial fraction of serous ovarian cancers and could provide a molecular marker for detection of the cancer. Gene fusions involving adjacent or nearby genes can readily escape detection but may play important roles in the development and progression of cancer.

  View details for DOI 10.1371/journal.pbio.1001156

  View details for Web of Science ID 000295372800012

  View details for PubMedID 21949640

  View details for PubMedCentralID PMC3176749

• Defining the Specificity of Cotranslationally Acting Chaperones by Systematic Analysis of mRNAs Associated with Ribosome-Nascent Chain Complexes PLOS BIOLOGY del Alamo, M., Hogan, D. J., Pechmann, S., Albanese, V., Brown, P. O., Frydman, J. 2011; 9 (7)

  Abstract

  Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

  View details for DOI 10.1371/journal.pbio.1001100

  View details for Web of Science ID 000293219800007

  View details for PubMedID 21765803

• Identification of RNA recognition elements in the Saccharomyces cerevisiae transcriptome NUCLEIC ACIDS RESEARCH Riordan, D. P., Herschlag, D., Brown, P. O. 2011; 39 (4): 1501-1509

  Abstract

  Post-transcriptional regulation of gene expression, including mRNA localization, translation and decay, is ubiquitous yet still largely unexplored. How is the post-transcriptional regulatory program of each mRNA encoded in its sequence? Hundreds of specific RNA-binding proteins (RBPs) appear to play roles in mediating the post-transcriptional regulatory program, akin to the roles of specific DNA-binding proteins in transcription. As a step toward decoding the regulatory programs encoded in each mRNA, we focused on specific mRNA-protein interactions. We computationally analyzed the sequences of Saccharomyces cerevisiae mRNAs bound in vivo by 29 specific RBPs, identifying eight novel candidate motifs and confirming or extending six earlier reported recognition elements. Biochemical selections for RNA sequences selectively recognized by 12 yeast RBPs yielded novel motifs bound by Pin4, Nsr1, Hrb1, Gbp2, Sgn1 and Mrn1, and recovered the known recognition elements for Puf3, She2, Vts1 and Whi3. Most of the RNA elements we uncovered were associated with coherent mRNA expression changes and were significantly conserved in related yeasts, supporting their functional importance and suggesting that the corresponding RNA-protein interactions are evolutionarily conserved.

  View details for DOI 10.1093/nar/gkq920

  View details for Web of Science ID 000288019400036

  View details for PubMedID 20959291

• Stunned Silence: Gene Expression Programs in Human Cells Infected with Monkeypox or Vaccinia Virus PLOS ONE Rubins, K. H., Hensley, L. E., Relman, D. A., Brown, P. O. 2011; 6 (1)

  Abstract

  Poxviruses use an arsenal of molecular weapons to evade detection and disarm host immune responses. We used DNA microarrays to investigate the gene expression responses to infection by monkeypox virus (MPV), an emerging human pathogen, and Vaccinia virus (VAC), a widely used model and vaccine organism, in primary human macrophages, primary human fibroblasts and HeLa cells. Even as the overwhelmingly infected cells approached their demise, with extensive cytopathic changes, their gene expression programs appeared almost oblivious to poxvirus infection. Although killed (gamma-irradiated) MPV potently induced a transcriptional program characteristic of the interferon response, no such response was observed during infection with either live MPV or VAC. Moreover, while the gene expression response of infected cells to stimulation with ionomycin plus phorbol 12-myristate 13-acetate (PMA), or poly (I-C) was largely unimpaired by infection with MPV, a cluster of pro-inflammatory genes were a notable exception. Poly(I-C) induction of genes involved in alerting the innate immune system to the infectious threat, including TNF-alpha, IL-1 alpha and beta, CCL5 and IL-6, were suppressed by infection with live MPV. Thus, MPV selectively inhibits expression of genes with critical roles in cell-signaling pathways that activate innate immune responses, as part of its strategy for stealthy infection.

  View details for DOI 10.1371/journal.pone.0015615

  View details for Web of Science ID 000286519500024

  View details for PubMedID 21267444

• Three-dimensional tracking of single mRNA particles in Saccharomyces cerevisiae using a double-helix point spread function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Thompson, M. A., Casolari, J. M., Badieirostami, M., Brown, P. O., Moerner, W. E. 2010; 107 (42): 17864-17871

  Abstract

  Optical imaging of single biomolecules and complexes in living cells provides a useful window into cellular processes. However, the three-dimensional dynamics of most important biomolecules in living cells remains essentially uncharacterized. The precise subcellular localization of mRNA-protein complexes plays a critical role in the spatial and temporal control of gene expression, and a full understanding of the control of gene expression requires precise characterization of mRNA transport dynamics beyond the optical diffraction limit. In this paper, we describe three-dimensional tracking of single mRNA particles with 25-nm precision in the x and y dimensions and 50-nm precision in the z dimension in live budding yeast cells using a microscope with a double-helix point spread function. Two statistical methods to detect intermittently confined and directed transport were used to quantify the three-dimensional trajectories of mRNA for the first time, using ARG3 mRNA as a model. Measurements and analysis show that the dynamics of ARG3 mRNA molecules are mostly diffusive, although periods of non-Brownian confinement and directed transport are observed. The quantitative methods detailed in this paper can be broadly applied to the study of mRNA localization and the dynamics of diverse other biomolecules in a wide variety of cell types.

  View details for DOI 10.1073/pnas.1012868107

  View details for Web of Science ID 000283184800008

  View details for PubMedID 20921361

  View details for PubMedCentralID PMC2964242

• Proteome-Wide Search Reveals Unexpected RNA-Binding Proteins in Saccharomyces cerevisiae PLOS ONE Tsvetanova, N. G., Klass, D. M., Salzman, J., Brown, P. O. 2010; 5 (9)

  Abstract

  The vast landscape of RNA-protein interactions at the heart of post-transcriptional regulation remains largely unexplored. Indeed it is likely that, even in yeast, a substantial fraction of the regulatory RNA-binding proteins (RBPs) remain to be discovered. Systematic experimental methods can play a key role in discovering these RBPs--most of the known yeast RBPs lack RNA-binding domains that might enable this activity to be predicted. We describe here a proteome-wide approach to identify RNA-protein interactions based on in vitro binding of RNA samples to yeast protein microarrays that represent over 80% of the yeast proteome. We used this procedure to screen for novel RBPs and RNA-protein interactions. A complementary mass spectrometry technique also identified proteins that associate with yeast mRNAs. Both the protein microarray and mass spectrometry methods successfully identify previously annotated RBPs, suggesting that other proteins identified in these assays might be novel RBPs. Of 35 putative novel RBPs identified by either or both of these methods, 12, including 75% of the eight most highly-ranked candidates, reproducibly associated with specific cellular RNAs. Surprisingly, most of the 12 newly discovered RBPs were enzymes. Functional characteristics of the RNA targets of some of the novel RBPs suggest coordinated post-transcriptional regulation of subunits of protein complexes and a possible link between mRNA trafficking and vesicle transport. Our results suggest that many more RBPs still remain to be identified and provide a set of candidates for further investigation.

  View details for DOI 10.1371/journal.pone.0012671

  View details for Web of Science ID 000281687300015

  View details for PubMedID 20844764

• Dissecting Interferon-Induced Transcriptional Programs in Human Peripheral Blood Cells PLOS ONE Waddell, S. J., Popper, S. J., Rubins, K. H., Griffiths, M. J., Brown, P. O., Levin, M., Relman, D. A. 2010; 5 (3)

  Abstract

  Interferons are key modulators of the immune system, and are central to the control of many diseases. The response of immune cells to stimuli in complex populations is the product of direct and indirect effects, and of homotypic and heterotypic cell interactions. Dissecting the global transcriptional profiles of immune cell populations may provide insights into this regulatory interplay. The host transcriptional response may also be useful in discriminating between disease states, and in understanding pathophysiology. The transcriptional programs of cell populations in health therefore provide a paradigm for deconvoluting disease-associated gene expression profiles.We used human cDNA microarrays to (1) compare the gene expression programs in human peripheral blood mononuclear cells (PBMCs) elicited by 6 major mediators of the immune response: interferons alpha, beta, omega and gamma, IL12 and TNFalpha; and (2) characterize the transcriptional responses of purified immune cell populations (CD4+ and CD8+ T cells, B cells, NK cells and monocytes) to IFNgamma stimulation. We defined a highly stereotyped response to type I interferons, while responses to IFNgamma and IL12 were largely restricted to a subset of type I interferon-inducible genes. TNFalpha stimulation resulted in a distinct pattern of gene expression. Cell type-specific transcriptional programs were identified, highlighting the pronounced response of monocytes to IFNgamma, and emergent properties associated with IFN-mediated activation of mixed cell populations. This information provides a detailed view of cellular activation by immune mediators, and contributes an interpretive framework for the definition of host immune responses in a variety of disease settings.

  View details for DOI 10.1371/journal.pone.0009753

  View details for Web of Science ID 000275894300006

  View details for PubMedID 20339534

  View details for PubMedCentralID PMC2842296

• Concordant Regulation of Translation and mRNA Abundance for Hundreds of Targets of a Human microRNA PLOS BIOLOGY Hendrickson, D. G., Hogan, D. J., McCullough, H. L., Myers, J. W., Herschlag, D., Ferrell, J. E., Brown, P. O. 2009; 7 (11)

  Abstract

  MicroRNAs (miRNAs) regulate gene expression posttranscriptionally by interfering with a target mRNA's translation, stability, or both. We sought to dissect the respective contributions of translational inhibition and mRNA decay to microRNA regulation. We identified direct targets of a specific miRNA, miR-124, by virtue of their association with Argonaute proteins, core components of miRNA effector complexes, in response to miR-124 transfection in human tissue culture cells. In parallel, we assessed mRNA levels and obtained translation profiles using a novel global approach to analyze polysomes separated on sucrose gradients. Analysis of translation profiles for approximately 8,000 genes in these proliferative human cells revealed that basic features of translation are similar to those previously observed in rapidly growing Saccharomyces cerevisiae. For approximately 600 mRNAs specifically recruited to Argonaute proteins by miR-124, we found reductions in both the mRNA abundance and inferred translation rate spanning a large dynamic range. The changes in mRNA levels of these miR-124 targets were larger than the changes in translation, with average decreases of 35% and 12%, respectively. Further, there was no identifiable subgroup of mRNA targets for which the translational response was dominant. Both ribosome occupancy (the fraction of a given gene's transcripts associated with ribosomes) and ribosome density (the average number of ribosomes bound per unit length of coding sequence) were selectively reduced for hundreds of miR-124 targets by the presence of miR-124. Changes in protein abundance inferred from the observed changes in mRNA abundance and translation profiles closely matched changes directly determined by Western analysis for 11 of 12 proteins, suggesting that our assays captured most of miR-124-mediated regulation. These results suggest that miRNAs inhibit translation initiation or stimulate ribosome drop-off preferentially near the start site and are not consistent with inhibition of polypeptide elongation, or nascent polypeptide degradation contributing significantly to miRNA-mediated regulation in proliferating HEK293T cells. The observation of concordant changes in mRNA abundance and translational rate for hundreds of miR-124 targets is consistent with a functional link between these two regulatory outcomes of miRNA targeting, and the well-documented interrelationship between translation and mRNA decay.

  View details for DOI 10.1371/journal.pbio.1000238

  View details for Web of Science ID 000272032000004

  View details for PubMedID 19901979

• The Preclinical Natural History of Serous Ovarian Cancer: Defining the Target for Early Detection PLOS MEDICINE Brown, P. O., Palmer, C. 2009; 6 (7)

  Abstract

  Ovarian cancer kills approximately 15,000 women in the United States every year, and more than 140,000 women worldwide. Most deaths from ovarian cancer are caused by tumors of the serous histological type, which are rarely diagnosed before the cancer has spread. Rational design of a potentially life-saving early detection and intervention strategy requires understanding the lesions we must detect in order to prevent lethal progression. Little is known about the natural history of lethal serous ovarian cancers before they become clinically apparent. We can learn about this occult period by studying the unsuspected serous cancers that are discovered in a small fraction of apparently healthy women who undergo prophylactic bilateral salpingo-oophorectomy (PBSO).We developed models for the growth, progression, and detection of occult serous cancers on the basis of a comprehensive analysis of published data on serous cancers discovered by PBSO in BRCA1 mutation carriers. Our analysis yielded several critical insights into the early natural history of serous ovarian cancer. First, these cancers spend on average more than 4 y as in situ, stage I, or stage II cancers and approximately 1 y as stage III or IV cancers before they become clinically apparent. Second, for most of the occult period, serous cancers are less than 1 cm in diameter, and not visible on gross examination of the ovaries and Fallopian tubes. Third, the median diameter of a serous ovarian cancer when it progresses to an advanced stage (stage III or IV) is about 3 cm. Fourth, to achieve 50% sensitivity in detecting tumors before they advance to stage III, an annual screen would need to detect tumors of 1.3 cm in diameter; 80% detection sensitivity would require detecting tumors less than 0.4 cm in diameter. Fifth, to achieve a 50% reduction in serous ovarian cancer mortality with an annual screen, a test would need to detect tumors of 0.5 cm in diameter.Our analysis has formalized essential conditions for successful early detection of serous ovarian cancer. Although the window of opportunity for early detection of these cancers lasts for several years, developing a test sufficiently sensitive and specific to take advantage of that opportunity will be a challenge. We estimated that the tumors we would need to detect to achieve even 50% sensitivity are more than 200 times smaller than the clinically apparent serous cancers typically used to evaluate performance of candidate biomarkers; none of the biomarker assays reported to date comes close to the required level of performance. Overcoming the signal-to-noise problem inherent in detection of tiny tumors will likely require discovery of truly cancer-specific biomarkers or development of novel approaches beyond traditional blood protein biomarkers. While this study was limited to ovarian cancers of serous histological type and to those arising in BRCA1 mutation carriers specifically, we believe that the results are relevant to other hereditary serous cancers and to sporadic ovarian cancers. A similar approach could be applied to other cancers to aid in defining their early natural history and to guide rational design of an early detection strategy.

  View details for DOI 10.1371/journal.pmed.1000114

  View details for Web of Science ID 000268452400011

  View details for PubMedID 19636370

• Diverse RNA-Binding Proteins Interact with Functionally Related Sets of RNAs, Suggesting an Extensive Regulatory System PLOS BIOLOGY Hogan, D. J., Riordan, D. P., Gerber, A. P., Herschlag, D., Brown, P. O. 2008; 6 (10): 2297-2313

  Abstract

  RNA-binding proteins (RBPs) have roles in the regulation of many post-transcriptional steps in gene expression, but relatively few RBPs have been systematically studied. We searched for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs, as well as several proteins not annotated as RBPs. At least 33 of these 40 proteins, including three of the four proteins that were not previously known or predicted to be RBPs, were reproducibly associated with specific sets of a few to several hundred RNAs. Remarkably, many of the RBPs we studied bound mRNAs whose protein products share identifiable functional or cytotopic features. We identified specific sequences or predicted structures significantly enriched in target mRNAs of 16 RBPs. These potential RNA-recognition elements were diverse in sequence, structure, and location: some were found predominantly in 3'-untranslated regions, others in 5'-untranslated regions, some in coding sequences, and many in two or more of these features. Although this study only examined a small fraction of the universe of yeast RBPs, 70% of the mRNA transcriptome had significant associations with at least one of these RBPs, and on average, each distinct yeast mRNA interacted with three of the RBPs, suggesting the potential for a rich, multidimensional network of regulation. These results strongly suggest that combinatorial binding of RBPs to specific recognition elements in mRNAs is a pervasive mechanism for multi-dimensional regulation of their post-transcriptional fate.

  View details for DOI 10.1371/journal.pbio.0060255

  View details for Web of Science ID 000260423900021

  View details for PubMedID 18959479

• Systematic Evaluation of Candidate Blood Markers for Detecting Ovarian Cancer PLOS ONE Palmer, C., Duan, X., Hawley, S., Scholler, N., Thorpe, J. D., Sahota, R. A., Wong, M. Q., Wray, A., Bergan, L. A., Drescher, C. W., McIntosh, M. W., Brown, P. O., Nelson, B. H., Urban, N. 2008; 3 (7)

  Abstract

  Epithelial ovarian cancer is a significant cause of mortality both in the United States and worldwide, due largely to the high proportion of cases that present at a late stage, when survival is extremely poor. Early detection of epithelial ovarian cancer, and of the serous subtype in particular, is a promising strategy for saving lives. The low prevalence of ovarian cancer makes the development of an adequately sensitive and specific test based on blood markers very challenging. We evaluated the performance of a set of candidate blood markers and combinations of these markers in detecting serous ovarian cancer.We selected 14 candidate blood markers of serous ovarian cancer for which assays were available to measure their levels in serum or plasma, based on our analysis of global gene expression data and on literature searches. We evaluated the performance of these candidate markers individually and in combination by measuring them in overlapping sets of serum (or plasma) samples from women with clinically detectable ovarian cancer and women without ovarian cancer. Based on sensitivity at high specificity, we determined that 4 of the 14 candidate markers--MUC16, WFDC2, MSLN and MMP7--warrant further evaluation in precious serum specimens collected months to years prior to clinical diagnosis to assess their utility in early detection. We also reported differences in the performance of these candidate blood markers across histological types of epithelial ovarian cancer.By systematically analyzing the performance of candidate blood markers of ovarian cancer in distinguishing women with clinically apparent ovarian cancer from women without ovarian cancer, we identified a set of serum markers with adequate performance to warrant testing for their ability to identify ovarian cancer months to years prior to clinical diagnosis. We argued for the importance of sensitivity at high specificity and of magnitude of difference in marker levels between cases and controls as performance metrics and demonstrated the importance of stratifying analyses by histological type of ovarian cancer. Also, we discussed the limitations of studies (like this one) that use samples obtained from symptomatic women to assess potential utility in detection of disease months to years prior to clinical detection.

  View details for DOI 10.1371/journal.pone.0002633

  View details for Web of Science ID 000264065800035

  View details for PubMedID 18612378

  View details for PubMedCentralID PMC2440813

• Comparative Analysis of Viral Gene Expression Programs during Poxvirus Infection: A Transcriptional Map of the Vaccinia and Monkeypox Genomes PLOS ONE Rubins, K. H., Hensley, L. E., Bell, G. W., Wang, C., Lefkowitz, E. J., Brown, P. O., Relman, D. A. 2008; 3 (7)

  Abstract

  Poxviruses engage in a complex and intricate dialogue with host cells as part of their strategy for replication. However, relatively little molecular detail is available with which to understand the mechanisms behind this dialogue.We designed a specialized microarray that contains probes specific to all predicted ORFs in the Monkeypox Zaire (MPXV) and Vaccinia Western Reserve (VACV) genomes, as well as >18,000 human genes, and used this tool to characterize MPXV and VACV gene expression responses in vitro during the course of primary infection of human monocytes, primary human fibroblasts and HeLa cells. The two viral transcriptomes show distinct features of temporal regulation and species-specific gene expression, and provide an early foundation for understanding global gene expression responses during poxvirus infection.The results provide a temporal map of the transcriptome of each virus during infection, enabling us to compare viral gene expression across species, and classify expression patterns of previously uncharacterized ORFs.

  View details for DOI 10.1371/journal.pone.0002628

  View details for Web of Science ID 000264065800030

  View details for PubMedID 18612436

• Systematic Identification of mRNAs Recruited to Argonaute 2 by Specific microRNAs and Corresponding Changes in Transcript Abundance PLOS ONE Hendrickson, D. G., Hogan, D. J., Herschlag, D., Ferrell, J. E., Brown, P. O. 2008; 3 (5)

  Abstract

  microRNAs (miRNAs) are small non-coding RNAs that regulate mRNA stability and translation through the action of the RNAi-induced silencing complex (RISC). Our current understanding of miRNA function is inferred largely from studies of the effects of miRNAs on steady-state mRNA levels and from seed match conservation and context in putative targets. Here we have taken a more direct approach to these issues by comprehensively assessing the miRNAs and mRNAs that are physically associated with Argonaute 2 (Ago2), which is a core RISC component. We transfected HEK293T cells with epitope-tagged Ago2, immunopurified Ago2 together with any associated miRNAs and mRNAs, and quantitatively determined the levels of these RNAs by microarray analyses. We found that Ago2 immunopurified samples contained a representative repertoire of the cell's miRNAs and a select subset of the cell's total mRNAs. Transfection of the miRNAs miR-1 and miR-124 caused significant changes in the association of scores of mRNAs with Ago2. The mRNAs whose association with Ago2 increased upon miRNA expression were much more likely to contain specific miRNA seed matches and to have their overall mRNA levels decrease in response to the miRNA transfection than expected by chance. Hundreds of mRNAs were recruited to Ago2 by each miRNA via seed sequences in 3'-untranslated regions and coding sequences and a few mRNAs appear to be targeted via seed sequences in 5'-untranslated regions. Microarray analysis of Ago2 immunopurified samples provides a simple, direct method for experimentally identifying the targets of miRNAs and for elucidating roles of miRNAs in cellular regulation.

  View details for DOI 10.1371/journal.pone.0002126

  View details for Web of Science ID 000261642400047

  View details for PubMedID 18461144

• The Stanford Tissue Microarray Database NUCLEIC ACIDS RESEARCH Marinelli, R. J., Montgomery, K., Liu, C. L., Shah, N. H., Prapong, W., Nitzberg, M., Zachariah, Z. K., Sherlock, G. J., Natkunam, Y., West, R. B., van de Rijn, M., Brown, P. O., Ball, C. A. 2008; 36: D871-D877

  Abstract

  The Stanford Tissue Microarray Database (TMAD; http://tma.stanford.edu) is a public resource for disseminating annotated tissue images and associated expression data. Stanford University pathologists, researchers and their collaborators worldwide use TMAD for designing, viewing, scoring and analyzing their tissue microarrays. The use of tissue microarrays allows hundreds of human tissue cores to be simultaneously probed by antibodies to detect protein abundance (Immunohistochemistry; IHC), or by labeled nucleic acids (in situ hybridization; ISH) to detect transcript abundance. TMAD archives multi-wavelength fluorescence and bright-field images of tissue microarrays for scoring and analysis. As of July 2007, TMAD contained 205 161 images archiving 349 distinct probes on 1488 tissue microarray slides. Of these, 31 306 images for 68 probes on 125 slides have been released to the public. To date, 12 publications have been based on these raw public data. TMAD incorporates the NCI Thesaurus ontology for searching tissues in the cancer domain. Image processing researchers can extract images and scores for training and testing classification algorithms. The production server uses the Apache HTTP Server, Oracle Database and Perl application code. Source code is available to interested researchers under a no-cost license.

  View details for DOI 10.1093/nar/gkm861

  View details for Web of Science ID 000252545400154

  View details for PubMedID 17989087

  View details for PubMedCentralID PMC2238948

• Parallels between global transcriptional programs of polarizing caco-2 intestinal epithelial cells in vitro and gene expression programs in normal colon and colon cancer MOLECULAR BIOLOGY OF THE CELL Saeaef, A. M., Halbleib, J. M., Chen, X., Yuen, S. T., Leung, S. Y., Nelson, W. J., Brown, P. O. 2007; 18 (11): 4245-4260

  Abstract

  Posttranslational mechanisms are implicated in the development of epithelial cell polarity, but little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized temporal patterns of gene expression during cell-cell adhesion-initiated polarization of cultured human Caco-2 cells, which develop structural and functional polarity resembling enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts. Comparison to gene expression patterns in normal human colon and colon tumors revealed that the pattern in proliferating, nonpolarized Caco-2 cells paralleled patterns seen in human colon cancer in vivo, including expression of genes involved in cell proliferation. The pattern switched in polarized Caco-2 cells to one more closely resembling that in normal colon tissue, indicating that regulation of transcription underlying Caco-2 cell polarization is similar to that during enterocyte differentiation in vivo. Surprisingly, the temporal program of gene expression in polarizing Caco-2 cells involved changes in signaling pathways (e.g., Wnt, Hh, BMP, FGF) in patterns similar to those during migration and differentiation of intestinal epithelial cells in vivo, despite the absence of morphogen gradients and interactions with stromal cells characteristic of enterocyte differentiation in situ. The full data set is available at http://microarray-pubs.stanford.edu/CACO2.

  View details for DOI 10.1091/mbc.E07-04-0309

  View details for Web of Science ID 000250740000005

  View details for PubMedID 17699589

• Transcriptional modulation of genes encoding structural characteristics of differentiating Enterocytes during development of a polarized epithelium in vitro MOLECULAR BIOLOGY OF THE CELL Halbleib, J. M., Saeaef, A. M., Brown, P. O., Nelson, W. J. 2007; 18 (11): 4261-4278

  Abstract

  Although there is considerable evidence implicating posttranslational mechanisms in the development of epithelial cell polarity, little is known about the patterns of gene expression and transcriptional regulation during this process. We characterized the temporal program of gene expression during cell-cell adhesion-initiated polarization of human Caco-2 cells in tissue culture, which develop structural and functional polarity similar to that of enterocytes in vivo. A distinctive switch in gene expression patterns occurred upon formation of cell-cell contacts between neighboring cells. Expression of genes involved in cell proliferation was down-regulated concomitant with induction of genes necessary for functional specialization of polarized epithelial cells. Transcriptional up-regulation of these latter genes correlated with formation of important structural and functional features in enterocyte differentiation and establishment of structural and functional cell polarity; components of the apical microvilli were induced as the brush border formed during polarization; as barrier function was established, expression of tight junction transmembrane proteins peaked; transcripts encoding components of the apical, but not the basal-lateral trafficking machinery were increased during polarization. Coordinated expression of genes encoding components of functional cell structures were often observed indicating temporal control of expression and assembly of multiprotein complexes.

  View details for DOI 10.1091/mbc.E07-04-0308

  View details for Web of Science ID 000250740000006

  View details for PubMedID 17699590

• Transcriptional Program Induced by Wnt Protein in Human Fibroblasts Suggests Mechanisms for Cell Cooperativity in Defining Tissue Microenvironments PLOS ONE Klapholz-Brown, Z., Walmsley, G. G., Nusse, Y. M., Nusse, R., Brown, P. O. 2007; 2 (9)

  Abstract

  The Wnt signaling system plays key roles in development, regulation of stem cell self-renewal and differentiation, cell polarity, morphogenesis and cancer. Given the multifaceted roles of Wnt signaling in these processes, its transcriptional effects on the stromal cells that make up the scaffold and infrastructure of epithelial tissues are of great interest.To begin to investigate these effects, we used DNA microarrays to identify transcriptional targets of the Wnt pathway in human lung fibroblasts. Cells were treated with active Wnt3a protein in culture, and RNA was harvested at 4 hours and 24 hours. Nuclear accumulation of ss-Catenin, as shown by immunofluorescence, and induction of AXIN2 demonstrate that fibroblasts are programmed to respond to extracellular Wnt signals. In addition to several known Wnt targets, we found many new Wnt induced genes, including many transcripts encoding regulatory proteins. Transcription factors with important developmental roles, including HOX genes, dominated the early transcriptional response. Furthermore, we found differential expression of several genes that play direct roles in the Wnt signaling pathway, as well as genes involved in other cell signaling pathways including fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) signaling. The gene most highly induced by Wnt3a was GREMLIN2, which encodes a secreted BMP antagonist.Elevated expression of GREMLIN2 suggests a new role for Wnt signals in the maintenance of stem cell niches, whereby Wnt signals induce nearby fibroblasts to produce a BMP antagonist, inhibiting differentiation and promoting expansion of stem cells in their microenvironment. We suggest that Wnt-induced changes in the gene expression program of local stromal cells may play an important role in the establishment of specialized niches hospitable to the self-renewal of normal or malignant epithelial stem cells in vivo.

  View details for DOI 10.1371/journal.pone.0000945

  View details for Web of Science ID 000207455800024

  View details for PubMedID 17895986

• Gene expression programs of human smooth muscle cells: Tissue-specific differentiation and prognostic significance in breast cancers PLOS GENETICS Chi, J., Rodriguez, E. H., Wang, Z., Nuyten, D. S., Mukherjee, S., van de Rijn, M., van de Vijver, M. J., Hastie, T., Brown, P. O. 2007; 3 (9): 1770-1784

  Abstract

  Smooth muscle is present in a wide variety of anatomical locations, such as blood vessels, various visceral organs, and hair follicles. Contraction of smooth muscle is central to functions as diverse as peristalsis, urination, respiration, and the maintenance of vascular tone. Despite the varied physiological roles of smooth muscle cells (SMCs), we possess only a limited knowledge of the heterogeneity underlying their functional and anatomic specializations. As a step toward understanding the intrinsic differences between SMCs from different anatomical locations, we used DNA microarrays to profile global gene expression patterns in 36 SMC samples from various tissues after propagation under defined conditions in cell culture. Significant variations were found between the cells isolated from blood vessels, bronchi, and visceral organs. Furthermore, pervasive differences were noted within the visceral organ subgroups that appear to reflect the distinct molecular pathways essential for organogenesis as well as those involved in organ-specific contractile and physiological properties. Finally, we sought to understand how this diversity may contribute to SMC-involving pathology. We found that a gene expression signature of the responses of vascular SMCs to serum exposure is associated with a significantly poorer prognosis in human cancers, potentially linking vascular injury response to tumor progression.

  View details for DOI 10.1371/journal.pgen.0030164

  View details for Web of Science ID 000249767800019

  View details for PubMedID 17907811

• Development of the human infant intestinal microbiota PLOS BIOLOGY Palmer, C., Bik, E. M., DiGiulio, D. B., Relman, D. A., Brown, P. O. 2007; 5 (7): 1556-1573

  Abstract

  Almost immediately after a human being is born, so too is a new microbial ecosystem, one that resides in that person's gastrointestinal tract. Although it is a universal and integral part of human biology, the temporal progression of this process, the sources of the microbes that make up the ecosystem, how and why it varies from one infant to another, and how the composition of this ecosystem influences human physiology, development, and disease are still poorly understood. As a step toward systematically investigating these questions, we designed a microarray to detect and quantitate the small subunit ribosomal RNA (SSU rRNA) gene sequences of most currently recognized species and taxonomic groups of bacteria. We used this microarray, along with sequencing of cloned libraries of PCR-amplified SSU rDNA, to profile the microbial communities in an average of 26 stool samples each from 14 healthy, full-term human infants, including a pair of dizygotic twins, beginning with the first stool after birth and continuing at defined intervals throughout the first year of life. To investigate possible origins of the infant microbiota, we also profiled vaginal and milk samples from most of the mothers, and stool samples from all of the mothers, most of the fathers, and two siblings. The composition and temporal patterns of the microbial communities varied widely from baby to baby. Despite considerable temporal variation, the distinct features of each baby's microbial community were recognizable for intervals of weeks to months. The strikingly parallel temporal patterns of the twins suggested that incidental environmental exposures play a major role in determining the distinctive characteristics of the microbial community in each baby. By the end of the first year of life, the idiosyncratic microbial ecosystems in each baby, although still distinct, had converged toward a profile characteristic of the adult gastrointestinal tract.

  View details for Web of Science ID 000249124400020

  View details for PubMedID 17594176

• Virtual Northern Analysis of the Human Genome PLOS ONE Hurowitz, E. H., Drori, I., Stodden, V. C., Donoho, D. L., Brown, P. O. 2007; 2 (5)

  Abstract

  We applied the Virtual Northern technique to human brain mRNA to systematically measure human mRNA transcript lengths on a genome-wide scale.We used separation by gel electrophoresis followed by hybridization to cDNA microarrays to measure 8,774 mRNA transcript lengths representing at least 6,238 genes at high (>90%) confidence. By comparing these transcript lengths to the Refseq and H-Invitational full-length cDNA databases, we found that nearly half of our measurements appeared to represent novel transcript variants. Comparison of length measurements determined by hybridization to different cDNAs derived from the same gene identified clones that potentially correspond to alternative transcript variants. We observed a close linear relationship between ORF and mRNA lengths in human mRNAs, identical in form to the relationship we had previously identified in yeast. Some functional classes of protein are encoded by mRNAs whose untranslated regions (UTRs) tend to be longer or shorter than average; these functional classes were similar in both human and yeast.Human transcript diversity is extensive and largely unannotated. Our length dataset can be used as a new criterion for judging the completeness of cDNAs and annotating mRNA sequences. Similar relationships between the lengths of the UTRs in human and yeast mRNAs and the functions of the proteins they encode suggest that UTR sequences serve an important regulatory role among eukaryotes.

  View details for DOI 10.1371/journal.pone.0000460

  View details for Web of Science ID 000207446000008

  View details for PubMedID 17520019

• Gene Expression Patterns in Pancreatic Tumors, Cells and Tissues PLOS ONE Lowe, A. W., Olsen, M., Hao, Y., Lee, S. P., Lee, K. T., Chen, X., van de Rijn, M., Brown, P. O. 2007; 2 (3)

  Abstract

  Cancers of the pancreas originate from both the endocrine and exocrine elements of the organ, and represent a major cause of cancer-related death. This study provides a comprehensive assessment of gene expression for pancreatic tumors, the normal pancreas, and nonneoplastic pancreatic disease.DNA microarrays were used to assess the gene expression for surgically derived pancreatic adenocarcinomas, islet cell tumors, and mesenchymal tumors. The addition of normal pancreata, isolated islets, isolated pancreatic ducts, and pancreatic adenocarcinoma cell lines enhanced subsequent analysis by increasing the diversity in gene expression profiles obtained. Exocrine, endocrine, and mesenchymal tumors displayed unique gene expression profiles. Similarities in gene expression support the pancreatic duct as the origin of adenocarcinomas. In addition, genes highly expressed in other cancers and associated with specific signal transduction pathways were also found in pancreatic tumors.The scope of the present work was enhanced by the inclusion of publicly available datasets that encompass a wide spectrum of human tissues and enabled the identification of candidate genes that may serve diagnostic and therapeutic goals.

  View details for DOI 10.1371/journal.pone.0000323

  View details for Web of Science ID 000207445200006

  View details for PubMedID 17389914

  View details for PubMedCentralID PMC1824711

• Characterization of heterotypic interaction effects in vitro to deconvolute global gene expression profiles in cancer GENOME BIOLOGY Buess, M., Nuyten, D. S., Hastie, T., Nielsen, T., Pesich, R., Brown, P. O. 2007; 8 (9)

  Abstract

  Perturbations in cell-cell interactions are a key feature of cancer. However, little is known about the systematic effects of cell-cell interaction on global gene expression in cancer.We used an ex vivo model to simulate tumor-stroma interaction by systematically co-cultivating breast cancer cells with stromal fibroblasts and determined associated gene expression changes with cDNA microarrays. In the complex picture of epithelial-mesenchymal interaction effects, a prominent characteristic was an induction of interferon-response genes (IRGs) in a subset of cancer cells. In close proximity to these cancer cells, the fibroblasts secreted type I interferons, which, in turn, induced expression of the IRGs in the tumor cells. Paralleling this model, immunohistochemical analysis of human breast cancer tissues showed that STAT1, the key transcriptional activator of the IRGs, and itself an IRG, was expressed in a subset of the cancers, with a striking pattern of elevated expression in the cancer cells in close proximity to the stroma. In vivo, expression of the IRGs was remarkably coherent, providing a basis for segregation of 295 early-stage breast cancers into two groups. Tumors with high compared to low expression levels of IRGs were associated with significantly shorter overall survival; 59% versus 80% at 10 years (log-rank p = 0.001).In an effort to deconvolute global gene expression profiles of breast cancer by systematic characterization of heterotypic interaction effects in vitro, we found that an interaction between some breast cancer cells and stromal fibroblasts can induce an interferon-response, and that this response may be associated with a greater propensity for tumor progression.

  View details for DOI 10.1186/gb-2007-8-9-r191

  View details for Web of Science ID 000252100800017

  View details for PubMedID 17868458

• Gene-expression patterns reveal underlying biological processes in Kawasaki disease GENOME BIOLOGY Popper, S. J., Shimizu, C., Shike, H., Kanegaye, J. T., Newburger, J. W., Sundel, R. P., Brown, P. O., Burns, J. C., Relman, D. A. 2007; 8 (12)

  Abstract

  Kawasaki disease (KD) is an acute self-limited vasculitis and the leading cause of acquired heart disease in children in developed countries. No etiologic agent(s) has been identified, and the processes that mediate formation of coronary artery aneurysms and abatement of fever following treatment with intravenous immunoglobulin (IVIG) remain poorly understood.In an initial survey, we used DNA microarrays to examine patterns of gene expression in peripheral whole blood from 20 children with KD; each was sampled during the acute, subacute, and convalescent phases of the illness. Acute KD was characterized by increased relative abundance of gene transcripts associated with innate immune and proinflammatory responses and decreased abundance of transcripts associated with natural killer cells and CD8+ lymphocytes. There was significant temporal variation in transcript levels during the acute disease phase and stabilization thereafter. We confirmed these temporal patterns in a second cohort of 64 patients, and identified additional inter-individual differences in transcript abundance. Notably, higher levels of transcripts of the gene for carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) were associated with an increased percentage of unsegmented neutrophils, fewer days of illness, higher levels of C-reactive protein, and subsequent non-response to IVIG; this last association was confirmed by quantitative reverse transcription PCR in a third cohort of 33 patients, and was independent of day of illness.Acute KD is characterized by dynamic and variable gene-expression programs that highlight the importance of neutrophil activation state and apoptosis in KD pathogenesis. Our findings also support the feasibility of extracting biomarkers associated with clinical prognosis from gene-expression profiles of individuals with systemic inflammatory illnesses.

  View details for DOI 10.1186/gb-2007-8-12-r261

  View details for Web of Science ID 000253451800009

  View details for PubMedID 18067656

• The temporal program of peripheral blood gene expression in the response of nonhuman primates to Ebola hemorrhagic fever GENOME BIOLOGY Rubins, K. H., Hensley, L. E., Wahl-Jensen, V., DiCaprio, K. M., Young, H. A., Reed, D. S., Jahrling, P. B., Brown, P. O., Relman, D. A., Geisbert, T. W. 2007; 8 (8)

  Abstract

  Infection with Ebola virus (EBOV) causes a fulminant and often fatal hemorrhagic fever. In order to improve our understanding of EBOV pathogenesis and EBOV-host interactions, we examined the molecular features of EBOV infection in vivo.Using high-density cDNA microarrays, we analyzed genome-wide host expression patterns in sequential blood samples from nonhuman primates infected with EBOV. The temporal program of gene expression was strikingly similar between animals. Of particular interest were features of the data that reflect the interferon response, cytokine signaling, and apoptosis. Transcript levels for tumor necrosis factor-alpha converting enzyme (TACE)/alpha-disintegrin and metalloproteinase (ADAM)-17 increased during days 4 to 6 after infection. In addition, the serum concentration of cleaved Ebola glycoprotein (GP2 delta) was elevated in late-stage EBOV infected animals. Of note, we were able to detect changes in gene expression of more than 300 genes before symptoms appeared.These results provide the first genome-wide ex vivo analysis of the host response to systemic filovirus infection and disease. These data may elucidate mechanisms of viral pathogenesis and host defense, and may suggest targets for diagnostic and therapeutic development.

  View details for DOI 10.1186/gb-2007-8-8-r174

  View details for Web of Science ID 000253938500026

  View details for PubMedID 17725815

• The Stanford Microarray Database: implementation of new analysis tools and open source release of software NUCLEIC ACIDS RESEARCH Demeter, J., Beauheim, C., Gollub, J., Hernandez-Boussard, T., Jin, H., Maier, D., Matese, J. C., Nitzberg, M., Wymore, F., Zachariah, Z. K., Brown, P. O., Sherlock, G., Ball, C. A. 2007; 35: D766-D770

  Abstract

  The Stanford Microarray Database (SMD; http://smd.stanford.edu/) is a research tool and archive that allows hundreds of researchers worldwide to store, annotate, analyze and share data generated by microarray technology. SMD supports most major microarray platforms, and is MIAME-supportive and can export or import MAGE-ML. The primary mission of SMD is to be a research tool that supports researchers from the point of data generation to data publication and dissemination, but it also provides unrestricted access to analysis tools and public data from 300 publications. In addition to supporting ongoing research, SMD makes its source code fully and freely available to others under an Open Source license, enabling other groups to create a local installation of SMD. In this article, we describe several data analysis tools implemented in SMD and we discuss features of our software release.

  View details for DOI 10.1093/nar/gkl1019

  View details for Web of Science ID 000243494600151

  View details for PubMedID 17182626

• Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sneddon, J. B., Zhen, H. H., Montgomery, K., van de Rijn, M., Tward, A. D., West, R., Gladstone, H., Chang, H. Y., Morganroth, G. S., Oro, A. E., Brown, P. O. 2006; 103 (40): 14842-14847

  Abstract

  Although tissue microenvironments play critical roles in epithelial development and tumorigenesis, the factors mediating these effects are poorly understood. In this work, we used a genomic approach to identify factors produced by cells in the microenvironment of basal cell carcinoma (BCC) of the skin, one of the most common human cancers. The global gene expression programs of stromal cell cultures derived from human BCCs showed consistent, systematic differences from those derived from nontumor skin. The gene most consistently expressed at a higher level in BCC tumor stromal cells compared with those from nontumor skin was GREMLIN 1, which encodes a secreted antagonist of the bone morphogenetic protein (BMP) pathway. BMPs and their antagonists are known to play a crucial role in stem and progenitor cell biology as regulators of the balance between expansion and differentiation. Consistent with the hypothesis that BMP antagonists might have a similar role in cancer, we found GREMLIN 1 expression in the stroma of human BCC tumors but not in normal skin in vivo. Furthermore, BMP 2 and 4 are expressed by BCC cells. Ex vivo, BMP inhibits, and Gremlin 1 promotes, proliferation of cultured BCC cells. We further found that GREMLIN 1 is expressed by stromal cells in many carcinomas but not in the corresponding normal tissue counterparts that we examined. Our data suggest that BMP antagonists may be important constituents of tumor stroma, providing a favorable microenvironment for cancer cell survival and expansion in many cancers.

  View details for DOI 10.1073/pnas.0606857103

  View details for Web of Science ID 000241069300037

  View details for PubMedID 17003113

  View details for PubMedCentralID PMC1578503

• Discovery and validation of breast cancer subtypes BMC GENOMICS Kapp, A. V., Jeffrey, S. S., Langerod, A., Borresen-Dale, A., Han, W., Noh, D., Bukholm, I. R., Nicolau, M., Brown, P. O., Tibshirani, R. 2006; 7

  Abstract

  Previous studies demonstrated breast cancer tumor tissue samples could be classified into different subtypes based upon DNA microarray profiles. The most recent study presented evidence for the existence of five different subtypes: normal breast-like, basal, luminal A, luminal B, and ERBB2+.Based upon the analysis of 599 microarrays (five separate cDNA microarray datasets) using a novel approach, we present evidence in support of the most consistently identifiable subtypes of breast cancer tumor tissue microarrays being: ESR1+/ERBB2-, ESR1-/ERBB2-, and ERBB2+ (collectively called the ESR1/ERBB2 subtypes). We validate all three subtypes statistically and show the subtype to which a sample belongs is a significant predictor of overall survival and distant-metastasis free probability.As a consequence of the statistical validation procedure we have a set of centroids which can be applied to any microarray (indexed by UniGene Cluster ID) to classify it to one of the ESR1/ERBB2 subtypes. Moreover, the method used to define the ESR1/ERBB2 subtypes is not specific to the disease. The method can be used to identify subtypes in any disease for which there are at least two independent microarray datasets of disease samples.

  View details for DOI 10.1186/1471-2164-7-231

  View details for Web of Science ID 000240732900001

  View details for PubMedID 16965636

• 2005 Curt Stern Award address. Exploring along a crooked path. American journal of human genetics Brown, P. O. 2006; 79 (3): 429-433

  View details for PubMedID 16909380

• Anatomic demarcation by positional variation in fibroblast gene expression programs PLOS GENETICS Rinn, J. L., Bondre, C., Gladstone, H. B., Brown, P. O., Chang, H. Y. 2006; 2 (7): 1084-1096

  Abstract

  Fibroblasts are ubiquitous mesenchymal cells with many vital functions during development, tissue repair, and disease. Fibroblasts from different anatomic sites have distinct and characteristic gene expression patterns, but the principles that govern their molecular specialization are poorly understood. Spatial organization of cellular differentiation may be achieved by unique specification of each cell type; alternatively, organization may arise by cells interpreting their position along a coordinate system. Here we test these models by analyzing the genome-wide gene expression profiles of primary fibroblast populations from 43 unique anatomical sites spanning the human body. Large-scale differences in the gene expression programs were related to three anatomic divisions: anterior-posterior (rostral-caudal), proximal-distal, and dermal versus nondermal. A set of 337 genes that varied according to these positional divisions was able to group all 47 samples by their anatomic sites of origin. Genes involved in pattern formation, cell-cell signaling, and matrix remodeling were enriched among this minimal set of positional identifier genes. Many important features of the embryonic pattern of HOX gene expression were retained in fibroblasts and were confirmed both in vitro and in vivo. Together, these findings suggest that site-specific variations in fibroblast gene expression programs are not idiosyncratic but rather are systematically related to their positional identities relative to major anatomic axes.

  View details for DOI 10.1371/journal.pgen.0020119

  View details for Web of Science ID 000239494800018

  View details for PubMedID 16895450

• Cell-type specific gene expression profiles of leukocytes in human peripheral blood BMC GENOMICS Palmer, C., Diehn, M., Alizadeh, A. A., Brown, P. O. 2006; 7

  Abstract

  Blood is a complex tissue comprising numerous cell types with distinct functions and corresponding gene expression profiles. We attempted to define the cell type specific gene expression patterns for the major constituent cells of blood, including B-cells, CD4+ T-cells, CD8+ T-cells, lymphocytes and granulocytes. We did this by comparing the global gene expression profiles of purified B-cells, CD4+ T-cells, CD8+ T-cells, granulocytes, and lymphocytes using cDNA microarrays.Unsupervised clustering analysis showed that similar cell populations from different donors share common gene expression profiles. Supervised analyses identified gene expression signatures for B-cells (427 genes), T-cells (222 genes), CD8+ T-cells (23 genes), granulocytes (411 genes), and lymphocytes (67 genes). No statistically significant gene expression signature was identified for CD4+ cells. Genes encoding cell surface proteins were disproportionately represented among the genes that distinguished among the lymphocyte subpopulations. Lymphocytes were distinguishable from granulocytes based on their higher levels of expression of genes encoding ribosomal proteins, while granulocytes exhibited characteristic expression of various cell surface and inflammatory proteins.The genes comprising the cell-type specific signatures encompassed many of the genes already known to be involved in cell-type specific processes, and provided clues that may prove useful in discovering the functions of many still unannotated genes. The most prominent feature of the cell type signature genes was the enrichment of genes encoding cell surface proteins, perhaps reflecting the importance of specialized systems for sensing the environment to the physiology of resting leukocytes.

  View details for DOI 10.1186/1471-2164-7-115

  View details for Web of Science ID 000238364000001

  View details for PubMedID 16704732

• Gene expression patterns in human placenta PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sood, R., Zehnder, J. L., Druzin, M. L., Brown, P. O. 2006; 103 (14): 5478-5483

  Abstract

  The placenta is the principal metabolic, respiratory, excretory, and endocrine organ for the first 9 months of fetal life. Its role in fetal and maternal physiology is remarkably diverse. Because of the central role that the placenta has in fetal and maternal physiology and development, the possibility that variation in placental gene expression patterns might be linked to important abnormalities in maternal or fetal health, or even variations in later life, warrants investigation. As an initial step, we used DNA microarrays to analyze gene expression patterns in 72 samples of amnion, chorion, umbilical cord, and sections of villus parenchyma from 19 human placentas from successful full-term pregnancies. The umbilical cord, chorion, amnion, and villus parenchyma samples were readily distinguished by differences in their global gene-expression patterns, many of which seemed to be related to physiology and histology. Differentially expressed genes have roles that include placental trophoblast secretion, signal transduction, metabolism, immune regulation, cell adhesion, and structure. We found interindividual differences in expression patterns in villus parenchyma and systematic differences between the maternal, fetal, and intermediate layers. A group of genes that was expressed in both the maternal and fetal villus parenchyma sections of placenta included genes that may be associated with preeclampsia. We identified sets of genes whose expression in placenta was significantly correlated with the sex of the fetus. This study provides a rich and diverse picture of the molecular variation in the placenta from healthy pregnancies.

  View details for DOI 10.1073/pnas.0508035103

  View details for Web of Science ID 000236636400044

  View details for PubMedID 16567644

  View details for PubMedCentralID PMC1414632

• Genome-wide identification of mRNAs associated with the translational regulator PUMILIO in Drosophila melanogaster PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gerber, A. P., Luschnig, S., Krasnow, M. A., Brown, P. O., Herschlag, D. 2006; 103 (12): 4487-4492

  Abstract

  Genome-wide identification of RNAs associated with RNA-binding proteins is crucial for deciphering posttranscriptional regulatory systems. PUMILIO is a member of the evolutionary conserved Puf-family of RNA-binding proteins that repress gene expression posttranscriptionally. We generated transgenic flies expressing affinity-tagged PUMILIO under the control of an ovary-specific promoter, and we purified PUMILIO from whole adult flies and embryos and analyzed associated mRNAs by using DNA microarrays. Distinct sets comprising hundreds of mRNAs were associated with PUMILIO at the two developmental stages. Many of these mRNAs encode functionally related proteins, supporting a model for coordinated regulation of posttranscriptional modules by specific RNA-binding proteins. We identified a characteristic sequence motif in the 3'-untranslated regions of mRNAs associated with PUMILIO, and the sufficiency of this motif for interaction with PUMILIO was confirmed by RNA pull-down experiments with biotinylated synthetic RNAs. The RNA motif strikingly resembles the one previously identified for Puf3p, one of five Saccharomyces cerevisiae Puf proteins; however, proteins encoded by the associated mRNAs in yeast and Drosophila do not appear to be related. The results suggest extensive posttranscriptional regulation by PUMILIO and uncover evolutionary features of this conserved family of RNA-binding proteins.

  View details for DOI 10.1073/pnas.0509260103

  View details for Web of Science ID 000236362600031

  View details for PubMedID 16537387

• Gene expression programs in response to hypoxia: Cell type specificity and prognostic significance in human cancers PLOS MEDICINE Chi, J. T., Wang, Z., Nuyten, D. S., Rodriguez, E. H., Schaner, M. E., Salim, A., Wang, Y., Kristensen, G. B., Helland, A., Borresen-Dale, A. L., Giaccia, A., Longaker, M. T., Hastie, T., Yang, G. P., Van de Vijver, M. J., Brown, P. O. 2006; 3 (3): 395-409

  Abstract

  Inadequate oxygen (hypoxia) triggers a multifaceted cellular response that has important roles in normal physiology and in many human diseases. A transcription factor, hypoxia-inducible factor (HIF), plays a central role in the hypoxia response; its activity is regulated by the oxygen-dependent degradation of the HIF-1alpha protein. Despite the ubiquity and importance of hypoxia responses, little is known about the variation in the global transcriptional response to hypoxia among different cell types or how this variation might relate to tissue- and cell-specific diseases.We analyzed the temporal changes in global transcript levels in response to hypoxia in primary renal proximal tubule epithelial cells, breast epithelial cells, smooth muscle cells, and endothelial cells with DNA microarrays. The extent of the transcriptional response to hypoxia was greatest in the renal tubule cells. This heightened response was associated with a uniquely high level of HIF-1alpha RNA in renal cells, and it could be diminished by reducing HIF-1alpha expression via RNA interference. A gene-expression signature of the hypoxia response, derived from our studies of cultured mammary and renal tubular epithelial cells, showed coordinated variation in several human cancers, and was a strong predictor of clinical outcomes in breast and ovarian cancers. In an analysis of a large, published gene-expression dataset from breast cancers, we found that the prognostic information in the hypoxia signature was virtually independent of that provided by the previously reported wound signature and more predictive of outcomes than any of the clinical parameters in current use.The transcriptional response to hypoxia varies among human cells. Some of this variation is traceable to variation in expression of the HIF1A gene. A gene-expression signature of the cellular response to hypoxia is associated with a significantly poorer prognosis in breast and ovarian cancer.

  View details for DOI 10.1371/journal.pmed.0030047

  View details for Web of Science ID 000236897500020

  View details for PubMedID 16417408

• A landscape effect in tenosynovial giant-cell tumor from activation of CSF1 expression by a translocation in a minority of tumor cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA West, R. B., Rubin, B. P., Miller, M. A., Subramanian, S., Kaygusuz, G., Montgomery, K., Zhu, S., Marinelli, R. J., De Luca, A., Downs-Kelly, E., Goldblum, J. R., Corless, C. L., Brown, P. O., Gilks, C. B., Nielsen, T. O., Huntsman, D., van de Rijn, M. 2006; 103 (3): 690-695

  Abstract

  Tenosynovial giant-cell tumor (TGCT) and pigmented villonodular synovitis (PVNS) are related conditions with features of both reactive inflammatory disorders and clonal neoplastic proliferations. Chromosomal translocations involving chromosome 1p13 have been reported in both TGCT and PVNS. We confirm that translocations involving 1p13 are present in a majority of cases of TGCT and PVNS and show that CSF1 is the gene at the chromosome 1p13 breakpoint. In some cases of both TGCT and PVNS, CSF1 is fused to COL6A3 (2q35). The CSF1 translocations result in overexpression of CSF1. In cases of TGCT and PVNS carrying this translocation, it is present in a minority of the intratumoral cells, leading to CSF1 expression only in these cells, whereas the majority of cells express CSF1R but not CSF1, suggesting a tumor-landscaping effect with aberrant CSF1 expression in the neoplastic cells, leading to the abnormal accumulation of nonneoplastic cells that form a tumorous mass.

  View details for DOI 10.1073/pnas.0507321103

  View details for Web of Science ID 000234727800035

  View details for PubMedID 16407111

• Minimizing off-target effects by using diced siRNAs for RNA interference. Journal of RNAi and gene silencing : an international journal of RNA and gene targeting research Myers, J. W., Chi, J., Gong, D., Schaner, M. E., Brown, P. O., Ferrell, J. E. 2006; 2 (2): 181-194

  Abstract

  Microarray studies have shown that individual synthetic small interfering RNAs (siRNAs) can have substantial off-target effects. Pools of siRNAs, produced by incubation of dsRNAs with recombinant Dicer or RNase III, can also be used to silence genes. Here we show that diced siRNA pools are highly complex, containing hundreds of different individual siRNAs. This high complexity could either compound the problem of off-target effects, since the number of potentially problematic siRNAs is high, or it could diminish the problem, since the concentration of any individual problematic siRNA is low. We therefore compared the off-target effects of diced siRNAs to chemically synthesized siRNAs. In agreement with previous reports, we found that two chemically synthesized siRNAs targeted against p38alpha MAPK (MAPK14) induced off-target changes in the abundance of hundreds of mRNAs. In contrast, three diced siRNA pools against p38alpha MAPK had almost no off-target effects. The off-target effects of a synthetic siRNA were reduced when the siRNA was diluted 3-fold in a diced pool and completely alleviated when it was diluted 30- or 300-fold, suggesting that when problematic siRNAs are present within a diced pool, their absolute concentration is too low to result in significant off-target effects. These data rationalize the observed high specificity of RNA interference in C. elegans and D. melanogaster, where gene suppression is mediated by endogenously-generated diced siRNA pools, and provide a strategy for improving the specificity of RNA interference experiments and screens in mammalian cells.

  View details for PubMedID 19771225

• Genome-scale identification of membrane-associated human mRNAs PLOS GENETICS Diehn, M., Bhattacharya, R., Botstein, D., Brown, P. O. 2006; 2 (1): 39-50

  Abstract

  The subcellular localization of proteins is critical to their biological roles. Moreover, whether a protein is membrane-bound, secreted, or intracellular affects the usefulness of, and the strategies for, using a protein as a diagnostic marker or a target for therapy. We employed a rapid and efficient experimental approach to classify thousands of human gene products as either "membrane-associated/secreted" (MS) or "cytosolic/nuclear" (CN). Using subcellular fractionation methods, we separated mRNAs associated with membranes from those associated with the soluble cytosolic fraction and analyzed these two pools by comparative hybridization to DNA microarrays. Analysis of 11 different human cell lines, representing lymphoid, myeloid, breast, ovarian, hepatic, colon, and prostate tissues, identified more than 5,000 previously uncharacterized MS and more than 6,400 putative CN genes at high confidence levels. The experimentally determined localizations correlated well with in silico predictions of signal peptides and transmembrane domains, but also significantly increased the number of human genes that could be cataloged as encoding either MS or CN proteins. Using gene expression data from a variety of primary human malignancies and normal tissues, we rationally identified hundreds of MS gene products that are significantly overexpressed in tumors compared to normal tissues and thus represent candidates for serum diagnostic tests or monoclonal antibody-based therapies. Finally, we used the catalog of CN gene products to generate sets of candidate markers of organ-specific tissue injury. The large-scale annotation of subcellular localization reported here will serve as a reference database and will aid in the rational design of diagnostic tests and molecular therapies for diverse diseases.

  View details for DOI 10.1371/journal.pgen.0020011

  View details for Web of Science ID 000239481100004

  View details for PubMedID 16415983

• Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments MOLECULAR SYSTEMS BIOLOGY Soen, Y., Mori, A., Palmer, T. D., Brown, P. O. 2006; 2

  Abstract

  Cells of a developing embryo integrate a complex array of local and long-range signals that act in concert with cell-intrinsic determinants to influence developmental decisions. To systematically investigate the effects of molecular microenvironments on cell fate decisions, we developed an experimental method based on parallel exposure of cells to diverse combinations of extracellular signals followed by quantitative, multi-parameter analysis of cellular responses. Primary human neural precursor cells were captured and cultured on printed microenvironment arrays composed of mixtures of extracellular matrix components, morphogens, and other signaling proteins. Quantitative single cell analysis revealed striking effects of some of these signals on the extent and direction of differentiation. We found that Wnt and Notch co-stimulation could maintain the cells in an undifferentiated-like, proliferative state, whereas bone morphogenetic protein 4 induced an 'indeterminate' differentiation phenotype characterized by simultaneous expression of glial and neuronal markers. Multi-parameter analysis of responses to conflicting signals revealed interactions more complex than previously envisaged including dominance relations that may reflect a cell-intrinsic system for robust specification of responses in complex microenvironments.

  View details for DOI 10.1038/msb4100076

  View details for Web of Science ID 000243245400037

  View details for PubMedID 16820778

• Predicting a local recurrence after breast-conserving therapy by gene expression profiling BREAST CANCER RESEARCH Nuyten, D. S., Kreike, B., Hart, A. A., Chi, J. A., Sneddon, J. B., Wessels, L. F., Peterse, H. J., Bartelink, H., Brown, P. O., Chang, H. Y., van de Vijver, M. J. 2006; 8 (5)

  Abstract

  To tailor local treatment in breast cancer patients there is a need for predicting ipsilateral recurrences after breast-conserving therapy. After adequate treatment (excision with free margins and radiotherapy), young age and incompletely excised extensive intraductal component are predictors for local recurrence, but many local recurrences can still not be predicted. Here we have used gene expression profiling by microarray analysis to identify gene expression profiles that can help to predict local recurrence in individual patients.By using previously established gene expression profiles with proven value in predicting metastasis-free and overall survival (wound-response signature, 70-gene prognosis profile and hypoxia-induced profile) and training towards an optimal prediction of local recurrences in a training series, we establish a classifier for local recurrence after breast-conserving therapy.Validation of the different gene lists shows that the wound-response signature is able to separate patients with a high (29%) or low (5%) risk of a local recurrence at 10 years (sensitivity 87.5%, specificity 75%). In multivariable analysis the classifier is an independent predictor for local recurrence.Our findings indicate that gene expression profiling can identify subgroups of patients at increased risk of developing a local recurrence after breast-conserving therapy.

  View details for DOI 10.1186/bcr1614

  View details for Web of Science ID 000243169100015

  View details for PubMedID 17069664

  View details for PubMedCentralID PMC1779489

• Rapid quantitative profiling of complex microbial populations NUCLEIC ACIDS RESEARCH PALMER, C., Bik, E. M., Eisen, M. B., Eckburg, P. B., Sana, T. R., Wolber, P. K., Relman, D. A., Brown, P. O. 2006; 34 (1)

  Abstract

  Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples--the current 'gold standard' method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.

  View details for DOI 10.1093/nar/gnj007

  View details for Web of Science ID 000234782300005

  View details for PubMedID 16407321

• Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray PLOS MEDICINE Chen, D. S., Soen, Y., Stuge, T. B., Lee, P. P., Weber, J. S., Brown, P. O., Davis, M. M. 2005; 2 (10): 1018-1030

  Abstract

  In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC) together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.

  View details for DOI 10.1371/journal.pmed.0020265

  View details for Web of Science ID 000233182500019

  View details for PubMedID 16162034

• Determination of stromal signatures in breast carcinoma PLOS BIOLOGY West, R. B., Nuyten, D. S., Subramanian, S., Nielsen, T. O., Corless, C. L., Rubin, B. P., Montgomery, K., Zhu, S., Patel, R., Hernandez-Boussard, T., Goldblum, J. R., Brown, P. O., van De Vijver, M., van de Rijn, M. 2005; 3 (6): 1101-1110

  Abstract

  Many soft tissue tumors recapitulate features of normal connective tissue. We hypothesize that different types of fibroblastic tumors are representative of different populations of fibroblastic cells or different activation states of these cells. We examined two tumors with fibroblastic features, solitary fibrous tumor (SFT) and desmoid-type fibromatosis (DTF), by DNA microarray analysis and found that they have very different expression profiles, including significant differences in their patterns of expression of extracellular matrix genes and growth factors. Using immunohistochemistry and in situ hybridization on a tissue microarray, we found that genes specific for these two tumors have mutually specific expression in the stroma of nonneoplastic tissues. We defined a set of 786 gene spots whose pattern of expression distinguishes SFT from DTF. In an analysis of DNA microarray gene expression data from 295 previously published breast carcinomas, we found that expression of this gene set defined two groups of breast carcinomas with significant differences in overall survival. One of the groups had a favorable outcome and was defined by the expression of DTF genes. The other group of tumors had a poor prognosis and showed variable expression of genes enriched for SFT type. Our findings suggest that the host stromal response varies significantly among carcinomas and that gene expression patterns characteristic of soft tissue tumors can be used to discover new markers for normal connective tissue cells.

  View details for DOI 10.1371/journal.pbio.0030187

  View details for Web of Science ID 000229992900019

  View details for PubMedID 15869330

• Gene expression profiling reveals molecularly and clinically distinct subtypes of glioblastoma multiforme PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Liang, Y., Diehn, M., Watson, N., Bollen, A. W., Aldape, K. D., Nicholas, M. K., Lamborn, K. R., Berger, M. S., Botstein, D., Brown, P. O., Israel, M. A. 2005; 102 (16): 5814-5819

  Abstract

  Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by genetic instability, intratumoral histopathological variability, and unpredictable clinical behavior. We investigated global gene expression in surgical samples of brain tumors. Gene expression profiling revealed large differences between normal brain samples and tumor tissues and between GBMs and lower-grade oligodendroglial tumors. Extensive differences in gene expression were found among GBMs, particularly in genes involved in angiogenesis, immune cell infiltration, and extracellular matrix remodeling. We found that the gene expression patterns in paired specimens from the same GBM invariably were more closely related to each other than to any other tumor, even when the paired specimens had strikingly divergent histologies. Survival analyses revealed a set of approximately 70 genes more highly expressed in rapidly progressing tumors that stratified GBMs into two groups that differed by >4-fold in median duration of survival. We further investigated one gene from the group, FABP7, and confirmed its association with survival in two unrelated cohorts totaling 105 patients. Expression of FABP7 enhanced the motility of glioma-derived cells in vitro. Our analyses thus identify and validate a prognostic marker of both biologic and clinical significance and provide a series of putative markers for additional evaluation.

  View details for DOI 10.1073/pnas.0402870102

  View details for Web of Science ID 000228565200034

  View details for PubMedID 15827123

• Robustness, scalability, and integration of a wound-response gene expression signature in predicting breast cancer survival PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Nuyten, D. S., Sneddon, J. B., Hastie, T., Tibshirani, R., Sorlie, T., Dai, H. Y., He, Y. D., Van't Veer, L. J., Bartelink, H., van de Rijn, M., Brown, P. O., van de Vijver, M. J. 2005; 102 (10): 3738-3743

  Abstract

  Based on the hypothesis that features of the molecular program of normal wound healing might play an important role in cancer metastasis, we previously identified consistent features in the transcriptional response of normal fibroblasts to serum, and used this "wound-response signature" to reveal links between wound healing and cancer progression in a variety of common epithelial tumors. Here, in a consecutive series of 295 early breast cancer patients, we show that both overall survival and distant metastasis-free survival are markedly diminished in patients whose tumors expressed this wound-response signature compared to tumors that did not express this signature. A gene expression centroid of the wound-response signature provides a basis for prospectively assigning a prognostic score that can be scaled to suit different clinical purposes. The wound-response signature improves risk stratification independently of known clinico-pathologic risk factors and previously established prognostic signatures based on unsupervised hierarchical clustering ("molecular subtypes") or supervised predictors of metastasis ("70-gene prognosis signature").

  View details for DOI 10.1073/pnas.0409462102

  View details for Web of Science ID 000227533100040

  View details for PubMedID 15701700

• A DNA microarray survey of gene expression in normal human tissues GENOME BIOLOGY Shyamsundar, R., Kim, Y. H., Higgins, J. P., Montgomery, K., Jorden, M., Sethuraman, A., van de Rijn, M., Botstein, D., Brown, P. O., Pollack, J. R. 2005; 6 (3)

  Abstract

  Numerous studies have used DNA microarrays to survey gene expression in cancer and other disease states. Comparatively little is known about the genes expressed across the gamut of normal human tissues. Systematic studies of global gene-expression patterns, by linking variation in the expression of specific genes to phenotypic variation in the cells or tissues in which they are expressed, provide clues to the molecular organization of diverse cells and to the potential roles of the genes.Here we describe a systematic survey of gene expression in 115 human tissue samples representing 35 different tissue types, using cDNA microarrays representing approximately 26,000 different human genes. Unsupervised hierarchical cluster analysis of the gene-expression patterns in these tissues identified clusters of genes with related biological functions and grouped the tissue specimens in a pattern that reflected their anatomic locations, cellular compositions or physiologic functions. In unsupervised and supervised analyses, tissue-specific patterns of gene expression were readily discernable. By comparative hybridization to normal genomic DNA, we were also able to estimate transcript abundances for expressed genes.Our dataset provides a baseline for comparison to diseased tissues, and will aid in the identification of tissue-specific functions. In addition, our analysis identifies potential molecular markers for detection of injury to specific organs and tissues, and provides a foundation for selection of potential targets for selective anticancer therapy.

  View details for Web of Science ID 000228246400007

  View details for PubMedID 15774023

  View details for PubMedCentralID PMC1088941

• Dissecting eukaryotic translation and its control by ribosome density mapping NUCLEIC ACIDS RESEARCH Arava, Y., Boas, F. E., Brown, P. O., Herschlag, D. 2005; 33 (8): 2421-2432

  Abstract

  Translation of an mRNA is generally divided into three stages: initiation, elongation and termination. The relative rates of these steps determine both the number and position of ribosomes along the mRNA, but traditional velocity sedimentation assays for the translational status of mRNA determine only the number of bound ribosomes. We developed a procedure, termed Ribosome Density Mapping (RDM), that uses site-specific cleavage of polysomal mRNA followed by separation on a sucrose gradient and northern analysis, to determine the number of ribosomes associated with specified portions of a particular mRNA. This procedure allows us to test models for translation and its control, and to examine properties of individual steps of translation in vivo. We tested specific predictions from the current model for translational control of GCN4 expression in yeast and found that ribosomes were differentially associated with the uORFs elements and coding region under different growth conditions, consistent with this model. We also mapped ribosome density along the ORF of several mRNAs, to probe basic kinetic properties of translational steps in yeast. We found no detectable decline in ribosome density between the 5' and 3' ends of the ORFs, suggesting that the average processivity of elongation is very high. Conversely, there was no queue of ribosomes at the termination site, suggesting that termination is not very slow relative to elongation and initiation. Finally, the RDM results suggest that less frequent initiation of translation on mRNAs with longer ORFs is responsible for the inverse correlation between ORF length and ribosomal density that we observed in a global analysis of translation. These results provide new insights into eukaryotic translation in vivo.

  View details for DOI 10.1093/nar/gki331

  View details for Web of Science ID 000229113100017

  View details for PubMedID 15860778

• Differential gene expression in anatomical compartments of the human eye GENOME BIOLOGY Diehn, J. J., Diehn, M., Marmor, M. F., Brown, P. O. 2005; 6 (9)

  Abstract

  The human eye is composed of multiple compartments, diverse in form, function, and embryologic origin, that work in concert to provide us with our sense of sight. We set out to systematically characterize the global gene expression patterns that specify the distinctive characteristics of the various eye compartments.We used DNA microarrays representing approximately 30,000 human genes to analyze gene expression in the cornea, lens, iris, ciliary body, retina, and optic nerve. The distinctive patterns of expression in each compartment could be interpreted in relation to the physiology and cellular composition of each tissue. Notably, the sets of genes selectively expressed in the retina and in the lens were particularly large and diverse. Genes with roles in immune defense, particularly complement components, were expressed at especially high levels in the anterior segment tissues. We also found consistent differences between the gene expression patterns of the macula and peripheral retina, paralleling the differences in cell layer densities between these regions. Based on the hypothesis that genes responsible for diseases that affect a particular eye compartment are likely to be selectively expressed in that compartment, we compared our gene expression signatures with genetic mapping studies to identify candidate genes for diseases affecting the cornea, lens, and retina.Through genome-scale gene expression profiling, we were able to discover distinct gene expression 'signatures' for each eye compartment and identified candidate disease genes that can serve as a reference database for investigating the physiology and pathophysiology of the eye.

  View details for DOI 10.1186/gb-2005-6-9-r74

  View details for Web of Science ID 000232301600008

  View details for PubMedID 16168081

• The Stanford Microarray Database accommodates additional microarray platforms and data formats NUCLEIC ACIDS RESEARCH Ball, C. A., Awad, I. A., Demeter, J., Gollub, J., Hebert, J. M., Hernandez-Boussard, T., Jin, H., Matese, J. C., Nitzberg, M., Wymore, F., Zachariah, Z. K., Brown, P. O., Sherlock, G. 2005; 33: D580-D582

  Abstract

  The Stanford Microarray Database (SMD) (http://smd.stanford.edu) is a research tool for hundreds of Stanford researchers and their collaborators. In addition, SMD functions as a resource for the entire biological research community by providing unrestricted access to microarray data published by SMD users and by disseminating its source code. In addition to storing GenePix (Axon Instruments) and ScanAlyze output from spotted microarrays, SMD has recently added the ability to store, retrieve, display and analyze the complete raw data produced by several additional microarray platforms and image analysis software packages, so that we can also now accept data from Affymetrix GeneChips (MAS5/GCOS or dChip), Agilent Catalog or Custom arrays (using Agilent's Feature Extraction software) or data created by SpotReader (Niles Scientific). We have implemented software that allows us to accept MAGE-ML documents from array manufacturers and to submit MIAME-compliant data in MAGE-ML format directly to ArrayExpress and GEO, greatly increasing the ease with which data from SMD can be published adhering to accepted standards and also increasing the accessibility of published microarray data to the general public. We have introduced a new tool to facilitate data sharing among our users, so that datasets can be shared during, before or after the completion of data analysis. The latest version of the source code for the complete database package was released in November 2004 (http://smd.stanford.edu/download/), allowing researchers around the world to deploy their own installations of SMD.

  View details for Web of Science ID 000226524300119

  View details for PubMedID 15608265

• The host response to smallpox: Analysis of the gene expression program in peripheral blood cells in a nonhuman primate model PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rubins, K. H., Hensley, L. E., JAHRLING, P. B., Whitney, A. R., Geisbert, T. W., HUGGINS, J. W., Owen, A., LEDUC, J. W., Brown, P. O., Relman, D. A. 2004; 101 (42): 15190-15195

  Abstract

  Smallpox has played an unparalleled role in human history and remains a significant potential threat to public health. Despite the historical significance of this disease, we know little about the underlying pathophysiology or the virulence mechanisms of the causative agent, variola virus. To improve our understanding of variola pathogenesis and variola-host interactions, we examined the molecular and cellular features of hemorrhagic smallpox in cynomolgus macaques. We used cDNA microarrays to analyze host gene expression patterns in sequential blood samples from each of 22 infected animals. Variola infection elicited striking and temporally coordinated patterns of gene expression in peripheral blood. Of particular interest were features that appear to represent an IFN response, cell proliferation, immunoglobulin gene expression, viral dose-dependent gene expression patterns, and viral modulation of the host immune response. The virtual absence of a tumor necrosis factor alpha/NF-kappaB-activated transcriptional program in the face of an overwhelming systemic infection suggests that variola gene products may ablate this response. These results provide a detailed picture of the host transcriptional response during smallpox infection, and may help guide the development of diagnostic, therapeutic, and prophylactic strategies.

  View details for DOI 10.1073/pnas.0405759101

  View details for Web of Science ID 000224688700039

  View details for PubMedID 15477590

• A method for detecting and correcting feature misidentification on expression microarrays BMC GENOMICS Tu, I. P., Schaner, M., Diehn, M., Sikic, B. I., Brown, P. O., Botstein, D., Fero, M. J. 2004; 5

  Abstract

  Much of the microarray data published at Stanford is based on mouse and human arrays produced under controlled and monitored conditions at the Brown and Botstein laboratories and at the Stanford Functional Genomics Facility (SFGF). Nevertheless, as large datasets based on the Stanford Human array began to accumulate, a small but significant number of discrepancies were detected that required a serious attempt to track down the original source of error. Due to a controlled process environment, sufficient data was available to accurately track the entire process leading to up to the final expression data. In this paper, we describe our statistical methods to detect the inconsistencies in microarray data that arise from process errors, and discuss our technique to locate and fix these errors.To date, the Brown and Botstein laboratories and the Stanford Functional Genomics Facility have together produced 40,000 large-scale (10-50,000 feature) cDNA microarrays. By applying the heuristic described here, we have been able to check most of these arrays for misidentified features, and have been able to confidently apply fixes to the data where needed. Out of the 265 million features checked in our database, problems were detected and corrected on 1.3 million of them.Process errors in any genome scale high throughput production regime can lead to subsequent errors in data analysis. We show the value of tracking multi-step high throughput operations by using this knowledge to detect and correct misidentified data on gene expression microarrays.

  View details for DOI 10.1186/1471-2164-5-64

  View details for Web of Science ID 000224203400001

  View details for PubMedID 15357875

• A specific gene expression program triggered by gram-positive bacteria in the cytosol PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA McCaffrey, R. L., Fawcett, P., O'Riordan, M., Lee, K. D., Havell, E. A., Brown, P. O., Portnoy, D. A. 2004; 101 (31): 11386-11391

  Abstract

  Innate and adaptive immunity depends critically on host recognition of pathogen-associated molecules. Toll-like receptors (TLRs) are key mediators of pathogen surveillance at the cell or phagocytic vacuole surface. However, mechanisms underlying recognition of pathogens in other cellular compartments remain unclear, and responses elicited by cytosolic challenge are poorly characterized. We therefore used mouse cDNA microarrays to investigate gene expression triggered by infection of bone marrow-derived macrophages with cytosol- and vacuole-localized Listeria monocytogenes (Lm), a model cytosolic pathogen. The resulting gene expression program included two basic categories of induced genes: an "early/persistent" cluster consistent with NF-kappaB-dependent responses downstream of TLRs, and a subsequent "late response" cluster largely composed of IFN-responsive genes (IRGs). The early/persistent cluster was observed upon infection with WT, heat-killed, or mutant Lm lacking listeriolysin O, the pore-forming hemolysin that promotes escape from phagocytic vacuoles. However, the IRG cluster depended on entry of WT Lm into the cytosol. Infection with listeriolysin O-expressing, cytosolic Bacillus subtilis (Bs) strikingly recapitulated the expression profile associated with WT Lm, including IRG induction. IRG up-regulation was associated with MyD88-independent induction of IFN-beta transcription and activity. Whereas Staphylococcus aureus (Sa) lipoteichoic acid treatment confirmed that many late-response genes could also be stimulated through TLRs, our study identified a cytosol-specific transcriptional program independent of TLR signaling through MyD88. Further characterization of cytosolic surveillance pathway(s) and their points of convergence with TLR- and IFN-dependent pathways will enhance our understanding of the means by which mammals detect and respond to pathogens.

  View details for DOI 10.1073/pnas.0403215101

  View details for Web of Science ID 000223134400037

  View details for PubMedID 15269347

• Apo D in soft tissue tumors - A novel marker for dermatofibrosarcoma protuberans AMERICAN JOURNAL OF SURGICAL PATHOLOGY West, R. B., Harvell, J., Linn, S. C., Lui, C. L., Prapong, W., Hernandez-Boussard, T., Montgomery, K., Nielsen, T. O., Rubin, B. P., Patel, R., Goldblum, J. R., Brown, P. O., van de Rijn, M. 2004; 28 (8): 1063-1069

  Abstract

  Using gene microarray expression profiling, we previously found that apolipoprotein D (Apo D) was highly expressed in dermatofibrosarcoma protuberans (DFSP). In this study, we confirm that Apo D is highly and relatively specifically expressed in DFSP using immunohistochemistry. A tissue microarray containing 421 soft tissue tumors was constructed and stained with antibodies against Apo D and CD34. Cytoplasmic immunostaining for Apo D was found in 9 of 10 typical DFSPs. In addition, 3 of 3 Bednar tumors and 2 of 3 giant cell fibroblastomas stained in conventional sections. In contrast, Apo D was immunoreactive in only a very small subset of a diverse collection of other soft tissue tumors, including Malignant Fibrous Histiocytoma (MFH), glomus tumor, neurofibroma, and malignant peripheral nerve sheath tumors. Immunostains for Apo D were negative in conventional sections of 16 fibrous histiocytomas, and an additional 12 variants of fibrous histiocytoma. Digital images of all immunohistochemical and hematoxylin and eosin tissue microarray stains are available at the accompanying website (http://microarray-pubs.stanford.edu/tma_portal/apod/). We conclude that Apo D is strongly expressed in DFSPs and neural lesions and may be useful in differentiating DFSP from fibrous histiocytoma.

  View details for Web of Science ID 000222891400012

  View details for PubMedID 15252314

• The novel marker, DOG1, is expressed ubiquitously in gastrointestinal stromal tumors irrespective of KIT or PDGFRA mutation status AMERICAN JOURNAL OF PATHOLOGY West, R. B., Corless, C. L., Chen, X., Rubin, B. P., Subramanian, S., Montgomery, K., Zhu, S., Ball, C. A., Nielsen, T. O., Patel, R., Goldblum, J. R., Brown, P. O., Heinrich, M. C., van de Rijn, M. 2004; 165 (1): 107-113

  Abstract

  We recently characterized gene expression patterns in gastrointestinal stromal tumors (GISTs) using cDNA microarrays, and found that the gene FLJ10261 (DOG1, discovered on GIST-1), encoding a hypothetical protein, was specifically expressed in GISTs. The immunoreactivity of a rabbit antiserum to synthetic DOG1 peptides was assessed on two soft tissue tumor microarrays. The tissue microarrays included 587 soft tissue tumors, with 149 GISTs, including 127 GIST cases for which the KIT and PDGFRA mutation status was known. Immunoreactivity for DOG1 was found in 136 of 139 (97.8%) of scorable GISTs. All seven GIST cases with a PDGFRA mutation were DOG1-positive, while most of these failed to react for KIT. The immunohistochemical findings were confirmed with in situ hybridization probes for DOG1, KIT, and PDGFRA. Other neoplasms in the differential diagnosis of GIST, including desmoid fibromatosis (0 of 17) and Schwannoma (0 of 3), were immunonegative for DOG1. Only 4 of 438 non-GIST cases were immunoreactive for DOG1. DOG1, a protein of unknown function, is expressed strongly on the cell surface of GISTs and is rarely expressed in other soft tissue tumors. Reactivity for DOG1 may aid in the diagnosis of GISTs, including PDGFRA mutants that fail to express KIT antigen, and lead to appropriate treatment with imatinib mesylate, an inhibitor of the KIT tyrosine kinase.

  View details for Web of Science ID 000222216000010

  View details for PubMedID 15215166

• Diverse and specific gene expression responses to stresses in cultured human cells MOLECULAR BIOLOGY OF THE CELL Murray, J. I., Whitfield, M. L., Trinklein, N. D., Myers, R. M., Brown, P. O., Botstein, D. 2004; 15 (5): 2361-2374

  Abstract

  We used cDNA microarrays in a systematic study of the gene expression responses of HeLa cells and primary human lung fibroblasts to heat shock, endoplasmic reticulum stress, oxidative stress, and crowding. Hierarchical clustering of the data revealed groups of genes with coherent biological themes, including genes that responded to specific stresses and others that responded to multiple types of stress. Fewer genes increased in expression after multiple stresses than in free-living yeasts, which have a large general stress response program. Most of the genes induced by multiple diverse stresses are involved in cell-cell communication and other processes specific to higher organisms. We found substantial differences between the stress responses of HeLa cells and primary fibroblasts. For example, many genes were induced by oxidative stress and dithiothreitol in fibroblasts but not HeLa cells; conversely, a group of transcription factors, including c-fos and c-jun, were induced by heat shock in HeLa cells but not in fibroblasts. The dataset is freely available for search and download at http://microarray-pubs.stanford.edu/human_stress/Home.shtml.

  View details for DOI 10.1091/mbc.E03-11-0799

  View details for Web of Science ID 000221189300027

  View details for PubMedID 15004229

• Presynaptic homeostasis at CNS nerve terminals compensates for lack of a key Ca2+ entry pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Piedras-Renteria, E. S., Pyle, J. L., Diehn, M., Glickfeld, L. L., Harata, N. C., Cao, Y. Q., Kavalali, E. T., Brown, P. O., Tsien, R. W. 2004; 101 (10): 3609-3614

  Abstract

  At central synapses, P/Q-type Ca(2+) channels normally provide a critical Ca(2+) entry pathway for neurotransmission. Nevertheless, we found that nerve terminals lacking alpha(1A) (Ca(V)2.1), the pore-forming subunit of P/Q-type channels, displayed a remarkable preservation of synaptic function. Two consistent physiological changes reflective of synaptic homeostasis were observed in cultured hippocampal neurons derived from alpha(1A) (-/-) mice. First, the presynaptic response to an ionophore-mediated Ca(2+) elevation was 50% greater, indicating an enhanced Ca(2+) sensitivity of the release machinery. Second, basal miniature excitatory postsynaptic current frequency in alpha(1A) (-/-) neurons was increased 2-fold compared with WT neurons and occluded the normal response of presynaptic terminals to cAMP elevation, suggesting that the compensatory mechanism in alpha(1A) (-/-) synapses and the modulation of presynaptic function by PKA might share a final common pathway. We used cDNA microarray analysis to identify molecular changes underlying homeostatic regulation in the alpha(1A) (-/-) hippocampus. The 40,000 entries in our custom-made array included likely targets of presynaptic homeostasis, along with many other transcripts, allowing a wide-ranging examination of gene expression. The developmental pattern of changes in transcript levels relative to WT was striking; mRNAs at 5 and 11 days postnatal showed little deviation, but clear differences emerged by 22 days. Many of the transcripts that differed significantly in abundance corresponded to known genes that could be incorporated within a logical pattern consistent with the modulation of presynaptic function. Changes in endocytotic proteins, signal transduction kinases, and candidates for Ca(2+)-sensing molecules were consistent with implications of the direct physiological experiments.

  View details for DOI 10.1073/pnas.0308188100

  View details for Web of Science ID 000220163800052

  View details for PubMedID 14990796

• Cancer characterization and feature set extraction by discriminative margin clustering BMC BIOINFORMATICS Munagala, K., Tibshirani, R., O Brown, P. 2004; 5

  Abstract

  A central challenge in the molecular diagnosis and treatment of cancer is to define a set of molecular features that, taken together, distinguish a given cancer, or type of cancer, from all normal cells and tissues.Discriminative margin clustering is a new technique for analyzing high dimensional quantitative datasets, specially applicable to gene expression data from microarray experiments related to cancer. The goal of the analysis is find highly specialized sub-types of a tumor type which are similar in having a small combination of genes which together provide a unique molecular portrait for distinguishing the sub-type from any normal cell or tissue. Detection of the products of these genes can then, in principle, provide a basis for detection and diagnosis of a cancer, and a therapy directed specifically at the distinguishing constellation of molecular features can, in principle, provide a way to eliminate the cancer cells, while minimizing toxicity to any normal cell.The new methodology yields highly specialized tumor subtypes which are similar in terms of potential diagnostic markers.

  View details for Web of Science ID 000220984700002

  View details for PubMedID 15070405

• Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast. PLoS biology Gerber, A. P., Herschlag, D., Brown, P. O. 2004; 2 (3): E79-?

  Abstract

  Genes encoding RNA-binding proteins are diverse and abundant in eukaryotic genomes. Although some have been shown to have roles in post-transcriptional regulation of the expression of specific genes, few of these proteins have been studied systematically. We have used an affinity tag to isolate each of the five members of the Puf family of RNA-binding proteins in Saccharomyces cerevisiae and DNA microarrays to comprehensively identify the associated mRNAs. Distinct groups of 40-220 different mRNAs with striking common themes in the functions and subcellular localization of the proteins they encode are associated with each of the five Puf proteins: Puf3p binds nearly exclusively to cytoplasmic mRNAs that encode mitochondrial proteins; Puf1p and Puf2p interact preferentially with mRNAs encoding membrane-associated proteins; Puf4p preferentially binds mRNAs encoding nucleolar ribosomal RNA-processing factors; and Puf5p is associated with mRNAs encoding chromatin modifiers and components of the spindle pole body. We identified distinct sequence motifs in the 3'-untranslated regions of the mRNAs bound by Puf3p, Puf4p, and Puf5p. Three-hybrid assays confirmed the role of these motifs in specific RNA-protein interactions in vivo. The results suggest that combinatorial tagging of transcripts by specific RNA-binding proteins may be a general mechanism for coordinated control of the localization, translation, and decay of mRNAs and thus an integral part of the global gene expression program.

  View details for PubMedID 15024427

• Extensive association of functionally and cytotopically related mRNAs with Puf family RNA-binding proteins in yeast PLOS BIOLOGY Gerber, A. P., Herschlag, D., Brown, P. O. 2004; 2 (3): 342-354

  View details for DOI 10.1371/journal.pbio.0020079

  View details for Web of Science ID 000220512000010

• Transcriptional remodeling in response to iron deprivation in Saccharomyces cerevisiae MOLECULAR BIOLOGY OF THE CELL Shakoury-Elizeh, M., Tiedeman, J., Rashford, J., Ferea, T., Demeter, J., Garcia, E., Rolfes, R., Brown, P. O., Botstein, D., Philpott, C. C. 2004; 15 (3): 1233-1243

  Abstract

  The budding yeast Saccharomyces cerevisiae responds to depletion of iron in the environment by activating Aft1p, the major iron-dependent transcription factor, and by transcribing systems involved in the uptake of iron. Here, we have studied the transcriptional response to iron deprivation and have identified new Aft1p target genes. We find that other metabolic pathways are regulated by iron: biotin uptake and biosynthesis, nitrogen assimilation, and purine biosynthesis. Two enzymes active in these pathways, biotin synthase and glutamate synthase, require an iron-sulfur cluster for activity. Iron deprivation activates transcription of the biotin importer and simultaneously represses transcription of the entire biotin biosynthetic pathway. Multiple genes involved in nitrogen assimilation and amino acid metabolism are induced by iron deprivation, whereas glutamate synthase, a key enzyme in nitrogen assimilation, is repressed. A CGG palindrome within the promoter of glutamate synthase confers iron-regulated expression, suggesting control by a transcription factor of the binuclear zinc cluster family. We provide evidence that yeast subjected to iron deprivation undergo a transcriptional remodeling, resulting in a shift from iron-dependent to parallel, but iron-independent, metabolic pathways.

  View details for DOI 10.1091/mbc.E03-09-0642

  View details for Web of Science ID 000220051900027

  View details for PubMedID 14668481

• Gene expression signature of fibroblast serum response predicts human cancer progression: similarities between tumors and wounds. PLoS biology Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): E7-?

  Abstract

  Cancer invasion and metastasis have been likened to wound healing gone awry. Despite parallels in cellular behavior between cancer progression and wound healing, the molecular relationships between these two processes and their prognostic implications are unclear. In this study, based on gene expression profiles of fibroblasts from ten anatomic sites, we identify a stereotyped gene expression program in response to serum exposure that appears to reflect the multifaceted role of fibroblasts in wound healing. The genes comprising this fibroblast common serum response are coordinately regulated in many human tumors, allowing us to identify tumors with gene expression signatures suggestive of active wounds. Genes induced in the fibroblast serum-response program are expressed in tumors by the tumor cells themselves, by tumor-associated fibroblasts, or both. The molecular features that define this wound-like phenotype are evident at an early clinical stage, persist during treatment, and predict increased risk of metastasis and death in breast, lung, and gastric carcinomas. Thus, the transcriptional signature of the response of fibroblasts to serum provides a possible link between cancer progression and wound healing, as well as a powerful predictor of the clinical course in several common carcinomas.

  View details for PubMedID 14737219

  View details for PubMedCentralID PMC314300

• Gene expression signature of fibroblast serum response predicts human cancer progression: Similarities between tumors and wounds PLOS BIOLOGY Chang, H. Y., Sneddon, J. B., Alizadeh, A. A., Sood, R., West, R. B., Montgomery, K., Chi, J. T., van de Rijn, M., Botstein, D., Brown, P. O. 2004; 2 (2): 206-214

  View details for DOI 10.1371/journal.pbio.0020007

  View details for Web of Science ID 000189314400013

• Gene expression in the normal adult human kidney assessed by complementary DNA microarray MOLECULAR BIOLOGY OF THE CELL Higgins, J. P., Wang, L. L., Kambham, N., Montgomery, K., Mason, V., Vogelmann, S. U., Lemley, K. V., Brown, P. O., Brooks, J. D., van de Rijn, M. 2004; 15 (2): 649-656

  Abstract

  The kidney is a highly specialized organ with a complex, stereotyped architecture and a great diversity of functions and cell types. Because the microscopic organization of the nephron, the functional unit of the kidney, has a consistent relationship to the macroscopic anatomy of the kidney, knowledge of the characteristic patterns of gene expression in different compartments of the kidney could provide insight into the functions and functional organization of the normal nephron. We studied gene expression in dissected renal lobes of five adult human kidneys using cDNA microarrays representing approximately 30,000 different human genes. Total RNA was isolated from sections of the inner and outer cortex, inner and outer medulla, papillary tips, and renal pelvis and from glomeruli isolated by sieving. The results revealed unique and highly distinctive patterns of gene expression for glomeruli, cortex, medulla, papillary tips, and pelvic samples. Immunohistochemical staining using selected antisera confirmed differential expression of several cognate proteins and provided histological localization of expression within the nephron. The distinctive patterns of gene expression in discrete portions of the kidney may serve as a resource for further understanding of renal physiology and the molecular and cellular organization of the nephron.

  View details for DOI 10.1091/mbc.E03-06-0432

  View details for Web of Science ID 000188718900024

  View details for PubMedID 14657249

  View details for PubMedCentralID PMC329285

• Gene expression profiling identifies clinically relevant subtypes of prostate cancer PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lapointe, J., Li, C., Higgins, J. P., van de Rijn, M., Bair, E., Montgomery, K., Ferrari, M., Egevad, L., Rayford, W., Bergerheim, U., Ekman, P., DeMarzo, A. M., Tibshirani, R., Botstein, D., Brown, P. O., Brooks, J. D., Pollack, J. R. 2004; 101 (3): 811-816

  Abstract

  Prostate cancer, a leading cause of cancer death, displays a broad range of clinical behavior from relatively indolent to aggressive metastatic disease. To explore potential molecular variation underlying this clinical heterogeneity, we profiled gene expression in 62 primary prostate tumors, as well as 41 normal prostate specimens and nine lymph node metastases, using cDNA microarrays containing approximately 26,000 genes. Unsupervised hierarchical clustering readily distinguished tumors from normal samples, and further identified three subclasses of prostate tumors based on distinct patterns of gene expression. High-grade and advanced stage tumors, as well as tumors associated with recurrence, were disproportionately represented among two of the three subtypes, one of which also included most lymph node metastases. To further characterize the clinical relevance of tumor subtypes, we evaluated as surrogate markers two genes differentially expressed among tumor subgroups by using immunohistochemistry on tissue microarrays representing an independent set of 225 prostate tumors. Positive staining for MUC1, a gene highly expressed in the subgroups with "aggressive" clinicopathological features, was associated with an elevated risk of recurrence (P = 0.003), whereas strong staining for AZGP1, a gene highly expressed in the other subgroup, was associated with a decreased risk of recurrence (P = 0.0008). In multivariate analysis, MUC1 and AZGP1 staining were strong predictors of tumor recurrence independent of tumor grade, stage, and preoperative prostate-specific antigen levels. Our results suggest that prostate tumors can be usefully classified according to their gene expression patterns, and these tumor subtypes may provide a basis for improved prognostication and treatment stratification.

  View details for DOI 10.1073/pnas.0304146101

  View details for Web of Science ID 000188555400023

  View details for PubMedID 14711987

  View details for PubMedCentralID PMC321763

• Genomic transcriptional response to loss of chromosomal supercoiling in Escherichia coli GENOME BIOLOGY Peter, B. J., Arsuaga, J., Breier, A. M., Khodursky, A. B., Brown, P. O., Cozzarelli, N. R. 2004; 5 (11)

  Abstract

  The chromosome of Escherichia coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. In turn, supercoiling influences local DNA structure and can affect gene expression. We used microarrays representing nearly the entire genome of Escherichia coli MG1655 to examine the dynamics of chromosome structure.We measured the transcriptional response to a loss of supercoiling caused either by genetic impairment of a topoisomerase or addition of specific topoisomerase inhibitors during log-phase growth and identified genes whose changes are statistically significant. Transcription of 7% of the genome (306 genes) was rapidly and reproducibly affected by changes in the level of supercoiling; the expression of 106 genes increased upon chromosome relaxation and the expression of 200 decreased. These changes are most likely to be direct effects, as the kinetics of their induction or repression closely follow the kinetics of DNA relaxation in the cells. Unexpectedly, the genes induced by relaxation have a significantly enriched AT content in both upstream and coding regions.The 306 supercoiling-sensitive genes are functionally diverse and widely dispersed throughout the chromosome. We propose that supercoiling acts as a second messenger that transmits information about the environment to many regulatory networks in the cell.

  View details for Web of Science ID 000224855300008

  View details for PubMedID 15535863

• Genome-wide analysis of mRNA lengths in Saccharomyces cerevisiae GENOME BIOLOGY Hurowitz, E. H., Brown, P. O. 2004; 5 (1)

  View details for Web of Science ID 000187747000008

• PLoS Medicine - a medical journal for the internet age PLoS Medicine Eisen MB, Brown PO, Varmus HE 2004; 1 (1): e31
• Gene expression patterns and gene copy number changes in dermatofibrosarcoma protuberans AMERICAN JOURNAL OF PATHOLOGY Linn, S. C., West, R. B., Pollack, J. R., Zhu, S., Hernandez-Boussard, T., Nielsen, T. O., Rubin, B. P., Patel, R., Goldblum, J. R., Siegmund, D., Botstein, D., Brown, P. O., Gilks, C. B., van de Rijn, M. 2003; 163 (6): 2383-2395

  Abstract

  Dermatofibrosarcoma protuberans (DFSP) is an aggressive spindle cell neoplasm. It is associated with the chromosomal translocation, t(17:22), which fuses the COL1A1 and PDGFbeta genes. We determined the characteristic gene expression profile of DFSP and characterized DNA copy number changes in DFSP by array-based comparative genomic hybridization (array CGH). Fresh frozen and formalin-fixed, paraffin-embedded samples of DFSP were analyzed by array CGH (four cases) and DNA microarray analysis of global gene expression (nine cases). The nine DFSPs were readily distinguished from 27 other diverse soft tissue tumors based on their gene expression patterns. Genes characteristically expressed in the DFSPs included PDGF beta and its receptor, PDGFRB, APOD, MEOX1, PLA2R, and PRKCA. Array CGH of DNA extracted either from frozen tumor samples or from paraffin blocks yielded equivalent results. Large areas of chromosomes 17q and 22q, bounded by COL1A1 and PDGF beta, respectively, were amplified in DFSP. Expression of genes in the amplified regions was significantly elevated. Our data shows that: 1) DFSP has a distinctive gene expression profile; 2) array CGH can be applied successfully to frozen or formalin-fixed, paraffin-embedded tumor samples; 3) a characteristic amplification of sequences from chromosomes 17q and 22q, demarcated by the COL1A1 and PDGF beta genes, respectively, was associated with elevated expression of the amplified genes.

  View details for Web of Science ID 000186769800024

  View details for PubMedID 14633610

• Detection and characterization of cellular immune responses using peptide-MHC microarrays. PLoS biology Soen, Y., Chen, D. S., Kraft, D. L., Davis, M. M., Brown, P. O. 2003; 1 (3): E65-?

  Abstract

  The detection and characterization of antigen-specific T cell populations is critical for understanding the development and physiology of the immune system and its responses in health and disease. We have developed and tested a method that uses arrays of peptide-MHC complexes for the rapid identification, isolation, activation, and characterization of multiple antigen-specific populations of T cells. CD4(+) or CD8(+) lymphocytes can be captured in accordance with their ligand specificity using an array of peptide-MHC complexes printed on a film-coated glass surface. We have characterized the specificity and sensitivity of a peptide-MHC array using labeled lymphocytes from T cell receptor transgenic mice. In addition, we were able to use the array to detect a rare population of antigen-specific T cells following vaccination of a normal mouse. This approach should be useful for epitope discovery, as well as for characterization and analysis of multiple epitope-specific T cell populations during immune responses associated with viral and bacterial infection, cancer, autoimmunity, and vaccination.

  View details for PubMedID 14691537

• Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sperger, J. M., Chen, X., Draper, J. S., Antosiewicz, J. E., Chon, C. H., Jones, S. B., Brooks, J. D., Andrews, P. W., Brown, P. O., Thomson, J. A. 2003; 100 (23): 13350-13355

  Abstract

  Remarkably little is known about the transcriptional profiles of human embryonic stem (ES) cells or the molecular mechanisms that underlie their pluripotency. To identify commonalties among the transcriptional profiles of different human pluripotent cells and to search for clues into the genesis of human germ cell tumors, we compared the expression profiles of human ES cell lines, human germ cell tumor cell lines and tumor samples, somatic cell lines, and testicular tissue samples by using cDNA microarray analysis. Hierarchical cluster analysis of gene expression profiles showed that the five independent human ES cell lines clustered tightly together, reflecting highly similar expression profiles. The gene expression patterns of human ES cell lines showed many similarities with the human embryonal carcinoma cell samples and more distantly with the seminoma samples. We identified 895 genes that were expressed at significantly greater levels in human ES and embryonal carcinoma cell lines than in control samples. These genes are candidates for involvement in the maintenance of a pluripotent, undifferentiated phenotype.

  View details for DOI 10.1073/pnas.2235735100

  View details for Web of Science ID 000186573700044

  View details for PubMedID 14595015

  View details for PubMedCentralID PMC263817

• Gene expression patterns in ovarian carcinomas MOLECULAR BIOLOGY OF THE CELL Schaner, M. E., Ross, D. T., Ciaravino, G., Sorlie, T., Troyanskaya, O., Diehn, M., Wang, Y. C., Duran, G. E., Sikic, T. L., Caldeira, S., Skomedal, H., Tu, I. P., Hernandez-Boussard, T., Johnson, S. W., O'Dwyer, P. J., Fero, M. J., Kristensen, G. B., Borresen-Dale, A. L., Hastie, T., Tibshirani, R., van de Rijn, M., Teng, N. N., Longacre, T. A., Botstein, D., Brown, P. O., Sikic, B. I. 2003; 14 (11): 4376-4386

  Abstract

  We used DNA microarrays to characterize the global gene expression patterns in surface epithelial cancers of the ovary. We identified groups of genes that distinguished the clear cell subtype from other ovarian carcinomas, grade I and II from grade III serous papillary carcinomas, and ovarian from breast carcinomas. Six clear cell carcinomas were distinguished from 36 other ovarian carcinomas (predominantly serous papillary) based on their gene expression patterns. The differences may yield insights into the worse prognosis and therapeutic resistance associated with clear cell carcinomas. A comparison of the gene expression patterns in the ovarian cancers to published data of gene expression in breast cancers revealed a large number of differentially expressed genes. We identified a group of 62 genes that correctly classified all 125 breast and ovarian cancer specimens. Among the best discriminators more highly expressed in the ovarian carcinomas were PAX8 (paired box gene 8), mesothelin, and ephrin-B1 (EFNB1). Although estrogen receptor was expressed in both the ovarian and breast cancers, genes that are coregulated with the estrogen receptor in breast cancers, including GATA-3, LIV-1, and X-box binding protein 1, did not show a similar pattern of coexpression in the ovarian cancers.

  View details for Web of Science ID 000186738300005

  View details for PubMedID 12960427

• T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression PLOS BIOLOGY Roose, J. P., Diehn, M., Tomlinson, M. G., Lin, J., Alizadeh, A. A., Botstein, D., Brown, P. O., Weiss, A. 2003; 1 (2): 271-287

  View details for DOI 10.1371/journal.pbio.0000053

  View details for Web of Science ID 000188835200018

• T cell receptor-independent basal signaling via Erk and Abl kinases suppresses RAG gene expression. PLoS biology Roose, J. P., Diehn, M., Tomlinson, M. G., Lin, J., Alizadeh, A. A., Botstein, D., Brown, P. O., Weiss, A. 2003; 1 (2): E53-?

  Abstract

  Signal transduction pathways guided by cellular receptors commonly exhibit low-level constitutive signaling in a continuous, ligand-independent manner. The dynamic equilibrium of positive and negative regulators establishes such a tonic signal. Ligand-independent signaling by the precursors of mature antigen receptors regulates development of B and T lymphocytes. Here we describe a basal signal that controls gene expression profiles in the Jurkat T cell line and mouse thymocytes. Using DNA microarrays and Northern blots to analyze unstimulated cells, we demonstrate that expression of a cluster of genes, including RAG-1 and RAG-2, is repressed by constitutive signals requiring the adapter molecules LAT and SLP-76. This TCR-like pathway results in constitutive low-level activity of Erk and Abl kinases. Inhibition of Abl by the drug STI-571 or inhibition of signaling events upstream of Erk increases RAG-1 expression. Our data suggest that physiologic gene expression programs depend upon tonic activity of signaling pathways independent of receptor ligation.

  View details for PubMedID 14624253

• Systemic and cell type-specific gene expression patterns in scleroderma skin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Whitfield, M. L., Finlay, D. R., Murray, J. I., Troyanskaya, O. G., Chi, J. T., Pergamenschikov, A., McCalmont, T. H., Brown, P. O., Botstein, D., Connolly, M. K. 2003; 100 (21): 12319-12324

  Abstract

  We used DNA microarrays representing >12,000 human genes to characterize gene expression patterns in skin biopsies from individuals with a diagnosis of systemic sclerosis with diffuse scleroderma. We found consistent differences in the patterns of gene expression between skin biopsies from individuals with scleroderma and those from normal, unaffected individuals. The biopsies from affected individuals showed nearly indistinguishable patterns of gene expression in clinically affected and clinically unaffected tissue, even though these were clearly distinguishable from the patterns found in similar tissue from unaffected individuals. Genes characteristically expressed in endothelial cells, B lymphocytes, and fibroblasts showed differential expression between scleroderma and normal biopsies. Analysis of lymphocyte populations in scleroderma skin biopsies by immunohistochemistry suggest the B lymphocyte signature observed on our arrays is from CD20+ B cells. These results provide evidence that scleroderma has systemic manifestations that affect multiple cell types and suggests genes that could be used as potential markers for the disease.

  View details for DOI 10.1073/pnas.1635114100

  View details for Web of Science ID 000186024300070

  View details for PubMedID 14530402

• Tissue microarray validation of epidermal growth factor receptor and SALL2 in synovial sarcoma with comparison to tumors of similar histology AMERICAN JOURNAL OF PATHOLOGY Nielsen, T. O., Hsu, F. D., O'Connell, J. X., Gilks, C. B., Sorensen, P. H., Linn, S., West, R. B., Liu, C. L., Botstein, D., Brown, P. O., van de Rijn, M. 2003; 163 (4): 1449-1456

  Abstract

  Histological diagnosis of synovial sarcoma can be difficult. Genome-wide expression profiling has identified a number of genes expressed at higher levels in synovial sarcoma than in other soft tissue tumors, representing excellent candidates for diagnostic immunohistochemical markers. A tissue microarray comprising 77 sarcomas, including 46 synovial sarcomas, was constructed to validate identified markers and investigate their expression in tumors in the differential diagnosis of synovial sarcoma. Immunostaining was performed for two such markers, epidermal growth factor receptor and SAL (drosophila)-like 2 (SALL2), and for fifteen established markers used in the differential diagnosis of sarcomas. As predicted by expression profiling, epidermal growth factor receptor (a potential therapeutic target) and SALL2 stained most cases of synovial sarcoma; staining was significantly less common among other tested sarcomas. Hierarchical clustering analysis applied to immunostaining results for all 18 antibodies showed that synovial sarcomas, leiomyosarcomas, hemangiopericytomas, and solitary fibrous tumors cluster distinctly, and assigned one case with indeterminate histology as a Ewing sarcoma. Digital images from over 2500 immunostained cores analyzed in this study were captured and are made accessible through the accompanying website: http://microarray-pubs.stanford.edu/tma_portal/synsarc.

  View details for Web of Science ID 000185517500022

  View details for PubMedID 14507652

• Why PLoS became a publisher. PLoS biology Brown, P. O., Eisen, M. B., Varmus, H. E. 2003; 1 (1): E36-?

  View details for PubMedID 14551926

• Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts using DNA microarray analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shepard, K. A., Gerber, A. P., Jambhekar, A., Takizawa, P. A., Brown, P. O., Herschlag, D., DeRisi, J. L., Vale, R. D. 2003; 100 (20): 11429-11434

  Abstract

  Cytoplasmic mRNA localization provides a means of generating cell asymmetry and segregating protein activity. Previous studies have identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae. To identify additional localized mRNAs, we immunoprecipitated the RNA transport components She2p, She3p, and Myo4p and performed DNA microarray analysis of their associated RNAs. A secondary screen, using a GFP-tagged RNA reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety of proteins, including several involved in stress responses and cell wall maintenance. Many of these proteins are asymmetrically localized to buds. However, asymmetric localization also occurs in the absence of RNA transport, suggesting the existence of redundant protein localization mechanisms. In contrast to findings in metazoans, the untranslated regions are dispensable for mRNA localization in yeast. This study reveals an unanticipated widespread use of RNA transport in budding yeast.

  View details for DOI 10.1073/pnas.2033246100

  View details for Web of Science ID 000185685700047

  View details for PubMedID 13679573

• Endothelial cell diversity revealed by global expression profiling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Haraldsen, G., Jahnsen, F. L., Troyanskaya, O. G., Chang, D. S., Wang, Z., Rockson, S. G., van de Rijn, M., Botstein, D., Brown, P. O. 2003; 100 (19): 10623-10628

  Abstract

  The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues.

  View details for DOI 10.1073/pnas.1434429100

  View details for Web of Science ID 000185415300012

  View details for PubMedID 12963823

• Variation in gene expression patterns in human gastric cancers MOLECULAR BIOLOGY OF THE CELL Chen, X., Leung, S. Y., Yuen, S. T., Chu, K. M., Ji, J. F., Li, R., Chan, A. S., Law, S., Troyanskaya, O. G., Wong, J., So, S., Botstein, D., Brown, P. O. 2003; 14 (8): 3208-3215

  Abstract

  Gastric cancer is the world's second most common cause of cancer death. We analyzed gene expression patterns in 90 primary gastric cancers, 14 metastatic gastric cancers, and 22 nonneoplastic gastric tissues, using cDNA microarrays representing approximately 30,300 genes. Gastric cancers were distinguished from nonneoplastic gastric tissues by characteristic differences in their gene expression patterns. We found a diversity of gene expression patterns in gastric cancer, reflecting variation in intrinsic properties of tumor and normal cells and variation in the cellular composition of these complex tissues. We identified several genes whose expression levels were significantly correlated with patient survival. The variations in gene expression patterns among cancers in different patients suggest differences in pathogenetic pathways and potential therapeutic strategies.

  View details for Web of Science ID 000184877400012

  View details for PubMedID 12925757

• Repeated observation of breast tumor subtypes in independent gene expression data sets PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sorlie, T., Tibshirani, R., Parker, J., Hastie, T., Marron, J. S., Nobel, A., Deng, S., Johnsen, H., Pesich, R., Geisler, S., Demeter, J., Perou, C. M., Lonning, P. E., Brown, P. O., Borresen-Dale, A. L., Botstein, D. 2003; 100 (14): 8418-8423

  Abstract

  Characteristic patterns of gene expression measured by DNA microarrays have been used to classify tumors into clinically relevant subgroups. In this study, we have refined the previously defined subtypes of breast tumors that could be distinguished by their distinct patterns of gene expression. A total of 115 malignant breast tumors were analyzed by hierarchical clustering based on patterns of expression of 534 "intrinsic" genes and shown to subdivide into one basal-like, one ERBB2-overexpressing, two luminal-like, and one normal breast tissue-like subgroup. The genes used for classification were selected based on their similar expression levels between pairs of consecutive samples taken from the same tumor separated by 15 weeks of neoadjuvant treatment. Similar cluster analyses of two published, independent data sets representing different patient cohorts from different laboratories, uncovered some of the same breast cancer subtypes. In the one data set that included information on time to development of distant metastasis, subtypes were associated with significant differences in this clinical feature. By including a group of tumors from BRCA1 carriers in the analysis, we found that this genotype predisposes to the basal tumor subtype. Our results strongly support the idea that many of these breast tumor subtypes represent biologically distinct disease entities.

  View details for DOI 10.1073/pnas.0932692100

  View details for Web of Science ID 000184222500069

  View details for PubMedID 12829800

• Genomewide demarcation of RNA polymerase II transcription units revealed by physical fractionation of chromatin PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Nagy, P. L., Cleary, M. L., Brown, P. O., Lieb, J. D. 2003; 100 (11): 6364-6369

  Abstract

  Epigenetic modifications of chromatin serve an important role in regulating the expression and accessibility of genomic DNA. We report here a genomewide approach for fractionating yeast chromatin into two functionally distinct parts, one containing RNA polymerase II transcribed sequences, and the other comprising noncoding sequences and genes transcribed by RNA polymerases I and III. Noncoding regions could be further fractionated into promoters and segments lacking promoters. The observed separations were apparently based on differential crosslinking efficiency of chromatin in different genomic regions. The results reveal a genomewide molecular mechanism for marking promoters and genomic regions that have a license to be transcribed by RNA polymerase II, a previously unrecognized level of genomic complexity that may exist in all eukaryotes. Our approach has broad potential use as a tool for genome annotation and for the characterization of global changes in chromatin structure that accompany different genetic, environmental, and disease states.

  View details for DOI 10.1073/pnas.1131966100

  View details for Web of Science ID 000183190700016

  View details for PubMedID 12750471

• Genomewide view of gene silencing by small interfering RNAs PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chi, J. T., Chang, H. Y., Wang, N. N., Chang, D. S., Dunphy, N., Brown, P. O. 2003; 100 (11): 6343-6346

  Abstract

  RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells.

  View details for DOI 10.1073/pnas.1037853100

  View details for Web of Science ID 000183190700012

  View details for PubMedID 12730368

• Genome-wide analysis of mRNA translation profiles in Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Arava, Y., Wang, Y. L., Storey, J. D., Liu, C. L., Brown, P. O., Herschlag, D. 2003; 100 (7): 3889-3894

  Abstract

  We have analyzed the translational status of each mRNA in rapidly growing Saccharomyces cerevisiae. mRNAs were separated by velocity sedimentation on a sucrose gradient, and 14 fractions across the gradient were analyzed by quantitative microarray analysis, providing a profile of ribosome association with mRNAs for thousands of genes. For most genes, the majority of mRNA molecules were associated with ribosomes and presumably engaged in translation. This systematic approach enabled us to recognize genes with unusual behavior. For 43 genes, most mRNA molecules were not associated with ribosomes, suggesting that they may be translationally controlled. For 53 genes, including GCN4, CPA1, and ICY2, three genes for which translational control is known to play a key role in regulation, most mRNA molecules were associated with a single ribosome. The number of ribosomes associated with mRNAs increased with increasing length of the putative protein-coding sequence, consistent with longer transit times for ribosomes translating longer coding sequences. The density at which ribosomes were distributed on each mRNA (i.e., the number of ribosomes per unit ORF length) was well below the maximum packing density for nearly all mRNAs, consistent with initiation as the rate-limiting step in translation. Global analysis revealed an unexpected correlation: Ribosome density decreases with increasing ORF length. Models to account for this surprising observation are discussed.

  View details for DOI 10.1073/pnas.0635171100

  View details for Web of Science ID 000182058400066

  View details for PubMedID 12660367

  View details for PubMedCentralID PMC153018

• Exploration of global gene expression patterns in pancreatic adenocarcinoma using cDNA microarrays AMERICAN JOURNAL OF PATHOLOGY Iacobuzio-Donahue, C. A., Maitra, A., Olsen, M., Lowe, A. W., van Heek, N. T., Rosty, C., Walter, K., Sato, N., Parker, A., Ashfaq, R., Jaffee, E., Ryu, B., Jones, J., Eshleman, J. R., Yeo, C. J., Cameron, J. L., Kern, S. E., Hruban, R. H., Brown, P. O., Goggins, M. 2003; 162 (4): 1151-1162

  Abstract

  Pancreatic cancer is the fifth leading cause of cancer death in the United States. We used cDNA microarrays to analyze global gene expression patterns in 14 pancreatic cancer cell lines, 17 resected infiltrating pancreatic cancer tissues, and 5 samples of normal pancreas to identify genes that are differentially expressed in pancreatic cancer. We found more than 400 cDNAs corresponding to genes that were differentially expressed in the pancreatic cancer tissues and cell lines as compared to normal pancreas. These genes that tended to be expressed at higher levels in pancreatic cancers were associated with a variety of processes, including cell-cell and cell-matrix interactions, cytoskeletal remodeling, proteolytic activity, and Ca(++) homeostasis. Two prominent clusters of genes were related to the high rates of cellular proliferation in pancreatic cancer cell lines and the host desmoplastic response in the resected pancreatic cancer tissues. Of 149 genes identified as more highly expressed in the pancreatic cancers compared with normal pancreas, 103 genes have not been previously reported in association with pancreatic cancer. The expression patterns of 14 of these highly expressed genes were validated by either immunohistochemistry or reverse transcriptase-polymerase chain reaction as being expressed in pancreatic cancer. The overexpression of one gene in particular, 14-3-3 sigma, was found to be associated with aberrant hypomethylation in the majority of pancreatic cancers analyzed. The genes and expressed sequence tags presented in this study provide clues to the pathobiology of pancreatic cancer and implicate a large number of potentially new molecular markers for the detection and treatment of pancreatic cancer.

  View details for Web of Science ID 000181748300013

  View details for PubMedID 12651607

  View details for PubMedCentralID PMC1851213

• Generalized singular value decomposition for comparative analysis of genome-scale expression data sets of two different organisms PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Alter, O., Brown, P. O., Botstein, D. 2003; 100 (6): 3351-3356

  Abstract

  We describe a comparative mathematical framework for two genome-scale expression data sets. This framework formulates expression as superposition of the effects of regulatory programs, biological processes, and experimental artifacts common to both data sets, as well as those that are exclusive to one data set or the other, by using generalized singular value decomposition. This framework enables comparative reconstruction and classification of the genes and arrays of both data sets. We illustrate this framework with a comparison of yeast and human cell-cycle expression data sets.

  View details for DOI 10.1073/pnas.0530258100

  View details for Web of Science ID 000181675200068

  View details for PubMedID 12631705

• Variation in gene expression patterns in follicular lymphoma and the response to rituximab PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bohen, S. P., Troyanskaya, O. G., Alter, O., Warnke, R., Botstein, D., Brown, P. O., Levy, R. 2003; 100 (4): 1926-1930

  Abstract

  Analysis of the patterns of gene expression in follicular lymphomas from 24 patients suggested that two groups of tumors might be distinguished. All patients, whose biopsies were obtained before any treatment, were treated with rituximab, a monoclonal antibody directed against the B cell antigen, CD20. Gene expression patterns in the tumors that subsequently failed to respond to rituximab appeared more similar to those of normal lymphoid tissues than to gene expression patterns of tumors from rituximab responders. These findings suggest the possibility that the response of follicular lymphoma to rituximab treatment may be predicted from the gene expression pattern of tumors.

  View details for DOI 10.1073/pnas.0437875100

  View details for Web of Science ID 000181073000087

  View details for PubMedID 12571354

• Individuality and variation in gene expression patterns in human blood PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Whitney, A. R., Diehn, M., Popper, S. J., Alizadeh, A. A., Boldrick, J. C., Relman, D. A., Brown, P. O. 2003; 100 (4): 1896-1901

  Abstract

  The nature and extent of interindividual and temporal variation in gene expression patterns in specific cells and tissues is an important and relatively unexplored issue in human biology. We surveyed variation in gene expression patterns in peripheral blood from 75 healthy volunteers by using cDNA microarrays. Characterization of the variation in gene expression in healthy tissue is an essential foundation for the recognition and interpretation of the changes in these patterns associated with infections and other diseases, and peripheral blood was selected because it is a uniquely accessible tissue in which to examine this variation in patients or healthy volunteers in a clinical setting. Specific features of interindividual variation in gene expression patterns in peripheral blood could be traced to variation in the relative proportions of specific blood cell subsets; other features were correlated with gender, age, and the time of day at which the sample was taken. An analysis of multiple sequential samples from the same individuals allowed us to discern donor-specific patterns of gene expression. These data help to define human individuality and provide a database with which disease-associated gene expression patterns can be compared.

  View details for DOI 10.1073/pnas.252784499

  View details for Web of Science ID 000181073000082

  View details for PubMedID 12578971

  View details for PubMedCentralID PMC149930

• The Stanford Microarray Database: data access and quality assessment tools NUCLEIC ACIDS RESEARCH Gollub, J., Ball, C. A., Binkley, G., Demeter, J., Finkelstein, D. B., Hebert, J. M., Hernandez-Boussard, T., Jin, H., Kaloper, M., Matese, J. C., Schroeder, M., Brown, P. O., Botstein, D., Sherlock, G. 2003; 31 (1): 94-96

  Abstract

  The Stanford Microarray Database (SMD; http://genome-www.stanford.edu/microarray/) serves as a microarray research database for Stanford investigators and their collaborators. In addition, SMD functions as a resource for the entire scientific community, by making freely available all of its source code and providing full public access to data published by SMD users, along with many tools to explore and analyze those data. SMD currently provides public access to data from 3500 microarrays, including data from 85 publications, and this total is increasing rapidly. In this article, we describe some of SMD's newer tools for accessing public data, assessing data quality and for data analysis.

  View details for DOI 10.1093/nar/gkg078

  View details for Web of Science ID 000181079700020

  View details for PubMedID 12519956

• Genome-wide analysis of mRNA lengths in Saccharomyces cerevisiae. Genome biology Hurowitz, E. H., Brown, P. O. 2003; 5 (1): R2-?

  Abstract

  Although the protein-coding sequences in the Saccharomyces cerevisiae genome have been studied and annotated extensively, much less is known about the extent and characteristics of the untranslated regions of yeast mRNAs.We developed a 'Virtual Northern' method, using DNA microarrays for genome-wide systematic analysis of mRNA lengths. We used this method to measure mRNAs corresponding to 84% of the annotated open reading frames (ORFs) in the S. cerevisiae genome, with high precision and accuracy (measurement errors +/- 6-7%). We found a close linear relationship between mRNA lengths and the lengths of known or predicted translated sequences; mRNAs were typically around 300 nucleotides longer than the translated sequences. Analysis of genes deviating from that relationship identified ORFs with annotation errors, ORFs that appear not to be bona fide genes, and potentially novel genes. Interestingly, we found that systematic differences in the total length of the untranslated sequences in mRNAs were related to the functions of the encoded proteins.The Virtual Northern method provides a practical and efficient method for genome-scale analysis of transcript lengths. Approximately 12-15% of the yeast genome is represented in untranslated sequences of mRNAs. A systematic relationship between the lengths of the untranslated regions in yeast mRNAs and the functions of the proteins they encode may point to an important regulatory role for these sequences.

  View details for PubMedID 14709174

• A gene-expression program reflecting the innate immune response of cultured intestinal epithelial cells to infection by Listeria monocytogenes GENOME BIOLOGY Baldwin, D. N., Vanchinathan, V., Brown, P. O., Theriot, J. A. 2003; 4 (1)

  Abstract

  Listeria monocytogenes is a Gram-positive, facultative, intracellular bacterial pathogen found in soil, which occasionally causes serious food-borne disease in humans. The outcome of an infection is dependent on the state of the infected individual's immune system, neutrophils being key players in clearing the microorganism from the body. The first line of host defense, however, is the intestinal epithelium.We have examined the transcriptional response of cultured human intestinal epithelial cells to infection by L. monocytogenes, which replicates in the host cell cytoplasm and spreads from cell to cell using a form of actin-based motility. We found that the predominant host response to infection was mediated by NFkappaB. To determine whether any host responses were due to recognition of specific virulence factors during infection, we also examined the transcriptional response to two bacterial mutants; actA which is defective in actin-based motility, and prfA, which is defective in the expression of all L. monocytogenes virulence genes. Remarkably, we found no detectable difference in the host transcriptional response to the wild-type and mutant bacteria.These results suggest that cultured intestinal epithelial cells are capable of mounting and recruiting a powerful innate immune response to L. monocytogenes infection. Our results imply that L. monocytogenes is not specifically detected in the host cytoplasm of Caco-2 cells by intracellular signals. This suggests that entry of bacteria is mediated in the host cell post-translationally, and that these bacteria seek the cytosol not only for the nutrient-rich environment, but also for protection from detection by the immune system.

  View details for Web of Science ID 000182677900007

  View details for PubMedID 12537547

• SOURCE: a unified genomic resource of functional annotations, ontologies, and gene expression data NUCLEIC ACIDS RESEARCH Diehn, M., Sherlock, G., Binkley, G., Jin, H., Matese, J. C., Hernandez-Boussard, T., Rees, C. A., Cherry, J. M., Botstein, D., Brown, P. O., Alizadeh, A. A. 2003; 31 (1): 219-223

  Abstract

  The explosion in the number of functional genomic datasets generated with tools such as DNA microarrays has created a critical need for resources that facilitate the interpretation of large-scale biological data. SOURCE is a web-based database that brings together information from a broad range of resources, and provides it in manner particularly useful for genome-scale analyses. SOURCE's GeneReports include aliases, chromosomal location, functional descriptions, GeneOntology annotations, gene expression data, and links to external databases. We curate published microarray gene expression datasets and allow users to rapidly identify sets of co-regulated genes across a variety of tissues and a large number of conditions using a simple and intuitive interface. SOURCE provides content both in gene and cDNA clone-centric pages, and thus simplifies analysis of datasets generated using cDNA microarrays. SOURCE is continuously updated and contains the most recent and accurate information available for human, mouse, and rat genes. By allowing dynamic linking to individual gene or clone reports, SOURCE facilitates browsing of large genomic datasets. Finally, SOURCEs batch interface allows rapid extraction of data for thousands of genes or clones at once and thus facilitates statistical analyses such as assessing the enrichment of functional attributes within clusters of genes. SOURCE is available at http://source.stanford.edu.

  View details for DOI 10.1093/nar/gkg014

  View details for Web of Science ID 000181079700050

  View details for PubMedID 12519986

• Differential gene-expression patterns in genital fibroblasts of normal males and 46,XY females with androgen insensitivity syndrome: evidence for early programming involving the androgen receptor GENOME BIOLOGY Holterhus, P. M., Hiort, O., Demeter, J., Brown, P. O., Brooks, J. D. 2003; 4 (6)

  Abstract

  Androgen insensitivity syndrome (AIS) comprises a range of phenotypes from male infertility to complete feminization. Most individuals with AIS carry germline mutations of the androgen receptor (AR) that interfere with or ablate its function. As genital fibroblasts retain expression of the AR in vitro, we used genital skin fibroblasts from normal males and 46,XY females with complete AIS due to known AR mutations to gain insights into the role of the AR in human genital differentiation.Using DNA microarrays representing 32,968 different genes, we identified 404 transcripts with significant differences in transcription levels between genital skin fibroblasts cultured from normal and AIS-affected individuals. Gene-cluster analyses uncovered coordinated expression of genes involved in key processes of morphogenesis. On the basis of animal studies and human genetic syndromes, several of these genes are known to have specific roles in genital differentiation. Remarkably, genital fibroblasts from both normal and AIS-affected individuals showed no transcriptional response to dihydrotestosterone treatment despite expression of the AR.The results suggest that in addition to differences in the anatomic origin of the cells, androgen signaling during prenatal development contributes to setting long-lasting, androgen-independent transcriptional programs in genital fibroblasts. Our findings have broad implications in understanding the establishment and the stability of sexual dimorphism in human genital development.

  View details for Web of Science ID 000183720100007

  View details for PubMedID 12801411

• Phospholipase A2 group IIA expression in gastric adenocarcinoma is associated with prolonged survival and less frequent metastasis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Leung, S. Y., Chen, X., Chu, K. M., Yuen, S. T., Mathy, J., Ji, J. F., Chan, A. S., Li, R., Law, S., Troyanskaya, O. G., Tu, I. P., Wong, J., So, S., Botstein, D., Brown, P. O. 2002; 99 (25): 16203-16208

  Abstract

  We analyzed gene expression patterns in human gastric cancers by using cDNA microarrays representing approximately equal 30,300 genes. Expression of PLA2G2A, a gene previously implicated as a modifier of the Apc(Min/+) (multiple intestinal neoplasia 1) mutant phenotype in the mouse, was significantly correlated with patient survival. We confirmed this observation in an independent set of patient samples by using quantitative RT-PCR. Beyond its potential diagnostic and prognostic significance, this result suggests the intriguing possibility that the activity of PLA2G2A may suppress progression or metastasis of human gastric cancer.

  View details for DOI 10.1073/pnas.212646299

  View details for Web of Science ID 000179783400069

  View details for PubMedID 12456890

• Characteristic genome rearrangements in experimental evolution of Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dunham, M. J., Badrane, H., Ferea, T., Adams, J., Brown, P. O., Rosenzweig, F., Botstein, D. 2002; 99 (25): 16144-16149

  Abstract

  Genome rearrangements, especially amplifications and deletions, have regularly been observed as responses to sustained application of the same strong selective pressure in microbial populations growing in continuous culture. We studied eight strains of budding yeast (Saccharomyces cerevisiae) isolated after 100-500 generations of growth in glucose-limited chemostats. Changes in DNA copy number were assessed at single-gene resolution by using DNA microarray-based comparative genomic hybridization. Six of these evolved strains were aneuploid as the result of gross chromosomal rearrangements. Most of the aneuploid regions were the result of translocations, including three instances of a shared breakpoint on chromosome 14 immediately adjacent to CIT1, which encodes the citrate synthase that performs a key regulated step in the tricarboxylic acid cycle. Three strains had amplifications in a region of chromosome 4 that includes the high-affinity hexose transporters; one of these also had the aforementioned chromosome 14 break. Three strains had extensive overlapping deletions of the right arm of chromosome 15. Further analysis showed that each of these genome rearrangements was bounded by transposon-related sequences at the breakpoints. The observation of repeated, independent, but nevertheless very similar, chromosomal rearrangements in response to persistent selection of growing cells parallels the genome rearrangements that characteristically accompany tumor progression.

  View details for DOI 10.1073/pnas.242624799

  View details for Web of Science ID 000179783400059

  View details for PubMedID 12446845

• Expression of cytokeratins 17 and 5 identifies a group of breast carcinomas with poor clinical outcome AMERICAN JOURNAL OF PATHOLOGY van de Rijn, M., Perou, C. M., Tibshirani, R., Haas, P., Kallioniemi, C., Kononen, J., Torhorst, J., Sauter, G., Zuber, M., Kochli, O. R., Mross, F., Dieterich, H., Seitz, R., Ross, D., Botstein, D., BROWN, P. 2002; 161 (6): 1991-1996

  Abstract

  While several prognostic factors have been identified in breast carcinoma, the clinical outcome remains hard to predict for individual patients. Better predictive markers are needed to help guide difficult treatment decisions. In a previous study of 78 breast carcinoma specimens, we noted an association between poor clinical outcome and the expression of cytokeratin 17 and/or cytokeratin 5 mRNAs. Here we describe the results of immunohistochemistry studies using monoclonal antibodies against these markers to analyze more than 600 paraffin-embedded breast tumors in tissue microarrays. We found that expression of cytokeratin 17 and/or cytokeratin 5/6 in tumor cells was associated with a poor clinical outcome. Moreover, multivariate analysis showed that in node-negative breast carcinoma, expression of these cytokeratins was a prognostic factor independent of tumor size and tumor grade.

  View details for Web of Science ID 000179663400005

  View details for PubMedID 12466114

• Nonparametric methods for identifying differentially expressed genes in microarray data BIOINFORMATICS Troyanskaya, O. G., Garber, M. E., Brown, P. O., Botstein, D., Altman, R. B. 2002; 18 (11): 1454-1461

  Abstract

  Gene expression experiments provide a fast and systematic way to identify disease markers relevant to clinical care. In this study, we address the problem of robust identification of differentially expressed genes from microarray data. Differentially expressed genes, or discriminator genes, are genes with significantly different expression in two user-defined groups of microarray experiments. We compare three model-free approaches: (1). nonparametric t-test, (2). Wilcoxon (or Mann-Whitney) rank sum test, and (3). a heuristic method based on high Pearson correlation to a perfectly differentiating gene ('ideal discriminator method'). We systematically assess the performance of each method based on simulated and biological data under varying noise levels and p-value cutoffs.All methods exhibit very low false positive rates and identify a large fraction of the differentially expressed genes in simulated data sets with noise level similar to that of actual data. Overall, the rank sum test appears most conservative, which may be advantageous when the computationally identified genes need to be tested biologically. However, if a more inclusive list of markers is desired, a higher p-value cutoff or the nonparametric t-test may be appropriate. When applied to data from lung tumor and lymphoma data sets, the methods identify biologically relevant differentially expressed genes that allow clear separation of groups in question. Thus the methods described and evaluated here provide a convenient and robust way to identify differentially expressed genes for further biological and clinical analysis.

  View details for Web of Science ID 000179249800008

  View details for PubMedID 12424116

• Adaptation to famine: A family of stationary-phase genes revealed by microarray analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tani, T. H., Khodursky, A., Blumenthal, R. M., Brown, P. O., Matthews, R. G. 2002; 99 (21): 13471-13476

  Abstract

  Bacterial adaptation to nutrient limitation and increased population densities is central to survival and virulence. Surprisingly, <3% of Escherichia coli genes are known to play roles specific to the stationary phase. There is evidence that the leucine-responsive regulatory protein (Lrp) may play an important role in stationary phase, so this study used microarrays representing >98% of E. coli genes to more comprehensively identify those controlled by Lrp. The primary analysis compared isogenic Lrp(+) and Lrp(-) strains in cells growing in steady state in glucose minimal medium, either in the presence or absence of leucine. More than 400 genes were significantly Lrp-responsive under the conditions used. Transcription of 147 genes was lower in Lrp(+) than in Lrp(-) cells whether or not leucine was present; most of these genes were tightly coregulated under several conditions, including a burst of synthesis on transition to stationary phase. This cluster includes 56 of 115 genes already known to play roles in stationary phase. Our results suggest that the actual number of genes induced on entrance into stationary phase is closer to 200 and that Lrp affects nearly three-quarters of them, including genes involved in response to nutrient limitation, high concentrations of organic acids, and osmotic stress.

  View details for DOI 10.1073/pnas.212510999

  View details for Web of Science ID 000178635700025

  View details for PubMedID 12374860

• Microarray analysis reveals a major direct role of DNA copy number alteration in the transcriptional program of human breast tumors PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Pollack, J. R., Sorlie, T., Perou, C. M., Rees, C. A., Jeffrey, S. S., Lonning, P. E., Tibshirani, R., Botstein, D., Borresen-Dale, A. L., Brown, P. O. 2002; 99 (20): 12963-12968

  Abstract

  Genomic DNA copy number alterations are key genetic events in the development and progression of human cancers. Here we report a genome-wide microarray comparative genomic hybridization (array CGH) analysis of DNA copy number variation in a series of primary human breast tumors. We have profiled DNA copy number alteration across 6,691 mapped human genes, in 44 predominantly advanced, primary breast tumors and 10 breast cancer cell lines. While the overall patterns of DNA amplification and deletion corroborate previous cytogenetic studies, the high-resolution (gene-by-gene) mapping of amplicon boundaries and the quantitative analysis of amplicon shape provide significant improvement in the localization of candidate oncogenes. Parallel microarray measurements of mRNA levels reveal the remarkable degree to which variation in gene copy number contributes to variation in gene expression in tumor cells. Specifically, we find that 62% of highly amplified genes show moderately or highly elevated expression, that DNA copy number influences gene expression across a wide range of DNA copy number alterations (deletion, low-, mid- and high-level amplification), that on average, a 2-fold change in DNA copy number is associated with a corresponding 1.5-fold change in mRNA levels, and that overall, at least 12% of all the variation in gene expression among the breast tumors is directly attributable to underlying variation in gene copy number. These findings provide evidence that widespread DNA copy number alteration can lead directly to global deregulation of gene expression, which may contribute to the development or progression of cancer.

  View details for DOI 10.1073/pnas.162471999

  View details for Web of Science ID 000178391700085

  View details for PubMedID 12297621

• Diversity, topographic differentiation, and positional memory in human fibroblasts PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, H. Y., Chi, J. T., Dudoit, S., Bondre, C., van de Rijn, M., Botstein, D., Brown, P. O. 2002; 99 (20): 12877-12882

  Abstract

  A fundamental feature of the architecture and functional design of vertebrate animals is a stroma, composed of extracellular matrix and mesenchymal cells, which provides a structural scaffold and conduit for blood and lymphatic vessels, nerves, and leukocytes. Reciprocal interactions between mesenchymal and epithelial cells are known to play a critical role in orchestrating the development and morphogenesis of tissues and organs, but the roles played by specific stromal cells in controlling the design and function of tissues remain poorly understood. The principal cells of stromal tissue are called fibroblasts, a catch-all designation that belies their diversity. We characterized genome-wide patterns of gene expression in cultured fetal and adult human fibroblasts derived from skin at different anatomical sites. Fibroblasts from each site displayed distinct and characteristic transcriptional patterns, suggesting that fibroblasts at different locations in the body should be considered distinct differentiated cell types. Notable groups of differentially expressed genes included some implicated in extracellular matrix synthesis, lipid metabolism, and cell signaling pathways that control proliferation, cell migration, and fate determination. Several genes implicated in genetic diseases were found to be expressed in fibroblasts in an anatomic pattern that paralleled the phenotypic defects. Finally, adult fibroblasts maintained key features of HOX gene expression patterns established during embryogenesis, suggesting that HOX genes may direct topographic differentiation and underlie the detailed positional memory in fibroblasts.

  View details for DOI 10.1073/pnas.162488599

  View details for Web of Science ID 000178391700069

  View details for PubMedID 12297622

• Public-access group supports PubMed Central NATURE Eisen, M. B., Brown, P. O., Varmus, H. E. 2002; 419 (6903): 111-111

  View details for Web of Science ID 000177931200016

  View details for PubMedID 12226637

• Genomic expression programs and the integration of the CD28 costimulatory signal in T cell activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Diehn, M., Alizadeh, A. A., Rando, O. J., Liu, C. L., Stankunas, K., Botstein, D., Crabtree, G. R., Brown, P. O. 2002; 99 (18): 11796-11801

  Abstract

  Optimal activation of T cells requires effective occupancy of both the antigen-specific T cell receptor and a second coreceptor such as CD28. We used cDNA microarrays to characterize the genomic expression program in human peripheral T cells responding to stimulation of these receptors. We found that CD28 agonists alone elicited few, but reproducible, changes in gene expression, whereas CD3 agonists elicited a multifaceted temporally choreographed gene expression program. The principal effect of simultaneous engagement of CD28 was to increase the amplitude of the CD3 transcriptional response. The induced genes whose expression was most enhanced by costimulation were significantly enriched for known targets of nuclear factor of activated T cells (NFAT) transcription factors. This enhancement was nearly abolished by blocking the nuclear translocation of NFATc by using the calcineurin inhibitor FK506. CD28 signaling promoted phosphorylation, and thus inactivation, of the NFAT nuclear export kinase glycogen synthase kinase-3 (GSK3), coincident with enhanced dephosphorylation of NFATc proteins. These results provide a detailed picture of the transcriptional program of T cell activation and suggest that enhancement of transcriptional activation by NFAT, through inhibition of its nuclear export, plays a key role in mediating the CD28 costimulatory signal.

  View details for DOI 10.1073/pnas.092284399

  View details for Web of Science ID 000177843100048

  View details for PubMedID 12195013

  View details for PubMedCentralID PMC129348

• Genome-wide analysis of gene expression regulated by the calcineurin/Crz1p signaling pathway in Saccharomyces cerevisiae JOURNAL OF BIOLOGICAL CHEMISTRY Yoshimoto, H., Saltsman, K., Gasch, A. P., Li, H. X., Ogawa, N., Botstein, D., Brown, P. O., Cyert, M. S. 2002; 277 (34): 31079-31088

  Abstract

  In Saccharomyces cerevisiae, the Ca(2+)/calmodulin-dependent protein phosphatase, calcineurin, is activated by specific environmental conditions, including exposure to Ca(2+) and Na(+), and induces gene expression by regulating the Crz1p/Tcn1p transcription factor. We used DNA microarrays to perform a comprehensive analysis of calcineurin/Crz1p-dependent gene expression following addition of Ca(2+) (200 mm) or Na(+) (0.8 m) to yeast. 163 genes exhibited increased expression that was reduced 50% or more by calcineurin inhibition. These calcineurin-dependent genes function in signaling pathways, ion/small molecule transport, cell wall maintenance, and vesicular transport, and include many open reading frames of previously unknown function. Three distinct gene classes were defined as follows: 28 genes displayed calcineurin-dependent induction in response to Ca(2+) and Na(+), 125 showed calcineurin-dependent expression following Ca(2+) but not Na(+) addition, and 10 were regulated by calcineurin in response to Na(+) but not Ca(2+). Analysis of crz1Delta cells established Crz1p as the major effector of calcineurin-regulated gene expression in yeast. We identified the Crz1p-binding site as 5'-GNGGC(G/T)CA-3' by in vitro site selection. A similar sequence, 5'-GAGGCTG-3', was identified as a common sequence motif in the upstream regions of calcineurin/ Crz1p-dependent genes. This finding is consistent with direct regulation of these genes by Crz1p.

  View details for DOI 10.1074/jbc.M202718200

  View details for Web of Science ID 000177579800087

  View details for PubMedID 12058033

• A transcriptional response to Wnt protein in human embryonic carcinoma cells. BMC developmental biology Willert, J., Epping, M., Pollack, J. R., Brown, P. O., Nusse, R. 2002; 2: 8-?

  Abstract

  Wnt signaling is implicated in many developmental decisions, including stem cell control, as well as in cancer. There are relatively few target genes known of the Wnt pathway.We have identified target genes of Wnt signaling using microarray technology and human embryonic carcinoma cells stimulated with active Wnt protein. The ~50 genes upregulated early after Wnt addition include the previously known Wnt targets Cyclin D1, MYC, ID2 and betaTRCP. The newly identified targets, which include MSX1, MSX2, Nucleophosmin, Follistatin, TLE/Groucho, Ubc4/5E2, CBP/P300, Frizzled and REST/NRSF, have important implications for understanding the roles of Wnts in development and cancer. The protein synthesis inhibitor cycloheximide blocks induction by Wnt, consistent with a requirement for newly synthesized beta-catenin protein prior to target gene activation. The promoters of nearly all the target genes we identified have putative TCF binding sites, and we show that the TCF binding site is required for induction of Follistatin. Several of the target genes have a cooperative response to a combination of Wnt and BMP.Wnt signaling activates genes that promote stem cell fate and inhibit cellular differentiation and regulates a remarkable number of genes involved in its own signaling system.

  View details for PubMedID 12095419

• Transformation of follicular lymphoma to diffuse large-cell lymphoma: Alternative patterns with increased or decreased expression of c-myc and its regulated genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lossos, I. S., Alizadeh, A. A., Diehn, M., Warnke, R., Thorstenson, Y., Oefner, P. J., Brown, P. O., Botstein, D., Levy, R. 2002; 99 (13): 8886-8891

  Abstract

  The natural history of follicular lymphoma (FL) is frequently characterized by transformation to a more aggressive diffuse large B cell lymphoma (DLBCL). We compared the gene-expression profiles between transformed DLBCL and their antecedent FL. No genes were observed to increase or decrease their expression in all of the cases of histological transformation. However, two different gene-expression profiles associated with the transformation process were defined, one in which c-myc and genes regulated by c-myc showed increased expression and one in which these same genes showed decreased expression. Further, there was a striking difference in gene-expression profiles between transformed DLBCL and de novo DLBCL, because the gene-expression profile of transformed DLBCL was more similar to their antecedent FL than to de novo DLBCL. This study demonstrates that transformation from FL to DLBCL can occur by alternative pathways and that transformed DLBCL and de novo DLBCL have very different gene-expression profiles that may underlie the different clinical behaviors of these two types of morphologically similar lymphomas.

  View details for DOI 10.1073/pnas.132253599

  View details for Web of Science ID 000176478200075

  View details for PubMedID 12077300

• Transcriptional response of human mast cells stimulated via the Fc(epsilon)RI and identification of mast cells as a source of IL-11. BMC immunology Sayama, K., Diehn, M., Matsuda, K., Lunderius, C., Tsai, M., Tam, S., Botstein, D., Brown, P. O., Galli, S. J. 2002; 3: 5-?

  Abstract

  In asthma and other allergic disorders, the activation of mast cells by IgE and antigen induces the cells to release histamine and other mediators of inflammation, as well as to produce certain cytokines and chemokines. To search for new mast cell products, we used complementary DNA microarrays to analyze gene expression in human umbilical cord blood-derived mast cells stimulated via the high-affinity IgE receptor (Fc(epsilon)RI).One to two hours after Fc(epsilon)RI-dependent stimulation, more than 2,400 genes (about half of which are of unknown function) exhibited 2-200 fold changes in expression. The transcriptional program included changes in the expression of IL-11 and at least 30 other cytokines and chemokines. Human mast cells secreted 130-529 pg of IL-11/106 cells by 6 h after stimulation with anti-IgE.Our initial analysis of the transcriptional program induced in in vitro-derived human mast cells stimulated via the Fc(epsilon)RI has identified many products that heretofore have not been associated with this cell type, but which may significantly influence mast cell function in IgE-associated host responses. We also have demonstrated that mast cells stimulated via the Fc(epsilon)RI can secrete IL-11. Based on the previously reported biological effects of IL-11, our results suggest that production of IL-11 may represent one link between IgE-dependent mast cell activation in subjects with allergic asthma and the development of a spectrum of structural changes in the airways of these individuals; such changes, collectively termed "airway remodeling," can constitute an important long term consequence of asthma.

  View details for PubMedID 12079505

• Gene expression patterns in human liver cancers MOLECULAR BIOLOGY OF THE CELL Chen, X., Cheung, S. T., So, S., Fan, S. T., Barry, C., Higgins, J., Lai, K. M., Ji, J. F., Dudoit, S., Ng, I. O., van de Rijn, M., Botstein, D., Brown, P. O. 2002; 13 (6): 1929-1939

  Abstract

  Hepatocellular carcinoma (HCC) is a leading cause of death worldwide. Using cDNA microarrays to characterize patterns of gene expression in HCC, we found consistent differences between the expression patterns in HCC compared with those seen in nontumor liver tissues. The expression patterns in HCC were also readily distinguished from those associated with tumors metastatic to liver. The global gene expression patterns intrinsic to each tumor were sufficiently distinctive that multiple tumor nodules from the same patient could usually be recognized and distinguished from all the others in the large sample set on the basis of their gene expression patterns alone. The distinctive gene expression patterns are characteristic of the tumors and not the patient; the expression programs seen in clonally independent tumor nodules in the same patient were no more similar than those in tumors from different patients. Moreover, clonally related tumor masses that showed distinct expression profiles were also distinguished by genotypic differences. Some features of the gene expression patterns were associated with specific phenotypic and genotypic characteristics of the tumors, including growth rate, vascular invasion, and p53 overexpression.

  View details for DOI 10.1091/mbc.02-02-0023

  View details for Web of Science ID 000176418800012

  View details for PubMedID 12058060

• Identification of genes periodically expressed in the human cell cycle and their expression in tumors MOLECULAR BIOLOGY OF THE CELL Whitfield, M. L., Sherlock, G., Saldanha, A. J., Murray, J. I., Ball, C. A., Alexander, K. E., Matese, J. C., Perou, C. M., Hurt, M. M., Brown, P. O., Botstein, D. 2002; 13 (6): 1977-2000

  Abstract

  The genome-wide program of gene expression during the cell division cycle in a human cancer cell line (HeLa) was characterized using cDNA microarrays. Transcripts of >850 genes showed periodic variation during the cell cycle. Hierarchical clustering of the expression patterns revealed coexpressed groups of previously well-characterized genes involved in essential cell cycle processes such as DNA replication, chromosome segregation, and cell adhesion along with genes of uncharacterized function. Most of the genes whose expression had previously been reported to correlate with the proliferative state of tumors were found herein also to be periodically expressed during the HeLa cell cycle. However, some of the genes periodically expressed in the HeLa cell cycle do not have a consistent correlation with tumor proliferation. Cell cycle-regulated transcripts of genes involved in fundamental processes such as DNA replication and chromosome segregation seem to be more highly expressed in proliferative tumors simply because they contain more cycling cells. The data in this report provide a comprehensive catalog of cell cycle regulated genes that can serve as a starting point for functional discovery. The full dataset is available at http://genome-www.stanford.edu/Human-CellCycle/HeLa/.

  View details for DOI 10.1091/mbc.02-02-0030

  View details for Web of Science ID 000176418800016

  View details for PubMedID 12058064

• Precision and functional specificity in mRNA decay PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, Y. L., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., Brown, P. O. 2002; 99 (9): 5860-5865

  Abstract

  Posttranscriptional processing of mRNA is an integral component of the gene expression program. By using DNA microarrays, we precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA polymerase II. The half-lives varied widely, ranging from approximately 3 min to more than 90 min. We found no simple correlation between mRNA half-lives and ORF size, codon bias, ribosome density, or abundance. However, the decay rates of mRNAs encoding groups of proteins that act together in stoichiometric complexes were generally closely matched, and other evidence pointed to a more general relationship between physiological function and mRNA turnover rates. The results provide strong evidence that precise control of the decay of each mRNA is a fundamental feature of the gene expression program in yeast.

  View details for DOI 10.1073/pnas.092538799

  View details for Web of Science ID 000175377800023

  View details for PubMedID 11972065

  View details for PubMedCentralID PMC122867

• Molecular characterisation of soft tissue tumours: a gene expression study LANCET Nielsen, T. O., West, R. B., Linn, S. C., Alter, O., Knowling, M. A., O'Connell, J. X., Zhu, S., Fero, M., Sherlock, G., Pollack, J. R., Brown, P. O., Botstein, D., van de Rijn, M. 2002; 359 (9314): 1301-1307

  Abstract

  Soft-tissue tumours are derived from mesenchymal cells such as fibroblasts, muscle cells, or adipocytes, but for many such tumours the histogenesis is controversial. We aimed to start molecular characterisation of these rare neoplasms and to do a genome-wide search for new diagnostic markers.We analysed gene-expression patterns of 41 soft-tissue tumours with spotted cDNA microarrays. After removal of errors introduced by use of different microarray batches, the expression patterns of 5520 genes that were well defined were used to separate tumours into discrete groups by hierarchical clustering and singular value decomposition.Synovial sarcomas, gastrointestinal stromal tumours, neural tumours, and a subset of the leiomyosarcomas, showed strikingly distinct gene-expression patterns. Other tumour categories--malignant fibrous histiocytoma, liposarcoma, and the remaining leiomyosarcomas--shared molecular profiles that were not predicted by histological features or immunohistochemistry. Strong expression of known genes, such as KIT in gastrointestinal stromal tumours, was noted within gene sets that distinguished the different sarcomas. However, many uncharacterised genes also contributed to the distinction between tumour types.These results suggest a new method for classification of soft-tissue tumours, which could improve on the method based on histological findings. Large numbers of uncharacterised genes contributed to distinctions between the tumours, and some of these could be useful markers for diagnosis, have prognostic significance, or prove possible targets for treatment.

  View details for Web of Science ID 000174989700013

  View details for PubMedID 11965276

• Autoantigen microarrays for multiplex characterization of autoantibody responses NATURE MEDICINE Robinson, W. H., DiGennaro, C., Hueber, W., Haab, B. B., KAMACHI, M., Dean, E. J., Fournel, S., Fong, D., Genovese, M. C., de Vegvar, H. E., Skriner, K., Hirschberg, D. L., Morris, R. I., Muller, S., Pruijn, G. J., van Venrooij, W. J., Smolen, J. S., Brown, P. O., Steinman, L., Utz, P. J. 2002; 8 (3): 295-301

  Abstract

  We constructed miniaturized autoantigen arrays to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens, using submicroliter quantities of clinical samples. Autoantigen microarrays were produced by attaching hundreds of proteins, peptides and other biomolecules to the surface of derivatized glass slides using a robotic arrayer. Arrays were incubated with patient serum, and spectrally resolvable fluorescent labels were used to detect autoantibody binding to specific autoantigens on the array. We describe and characterize arrays containing the major autoantigens in eight distinct human autoimmune diseases, including systemic lupus erythematosus and rheumatoid arthritis. This represents the first report of application of such technology to multiple human disease sera, and will enable validated detection of antibodies recognizing autoantigens including proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-translationally modified antigens. Autoantigen microarrays represent a powerful tool to study the specificity and pathogenesis of autoantibody responses, and to identify and define relevant autoantigens in human autoimmune diseases.

  View details for Web of Science ID 000174139500036

  View details for PubMedID 11875502

• In vivo regulation of human skeletal muscle gene expression by thyroid hormone GENOME RESEARCH Clement, K., Viguerie, N., Diehn, M., Alizadeh, A., Barbe, P., Thalamas, C., Storey, J. D., Brown, P. O., Barsh, G. S., Langin, D. 2002; 12 (2): 281-291

  Abstract

  Thyroid hormones are key regulators of metabolism that modulate transcription via nuclear receptors. Hyperthyroidism is associated with increased metabolic rate, protein breakdown, and weight loss. Although the molecular actions of thyroid hormones have been studied thoroughly, their pleiotropic effects are mediated by complex changes in expression of an unknown number of target genes. Here, we measured patterns of skeletal muscle gene expression in five healthy men treated for 14 days with 75 microg of triiodothyronine, using 24,000 cDNA element microarrays. To analyze the data, we used a new statistical method that identifies significant changes in expression and estimates the false discovery rate. The 381 up-regulated genes were involved in a wide range of cellular functions including transcriptional control, mRNA maturation, protein turnover, signal transduction, cellular trafficking, and energy metabolism. Only two genes were down-regulated. Most of the genes are novel targets of thyroid hormone. Cluster analysis of triiodothyronine-regulated gene expression among 19 different human tissues or cell lines revealed sets of coregulated genes that serve similar biologic functions. These results define molecular signatures that help to understand the physiology and pathophysiology of thyroid hormone action.

  View details for Web of Science ID 000173689600008

  View details for PubMedID 11827947

• Stereotyped and specific gene expression programs in human innate immune responses to bacteria PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boldrick, J. C., Alizadeh, A. A., Diehn, M., Dudoit, S., Liu, C. L., Belcher, C. E., Botstein, D., Staudt, L. M., Brown, P. O., Relman, D. A. 2002; 99 (2): 972-977

  Abstract

  The innate immune response is crucial for defense against microbial pathogens. To investigate the molecular choreography of this response, we carried out a systematic examination of the gene expression program in human peripheral blood mononuclear cells responding to bacteria and bacterial products. We found a remarkably stereotyped program of gene expression induced by bacterial lipopolysaccharide and diverse killed bacteria. An intricately choreographed expression program devoted to communication between cells was a prominent feature of the response. Other features suggested a molecular program for commitment of antigen-presenting cells to antigens captured in the context of bacterial infection. Despite the striking similarities, there were qualitative and quantitative differences in the responses to different bacteria. Modulation of this host-response program by bacterial virulence mechanisms was an important source of variation in the response to different bacteria.

  View details for Web of Science ID 000173450100078

  View details for PubMedID 11805339

  View details for PubMedCentralID PMC117415

• Exploratory screening of genes and clusters from microarray experiments STATISTICA SINICA Tibshirani, R., Hastie, T., Narasimhan, B., Eisen, M., Sherlock, G., Brown, P., Botstein, D. 2002; 12 (1): 47-59

  View details for Web of Science ID 000174372800004

• Software tools for high-throughput analysis and image retrieval of immunohistochemistry stains obtained on tissue microarrays Lui, C. L., Natkunam, Y., Prapong, W., Montgomery, K., Botstein, D., Brown, P. O., van de Rijn, M. NATURE PUBLISHING GROUP. 2002: 341A–341A

  View details for Web of Science ID 000173379701434

• Transcriptional programs activated by exposure of human prostate cancer cells to androgen GENOME BIOLOGY DePrimo, S. E., Diehn, M., Nelson, J. B., Reiter, R. E., Matese, J., Fero, M., Tibshirani, R., Brown, P. O., Brooks, J. D. 2002; 3 (7)

  Abstract

  Androgens are required for both normal prostate development and prostate carcinogenesis. We used DNA microarrays, representing approximately 18,000 genes, to examine the temporal program of gene expression following treatment of the human prostate cancer cell line LNCaP with a synthetic androgen.We observed statistically significant changes in levels of transcripts of more than 500 genes. Many of these genes were previously reported androgen targets, but most were not previously known to be regulated by androgens. The androgen-induced expression programs in three additional androgen-responsive human prostate cancer cell lines, and in four androgen-independent subclones derived from LNCaP, shared many features with those observed in LNCaP, but some differences were observed. A remarkable fraction of the genes induced by androgen appeared to be related to production of seminal fluid and these genes included many with roles in protein folding, trafficking, and secretion.Prostate cancer cell lines retain features of androgen responsiveness that reflect normal prostatic physiology. These results provide a broad view of the effect of androgen signaling on the transcriptional program in these cancer cells, and a foundation for further studies of androgen action.

  View details for Web of Science ID 000207581200008

  View details for PubMedID 12184806

  View details for PubMedCentralID PMC126237

• Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays GENOME BIOLOGY Lin, J. Y., Pollack, J. R., Chou, F., Rees, C. A., Christian, A. T., Bedford, J. S., Brown, P. O., Ginsberg, M. H. 2002; 3 (6)

  Abstract

  Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology.CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT.The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.

  View details for Web of Science ID 000207581100008

  View details for PubMedID 12093373

• Three cell wall mannoproteins facilitate the uptake of iron in Saccharomyces cerevisiae JOURNAL OF BIOLOGICAL CHEMISTRY Protchenko, O., Ferea, T., Rashford, J., Tiedeman, J., Brown, P. O., Botstein, D., Philpott, C. C. 2001; 276 (52): 49244-49250

  Abstract

  Analysis of iron-regulated gene expression in Saccharomyces cerevisiae using cDNA microarrays has identified three putative cell wall proteins that are directly regulated by Aft1p, the major iron-dependent transcription factor in yeast. FIT1, FIT2, and FIT3 (for facilitator of iron transport) were more highly expressed in strains grown in low concentrations of iron and in strains in which AFT1-1(up), a constitutively active allele of AFT1, was expressed. Northern blot analysis confirmed that FIT1, FIT2, and FIT3 mRNA transcript levels were increased 60-230-fold in response to iron deprivation in an Aft1p-dependent manner. Fit1p was localized exclusively to the cell wall by indirect immunofluorescence. Deletion of the FIT genes, individually or in combination, resulted in diminished uptake of iron bound to the siderophores ferrioxamine B and ferrichrome, without diminishing the uptake of ferric iron salts, or the siderophores triacetylfusarinine C and enterobactin. FIT-deletion strains exhibited increased expression of Aft1p target genes as measured by a FET3-lacZ reporter gene or by Arn1p Western blotting, indicating that cells respond to the absence of FIT genes by up-regulating systems of iron uptake. Aft1p activation in FIT-deleted strains occurred when either ferrichrome or ferric salts were used as sources of iron during growth, suggesting that the FIT genes enhance uptake of iron from both sources. Enzymatic digestion of the cell wall resulted in the release of significant amounts of iron from cells, and the relative quantity of iron released was reduced in FIT-deletion strains. Fit1p, Fit2p, and Fit3p may function by increasing the amount of iron associated with the cell wall and periplasmic space.

  View details for DOI 10.1074/jbc.M109220200

  View details for Web of Science ID 000173922100082

  View details for PubMedID 11673473

• A second iron-regulatory system in yeast independent of Aft1p PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Rutherford, J. C., Jaron, S., Ray, E., Brown, P. O., Winge, D. R. 2001; 98 (25): 14322-14327

  Abstract

  Iron homeostasis in the yeast Saccharomyces cerevisiae is regulated at the transcriptional level by Aft1p, which activates the expression of its target genes in response to low-iron conditions. The yeast genome contains a paralog of AFT1, which has been designated AFT2. To establish whether AFT1 and AFT2 have overlapping functions, a mutant containing a double aft1Deltaaft2Delta deletion was generated. Growth assays established that the single aft2Delta strain exhibited no iron-dependent phenotype. However, the double-mutant aft1Deltaaft2Delta strain was more sensitive to low-iron growth conditions than the single-mutant aft1Delta strain. A mutant allele of AFT2 (AFT2-1(up)), or overexpression of the wild-type AFT2 gene, led to partial complementation of the respiratory-deficient phenotype of the aft1Delta strain. The AFT2-1(up) allele also increased the uptake of (59)Fe in an aft1Delta strain. DNA microarrays were used to identify genes regulated by AFT2. Some of the AFT2-regulated genes are known to be regulated by Aft1p; however, AFT2-1(up)-dependent activation was independent of Aft1p. The kinetics of induction of two genes activated by the AFT2-1(up) allele are consistent with Aft2p acting as a direct transcriptional factor. Truncated forms of Aft1p and Aft2p bound to a DNA duplex containing the Aft1p binding site in vitro. The wild-type allele of AFT2 activated transcription in response to growth under low-iron conditions. Together, these data suggest that yeast has a second regulatory pathway for the iron regulon, with AFT1 and AFT2 playing partially redundant roles.

  View details for Web of Science ID 000172576900027

  View details for PubMedID 11734641

• Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia JOURNAL OF EXPERIMENTAL MEDICINE Rosenwald, A., Alizadeh, A. A., Widhopf, G., Simon, R., Davis, R. E., Yu, X., Yang, L. M., Pickeral, O. K., Rassenti, L. Z., Powell, J., Botstein, D., Byrd, J. C., Grever, M. R., Cheson, B. D., CHIORAZZI, N., Wilson, W. H., Kipps, T. J., Brown, P. O., Staudt, L. M. 2001; 194 (11): 1639-1647

  Abstract

  The most common human leukemia is B cell chronic lymphocytic leukemia (CLL), a malignancy of mature B cells with a characteristic clinical presentation but a variable clinical course. The rearranged immunoglobulin (Ig) genes of CLL cells may be either germ-line in sequence or somatically mutated. Lack of Ig mutations defined a distinctly worse prognostic group of CLL patients raising the possibility that CLL comprises two distinct diseases. Using genomic-scale gene expression profiling, we show that CLL is characterized by a common gene expression "signature," irrespective of Ig mutational status, suggesting that CLL cases share a common mechanism of transformation and/or cell of origin. Nonetheless, the expression of hundreds of other genes correlated with the Ig mutational status, including many genes that are modulated in expression during mitogenic B cell receptor signaling. These genes were used to build a CLL subtype predictor that may help in the clinical classification of patients with this disease.

  View details for Web of Science ID 000172659500009

  View details for PubMedID 11733578

• Comprehensive mutational analysis of the moloney murine leukemia virus envelope protein JOURNAL OF VIROLOGY Rothenberg, S. M., Olsen, M. N., Laurent, L. C., Crowley, R. A., Brown, P. O. 2001; 75 (23): 11851-11862

  Abstract

  The envelope (Env) protein of Moloney murine leukemia virus is the primary mediator of viral entry. We constructed a large pool of insertion mutations in the env gene and analyzed the fitness of each mutant in completing two critical steps in the virus life cycle: (i) the expression and delivery of the Env protein to the cell surface during virion assembly and (ii) the infectivity of virions displaying the mutant proteins. The majority of the mutants were poorly expressed at the producer cell surface, suggesting folding defects due to the presence of the inserted residues. The mutants with residual infectivity had insertions either in the amino-terminal signal sequence region, two disulfide-bonded loops in the receptor binding domain, discrete regions of the carboxy-terminal region of the surface subunit (SU), or the cytoplasmic tail. Insertions that allowed the mutants to reach the cell surface but not to mediate detectable infection were located within the amino-terminal sequence of the mature Env, within the SU carboxy-terminal region, near putative receptor binding residues, and throughout the fusion peptide. Independent analysis of select mutants in this group allowed more precise identification of the defect in Env function. Mapping of mutant phenotypes to a structural model of the receptor-binding domain provides insights into the protein's functional organization. The high-resolution functional map reported here will be valuable for the engineering of the Env protein for a variety of uses, including gene therapy.

  View details for Web of Science ID 000172033100061

  View details for PubMedID 11689666

• Diversity of gene expression in adenocarcinoma of the lung PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Garber, M. E., Troyanskaya, O. G., Schluens, K., Petersen, S., Thaesler, Z., Pacyna-Gengelbach, M., van de Rijn, M., Rosen, G. D., Perou, C. M., Whyte, R. I., Altman, R. B., Brown, P. O., Botstein, D., Petersen, I. 2001; 98 (24): 13784-13789

  Abstract

  The global gene expression profiles for 67 human lung tumors representing 56 patients were examined by using 24,000-element cDNA microarrays. Subdivision of the tumors based on gene expression patterns faithfully recapitulated morphological classification of the tumors into squamous, large cell, small cell, and adenocarcinoma. The gene expression patterns made possible the subclassification of adenocarcinoma into subgroups that correlated with the degree of tumor differentiation as well as patient survival. Gene expression analysis thus promises to extend and refine standard pathologic analysis.

  View details for Web of Science ID 000172328100058

  View details for PubMedID 11707590

  View details for PubMedCentralID PMC61119

• RERG is a novel ras-related, estrogen-regulated and growth-inhibitory gene in breast cancer JOURNAL OF BIOLOGICAL CHEMISTRY Finlin, B. S., Gau, C. L., Murphy, G. A., Shao, H. P., Kimel, T., Seitz, R. S., Chiu, Y. F., Botstein, D., Brown, P. O., Tamanoi, F., Andres, D. A., Perou, C. M. 2001; 276 (45): 42259-42267

  Abstract

  Using microarray analysis, we identified a unique ras superfamily gene, termed RERG (ras-related and estrogen-regulated growth inhibitor), whose expression was decreased or lost in a significant percentage of primary human breast tumors that show a poor clinical prognosis. Importantly, high RERG expression correlated with expression of a set of genes that define a breast tumor subtype that is estrogen receptor-positive and associated with a slow rate of tumor cell proliferation and a favorable prognosis for these cancer patients. RERG mRNA expression was induced rapidly in MCF-7 cells stimulated by beta-estradiol and repressed by tamoxifen treatment. Like Ras, RERG protein exhibited intrinsic GDP/GTP binding and GTP hydrolysis activity. Unlike Ras proteins, RERG lacks a known recognition signal for COOH-terminal prenylation and was localized primarily in the cytoplasm. Expression of RERG protein in MCF-7 breast carcinoma cells resulted in a significant inhibition of both anchorage-dependent and anchorage-independent growth in vitro and inhibited tumor formation in nude mice. These features of RERG are strikingly different from most Ras superfamily GTP-binding pro-teins and suggest that the loss of RERG expression may contribute to breast tumorigenesis.

  View details for Web of Science ID 000172450400096

  View details for PubMedID 11533059

• Haa1, a protein homologous to the copper-regulated transcription factor Ace1, is a novel transcriptional activator JOURNAL OF BIOLOGICAL CHEMISTRY Keller, G., Ray, E., Brown, P. O., Winge, D. R. 2001; 276 (42): 38697-38702

  Abstract

  The Saccharomyces cerevisiae genome contains a predicted gene, YPR008w, homologous to the gene encoding the copper-activated transcription factor Ace1. The product of the YPR008w gene, designated Haa1, regulates the transcription of a set of yeast genes, many of which encode membrane proteins. Two main target genes of Haa1 are the multidrug resistance gene YGR138c and the YRO2 homolog to the plasma membrane Hsp30. Haa1 is localized to the nucleus. Haa1-induced expression of YGR138c and YRO2 appears to be direct. Induction of HAA1 using a GAL1/HAA1 fusion gene resulted in rapid galactose-induced expression of both HAA1 and target genes. Although Haa1 has a sequence very similar to the Cu-activated DNA binding domain of Ace1, expression of Haa1 target genes was found to be independent of the copper status of cells. Haa1 does not exhibit metalloregulation in cells incubated with a range of transition metal salts. Haa1 does not exhibit any cross-talk with Ace1. Overexpression of Haa1 does not compensate for cells lacking a functional Ace1. The lack of metalloregulation of Haa1 despite the strong sequence similarity to the copper regulatory domain of Ace1 is discussed.

  View details for Web of Science ID 000171673200051

  View details for PubMedID 11504737

• Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p MOLECULAR BIOLOGY OF THE CELL Gasch, A. P., Huang, M. X., Metzner, S., Botstein, D., Elledge, S. J., Brown, P. O. 2001; 12 (10): 2987-3003

  Abstract

  Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1.

  View details for Web of Science ID 000171540300007

  View details for PubMedID 11598186

• Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sorlie, T., Perou, C. M., Tibshirani, R., Aas, T., Geisler, S., Johnsen, H., Hastie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Thorsen, T., Quist, H., Matese, J. C., Brown, P. O., Botstein, D., Lonning, P. E., Borresen-Dale, A. L. 2001; 98 (19): 10869-10874

  Abstract

  The purpose of this study was to classify breast carcinomas based on variations in gene expression patterns derived from cDNA microarrays and to correlate tumor characteristics to clinical outcome. A total of 85 cDNA microarray experiments representing 78 cancers, three fibroadenomas, and four normal breast tissues were analyzed by hierarchical clustering. As reported previously, the cancers could be classified into a basal epithelial-like group, an ERBB2-overexpressing group and a normal breast-like group based on variations in gene expression. A novel finding was that the previously characterized luminal epithelial/estrogen receptor-positive group could be divided into at least two subgroups, each with a distinctive expression profile. These subtypes proved to be reasonably robust by clustering using two different gene sets: first, a set of 456 cDNA clones previously selected to reflect intrinsic properties of the tumors and, second, a gene set that highly correlated with patient outcome. Survival analyses on a subcohort of patients with locally advanced breast cancer uniformly treated in a prospective study showed significantly different outcomes for the patients belonging to the various groups, including a poor prognosis for the basal-like subtype and a significant difference in outcome for the two estrogen receptor-positive groups.

  View details for Web of Science ID 000170966800067

  View details for PubMedID 11553815

• Microarrays in primary breast cancer - lessons from chemotherapy studies 4th International Forum on Current Topics in Breast Cancer Research Lonning, P. E., Sorlie, T., Perou, C. M., Brown, P. O., Botstein, D., Borresen-Dale, A. L. BIOSCIENTIFICA LTD. 2001: 259–63

  Abstract

  Current development in molecular techniques has extended the opportunities to explore genetic alterations in malignant tissue. There is a need to improve prognostication and, in particular, to understand the mechanisms of treatment resistance in different tumours. Gene analyses by microarrays allow concomitant analyses of several genes in concert, providing new opportunities for tumour classification and understanding of key biological disturbances. This paper outlines our continuing studies exploring prognostic and, we hope, predictive factors in breast cancer therapy.

  View details for Web of Science ID 000171231900013

  View details for PubMedID 11566617

• Regulation of CSF1 promoter by the SWI/SNF-like BAF complex CELL Liu, R., Liu, H., Chen, X., Kirby, M., Brown, P. O., Zhao, K. J. 2001; 106 (3): 309-318

  Abstract

  The mammalian BAF complex regulates gene expression by modifying chromatin structure. In this report, we identify 80 genes activated and 2 genes repressed by the BAF complex in SW-13 cells. We find that prior binding of NFI/CTF to the NFI/CTF binding site in CSF1 promoter is required for the recruitment of the BAF complex and the BAF-dependent activation of the promoter. Furthermore, the activation of the CSF1 promoter requires Z-DNA-forming sequences that are converted to Z-DNA structure upon activation by the BAF complex. The BAF complex facilitates Z-DNA formation in a nucleosomal template in vitro. We propose a model in which the BAF complex promotes Z-DNA formation which, in turn, stabilizes the open chromatin structure at the CSF1 promoter.

  View details for Web of Science ID 000170433300009

  View details for PubMedID 11509180

• Promoter-specific binding of Rap1 revealed by genome-wide maps of protein-DNA association NATURE GENETICS Lieb, J. D., Liu, X. L., Botstein, D., Brown, P. O. 2001; 28 (4): 327-334

  Abstract

  We determined the distribution of repressor-activator protein 1 (Rap1) and the accessory silencing proteins Sir2, Sir3 and Sir4 in vivo on the entire yeast genome, at a resolution of 2 kb. Rap1 is central to the cellular economy during rapid growth, targeting 294 loci, about 5% of yeast genes, and participating in the activation of 37% of all RNA polymerase II initiation events in exponentially growing cells. Although the DNA sequence recognized by Rap1 is found in both coding and intergenic sequences, the binding of Rap1 to the genome was highly specific to intergenic regions with the potential to act as promoters. This global phenomenon, which may be a general characteristic of sequence-specific transcriptional factors, indicates the existence of a genome-wide molecular mechanism for marking promoter regions.

  View details for Web of Science ID 000170174800012

  View details for PubMedID 11455386

• Use of cDNA microarrays to analyze dioxin-induced changes in human liver gene expression TOXICOLOGY LETTERS Frueh, F. W., Hayashibara, K. C., Brown, P. O., Whitlock, J. P. 2001; 122 (3): 189-203

  Abstract

  One mechanism by which cells adapt to environmental changes is by altering gene expression. Here, we have used cDNA microarrays to identify genes whose expression is altered by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The goal of our study was to enhance our understanding of toxicity mediated through the pathway by which TCDD stimulates gene expression. To model this toxicity response, we exposed human hepatoma (HepG2) cells to TCDD (10 nM for 18 h) and analyzed mRNA by two-color fluorescent hybridization to cDNA sequences immobilized on glass microscope slides (2.5 x 7.5 cm) covering a surface area of 2.25 cm(2). We analyzed approximately one-third of the genes expressed in HepG2 cells and found that TCDD up- or down-regulates 112 genes two-fold or more. Most changes are relatively subtle (two- to four-fold). We verified the regulation of protooncogene cot, XMP, and human enhancer of filamentation-1 (HEF1), genes involved in cellular proliferation, as well as metallothionein, plasminogen activator inhibitor (PAI1), and HM74, genes involved in cellular signaling and regeneration. To characterize the response in more detail, we performed time-course, dose-dependence studies, and cycloheximide experiments. We observed direct and indirect responses to TCDD implying that adaptation to TCDD (and other related environmental stimuli) is substantially more complex than we previously realized.

  View details for Web of Science ID 000170531500001

  View details for PubMedID 11489354

• Missing value estimation methods for DNA microarrays BIOINFORMATICS Troyanskaya, O., Cantor, M., Sherlock, G., BROWN, P., Hastie, T., Tibshirani, R., Botstein, D., Altman, R. B. 2001; 17 (6): 520-525

  Abstract

  Gene expression microarray experiments can generate data sets with multiple missing expression values. Unfortunately, many algorithms for gene expression analysis require a complete matrix of gene array values as input. For example, methods such as hierarchical clustering and K-means clustering are not robust to missing data, and may lose effectiveness even with a few missing values. Methods for imputing missing data are needed, therefore, to minimize the effect of incomplete data sets on analyses, and to increase the range of data sets to which these algorithms can be applied. In this report, we investigate automated methods for estimating missing data.We present a comparative study of several methods for the estimation of missing values in gene microarray data. We implemented and evaluated three methods: a Singular Value Decomposition (SVD) based method (SVDimpute), weighted K-nearest neighbors (KNNimpute), and row average. We evaluated the methods using a variety of parameter settings and over different real data sets, and assessed the robustness of the imputation methods to the amount of missing data over the range of 1--20% missing values. We show that KNNimpute appears to provide a more robust and sensitive method for missing value estimation than SVDimpute, and both SVDimpute and KNNimpute surpass the commonly used row average method (as well as filling missing values with zeros). We report results of the comparative experiments and provide recommendations and tools for accurate estimation of missing microarray data under a variety of conditions.

  View details for Web of Science ID 000169404700005

  View details for PubMedID 11395428

• Comparative gene expression profiles following UV exposure in wild-type and SOS-deficient Escherichia coli GENETICS Courcelle, J., Khodursky, A., Peter, B., Brown, P. O., Hanawalt, P. C. 2001; 158 (1): 41-64

  Abstract

  The SOS response in UV-irradiated Escherichia coli includes the upregulation of several dozen genes that are negatively regulated by the LexA repressor. Using DNA microarrays containing amplified DNA fragments from 95.5% of all open reading frames identified on the E. coli chromosome, we have examined the changes in gene expression following UV exposure in both wild-type cells and lexA1 mutants, which are unable to induce genes under LexA control. We report here the time courses of expression of the genes surrounding the 26 documented lexA-regulated regions on the E. coli chromosome. We observed 17 additional sites that responded in a lexA-dependent manner and a large number of genes that were upregulated in a lexA-independent manner although upregulation in this manner was generally not more than twofold. In addition, several transcripts were either downregulated or degraded following UV irradiation. These newly identified UV-responsive genes are discussed with respect to their possible roles in cellular recovery following exposure to UV irradiation.

  View details for Web of Science ID 000168637000005

  View details for PubMedID 11333217

• Comparative genome-scale analysis of gene expression profiles in T cell lymphoma cells during malignant progression using a complementary DNA microarray AMERICAN JOURNAL OF PATHOLOGY Li, S. Y., Ross, D. T., Kadin, M. E., Brown, P. O., Wasik, M. A. 2001; 158 (4): 1231-1237

  Abstract

  Using a cDNA microarray, we compared the expression of approximately 8000 genes between two unique, clonally related T cell lines derived from different stages of a progressive T cell lymphoma involving skin. A total of 180 genes was found to be differentially expressed at the RNA level by a factor of fivefold or greater. Compared with the cells from the earlier, clinically indolent stage of the lymphoma, 56 genes were up-regulated, whereas 124 genes were down-regulated in the cells from the advanced, clinically aggressive stage lymphoma. The functions of approximately 65% of these genes are currently unknown. The 22 genes with a known function that were up-regulated in the advanced lymphoma cells included several genes involved in promotion of cell proliferation and survival as well as drug resistance. The 42 functionally characterized genes that were down-regulated in the advanced lymphoma cells included negative regulators of cell activation and cell cycle, and mediators of cell adhesion, apoptosis, and genome integrity. The differential expression identified by the cDNA microarray analysis was confirmed for selected genes by reverse transcription-polymerase chain reaction and Northern blotting. The identified differences in gene expression may be related to the differences in behavior between the early and advanced stages of the T cell lymphoma and point to directions for further investigations into mechanisms of lymphoma progression.

  View details for Web of Science ID 000168134000008

  View details for PubMedID 11290540

• Information access - Building a "GenBank" of the published literature SCIENCE Roberts, R. J., Varmus, H. E., Ashburner, M., Brown, P. O., Eisen, M. B., Khosla, C., Kirschner, M., Nusse, R., Scott, M., Wold, B. 2001; 291 (5512): 2318-2319

  View details for Web of Science ID 000167618700024

  View details for PubMedID 11269300

• Delineating developmental and metabolic pathways in vivo by expression profiling using the RIKEN set of 18,816 full-length enriched mouse cDNA arrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Miki, R., Kadota, K., Bono, H., Mizuno, Y., Tomaru, Y., Carninci, P., Itoh, M., Shibata, K., Kawai, J., Konno, H., Watanabe, S., Sato, K., Tokusumi, Y., Kikuchi, N., Ishii, Y., Hamaguchi, Y., Nishizuka, I., Goto, H., Nitanda, H., Satomi, S., Yoshiki, A., Kusakabe, M., DeRisi, J. L., Eisen, M. B., Iyer, V. R., Brown, P. O., Muramatsu, M., Shimada, H., Okazaki, Y., Hayashizaki, Y. 2001; 98 (5): 2199-2204

  Abstract

  We have systematically characterized gene expression patterns in 49 adult and embryonic mouse tissues by using cDNA microarrays with 18,816 mouse cDNAs. Cluster analysis defined sets of genes that were expressed ubiquitously or in similar groups of tissues such as digestive organs and muscle. Clustering of expression profiles was observed in embryonic brain, postnatal cerebellum, and adult olfactory bulb, reflecting similarities in neurogenesis and remodeling. Finally, clustering genes coding for known enzymes into 78 metabolic pathways revealed a surprising coordination of expression within each pathway among different tissues. On the other hand, a more detailed examination of glycolysis revealed tissue-specific differences in profiles of key regulatory enzymes. Thus, by surveying global gene expression by using microarrays with a large number of elements, we provide insights into the commonality and diversity of pathways responsible for the development and maintenance of the mammalian body plan.

  View details for Web of Science ID 000167258900020

  View details for PubMedID 11226216

  View details for PubMedCentralID PMC30115

• Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source MOLECULAR AND CELLULAR BIOLOGY Kuhn, K. M., DeRisi, J. L., Brown, P. O., Sarnow, P. 2001; 21 (3): 916-927

  Abstract

  The global gene expression program that accompanies the adaptation of Saccharomyces cerevisiae to an abrupt transfer from a fermentable to a nonfermentable carbon source was characterized by using a cDNA microarray to monitor the relative abundances and polysomal distributions of mRNAs. Features of the program included a transient reduction in global translational activity and a severe decrease in polysome size of transcripts encoding ribosomal proteins. While the overall translation initiation of newly synthesized and preexisting mRNAs was generally repressed after the carbon source shift, the mRNA encoded by YPL250C was an exception in that it selectively mobilized into polysomes, although its relative abundance remained unchanged. In addition, splicing of HAC1 transcripts, which has previously been reported to occur during accumulation of unfolded proteins in the endoplasmic reticulum, was observed after the carbon shift. This finding suggests that the nonconventional splicing complex, composed of the kinase-endonuclease Ire1p and the tRNA ligase Rlg1p, was activated. While spliced HAC1 transcripts mobilized into polysomes, the vast majority of unspliced HAC1 RNA accumulated in nonpolysomal fractions before and after the carbon source shift, indicating that translation of unspliced HAC1 RNA is blocked at the translation initiation step, in addition to the previously reported elongation step. These findings reveal that S. cerevisiae reacts to the carbon source shift with a remarkable variety of responses, including translational regulation of specific mRNAs and activation of specific enzymes involved in a nonconventional splicing mechanism.

  View details for Web of Science ID 000166353700022

  View details for PubMedID 11154278

  View details for PubMedCentralID PMC86682

• Genomic analysis of renal allograft dysfunction using cDNA microarrays 18th World Congress of the Transplantation-Society Sarwal, M., Chang, S., Barry, C., Chen, X., Alizadeh, A., Salvatierra, O., BROWN, P. ELSEVIER SCIENCE INC. 2001: 297–98

  View details for Web of Science ID 000167629900133

  View details for PubMedID 11266826

• Role of thioredoxin reductase in the Yap1p-dependent response to oxidative stress in Saccharomyces cerevisiae MOLECULAR MICROBIOLOGY Carmel-Harel, O., Stearman, R., Gasch, A. P., Botstein, D., Brown, P. O., Storz, G. 2001; 39 (3): 595-605

  Abstract

  The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress. In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.

  View details for Web of Science ID 000167052100006

  View details for PubMedID 11169101

• Genomic binding sites of the yeast cell-cycle transcription factors SBF and MBF NATURE Iyer, V. R., Horak, C. E., Scafe, C. S., Botstein, D., Snyder, M., Brown, P. O. 2001; 409 (6819): 533-538

  Abstract

  Proteins interact with genomic DNA to bring the genome to life; and these interactions also define many functional features of the genome. SBF and MBF are sequence-specific transcription factors that activate gene expression during the G1/S transition of the cell cycle in yeast. SBF is a heterodimer of Swi4 and Swi6, and MBF is a heterodimer of Mbpl and Swi6 (refs 1, 3). The related Swi4 and Mbp1 proteins are the DNA-binding components of the respective factors, and Swi6 mayhave a regulatory function. A small number of SBF and MBF target genes have been identified. Here we define the genomic binding sites of the SBF and MBF transcription factors in vivo, by using DNA microarrays. In addition to the previously characterized targets, we have identified about 200 new putative targets. Our results support the hypothesis that SBF activated genes are predominantly involved in budding, and in membrane and cell-wall biosynthesis, whereas DNA replication and repair are the dominant functions among MBF activated genes. The functional specialization of these factors may provide a mechanism for independent regulation of distinct molecular processes that normally occur in synchrony during the mitotic cell cycle.

  View details for Web of Science ID 000166570500053

  View details for PubMedID 11206552

• Supervised harvesting of expression trees GENOME BIOLOGY Hastie, T., Tibshirani, R., Botstein, D., Brown, P. 2001; 2 (1)

  Abstract

  We propose a new method for supervised learning from gene expression data. We call it 'tree harvesting'. This technique starts with a hierarchical clustering of genes, then models the outcome variable as a sum of the average expression profiles of chosen clusters and their products. It can be applied to many different kinds of outcome measures such as censored survival times, or a response falling in two or more classes (for example, cancer classes). The method can discover genes that have strong effects on their own, and genes that interact with other genes.We illustrate the method on data from a lymphoma study, and on a dataset containing samples from eight different cancers. It identified some potentially interesting gene clusters. In simulation studies we found that the procedure may require a large number of experimental samples to successfully discover interactions.Tree harvesting is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worthy of further investigation.

  View details for Web of Science ID 000207583500011

  View details for PubMedID 11178280

• Modulation of cellular and viral gene expression by the latency-associated nuclear antigen of kaposi's sarcoma-associated herpesvirus JOURNAL OF VIROLOGY Renne, R., Barry, C., Dittmer, D., Compitello, N., Brown, P. O., Ganem, D. 2001; 75 (1): 458-468

  Abstract

  Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), is the likely etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Common to these malignancies is that tumor cells are latently infected with KSHV. Viral gene expression is limited to a few genes, one of which is the latency-associated nuclear antigen (LANA), the product of ORF73. Examination of the primary sequence of LANA reveals some structural features reminiscent of transcription factors, leading us to hypothesize that LANA may regulate viral and cellular transcription during latency. In reporter gene-based transient transfection assays, we found that LANA can have either positive or negative effects on gene expression. While expression of a reporter gene from several synthetic promoters was increased in the presence of LANA, expression from the human immunodeficiency virus (HIV) long terminal repeat (LTR)-and from NF-kappaB-dependent reporter genes-was reduced by LANA expression. In addition, the promoter of KSHV ORF73 itself is activated up to 5.5-fold by LANA. This autoregulation may be important in tumorigenesis, because two other genes (v-cyclin and v-FLIP) with likely roles in cell growth and survival are also controlled by this element. To identify cellular genes influenced by LANA, we employed cDNA array-based expression profiling. Six known genes (and nine expressed sequence tags) were found to be upregulated in LANA-expressing cell lines. One of these, Staf-50, is known to inhibit expression from the HIV LTR; most of the other known genes are interferon inducible, although the interferon genes themselves were not induced by LANA. These data demonstrate that LANA expression has effects on cellular and viral gene expression. We suggest that, whether direct or indirect in origin, these effects may play important roles in the pathobiology of KSHV infection.

  View details for Web of Science ID 000165863000048

  View details for PubMedID 11119614

• Visualizing associations between genome sequences and gene expression data using genome-mean expression profiles. Bioinformatics Chiang, D. Y., Brown, P. O., Eisen, M. B. 2001; 17: S49-55

  Abstract

  The combination of genome-wide expression patterns and full genome sequences offers a great opportunity to further our understanding of the mechanisms and logic of transcriptional regulation. Many methods have been described that identify sequence motifs enriched in transcription control regions of genes that share similar gene expression patterns. Here we present an alternative approach that evaluates the transcriptional information contained by specific sequence motifs by computing for each motif the mean expression profile of all genes that contain the motif in their transcription control regions. These genome-mean expression profiles (GMEP's) are valuable for visualizing the relationship between genome sequences and gene expression data, and for characterizing the transcriptional importance of specific sequence motifs. Analysis of GMEP's calculated from a dataset of 519 whole-genome microarray experiments in Saccharomyces cerevisiae show a significant correlation between GMEP's of motifs that are reverse complements, a result that supports the relationship between GMEP's and transcriptional regulation. Hierarchical clustering of GMEP's identifies clusters of motifs that correspond to binding sites of well-characterized transcription factors. The GMEP's of these clustered motifs have patterns of variation across conditions that reflect the known activities of these transcription factors. Software that computed GMEP's from sequence and gene expression data is available under the terms of the Gnu Public License from http://rana.lbl.gov/.

  View details for PubMedID 11472992

• Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions GENOME BIOLOGY Haab, B. B., Dunham, M. J., Brown, P. O. 2001; 2 (2)

  Abstract

  We have developed and tested a method for printing protein microarrays and using these microarrays in a comparative fluorescence assay to measure the abundance of many specific proteins in complex solutions. A robotic device was used to print hundreds of specific antibody or antigen solutions in an array on the surface of derivatized microscope slides. Two complex protein samples, one serving as a standard for comparative quantitation, the other representing an experimental sample in which the protein quantities were to be measured, were labeled by covalent attachment of spectrally resolvable fluorescent dyes.Specific antibody-antigen interactions localized specific components of the complex mixtures to defined cognate spots in the array, where the relative intensity of the fluorescent signal representing the experimental sample and the reference standard provided a measure of each protein's abundance in the experimental sample. To test the specificity, sensitivity and accuracy of this assay, we analyzed the performance of 115 antibody/antigen pairs. 50% of the arrayed antigens and 20% of the arrayed antibodies provided specific and accurate measurements of their cognate ligands at or below concentrations of 0.34 microg/ml and 1.6 microg/ml, respectively. Some of the antibody/antigen pairs allowed detection of the cognate ligands at absolute concentrations below 1 ng/ml, and partial concentrations of 1 part in 106, sensitivities sufficient for measurement of many clinically important proteins in patient blood samples.These results suggest that protein microarrays can provide a practical means to characterize patterns of variation in hundreds of thousands of different proteins in clinical or research applications.

  View details for Web of Science ID 000207583600009

  View details for PubMedID 11182887

• The Stanford Microarray Database NUCLEIC ACIDS RESEARCH Sherlock, G., Hernandez-Boussard, T., Kasarskis, A., Binkley, G., Matese, J. C., Dwight, S. S., Kaloper, M., Weng, S., Jin, H., Ball, C. A., Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D., Cherry, J. M. 2001; 29 (1): 152-155

  Abstract

  The Stanford Microarray Database (SMD) stores raw and normalized data from microarray experiments, and provides web interfaces for researchers to retrieve, analyze and visualize their data. The two immediate goals for SMD are to serve as a storage site for microarray data from ongoing research at Stanford University, and to facilitate the public dissemination of that data once published, or released by the researcher. Of paramount importance is the connection of microarray data with the biological data that pertains to the DNA deposited on the microarray (genes, clones etc.). SMD makes use of many public resources to connect expression information to the relevant biology, including SGD [Ball,C.A., Dolinski,K., Dwight,S.S., Harris,M.A., Issel-Tarver,L., Kasarskis,A., Scafe,C.R., Sherlock,G., Binkley,G., Jin,H. et al. (2000) Nucleic Acids Res., 28, 77-80], YPD and WormPD [Costanzo,M.C., Hogan,J.D., Cusick,M.E., Davis,B.P., Fancher,A.M., Hodges,P.E., Kondu,P., Lengieza,C., Lew-Smith,J.E., Lingner,C. et al. (2000) Nucleic Acids Res., 28, 73-76], Unigene [Wheeler,D.L., Chappey,C., Lash,A.E., Leipe,D.D., Madden,T.L., Schuler,G.D., Tatusova,T.A. and Rapp,B.A. (2000) Nucleic Acids Res., 28, 10-14], dbEST [Boguski,M.S., Lowe,T.M. and Tolstoshev,C.M. (1993) Nature Genet., 4, 332-333] and SWISS-PROT [Bairoch,A. and Apweiler,R. (2000) Nucleic Acids Res., 28, 45-48] and can be accessed at http://genome-www.stanford.edu/microarray.

  View details for Web of Science ID 000166360300039

  View details for PubMedID 11125075

• Coordinate regulation of yeast ribosomal protein genes is associated with targeted recruitment of Esa1 histone acetylase MOLECULAR CELL Reid, J. L., Iyer, V. R., Brown, P. O., Struhl, K. 2000; 6 (6): 1297-1307

  Abstract

  The Esa1-containing NuA4 histone acetylase complex can interact with activation domains in vitro and stimulate transcription on reconstituted chromatin templates. In yeast cells, Esa1 is targeted to a small subset of promoters in an activator-specific manner. Esa1 is specifically recruited to ribosomal protein (RP) promoters, and this recruitment appears to require binding by Rap1 or Abf1. Esa1 is important for RP transcription, and Esa1 recruitment to RP promoters correlates with coordinate regulation of RP genes in response to growth stimuli. However, following Esa1 depletion, H4 acetylation decreases dramatically at many loci, but transcription is not generally affected. Therefore, the transcription-associated targeted recruitment of Esa1 to RP promoters occurs in a background of more global nontargeted acetylation that is itself not required for transcription.

  View details for Web of Science ID 000166239500003

  View details for PubMedID 11163204

• Genomic expression programs in the response of yeast cells to environmental changes MOLECULAR BIOLOGY OF THE CELL Gasch, A. P., Spellman, P. T., Kao, C. M., Carmel-Harel, O., Eisen, M. B., Storz, G., Botstein, D., Brown, P. O. 2000; 11 (12): 4241-4257

  Abstract

  We explored genomic expression patterns in the yeast Saccharomyces cerevisiae responding to diverse environmental transitions. DNA microarrays were used to measure changes in transcript levels over time for almost every yeast gene, as cells responded to temperature shocks, hydrogen peroxide, the superoxide-generating drug menadione, the sulfhydryl-oxidizing agent diamide, the disulfide-reducing agent dithiothreitol, hyper- and hypo-osmotic shock, amino acid starvation, nitrogen source depletion, and progression into stationary phase. A large set of genes (approximately 900) showed a similar drastic response to almost all of these environmental changes. Additional features of the genomic responses were specialized for specific conditions. Promoter analysis and subsequent characterization of the responses of mutant strains implicated the transcription factors Yap1p, as well as Msn2p and Msn4p, in mediating specific features of the transcriptional response, while the identification of novel sequence elements provided clues to novel regulators. Physiological themes in the genomic responses to specific environmental stresses provided insights into the effects of those stresses on the cell.

  View details for Web of Science ID 000165955000015

  View details for PubMedID 11102521

• New components of a system for phosphate accumulation and polyphosphate metabolism in Saccharomyces cerevisiae revealed by genomic expression analysis MOLECULAR BIOLOGY OF THE CELL Ogawa, N., DeRisi, J., Brown, P. O. 2000; 11 (12): 4309-4321

  Abstract

  The PHO regulatory pathway is involved in the acquisition of phosphate (P(i)) in the yeast Saccharomyces cerevisiae. When extracellular P(i) concentrations are low, several genes are transcriptionally induced by this pathway, which includes the Pho4 transcriptional activator, the Pho80-Pho85 cyclin-CDK pair, and the Pho81 CDK inhibitor. In an attempt to identify all the components regulated by this system, a whole-genome DNA microarray analysis was employed, and 22 PHO-regulated genes were identified. The promoter regions of 21 of these genes contained at least one copy of a sequence that matched the Pho4 recognition site. Eight of these genes, PHM1-PHM8, had no previously defined function in phosphate metabolism. The amino acid sequences of PHM1 (YFL004w), PHM2 (YPL019c), PHM3 (YJL012c), and PHM4 (YER072w) are 32-56% identical. The phm3 and phm4 single mutants and the phm1 phm2 double mutant were each severely deficient in accumulation of inorganic polyphosphate (polyP) and P(i). The phenotype of the phm5 mutant suggests that PHM5 (YDR452w) is essential for normal catabolism of polyP in the yeast vacuole. Taken together, the results reveal important new features of a genetic system that plays a critical role in P(i) acquisition and polyP metabolism in yeast.

  View details for Web of Science ID 000165955000019

  View details for PubMedID 11102525

• DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Khodursky, A. B., Peter, B. J., Cozzarelli, N. R., Botstein, D., Brown, P. O., Yanofsky, C. 2000; 97 (22): 12170-12175

  Abstract

  We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism. To do so we printed DNA microarrays containing 95% of all annotated E. coli ORFs. We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor. Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile. The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels. Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan. TyrR can be activated by any one of the three aromatic amino acids. Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium. We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation.

  View details for Web of Science ID 000090071000084

  View details for PubMedID 11027315

• Identification of the copper regulon in Saccharomyces cerevisiae by DNA microarrays JOURNAL OF BIOLOGICAL CHEMISTRY Gross, C., Kelleher, M., Iyer, V. R., Brown, P. O., Winge, D. R. 2000; 275 (41): 32310-32316

  Abstract

  In Saccharomyces cerevisiae, copper ions regulate gene expression through the two transcriptional activators, Ace1 and Mac1. Ace1 mediates copper-induced gene expression in cells exposed to stressful levels of copper salts, whereas Mac1 activates a subset of genes under copper-deficient conditions. DNA microarray hybridization experiments revealed a limited set of yeast genes differentially expressed under growth conditions of excess copper or copper deficiency. Mac1 activates the expression of six S. cerevisiae genes, including CTR1, CTR3, FRE1, FRE7, YFR055w, and YJL217w. Two of the last three newly identified Mac1 target genes have no known function; the third, YFR055w, is homologous to cystathionine gamma-lyase encoded by CYS3. Several genes that are differentially expressed in cells containing a constitutively active Mac1, designated Mac1(up1), are not direct targets of Mac1. Induction or repression of these genes is likely a secondary effect of cells because of constitutive Mac1 activity. Elevated copper levels induced the expression of the metallothioneins CUP1 and CRS5 and two genes, FET3 and FTR1, in the iron uptake system. Copper-induced FET3 and FTR1 expression arises from an indirect copper effect on cellular iron pools.

  View details for Web of Science ID 000089858900103

  View details for PubMedID 10922376

• Global mapping of meiotic recombination hotspots and coldspots in the yeast Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gerton, J. L., DeRisi, J., Shroff, R., Lichten, M., Brown, P. O., Petes, T. D. 2000; 97 (21): 11383-11390

  Abstract

  In the yeast Saccharomyces cerevisiae, meiotic recombination is initiated by double-strand DNA breaks (DSBs). Meiotic DSBs occur at relatively high frequencies in some genomic regions (hotspots) and relatively low frequencies in others (coldspots). We used DNA microarrays to estimate variation in the level of nearby meiotic DSBs for all 6,200 yeast genes. Hotspots were nonrandomly associated with regions of high G + C base composition and certain transcriptional profiles. Coldspots were nonrandomly associated with the centromeres and telomeres.

  View details for Web of Science ID 000089825700049

  View details for PubMedID 11027339

• Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lossos, I. S., Alizadeh, A. A., Eisen, M. B., Chan, W. C., Brown, P. O., Botstein, D., Staudt, L. M., Levy, R. 2000; 97 (18): 10209-10213

  Abstract

  B cell diffuse large cell lymphoma (B-DLCL) is a heterogeneous group of tumors, based on significant variations in morphology, clinical presentation, and response to treatment. Gene expression profiling has revealed two distinct tumor subtypes of B-DLCL: germinal center B cell-like DLCL and activated B cell-like DLCL. In a separate study, we determined that B-DLCL can also be subdivided into two groups based on the presence or absence of ongoing Ig gene hypermutation. Here, we evaluated the correlation between these B-DLCL subtypes established by the two different methods. Fourteen primary B-DLCL cases were studied by gene expression profiling using DNA microarrays and for the presence of ongoing mutations in their Ig heavy chain gene. All seven cases classified as germinal center B cell-like DLCL by gene expression showed the presence of ongoing mutations in the Ig genes. Five of the seven cases classified by gene expression as activated B cell-like DLCL had no ongoing somatic mutations, whereas, in the remaining two cases, a single point mutation was observed in only 2 of 15 and 21 examined molecular clones of variable heavy (V(H)) chain gene, respectively. These two cases were distantly related to the rest of the activated B cell-like DLCL tumors by gene expression. Our findings validate the concept that lymphoid malignancies are derived from cells at discrete stages of normal lymphocyte maturation and that the malignant cells retain the genetic program of those normal cells.

  View details for Web of Science ID 000089067500071

  View details for PubMedID 10954754

• Singular value decomposition for genome-wide expression data processing and modeling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Alter, O., Brown, P. O., Botstein, D. 2000; 97 (18): 10101-10106

  Abstract

  We describe the use of singular value decomposition in transforming genome-wide expression data from genes x arrays space to reduced diagonalized "eigengenes" x "eigenarrays" space, where the eigengenes (or eigenarrays) are unique orthonormal superpositions of the genes (or arrays). Normalizing the data by filtering out the eigengenes (and eigenarrays) that are inferred to represent noise or experimental artifacts enables meaningful comparison of the expression of different genes across different arrays in different experiments. Sorting the data according to the eigengenes and eigenarrays gives a global picture of the dynamics of gene expression, in which individual genes and arrays appear to be classified into groups of similar regulation and function, or similar cellular state and biological phenotype, respectively. After normalization and sorting, the significant eigengenes and eigenarrays can be associated with observed genome-wide effects of regulators, or with measured samples, in which these regulators are overactive or underactive, respectively.

  View details for Web of Science ID 000089067500053

  View details for PubMedID 10963673

• Molecular portraits of human breast tumours NATURE Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., Pollack, J. R., Ross, D. T., Johnsen, H., Akslen, L. A., Fluge, O., Pergamenschikov, A., Williams, C., Zhu, S. X., Lonning, P. E., Borresen-Dale, A. L., Brown, P. O., Botstein, D. 2000; 406 (6797): 747-752

  Abstract

  Human breast tumours are diverse in their natural history and in their responsiveness to treatments. Variation in transcriptional programs accounts for much of the biological diversity of human cells and tumours. In each cell, signal transduction and regulatory systems transduce information from the cell's identity to its environmental status, thereby controlling the level of expression of every gene in the genome. Here we have characterized variation in gene expression patterns in a set of 65 surgical specimens of human breast tumours from 42 different individuals, using complementary DNA microarrays representing 8,102 human genes. These patterns provided a distinctive molecular portrait of each tumour. Twenty of the tumours were sampled twice, before and after a 16-week course of doxorubicin chemotherapy, and two tumours were paired with a lymph node metastasis from the same patient. Gene expression patterns in two tumour samples from the same individual were almost always more similar to each other than either was to any other sample. Sets of co-expressed genes were identified for which variation in messenger RNA levels could be related to specific features of physiological variation. The tumours could be classified into subtypes distinguished by pervasive differences in their gene expression patterns.

  View details for Web of Science ID 000088767700049

  View details for PubMedID 10963602

• Analysis of topoisomerase function in bacterial replication fork movement: Use of DNA microarrays PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Khodursky, A. B., Peter, B. J., Schmidt, M. B., DeRisi, J., Botstein, D., Brown, P. O., Cozzarelli, N. R. 2000; 97 (17): 9419-9424

  Abstract

  We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

  View details for Web of Science ID 000088840500017

  View details for PubMedID 10944214

• Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth NATURE Zhu, G. F., Spellman, P. T., Volpe, T., Brown, P. O., Botstein, D., Davis, T. N., Futcher, B. 2000; 406 (6791): 90-94

  Abstract

  There are about 800 genes in Saccharomyces cerevisiae whose transcription is cell-cycle regulated. Some of these form clusters of co-regulated genes. The 'CLB2' cluster contains 33 genes whose transcription peaks early in mitosis, including CLB1, CLB2, SWI5, ACE2, CDC5, CDC20 and other genes important for mitosis. Here we find that the genes in this cluster lose their cell cycle regulation in a mutant that lacks two forkhead transcription factors, Fkh1 and Fkh2. Fkh2 protein is associated with the promoters of CLB2, SWI5 and other genes of the cluster. These results indicate that Fkh proteins are transcription factors for the CLB2 cluster. The fkh1 fkh2 mutant also displays aberrant regulation of the 'SIC1' cluster, whose member genes are expressed in the M-G1 interval and are involved in mitotic exit. This aberrant regulation may be due to aberrant expression of the transcription factors Swi5 and Ace2, which are members of the CLB2 cluster and controllers of the SIC1 cluster. Thus, a cascade of transcription factors operates late in the cell cycle. Finally, the fkh1 fkh2 mutant displays a constitutive pseudohyphal morphology, indicating that Fkh1 and Fkh2 may help control the switch to this mode of growth.

  View details for Web of Science ID 000087991200051

  View details for PubMedID 10894548

• Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Lyons, T. J., Gasch, A. P., Gaither, L. A., Botstein, D., Brown, P. O., Eide, D. J. 2000; 97 (14): 7957-7962

  Abstract

  The Zap1p transcription factor senses cellular zinc status and increases expression of its target genes in response to zinc deficiency. Previously known Zap1p-regulated genes encode the Zrt1p, Zrt2p, and Zrt3p zinc transporter genes and Zap1p itself. To allow the characterization of additional genes in yeast important for zinc homeostasis, a systematic study of gene expression on the genome-wide scale was used to identify other Zap1p target genes. Using a combination of DNA microarrays and a computer-assisted analysis of shared motifs in the promoters of similarly regulated genes, we identified 46 genes that are potentially regulated by Zap1p. Zap1p-regulated expression of seven of these newly identified target genes was confirmed independently by using lacZ reporter fusions, suggesting that many of the remaining candidate genes are also Zap1p targets. Our studies demonstrate the efficacy of this combined approach to define the regulon of a specific eukaryotic transcription factor.

  View details for Web of Science ID 000088048400055

  View details for PubMedID 10884426

• Examining the living genome in health and disease with DNA microarrays JAMA-JOURNAL OF THE AMERICAN MEDICAL ASSOCIATION Diehn, M., Alizadeh, A. A., Brown, P. O. 2000; 283 (17): 2298-2299

  View details for Web of Science ID 000086671600042

  View details for PubMedID 10807394

• Large-scale identification of secreted and membrane-associated gene products using DNA microarrays NATURE GENETICS Diehn, M., Eisen, M. B., Botstein, D., Brown, P. O. 2000; 25 (1): 58-62

  Abstract

  Membrane-associated and secreted proteins are an important class of proteins and include receptors, transporters, adhesion molecules, hormones and cytokines. Although algorithms have been developed to recognize potential amino-terminal membrane-targeting signals or transmembrane domains in protein sequences, their accuracy is limited and they require knowledge of the entire coding sequence, including the N terminus, which is not currently available for most of the genes in most organisms, including human. Several experimental approaches for identifying secreted and membrane proteins have been described, but none have taken a comprehensive genomic approach. Furthermore, none of these methods allow easy classification of clones from arrayed cDNA libraries, for which large-scale gene-expression data are now becoming available through the use of DNA microarrays. We describe here a rapid and efficient method for identifying genes that encode secreted or membrane proteins. mRNA species bound to membrane-associated polysomes were separated from other mRNAs by sedimentation equilibrium or sedimentation velocity. The distribution of individual transcripts in the 'membrane-bound' and 'cytosolic' fractions was quantitated for thousands of genes by hybridization to DNA microarrays. Transcripts known to encode secreted or membrane proteins were enriched in the membrane-bound fractions, whereas those known to encode cytoplasmic proteins were enriched in the fractions containing mRNAs associated with free and cytoplasmic ribosomes. On this basis, we identified over 275 human genes and 285 yeast genes that are likely to encode previously unrecognized secreted or membrane proteins.

  View details for Web of Science ID 000086884000017

  View details for PubMedID 10802657

• Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae - Evidence for two pathways of iron uptake JOURNAL OF BIOLOGICAL CHEMISTRY Yun, C. W., Ferea, T., Rashford, J., Ardon, O., Brown, P. O., Botstein, D., Kaplan, J., Philpott, C. C. 2000; 275 (14): 10709-10715

  Abstract

  In the yeast Saccharomyces cerevisiae, uptake of iron is largely regulated by the transcription factor Aft1. cDNA microarrays were used to identify new iron and AFT1-regulated genes. Four homologous genes regulated as part of the AFT1-regulon (ARN1-4) were predicted to encode members of a subfamily of the major facilitator superfamily of transporters. These genes were predicted to encode proteins with 14 membrane spanning domains and were from 26 to 53% identical at the amino acid level. ARN3 is identical to SIT1, which is reported to encode a ferrioxamine B permease. Deletion of ARN3 did not prevent yeast from using ferrioxamine B as an iron source; however, deletion of ARN3 and FET3, a component of the high affinity ferrous iron transport system, did prevent uptake of ferrioxamine-bound iron and growth on ferrioxamine as an iron source. The siderophore-mediated transport system and the high affinity ferrous iron transport system were localized to separate cellular compartments. Epitope-tagged Arn3p was expressed in intracellular vesicles that co-sediment with the endosomal protein Pep12. In contrast, Fet3p was expressed on the plasma membrane and was digested by extracellular proteases. These data indicate that S. cerevisiae has two pathways for ferrrioxamine-mediated iron uptake, one occurring at the plasma membrane and the other occurring in an intracellular compartment.

  View details for Web of Science ID 000086345600110

  View details for PubMedID 10744769

• Degradation of proteins from the ER of S-cerevisiae requires an intact unfolded protein response pathway MOLECULAR CELL Casagrande, R., Stern, P., Diehn, M., Shamu, C., Osario, M., Zuniga, M., Brown, P. O., Ploegh, H. 2000; 5 (4): 729-735

  Abstract

  To dissect the requirements of membrane protein degradation from the ER, we expressed the mouse major histocompatibility complex class I heavy chain H-2K(b) in yeast. Like other proteins degraded from the ER, unassembled H-2K(b) heavy chains are not transported to the Golgi but are degraded in a proteasome-dependent manner. The overexpression of H-2K(b) heavy chains induces the unfolded protein response (UPR). In yeast mutants unable to mount the UPR, H-2K(b) heavy chains are greatly stabilized. This defect in degradation is suppressed by the expression of the active form of Hac1p, the transcription factor that upregulates UPR-induced genes. These results indicate that induction of the UPR is required for the degradation of protein substrates from the ER.

  View details for Web of Science ID 000086790000013

  View details for PubMedID 10882108

• Whole-genome expression analysis of snf/swi mutants of Saccharomyces cerevisiae PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sudarsanam, P., Iyer, V. R., Brown, P. O., Winston, F. 2000; 97 (7): 3364-3369

  Abstract

  The Saccharomyces cerevisiae Snf/Swi complex has been previously demonstrated to control transcription and chromatin structure of particular genes in vivo and to remodel nucleosomes in vitro. We have performed whole-genome expression analysis, using DNA microarrays, to study mutants deleted for a gene encoding one conserved (Snf2) or one unconserved (Swi1) Snf/Swi component. This analysis was performed on cells grown in both rich and minimal media. The microarray results, combined with Northern blot, computational, and genetic analyses, show that snf2Delta and swi1Delta mutations cause similar effects on mRNA levels, that Snf/Swi controls some genes differently in rich and minimal media, and that Snf/Swi control is exerted at the level of individual genes rather than over larger chromosomal domains. In addition, this work shows that Snf/Swi controls mRNA levels of MATalpha-specific genes, likely via controlling transcription of the regulators MATalpha1 and MCM1. Finally, we provide evidence that Snf/Swi acts both as an activator and as a repressor of transcription, and that neither mode of control is an indirect effect of the other.

  View details for Web of Science ID 000086195200074

  View details for PubMedID 10725359

• Genome microarray analysis of transcriptional activation in multidrug resistance yeast mutants FEBS LETTERS DeRisi, J., van den Hazel, B., Marc, P., Balzi, E., BROWN, P., Jacq, C., Goffeau, A. 2000; 470 (2): 156-160

  Abstract

  The cDNA from activated mutants of the homologous transcription factors Pdr1p and Pdr3p was used to screen DNA microarrays of the Saccharomyces cerevisiae complete genome. Twenty-six overexpressed targets of the PDR1-3 and/or PDR3-7 mutants were identified. Twenty-one are new targets, the majority of which are of unknown function. In addition to well known ABC transporters, these targets appear to be involved in transport or in membrane lipids and cell wall biosyntheses. Several of the targets seem to contribute to the cell defence against a variety of stresses. Pdr1p and Pdr3p do not act similarly on all targets. Unexpectedly, the expression of 23 other genes appeared to be repressed in the PDR1-3 and/or PDR3-7 mutants. In contrast to the majority of the activated genes, none of the repressed genes contains pleiotropic drug resistance binding sites in their promoter.

  View details for Web of Science ID 000086230300012

  View details for PubMedID 10734226

• Functional characterization of the human immunodeficiency virus type 1 genome by genetic footprinting JOURNAL OF VIROLOGY Laurent, L. C., Olsen, M. N., Crowley, R. A., Savilahti, H., Brown, P. O. 2000; 74 (6): 2760-2769

  Abstract

  We present a detailed and quantitative analysis of the functional characteristics of the 1,000-nucleotide segment at the 5' end of the human immunodeficiency virus type 1 (HIV-1) RNA genome. This segment of the viral genome contains several important cis-acting sequences, including the TAR, polyadenylation, viral att site, minus-strand primer-binding site, and 5' splice donor sequences, as well as coding sequences for the matrix protein and the N-terminal half of the capsid protein. The genetic footprinting technique was used to determine quantitatively the abilities of 134 independent insertion mutations to (i) make stable viral RNA, (ii) assemble and release viral RNA-containing viral particles, and (iii) enter host cells, complete reverse transcription, enter the nuclei of host cells, and generate proviruses in the host genome by integration. All of the mutants were constructed and analyzed en masse, greatly decreasing the labor typically involved in mutagenesis studies. The results confirmed the presence of several previously known functional features in this region of the HIV genome and provided evidence for several novel features, including newly identified cis-acting sequences that appeared to contribute to (i) the formation of stable viral transcripts, (ii) viral RNA packaging, and (iii) an early step in viral replication. The results also pointed to an unanticipated trans-acting role for the N-terminal portion of matrix in the formation of stable viral RNA transcripts. Finally, in contrast to previous reports, the results of this study suggested that detrimental mutations in the matrix and capsid proteins principally interfered with viral assembly.

  View details for Web of Science ID 000085447100030

  View details for PubMedID 10684292

• Systematic variation in gene expression patterns in human cancer cell lines NATURE GENETICS Ross, D. T., Scherf, U., Eisen, M. B., Perou, C. M., Rees, C., Spellman, P., Iyer, V., Jeffrey, S. S., van de Rijn, M., Waltham, M., Pergamenschikov, A., Lee, J. C., Lashkari, D., Shalon, D., Myers, T. G., Weinstein, J. N., Botstein, D., Brown, P. O. 2000; 24 (3): 227-235

  Abstract

  We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

  View details for Web of Science ID 000085590600011

  View details for PubMedID 10700174

• A gene expression database for the molecular pharmacology of cancer NATURE GENETICS Scherf, U., Ross, D. T., Waltham, M., Smith, L. H., Lee, J. K., Tanabe, L., Kohn, K. W., Reinhold, W. C., Myers, T. G., Andrews, D. T., Scudiero, D. A., Eisen, M. B., SAUSVILLE, E. A., Pommier, Y., Botstein, D., Brown, P. O., Weinstein, J. N. 2000; 24 (3): 236-244

  Abstract

  We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

  View details for Web of Science ID 000085590600012

  View details for PubMedID 10700175

• Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling NATURE Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. G., Sabet, H., Tran, T., Yu, X., Powell, J. I., Yang, L. M., Marti, G. E., Moore, T., Hudson, J., Lu, L. S., Lewis, D. B., Tibshirani, R., Sherlock, G., Chan, W. C., Greiner, T. C., Weisenburger, D. D., Armitage, J. O., Warnke, R., Levy, R., Wilson, W., Grever, M. R., Byrd, J. C., Botstein, D., Brown, P. O., Staudt, L. M. 2000; 403 (6769): 503-511

  Abstract

  Diffuse large B-cell lymphoma (DLBCL), the most common subtype of non-Hodgkin's lymphoma, is clinically heterogeneous: 40% of patients respond well to current therapy and have prolonged survival, whereas the remainder succumb to the disease. We proposed that this variability in natural history reflects unrecognized molecular heterogeneity in the tumours. Using DNA microarrays, we have conducted a systematic characterization of gene expression in B-cell malignancies. Here we show that there is diversity in gene expression among the tumours of DLBCL patients, apparently reflecting the variation in tumour proliferation rate, host response and differentiation state of the tumour. We identified two molecularly distinct forms of DLBCL which had gene expression patterns indicative of different stages of B-cell differentiation. One type expressed genes characteristic of germinal centre B cells ('germinal centre B-like DLBCL'); the second type expressed genes normally induced during in vitro activation of peripheral blood B cells ('activated B-like DLBCL'). Patients with germinal centre B-like DLBCL had a significantly better overall survival than those with activated B-like DLBCL. The molecular classification of tumours on the basis of gene expression can thus identify previously undetected and clinically significant subtypes of cancer.

  View details for Web of Science ID 000085227300039

  View details for PubMedID 10676951

• 'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns. Genome biology Hastie, T., Tibshirani, R., Eisen, M. B., Alizadeh, A., Levy, R., Staudt, L., Chan, W. C., Botstein, D., BROWN, P. 2000; 1 (2): RESEARCH0003-?

  Abstract

  Large gene expression studies, such as those conducted using DNA arrays, often provide millions of different pieces of data. To address the problem of analyzing such data, we describe a statistical method, which we have called 'gene shaving'. The method identifies subsets of genes with coherent expression patterns and large variation across conditions. Gene shaving differs from hierarchical clustering and other widely used methods for analyzing gene expression studies in that genes may belong to more than one cluster, and the clustering may be supervised by an outcome measure. The technique can be 'unsupervised', that is, the genes and samples are treated as unlabeled, or partially or fully supervised by using known properties of the genes or samples to assist in finding meaningful groupings.We illustrate the use of the gene shaving method to analyze gene expression measurements made on samples from patients with diffuse large B-cell lymphoma. The method identifies a small cluster of genes whose expression is highly predictive of survival.The gene shaving method is a potentially useful tool for exploration of gene expression data and identification of interesting clusters of genes worth further investigation.

  View details for PubMedID 11178228

  View details for PubMedCentralID PMC15015

• Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions GENOME BIOLOGY Haab, B. B., Dunham, M. J., Brown, P. O. 2000; 1 (6)

  View details for Web of Science ID 000207583400006

• Shotgun DNA microarrays and stage-specific gene expression in Plasmodium falciparum malaria MOLECULAR MICROBIOLOGY Hayward, R. E., DeRisi, J. L., Alfadhli, S., Kaslow, D. C., Brown, P. O., Rathod, P. K. 2000; 35 (1): 6-14

  Abstract

  Malaria infects over 200 million individuals and kills 2 million young children every year. Understanding the biology of malarial parasites will be facilitated by DNA microarray technology, which can track global changes in gene expression under different physiological conditions. However, genomes of Plasmodium sp. (and many other important pathogenic organisms) remain to be fully sequenced so, currently, it is not possible to construct gene-specific microarrays representing complete malarial genomes. In this study, 3648 random inserts from a Plasmodium falciparum mung bean nuclease genomic library were used to construct a shotgun DNA microarray. Through differential hybridization and sequencing of relevant clones, large differences in gene expression were identified between the blood stage trophozoite form of the malarial parasite and the sexual stage gametocyte form. The present study lengthens our list of stage-specific transcripts in malaria by at least an order of magnitude above all previous studies combined. The results offer an unprecedented number of leads for developing transmission blocking agents and for developing vaccines directed at blood stage antigens. A significant fraction of the stage-selective transcripts had no sequence homologues in the current genome data bases, thereby underscoring the importance of the shotgun approach. The malarial shotgun microarray will be useful for unravelling additional important aspects of malaria biology and the general approach may be applied to any organism, regardless of how much of its genome is sequenced.

  View details for Web of Science ID 000084867000002

  View details for PubMedID 10632873

• Arrest, adaptation, and recovery following a chromosome double-strand break in Saccharomyces cerevisiae Cold Spring Harbor Symposium on Quantitative Biology Lee, S. E., Pellicioli, A., Demeter, J., Vaze, M. P., Gasch, A. P., Malkova, A., Brown, P. O., Botstein, D., Stearns, T., Foiani, M., Haber, J. E. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 2000: 303–314

  View details for Web of Science ID 000169676800031

  View details for PubMedID 12760044

• Identification of clinically distinct types of diffuse large B-cell lymphoma based on gene expression patterns. Nature Brown, P.O., Alizadeh, A., Eisen, M.B., Davis, R.E., Ma, C., Rosenwald, A., Sherlock, G., Boldrick, J.C., Sabet, H., Tran, T., Yu, X., Powell, J.I., Yang, L., Marti, G.E., Moore, T., Hudson, J., Chan, W.C., Greiner, T., Weisenberger, D., Tibshirani, R., Armitage, J.O., Lossos, I., Levy, R., Wilson, W., Grever, M., Byrd, J., Botstein, D., Staudt, L. 2000: 503-511
• Genomic views of the immune system ANNUAL REVIEW OF IMMUNOLOGY Staudt, L. M., Brown, P. O. 2000; 18: 829-859

  Abstract

  Genomic-scale experimentation aims to view biological processes as a whole, yet with molecular precision. Using massively parallel DNA microarray technology, the mRNA expression of tens of thousands of genes can be measured simultaneously. Mathematical distillation of this flood of gene expression data reveals a deep molecular and biological logic underlying gene expression programs during cellular differentiation and activation. Genes that encode components of the same multi-subunit protein complex are often coordinately regulated. Coordinate regulation is also observed among genes whose products function in a common differentiation program or in the same physiological response pathway. Recent application of gene expression profiling to the immune system has shown that lymphocyte differentiation and activation are accompanied by changes of hundreds of genes in parallel. The databases of gene expression emerging from these studies of normal immune responses will be used to interpret the pathological changes in gene expression that accompany autoimmunity, immune deficiencies, and cancers of immune cells.

  View details for Web of Science ID 000087236500028

  View details for PubMedID 10837077

• Observing the living genome CURRENT OPINION IN GENETICS & DEVELOPMENT Ferea, T. L., Brown, P. O. 1999; 9 (6): 715-722

  Abstract

  Dynamic pictures of living genomes are now beginning to emerge from systematic studies of gene expression patterns using DNA microarrays. The rich information represented in the variation in each gene's expression provides the basis for a new kind of genomic map.

  View details for Web of Science ID 000084277900016

  View details for PubMedID 10607618

• Stereospecificity of reactions catalyzed by HIV-1 integrase JOURNAL OF BIOLOGICAL CHEMISTRY Gerton, J. L., Herschlag, D., Brown, P. O. 1999; 274 (47): 33480-33487

  Abstract

  The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.

  View details for Web of Science ID 000083745200048

  View details for PubMedID 10559232

• Identification of eukaryotic mRNAs that are translated at reduced cap binding complex eIF4F concentrations using a cDNA microarray PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Johannes, G., Carter, M. S., Eisen, M. B., Brown, P. O., Sarnow, P. 1999; 96 (23): 13118-13123

  Abstract

  Although most eukaryotic mRNAs need a functional cap binding complex eIF4F for efficient 5' end- dependent scanning to initiate translation, picornaviral, hepatitis C viral, and a few cellular RNAs have been shown to be translated by internal ribosome entry, a mechanism that can operate in the presence of low levels of functional eIF4F. To identify cellular mRNAs that can be translated when eIF4F is depleted or in low abundance and that, therefore, may contain internal ribosome entry sites, mRNAs that remained associated with polysomes were isolated from human cells after infection with poliovirus and were identified by using a cDNA microarray. Approximately 200 of the 7000 mRNAs analyzed remained associated with polysomes under these conditions. Among the gene products encoded by these polysome-associated mRNAs were immediate-early transcription factors, kinases, and phosphatases of the mitogen-activated protein kinase pathways and several protooncogenes, including c-myc and Pim-1. In addition, the mRNA encoding Cyr61, a secreted factor that can promote angiogenesis and tumor growth, was selectively mobilized into polysomes when eIF4F concentrations were reduced, although its overall abundance changed only slightly. Subsequent tests confirmed the presence of internal ribosome entry sites in the 5' noncoding regions of both Cyr61 and Pim-1 mRNAs. Overall, this study suggests that diverse mRNAs whose gene products have been implicated in a variety of stress responses, including inflammation, angiogenesis, and the response to serum, can use translational initiation mechanisms that require little or no intact cap binding protein complex eIF4F.

  View details for Web of Science ID 000083649400028

  View details for PubMedID 10557283

• Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wilson, W., DeRisi, J., Kristensen, H. H., Imboden, P., Rane, S., Brown, P. O., SCHOOLNIK, G. K. 1999; 96 (22): 12833-12838

  Abstract

  Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

  View details for Web of Science ID 000083373000113

  View details for PubMedID 10536008

• Genome-wide analysis of DNA copy-number changes using cDNA microarrays NATURE GENETICS Pollack, J. R., Perou, C. M., Alizadeh, A. A., Eisen, M. B., Pergamenschikov, A., Williams, C. F., Jeffrey, S. S., Botstein, D., Brown, P. O. 1999; 23 (1): 41-46

  Abstract

  Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.

  View details for Web of Science ID 000082337300013

  View details for PubMedID 10471496

• Systematic changes in gene expression patterns following adaptive evolution in yeast PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ferea, T. L., Botstein, D., Brown, P. O., Rosenzweig, R. F. 1999; 96 (17): 9721-9726

  Abstract

  Culturing a population of Saccharomyces cerevisiae for many generations under conditions to which it is not optimally adapted selects for fitter genetic variants. This simple experimental design provides a tractable model of adaptive evolution under natural selection. Beginning with a clonal, founding population, independently evolved strains were obtained from three independent cultures after continuous aerobic growth in glucose-limited chemostats for more than 250 generations. DNA microarrays were used to compare genome-wide patterns of gene expression in the evolved strains and the parental strain. Several hundred genes were found to have significantly altered expression in the evolved strains. Many of these genes showed similar alterations in their expression in all three evolved strains. Genes with altered expression in the three evolved strains included genes involved in glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and metabolite transport. These results are consistent with physiological observations and indicate that increased fitness is acquired by altering regulation of central metabolism such that less glucose is fermented and more glucose is completely oxidized.

  View details for Web of Science ID 000082098500052

  View details for PubMedID 10449761

• Distinctive gene expression patterns in human mammary epithelial cells and breast cancers PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Perou, C. M., Jeffrey, S. S., van de Rijn, M., Rees, C. A., Eisen, M. B., Ross, D. T., Pergamenschikov, A., Williams, C. F., Zhu, S. X., Lee, J. C., Lashkari, D., Shalon, D., Brown, P. O., Botstein, D. 1999; 96 (16): 9212-9217

  Abstract

  cDNA microarrays and a clustering algorithm were used to identify patterns of gene expression in human mammary epithelial cells growing in culture and in primary human breast tumors. Clusters of coexpressed genes identified through manipulations of mammary epithelial cells in vitro also showed consistent patterns of variation in expression among breast tumor samples. By using immunohistochemistry with antibodies against proteins encoded by a particular gene in a cluster, the identity of the cell type within the tumor specimen that contributed the observed gene expression pattern could be determined. Clusters of genes with coherent expression patterns in cultured cells and in the breast tumors samples could be related to specific features of biological variation among the samples. Two such clusters were found to have patterns that correlated with variation in cell proliferation rates and with activation of the IFN-regulated signal transduction pathway, respectively. Clusters of genes expressed by stromal cells and lymphocytes in the breast tumors also were identified in this analysis. These results support the feasibility and usefulness of this systematic approach to studying variation in gene expression patterns in human cancers as a means to dissect and classify solid tumors.

  View details for Web of Science ID 000081835500080

  View details for PubMedID 10430922

  View details for PubMedCentralID PMC17759

• Transduction of human progenitor hematopoietic stem cells by human immunodeficiency virus type 1-based vectors is cell cycle dependent JOURNAL OF VIROLOGY Sutton, R. E., Reitsma, M. J., Uchida, N., Brown, P. O. 1999; 73 (5): 3649-3660

  Abstract

  Human immunodeficiency virus (HIV) type 1 vectors are highly efficient in their ability to transduce human progenitor hematopoietic stem cells (PHSC). Although mitosis was not required for transduction of these cells, transduction rates were much greater once cells had been cultured in the presence of cytokines. Transduction rates, however, rarely exceeded 70%. We demonstrate here that there is a distinct subpopulation that is more easily transduced by HIV vectors. These cells were distinguished by a disproportionate population in the S/G2/M phases of the cell cycle. By sorting them prior to transduction, we found that those cells in either the G1 or S/G2/M fraction were more readily transduced than G0 cells. Maintaining the cells in G0 by omitting cytokines from the medium reduced transduction rates by up to 10-fold. Addition of cytokines to the medium immediately after transduction did not improve the transduction efficiency as measured by expression of the transgene. Analysis of replication intermediates indicated that the block to transduction of G0 cells operated near the time of initiation of reverse transcription. These results suggest that although lentivirus vectors can transduce nondividing PHSC, transduction efficiency is severalfold greater once the cells exit G0 and enter G1. Further characterization of these more transducible cells and identification of the cellular factors responsible may enhance transduction while maintaining the pluripotentiality of the PHSC.

  View details for Web of Science ID 000079701100019

  View details for PubMedID 10196257

• Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes NUCLEIC ACIDS RESEARCH Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O., Weigel, R. J. 1999; 27 (6): 1517-1523

  Abstract

  Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  View details for Web of Science ID 000079256800016

  View details for PubMedID 10037815

• Mediator protein mutations that selectively abolish activated transcription PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Myers, L. C., Gustafsson, C. M., Hayashibara, K. C., Brown, P. O., Kornberg, R. D. 1999; 96 (1): 67-72

  Abstract

  Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4-VP16 in vitro. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation in vivo were closely coincident with those in vitro. A whole genome expression profile of a Deltamed2 strain showed diminished transcription of a subset of inducible genes but only minor effects on "basal" transcription. These findings make an important connection between transcriptional activation in vitro and in vivo, and identify Mediator as a "global" transcriptional coactivator.

  View details for Web of Science ID 000078004400015

  View details for PubMedID 9874773

• DNA arrays for analysis of gene expression CDNA PREPARATION AND CHARACTERIZATION Eisen, M. B., Brown, P. O. 1999; 303: 179-205

  View details for Web of Science ID 000081913000012

  View details for PubMedID 10349646

• The transcriptional program in the response of human fibroblasts to serum SCIENCE Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson, J., Boguski, M. S., Lashkari, D., Shalon, D., Botstein, D., Brown, P. O. 1999; 283 (5398): 83-87

  Abstract

  The temporal program of gene expression during a model physiological response of human cells, the response of fibroblasts to serum, was explored with a complementary DNA microarray representing about 8600 different human genes. Genes could be clustered into groups on the basis of their temporal patterns of expression in this program. Many features of the transcriptional program appeared to be related to the physiology of wound repair, suggesting that fibroblasts play a larger and richer role in this complex multicellular response than had previously been appreciated.

  View details for Web of Science ID 000077976600055

  View details for PubMedID 9872747

• The lymphochip: A specialized cDNA microarray for the genomic-scale analysis of gene expression in normal and malignant lymphocytes 64th Symposia: Signaling and Gene Expression in the Immune System Alizadeh, A., Eisen, M., Davis, R. E., Ma, C., Sabet, H., Tran, T., Powell, J. I., Yang, L., Marti, G. E., Moore, D. T., Hudson, J. R., Chan, W. C., Greiner, T., Weisenburger, D., Armitage, J. O., Lossos, I., Levy, R., Botstein, D., Brown, P. O., Staudt, L. M. COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT. 1999: 71–78

  View details for Web of Science ID 000087225400011

  View details for PubMedID 11232339

• Exploring the new world of the genome with DNA microarrays NATURE GENETICS Brown, P. O., Botstein, D. 1999; 21: 33-37

  Abstract

  Thousands of genes are being discovered for the first time by sequencing the genomes of model organisms, an exhilarating reminder that much of the natural world remains to be explored at the molecular level. DNA microarrays provide a natural vehicle for this exploration. The model organisms are the first for which comprehensive genome-wide surveys of gene expression patterns or function are possible. The results can be viewed as maps that reflect the order and logic of the genetic program, rather than the physical order of genes on chromosomes. Exploration of the genome using DNA microarrays and other genome-scale technologies should narrow the gap in our knowledge of gene function and molecular biology between the currently-favoured model organisms and other species.

  View details for Web of Science ID 000078008200009

  View details for PubMedID 9915498

• Cluster analysis and display of genome-wide expression patterns PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. 1998; 95 (25): 14863-14868

  Abstract

  A system of cluster analysis for genome-wide expression data from DNA microarray hybridization is described that uses standard statistical algorithms to arrange genes according to similarity in pattern of gene expression. The output is displayed graphically, conveying the clustering and the underlying expression data simultaneously in a form intuitive for biologists. We have found in the budding yeast Saccharomyces cerevisiae that clustering gene expression data groups together efficiently genes of known similar function, and we find a similar tendency in human data. Thus patterns seen in genome-wide expression experiments can be interpreted as indications of the status of cellular processes. Also, coexpression of genes of known function with poorly characterized or novel genes may provide a simple means of gaining leads to the functions of many genes for which information is not available currently.

  View details for Web of Science ID 000077436700051

  View details for PubMedID 9843981

• Characterization of three related glucose repressors and genes they regulate in Saccharomyces cerevisiae GENETICS Lutfiyya, L. L., Iyer, V. R., DeRisi, J., DeVit, M. J., Brown, P. O., Johnston, M. 1998; 150 (4): 1377-1391

  Abstract

  Mig1 and Mig2 are proteins with similar zinc fingers that are required for glucose repression of SUC2 expression. Mig1, but not Mig2, is required for repression of some other glucose-repressed genes, including the GAL genes. A second homolog of Mig1, Yer028, appears to be a glucose-dependent transcriptional repressor that binds to the Mig1-binding sites in the SUC2 promoter, but is not involved in glucose repression of SUC2 expression. Despite their functional redundancy, we found several significant differences between Mig1 and Mig2: (1) in the absence of glucose, Mig1, but not Mig2, is inactivated by the Snf1 protein kinase; (2) nuclear localization of Mig1, but not Mig2, is regulated by glucose; (3) expression of MIG1, but not MIG2, is repressed by glucose; and (4) Mig1 and Mig2 bind to similar sites but with different relative affinities. By two approaches, we have identified many genes regulated by Mig1 and Mig2, and confirmed a role for Mig1 and Mig2 in repression of several of them. We found no genes repressed by Yer028. Also, we identified no genes repressed by only Mig1 or Mig2. Thus, Mig1 and Mig2 are redundant glucose repressors of many genes.

  View details for Web of Science ID 000077401300005

  View details for PubMedID 9832517

• Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization MOLECULAR BIOLOGY OF THE CELL Spellman, P. T., Sherlock, G., Zhang, M. Q., Iyer, V. R., Anders, K., Eisen, M. B., Brown, P. O., Botstein, D., Futcher, B. 1998; 9 (12): 3273-3297

  Abstract

  We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu

  View details for Web of Science ID 000077388600003

  View details for PubMedID 9843569

• Drug target validation and identification of secondary drug target effects using DNA microarrays NATURE MEDICINE Marton, M. J., DeRisi, J. L., Bennett, H. A., Iyer, V. R., Meyer, M. R., Roberts, C. J., Stoughton, R., Burchard, J., Slade, D., Dai, H. Y., Bassett, D. E., Hartwell, L. H., Brown, P. O., Friend, S. H. 1998; 4 (11): 1293-1301

  Abstract

  We describe here a method for drug target validation and identification of secondary drug target effects based on genome-wide gene expression patterns. The method is demonstrated by several experiments, including treatment of yeast mutant strains defective in calcineurin, immunophilins or other genes with the immunosuppressants cyclosporin A or FK506. Presence or absence of the characteristic drug 'signature' pattern of altered gene expression in drug-treated cells with a mutation in the gene encoding a putative target established whether that target was required to generate the drug signature. Drug dependent effects were seen in 'targetless' cells, showing that FK506 affects additional pathways independent of calcineurin and the immunophilins. The described method permits the direct confirmation of drug targets and recognition of drug-dependent changes in gene expression that are modulated through pathways distinct from the drug's intended target. Such a method may prove useful in improving the efficiency of drug development programs.

  View details for Web of Science ID 000076731000039

  View details for PubMedID 9809554

• Probing lymphocyte biology by genomic-scale gene expression analysis JOURNAL OF CLINICAL IMMUNOLOGY Alizadeh, A., Eisen, M., Botstein, D., Brown, P. O., Staudt, L. M. 1998; 18 (6): 373-379

  Abstract

  The identity and abundance of mRNA species within a cell dictate, to a large extent, the biological potential of that cell. Although posttranscriptional mechanisms modify protein expression in critical ways, cellular differentiation requires key changes in gene transcription, as evidenced by the potent phenotypes that result from disruption of transcription factor genes in mice. It is now possible to assess the mRNA profile of a cell globally using recently developed genomics techniques. This review focuses on the potential of cDNA microarrays to define gene expression in lymphoid cells, a field which is in its infancy. Examples of cellular activation genes and cytokine inducible genes discovered using this technology are presented but these represent only a taste of the fruit that this new technology will ultimately bear. Gene expression profiles should provide essential new insights into lymphocyte differentiation and activation, the pathogenesis of immune disorders, and the molecular abnormalities in lymphoid malignancies.

  View details for Web of Science ID 000077438400001

  View details for PubMedID 9857281

• The transcriptional program of sporulation in budding yeast SCIENCE Chu, S., DeRisi, J., Eisen, M., Mulholland, J., Botstein, D., Brown, P. O., Herskowitz, I. 1998; 282 (5389): 699-705

  Abstract

  Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis. DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation. At least seven distinct temporal patterns of induction were observed. The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase. Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes. The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

  View details for Web of Science ID 000076607500041

  View details for PubMedID 9784122

• Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells JOURNAL OF VIROLOGY Sutton, R. E., Wu, H. T., Rigg, R., Bohnlein, E., Brown, P. O. 1998; 72 (7): 5781-5788

  Abstract

  Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.

  View details for Web of Science ID 000074160500053

  View details for PubMedID 9621037

• Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yeager, M., Wilson-Kubalek, E. M., Weiner, S. G., Brown, P. O., Rein, A. 1998; 95 (13): 7299-7304

  Abstract

  We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR-) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR- MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR- nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR- MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of approximately 45 A and a length of approximately 200 A. Because of the pleomorphic shape and paracrystalline packing of the Gag-RNA complexes, we raise the possibility that assembly of MuLV is driven by protein-RNA, as well as protein-protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.

  View details for Web of Science ID 000074436400013

  View details for PubMedID 9636143

• Effects of mutations in residues near the active site of human immunodeficiency virus type 1 integrase on specific enzyme-substrate interactions JOURNAL OF VIROLOGY Gerton, J. L., Ohgi, S., Olsen, M., DeRisi, J., Brown, P. O. 1998; 72 (6): 5046-5055

  Abstract

  The phylogenetically conserved catalytic core domain of human immunodeficiency virus type 1 (HIV-1) integrase contains elements necessary for specific recognition of viral and target DNA features. In order to identify specific amino acids that determine substrate specificity, we mutagenized phylogenetically conserved residues that were located in close proximity to the active-site residues in the crystal structure of the isolated catalytic core domain of HIV-1 integrase. Residues composing the phylogenetically conserved DD(35)E active-site motif were also mutagenized. Purified mutant proteins were evaluated for their ability to recognize the phylogenetically conserved CA/TG base pairs near the viral DNA ends and the unpaired dinucleotide at the 5' end of the viral DNA, using disintegration substrates. Our findings suggest that specificity for the conserved A/T base pair depends on the active-site residue E152. The phenotype of IN(Q148L) suggested that Q148 may be involved in interactions with the 5' dinucleotide of the viral DNA end. The activities of some of the proteins with mutations in residues in close proximity to the active-site aspartic and glutamic acids were salt sensitive, suggesting that these mutations disrupted interactions with DNA.

  View details for Web of Science ID 000073497600058

  View details for PubMedID 9573274

• Photo-cross-linking studies suggest a model for the architecture of an active human immunodeficiency virus type 1 integrase-DNA complex BIOCHEMISTRY Heuer, T. S., Brown, P. O. 1998; 37 (19): 6667-6678

  Abstract

  The virally encoded integrase protein carries out retroviral integration, which requires specific interactions with the two ends of the viral DNA, and also with host DNA that is the target of integration. We attached a photo-cross-linking agent to specific viral and target DNA sites to identify regions of the integrase polypeptide that are in close proximity to those substrate features in the active integrase-DNA complex. The active form of integrase is a multimer. The higher-order organization of the active integration complex was therefore investigated by determining whether specific cross-links occurred to the active-site containing protomer. Both viral and target DNA cross-links to human immunodeficiency virus type 1 (HIV-1) integrase mapped predominantly to integrase protomers in trans to the active site, in a multimeric integrase complex. The results provide the basis for a model of the protein-DNA architecture of an active HIV-1 integration complex that suggests specific functions for the N-terminal, core, and C-terminal domains of retroviral integrase. One implication of this model is that the integrase multimer that mediates concerted integration of the viral DNA ends is composed of at least eight integrase protomers.

  View details for Web of Science ID 000073797300006

  View details for PubMedID 9578550

• Genomics and human disease - variations on variation NATURE GENETICS Brown, P. O., Hartwell, L. 1998; 18 (2): 91-93

  View details for Web of Science ID 000071779500002

  View details for PubMedID 9462728

• Enrichment for loci identical-by-descent between pairs of mouse or human genomes by genomic mismatch scanning GENOMICS McAllister, L., Penland, L., Brown, P. O. 1998; 47 (1): 7-11

  Abstract

  Mapping genes that underlie complex genetic traits, including genes that determine susceptibility to common diseases, requires an efficient method for high-resolution genotyping. Single-nucleotide differences between pairs of allelic sequences from unrelated individuals occur approximately once in every kilobase. Genomic mismatch scanning (GMS), by analyzing numerous single-nucleotide polymorphisms in a single genome-wide step, offers a potentially powerful and efficient approach to linkage analysis. GMS, originally developed in a yeast system, is shown here to be applicable to the more complex mouse and human genomes.

  View details for Web of Science ID 000071749500002

  View details for PubMedID 9465291

• Yeast microarrays for genome wide parallel genetic and gene expression analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA LASHKARI, D. A., DeRisi, J. L., McCusker, J. H., Namath, A. F., Gentile, C., Hwang, S. Y., Brown, P. O., Davis, R. W. 1997; 94 (24): 13057-13062

  Abstract

  We have developed high-density DNA microarrays of yeast ORFs. These microarrays can monitor hybridization to ORFs for applications such as quantitative differential gene expression analysis and screening for sequence polymorphisms. Automated scripts retrieved sequence information from public databases to locate predicted ORFs and select appropriate primers for amplification. The primers were used to amplify yeast ORFs in 96-well plates, and the resulting products were arrayed using an automated micro arraying device. Arrays containing up to 2,479 yeast ORFs were printed on a single slide. The hybridization of fluorescently labeled samples to the array were detected and quantitated with a laser confocal scanning microscope. Applications of the microarrays are shown for genetic and gene expression analysis at the whole genome level.

  View details for Web of Science ID A1997YJ45600066

  View details for PubMedID 9371799

• Exploring the metabolic and genetic control of gene expression on a genomic scale SCIENCE DeRisi, J. L., Iyer, V. R., Brown, P. O. 1997; 278 (5338): 680-686

  Abstract

  DNA microarrays containing virtually every gene of Saccharomyces cerevisiae were used to carry out a comprehensive investigation of the temporal program of gene expression accompanying the metabolic shift from fermentation to respiration. The expression profiles observed for genes with known metabolic functions pointed to features of the metabolic reprogramming that occur during the diauxic shift, and the expression patterns of many previously uncharacterized genes provided clues to their possible functions. The same DNA microarrays were also used to identify genes whose expression was affected by deletion of the transcriptional co-repressor TUP1 or overexpression of the transcriptional activator YAP1. These results demonstrate the feasibility and utility of this approach to genomewide exploration of gene expression patterns.

  View details for Web of Science ID A1997YC32300053

  View details for PubMedID 9381177

• The core domain of HIV-1 integrase recognizes key features of its DNA substrates JOURNAL OF BIOLOGICAL CHEMISTRY Gerton, J. L., Brown, P. O. 1997; 272 (41): 25809-25815

  Abstract

  We investigated which features of the substrate specificity of human immunodeficiency virus type 1 (HIV-1) integrase could be assigned to the central domain of the 288-residue HIV-1 integrase protein, composed of amino acids 50-212. This domain contains the active site and shares structural homology with a large family of polynucleotidyl transferases. Using model substrates with defined alterations in critical features we found that this domain alone is sufficient for recognition of: 1) the phylogenetically conserved CA/TG base pairs near the viral DNA end; 2) the 5'-terminal dinucleotide that is left unpaired after end processing; and 3) target DNA flanking the site of joining. Future efforts aimed at identifying specific amino acids involved in recognition of these key substrate features can now be targeted at this domain.

  View details for Web of Science ID A1997YA35800065

  View details for PubMedID 9325310

• Mapping features of HIV-1 integrase near selected sites on viral and target DNA molecules in an active enzyme-DNA complex by photo-cross-linking BIOCHEMISTRY Heuer, T. S., Brown, P. O. 1997; 36 (35): 10655-10665

  Abstract

  The virally encoded integrase protein carries out retroviral integration, and to do so, it must make specific interactions with both viral and target DNA sequences. The retroviral integrase has three domains: an amino-terminal region of about 50 amino acids that contains a zinc finger-like motif, a tightly folded, phylogenetically conserved core domain that contains the active site, and a carboxy-terminal domain that can bind DNA in a nonspecific manner. The complete roles of the amino- and carboxyl-terminal domains have not yet been determined, but they appear to participate in multimerization and nonspecific or target DNA binding, respectively. The number and identity of integrase's DNA binding sites have been difficult to determine by conventional mutagenesis studies. In this report, we describe a photo-cross-linking approach to address these issues. Our findings suggest that HIV-1 integrase contacts with conserved features of the viral DNA end are likely to be mediated by residues in two peptides within the conserved core domain. Additional cross-links were seen between viral DNA and the carboxy-terminal DNA binding domain. Numerous sites in integrase, including peptides in each of the three domains, could be cross-linked to target DNA features. Integrase is known to function as a multimer, and it remains to be determined which specific contacts are in cis or trans with respect to the active site.

  View details for Web of Science ID A1997XV68900007

  View details for PubMedID 9271496

• High-resolution functional mapping of a cloned gene by genetic footprinting PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Singh, I. R., Crowley, R. A., Brown, P. O. 1997; 94 (4): 1304-1309

  Abstract

  We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization. The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest. This mutant library is selected for gene function en masse. DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed. The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility. For the whole library, this results in a ladder of bands, each band representing a specific mutation. Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection. The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function. Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants. This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences.

  View details for Web of Science ID A1997WJ62100046

  View details for PubMedID 9037048

• 3'-end processing and kinetics of 5'-end joining during retroviral integration in vivo JOURNAL OF VIROLOGY Roe, T., Chow, S. A., Brown, P. O. 1997; 71 (2): 1334-1340

  Abstract

  Retroviral replication depends on integration of viral DNA into a host cell chromosome. Integration proceeds in three steps: 3'-end processing, the endonucleolytic removal of the two terminal nucleotides from each 3' end of the viral DNA; strand transfer, the joining of the 3' ends of viral DNA to host DNA; and 5'-end joining (or gap repair), the joining of the 5' ends of viral DNA to host DNA. The 5'-end joining step has never been investigated, either for retroviral integration or for any other transposition process. We have developed an assay for 5'-end joining in vivo and have examined the kinetics of 5'-end joining for Moloney murine leukemia virus (MLV). The interval between 3'-end and 5'-end joining is estimated to be less than 1 h. This assay will be a useful tool for examining whether viral or host components mediate 5'-end joining. MLV integrates its DNA only after its host cell has completed mitosis. We show that the extent of 3'-end processing is the same in unsynchronized and aphidicolin-arrested cells. 3'-end processing therefore does not depend on mitosis.

  View details for Web of Science ID A1997WC30500057

  View details for PubMedID 8995657

• Disruption of the terminal base pairs of retroviral DNA during integration Cold Spring Harbor Retroviruses Meeting SCOTTOLINE, B. P., Chow, S., Ellison, V., Brown, P. O. COLD SPRING HARBOR LAB PRESS. 1997: 371–82

  Abstract

  Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA. These two different polynucleotidyl transfer reactions are apparently carried out by a single active site. The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome. A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions. We propose that integrase distorts DNA substrates to accommodate both reactions within the active site. Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing. Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end. This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites. The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses.

  View details for Web of Science ID A1997WH81700008

  View details for PubMedID 9030689

• Functional analysis of the genes of yeast chromosome V by genetic footprinting SCIENCE Smith, V., Chou, K. N., Lashkari, D., Botstein, D., Brown, P. O. 1996; 274 (5295): 2069-2074

  Abstract

  Genetic footprinting was used to assess the phenotypic effects of Ty1 transposon insertions in 268 predicted genes of chromosome V of Saccharomyces cerevisiae. When seven selection protocols were used, Ty1 insertions in more than half the genes tested (157 of 268) were found to result in a detectable reduction in fitness. Results could not be obtained for fewer than 3 percent of the genes tested (7 of 268). Previously known mutant phenotypes were confirmed, and, for about 30 percent of the genes, new mutant phenotypes were identified.

  View details for Web of Science ID A1996VY97400046

  View details for PubMedID 8953036

• Use of a cDNA microarray to analyse gene expression patterns in human cancer NATURE GENETICS DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Y. D., Su, Y. A., Trent, J. M. 1996; 14 (4): 457-460

  Abstract

  The development and progression of cancer and the experimental reversal of tumorigenicity are accompanied by complex changes in patterns of gene expression. Microarrays of cDNA provide a powerful tool for studying these complex phenomena. The tumorigenic properties of a human melanoma cell line, UACC-903, can be suppressed by introduction of a normal human chromosome 6, resulting in a reduction of growth rate, restoration of contact inhibition, and suppression of both soft agar clonogenicity and tumorigenicity in nude mice. We used a high density microarray of 1,161 DNA elements to search for differences in gene expression associated with tumour suppression in this system. Fluorescent probes for hybridization were derived from two sources of cellular mRNA [UACC-903 and UACC-903(+6)] which were labelled with different fluors to provide a direct and internally controlled comparison of the mRNA levels corresponding to each arrayed gene. The fluorescence signals representing hybridization to each arrayed gene were analysed to determine the relative abundance in the two samples of mRNAs corresponding to each gene. Previously unrecognized alterations in the expression of specific genes provide leads for further investigation of the genetic basis of the tumorigenic phenotype of these cells.

  View details for Web of Science ID A1996VV73000022

  View details for PubMedID 8944026

• Parallel human genome analysis: Microarray-based expression monitoring of 1000 genes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P. O., Davis, R. W. 1996; 93 (20): 10614-10619

  Abstract

  Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery.

  View details for Web of Science ID A1996VL33300017

  View details for PubMedID 8855227

• A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization GENOME RESEARCH Shalon, D., Smith, S. J., Brown, P. O. 1996; 6 (7): 639-645

  Abstract

  Detecting and determining the relative abundance of diverse individual sequences in complex DNA samples is a recurring experimental challenge in analyzing genomes. We describe a general experimental approach to this problem, using microscopic arrays of DNA fragments on glass substrates for differential hybridization analysis of fluorescently labeled DNA samples. To test the system, 864 physically mapped lambda clones of yeast genomic DNA, together representing >75% of the yeast genome, were arranged into 1.8-cm x 1.8-cm arrays, each containing a total of 1744 elements. The microarrays were characterized by simultaneous hybridization of two different sets of isolated yeast chromosomes labeled with two different fluorophores. A laser fluorescent scanner was used to detect the hybridization signals from the two fluorophores. The results demonstrate the utility of DNA microarrays in the analysis of complex DNA samples. This system should find numerous applications in genome-wide genetic mapping, physical mapping, and gene expression studies.

  View details for Web of Science ID A1996UX13600009

  View details for PubMedID 8796352

• Monoclonal antibodies against human immunodeficiency virus type 1 integrase: Epitope mapping and differential effects on integrase activities in vitro JOURNAL OF VIROLOGY Nilsen, B. M., Haugan, I. R., Berg, K., Olsen, L., Brown, P. O., Helland, D. E. 1996; 70 (3): 1580-1587

  Abstract

  Human immunodeficiency virus type 1 (HIV-1) integrase (IN) catalyzes the integration of viral DNA into the host chromosome, an essential step in retroviral replication. As a tool to study the structure and function of this enzyme, monoclonal antibodies (MAbs) against HIV-1 IN were produced. Epitope mapping demonstrated that the 17 MAbs obtained could be divided into seven different groups, and the selection of MAbs representing these groups were tested for their effect on in vitro activities of IN. Four groups of MAbs recognized epitopes within the region of amino acids (aa) 1 to 16, 17 to 38, or 42 to 55 in and around the conserved HHCC motif near the N terminus of IN. MAbs binding to these epitopes inhibited end processing and DNA joining and either stimulated or had little effect on disintegration and reintegration activities of IN. Two MAbs binding to epitopes within the region of aa 56 to 102 in the central core or aa 186 to 250 in the C-terminal half of the protein showed only minor effects on the in vitro activities of IN. Three Mabs which recognized on epitope within the region of aa262 to 271 of HIV-1 IN cross-reacted with HIV-2 IN. MAbs binding to this epitope clearly inhibited end processing and DNA joining and stimulated or had little effect on disintegration. In contrast to the N-terminal-specific MAbs, these C-terminal-specific MAbs abolished reintegration activity of IN.

  View details for Web of Science ID A1996TV69600030

  View details for PubMedID 8627677

• QUANTITATIVE MONITORING OF GENE-EXPRESSION PATTERNS WITH A COMPLEMENTARY-DNA MICROARRAY SCIENCE Schena, M., Shalon, D., Davis, R. W., Brown, P. O. 1995; 270 (5235): 467-470

  Abstract

  A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

  View details for Web of Science ID A1995TA37400041

  View details for PubMedID 7569999

• GENETIC FOOTPRINTING - A GENOMIC STRATEGY FOR DETERMINING A GENES FUNCTION GIVEN ITS SEQUENCE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Smith, V., Botstein, D., Brown, P. O. 1995; 92 (14): 6479-6483

  Abstract

  This report describes an efficient strategy for determining the functions of sequenced genes in microorganisms. A large population of cells is subjected to insertional mutagenesis. The mutagenized population is then divided into representative samples, each of which is subjected to a different selection. DNA is prepared from each sample population after the selection. The polymerase chain reaction is then used to determine retrospectively whether insertions into a particular sequence affected the outcome of any selection. The method is efficient because the insertional mutagenesis and each selection need only to be performed once to enable the functions of thousands of genes to be investigated, rather than once for each gene. We tested this "genetic footprinting" strategy using the model organism Saccharomyces cerevisiae.

  View details for Web of Science ID A1995RG73600054

  View details for PubMedID 7604017

• STATISTICAL-METHODS FOR LINKAGE ANALYSIS OF COMPLEX TRAITS FROM HIGH-RESOLUTION MAPS OF IDENTITY BY DESCENT GENETICS Dupuis, J., Brown, P. O., Siegmund, D. 1995; 140 (2): 843-856

  Abstract

  A multilocus model for complex traits is described that generalizes the additive and multiplicative models and hence allows simultaneously for both heterogeneity and gene interaction (epistasis). Statistical methods of linkage analysis are discussed under the assumption that identity by descent data from a dense set of polymorphic markers are available. Three methods, single locus search, simultaneous search and conditional search, are described and compared.

  View details for Web of Science ID A1995RA36600035

  View details for PubMedID 7498758

• AN ESSENTIAL INTERACTION BETWEEN DISTINCT DOMAINS OF HIV-1 INTEGRASE MEDIATES ASSEMBLY OF THE ACTIVE MULTIMER JOURNAL OF BIOLOGICAL CHEMISTRY Ellison, V., Gerton, J., Vincent, K. A., Brown, P. O. 1995; 270 (7): 3320-3326

  Abstract

  Integrase mediates integration of the retroviral genome into a host cell chromosome, an essential step in the viral life cycle. In vitro, a stable complex containing only purified human immunodeficiency virus (HIV) integrase and a model viral DNA substrate processively executes the 3'-end processing and DNA joining steps in the integration reaction. We examined the relationship of three essential components of the HIV integrase: the HHCC domain, a putative zinc-finger near the N terminus; the phylogenetically conserved "DD35E" motif, which defines the catalytic domain; and a feature recognized by its sensitivity to the alkylating agent N-ethylmaleimide (NEM). HIV integrase is a multimer, and these three components can be distributed among at least two subunits of the multimeric enzyme. The components function asymmetrically in the active multimer; the DD35E motif and NEM-sensitive site are required in trans to the HHCC region. A divalent cation-dependent interaction involving the NEM-sensitive site of one integrase subunit and the HHCC region of another subunit points to a role for these two features of integrase in multimer assembly. Deletion of the HHCC domain, or modification of integrase with NEM, impaired the assembly of a stable complex between integrase and viral DNA, suggesting that this initial step in the integration pathway requires assembly of the active integrase multimer.

  View details for Web of Science ID A1995QG47100065

  View details for PubMedID 7852418

• CHARACTERIZATION OF RECOMBINANT MURINE LEUKEMIA-VIRUS INTEGRASE JOURNAL OF VIROLOGY Dotan, I., SCOTTOLINE, B. P., Heuer, T. S., Brown, P. O. 1995; 69 (1): 456-468

  Abstract

  Retroviral integration involves two DNA substrates that play different roles. The viral DNA substrate is recognized by virtue of specific nucleotide sequences near the end of a double-stranded DNA molecule. The target DNA substrate is recognized at internal sites with little sequence preference; nucleosomal DNA appears to be preferred for this role. Despite this apparent asymmetry in the sequence, structure, and roles of the DNA substrates in the integration reaction, the existence of distinct binding sites for viral and target DNA substrates has been controversial. In this report, we describe the expression in Escherichia coli and purification of Moloney murine leukemia virus integrase as a fusion protein with glutathione S-transferase, characterization of its activity by using several model DNA substrates, and the initial kinetic characterization of its interactions with a model viral DNA substrate. We provide evidence for functionally and kinetically distinct binding sites for viral and target DNA substrates and describe a cross-linking assay for DNA binding at a site whose specificity is consistent with the target DNA binding site.

  View details for Web of Science ID A1995PW81500053

  View details for PubMedID 7983742

• JUXTAPOSITION OF 2 VIRAL-DNA ENDS IN A BIMOLECULAR DISINTEGRATION REACTION MEDIATED BY MULTIMERS OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 OR MURINE LEUKEMIA-VIRUS INTEGRASE JOURNAL OF VIROLOGY Chow, S. A., Brown, P. O. 1994; 68 (12): 7869-7878

  Abstract

  Integration of retroviral DNA involves a coordinated joining of the two ends of a viral DNA molecule into precisely spaced sites on target DNA. In this study, we designed an assay that requires two separate oligonucleotides to be brought together via interactions between integrase promoters to form a "crossbones" substrate that mimics the integration intermediate. The crossbones substrate contains two viral DNA ends, each joined to one strand of target DNA and separated by a defined length of target DNA. We showed that purified integrases of human immunodeficiency virus type 1 (HIV-1) and murine leukemia virus (MLV) could mediate a concerted strand cleavage-ligation between the two half-substrates at one or both viral DNA joining sites (trans disintegration). Another major product, termed fold-back, resulted from an intramolecular attack on the phosphodiester bond at the viral-target DNA junction by the 3'-OH group of the same DNA molecule (cis disintegration). The activity of integrase on the crossbones substrate depended on the presence of viral DNA sequences. For trans disintegration, the optimal length of target DNA between the viral DNA joining sites of the crossbones substrate corresponded to the spacing between the staggered joints formed on two opposite strands of target DNA during retroviral DNA integration in vivo. The activity of integrases on crossbones did not require complementary base pairing between the two half-substrates, indicating that the half-substrates were juxtaposed solely through protein-DNA interactions. The crossbones assay, therefore, measures the ability of integrase to juxtapose two viral DNA ends, an activity which heretofore has been difficult to detect by using purified integrase in conventional assays. Certain mutant integrases that were otherwise inactive with the crossbones substrate could complement one another, indicating that no single protomer in the integrase multimer requires a complete set of functional domains either for catalytic activity or for juxtaposition of the two viral DNA ends by the active multimer.

  View details for Web of Science ID A1994PR43200024

  View details for PubMedID 7966577

• 2-DIMENSIONAL CRYSTALLIZATION OF HISTIDINE-TAGGED, HIV-1 REVERSE-TRANSCRIPTASE PROMOTED BY A NOVEL NICKEL-CHELATING LIPID JOURNAL OF STRUCTURAL BIOLOGY KUBALEK, E. W., LeGrice, S. F., Brown, P. O. 1994; 113 (2): 117-123

  Abstract

  Recombinant proteins containing a short stretch of contiguous histidine residues (approximately 6) ("a His-tag") can be specifically bound to N-nitrilotriacetic-acid-chelated nickel ions, providing a convenient general method for their purification. A lipid derivatized with a nickel-chelating head group may provide a general approach to two-dimensional crystallization of the His-tagged proteins, using the lipid layer technique. We have designed a synthetic phospholipid that carries a chelated nickel ion (Ni-NTA-DOPE). His-tagged recombinant HIV-1 reverse transcriptase (HIV-RT) bound specifically to lipid layers containing Ni-NTA-DOPE and formed crystals within minutes from a dilute protein solution. Two-dimensional crystals preserved in negative stain diffracted strongly to approximately 21 A. The projection map computed from averaged Fourier transforms revealed a structure similar in size and shape to a selected projection view of the 3-D structure that was previously determined for HIV-RT by X-ray crystallography.

  View details for Web of Science ID A1994QH69500003

  View details for PubMedID 7536435

• DNA STRAND EXCHANGE AND SELECTIVE DNA ANNEALING PROMOTED BY THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NUCLEOCAPSID PROTEIN JOURNAL OF VIROLOGY Tsuchihashi, Z., Brown, P. O. 1994; 68 (9): 5863-5870

  Abstract

  Nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) was expressed in Escherichia coli and purified. The protein displayed a variety of activities on DNA structure, all reflecting an ability to promote transition between double-helical and single-stranded conformations. We found that, in addition to its previously described ability to accelerate renaturation of complementary DNA strands, the HIV-1 NC protein could substantially lower the melting temperature of duplex DNA and could promote strand exchange between double-stranded and single-stranded DNA molecules. Moreover, in the presence of HIV-1 NC, annealing of a single-stranded DNA molecule to a complementary DNA strand that would yield a more stable double-stranded product was favored over annealing to alternative complementary DNA strands that would form less stable duplex products (selective annealing). NC thus appears to lower the kinetic barrier so that double-strand <==> single-strand equilibrium is rapidly reached to favor the lowest free-energy nucleic acid conformation. This activity of NC may be important for correct folding of viral genomic RNA and may have practical applications.

  View details for Web of Science ID A1994PB78500059

  View details for PubMedID 8057466

• A STABLE COMPLEX BETWEEN INTEGRASE AND VIRAL-DNA ENDS MEDIATES HUMAN-IMMUNODEFICIENCY-VIRUS INTEGRATION IN-VITRO PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ellison, V., Brown, P. O. 1994; 91 (15): 7316-7320

  Abstract

  Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.

  View details for Web of Science ID A1994NY34800117

  View details for PubMedID 8041787

• SUBSTRATE FEATURES IMPORTANT FOR RECOGNITION AND CATALYSIS BY HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE IDENTIFIED BY USING NOVEL DNA SUBSTRATES JOURNAL OF VIROLOGY Chow, S. A., Brown, P. O. 1994; 68 (6): 3896-3907

  Abstract

  The integrase encoded by human immunodeficiency virus type 1 (HIV-1) is required for integration of viral DNA into the host cell chromosome. In vitro, integrase mediates a concerted cleavage-ligation reaction (strand transfer) that results in covalent attachment of viral DNA to target DNA. With a substrate that mimics the strand transfer product, integrase carries out disintegration, the reverse of the strand transfer reaction, resolving this integration intermediate into its viral and target DNA parts. We used a set of disintegration substrates to study the catalytic mechanism of HIV-1 integrase and the interaction between the protein and the viral and target DNA sequence. One substrate termed dumbbell consists of a single oligonucleotide that can fold to form a structure that mimics the integration intermediate. Kinetic analysis using the dumbbell substrate showed that integrase turned over, establishing that HIV-1 integrase is an enzyme. Analysis of the disintegration activity on the dumbbell substrate and its derivatives showed that both the viral and target DNA parts of the molecule were required for integrase recognition. Integrase recognized target DNA asymmetrically: the target DNA upstream of the viral DNA joining site played a much more important role than the downstream target DNA in protein-DNA interaction. The site of transesterification was determined by both the DNA sequence of the viral DNA end and the structure of the branched substrate. Using a series of disintegration substrates with various base modifications, we found that integrase had relaxed structural specificity for the hydroxyl group used in transesterification and could tolerate distortion of the double-helical structure of these DNA substrates.

  View details for Web of Science ID A1994NL34900049

  View details for PubMedID 8189526

• Genome scanning methods. Current opinion in genetics & development Brown, P. O. 1994; 4 (3): 366-373

  Abstract

  Recently introduced methods can allow an entire genome to be scanned at one time for sequences linked to trait loci. Representational difference analysis and genomic mismatch scanning are designed, respectively, to isolate and map sequences that differ between genomes discordant for a trait, or that are identical between genomes concordant for a trait. Other methods allow the whole genome to be scanned for regions of increased or decreased copy number, or for differentially methylated sequences.

  View details for PubMedID 7919913

• GAUSSIAN MODELS FOR GENETIC-LINKAGE ANALYSIS USING COMPLETE HIGH-RESOLUTION MAPS OF IDENTITY BY DESCENT AMERICAN JOURNAL OF HUMAN GENETICS Feingold, E., Brown, P. O., Siegmund, D. 1993; 53 (1): 234-251

  Abstract

  Gaussian-process models are developed to detect genetic linkage using complete high-resolution maps of identity by descent between affected relative pairs. Approximations are given for the significance level and power of the likelihood-ratio test of no linkage and for likelihood-ratio confidence regions for trait loci. The sample sizes required to detect linkage by using different classes of affected relative pairs are compared, and the problem of combining data from different classes of relatives is discussed.

  View details for Web of Science ID A1993LJ38500027

  View details for PubMedID 8317489

• INTEGRATION OF MURINE LEUKEMIA-VIRUS DNA DEPENDS ON MITOSIS EMBO JOURNAL ROE, T. Y., Reynolds, T. C., Yu, G., Brown, P. O. 1993; 12 (5): 2099-2108

  Abstract

  In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.

  View details for Web of Science ID A1993LC94800038

  View details for PubMedID 8491198

• GENOMIC MISMATCH SCANNING - A NEW APPROACH TO GENETIC-LINKAGE MAPPING NATURE GENETICS Nelson, S. F., McCusker, J. H., SANDER, M. A., Kee, Y., Modrich, P., Brown, P. O. 1993; 4 (1): 11-18

  Abstract

  Genomic mismatch scanning (GMS) is a new method of genetic linkage analysis that does not require conventional polymorphic markers or gel electrophoresis. GMS is ideally suited to affected-relative-pair mapping. DNA fragments from all regions of identity-by-descent between two relatives are isolated based on their ability to form extensive mismatch-free hybrid molecules. The genomic origin of this selected pool of DNA fragments is then mapped in a single hybridization step. Here we demonstrate the practicality of GMS in a model organism, Saccharomyces cerevisiae. GMS is likely to be applicable to other organisms, including humans, and may be of particular value in mapping complex genetic traits.

  View details for Web of Science ID A1993LA33700006

  View details for PubMedID 8513319

• CHARACTERIZATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INTEGRASE EXPRESSED IN ESCHERICHIA-COLI AND ANALYSIS OF VARIANTS WITH AMINO-TERMINAL MUTATIONS JOURNAL OF VIROLOGY Vincent, K. A., Ellison, V., Chow, S. A., Brown, P. O. 1993; 67 (1): 425-437

  Abstract

  Replication of a retroviral genome depends upon integration of the viral DNA into a chromosome of the host cell. The integration reaction is mediated by integrase, a viral enzyme. Human immunodeficiency virus type 1 integrase was expressed in Escherichia coli and purified to near homogeneity. Optimum conditions for the integration and 3'-end-processing activities of integrase were characterized by using an in vitro assay with short, double-stranded oligonucleotide substrates. Mutants containing amino acid substitutions within the HHCC region, defined by phylogenetically conserved pairs of histidine and cysteine residues near the N terminus, were constructed and characterized by using three assays: 3'-end processing, integration, and the reverse of the integration reaction (or disintegration). Mutations in the conserved histidine and cysteine residues abolished both integration and processing activities. Weak activity in both assays was retained by two other mutants containing substitutions for less highly conserved amino acids in this region. All mutants retained activity in the disintegration assay, implying that the active site for DNA cleavage-ligation is not located in this domain and that the HHCC region is not the sole DNA-binding domain in the protein. However, the preferential impairment of processing and integration rather than disintegration by mutations in the HHCC region is consistent with a role for this domain in recognizing features of the viral DNA. This hypothesis is supported by the results of disintegration assays performed with altered substrates. The results support a model involving separate viral and target DNA-binding sites on integrase.

  View details for Web of Science ID A1993KB97000049

  View details for PubMedID 8416376

• SEQUENCE REQUIREMENTS FOR EFFICIENT TRANSLATIONAL FRAMESHIFTING IN THE ESCHERICHIA-COLI-DNAX GENE AND THE ROLE OF AN UNSTABLE INTERACTION BETWEEN TRANSFER RNA(LYS) AND AN AAG LYSINE CODON GENES & DEVELOPMENT Tsuchihashi, Z., Brown, P. O. 1992; 6 (3): 511-519

  Abstract

  Synthesis of the gamma-subunit of DNA polymerase III holoenzyme depends on precise and efficient translational frameshifting to the -1 frame at a specific site in the dnaX gene of Escherichia coli. In vitro mutagenesis of this frameshift site demonstrated the importance of an A AAA AAG heptanucleotide sequence, which allows two adjacent tRNAs to retain a stable interaction with mRNA after they slip to the -1 position. The AAG lysine codon present in the 3' half of this heptanucleotide was a key element for highly efficient frameshifting. A tRNA(Lys) with a CUU anticodon, which has a strong affinity for AAG lysine codons, is present in eukaryotic cells but absent in E. coli. Expression in E. coli of a mutant tRNA(Lys) with a CUU anticodon specifically inhibited the frameshifting at the AAG codon, suggesting that the absence of this tRNA in E. coli contributes to the efficiency of the dnaX frameshift.

  View details for Web of Science ID A1992HM44400015

  View details for PubMedID 1547945

• REVERSAL OF INTEGRATION AND DNA SPLICING MEDIATED BY INTEGRASE OF HUMAN-IMMUNODEFICIENCY-VIRUS SCIENCE Chow, S. A., Vincent, K. A., Ellison, V., Brown, P. O. 1992; 255 (5045): 723-726

  Abstract

  In retroviral integration, the viral integration protein (integrase) mediates a concerted DNA cleavage-ligation reaction in which the target DNA is cleaved and the resulting 5' ends of target DNA are joined to the 3' ends of viral DNA. Through an oligonucleotide substrate that mimics the recombination intermediate formed by this initial cleavage-ligation reaction, the purified integrase of human immunodeficiency virus was shown to promote the same reaction in reverse, a process called disintegration. Analysis of a set of structurally related substrates showed that integrase could promote a range of DNA cleavage-ligation reactions. When the viral DNA component of the disintegration substrate was single-stranded, integrase could mediate a DNA splicing reaction analogous to RNA splicing.

  View details for Web of Science ID A1992HC50600040

  View details for PubMedID 1738845

• HOST SEQUENCES FLANKING THE HIV PROVIRUS NUCLEIC ACIDS RESEARCH Vincent, K. A., YORKHIGGINS, D., Quiroga, M., Brown, P. O. 1990; 18 (20): 6045-6047

  Abstract

  A conserved property of retroviral proviruses is the presence of a direct repeat in the host DNA immediately flanking the viral sequence; each virus generates a repeat with a characteristic length. By sequencing the viral/host DNA junctions from five HIV-1 proviral clones, we have confirmed that integration of HIV results in the generation of a five basepair direct repeat. A target sequence in uninfected host DNA was analyzed to establish that the five basepair sequence flanking the provirus was present only once prior to integration. Of the five proviruses examined, two were found to have integrated in known repetitive sequence elements of the human genome; one in a Line-1 element and a second in satellite DNA.

  View details for Web of Science ID A1990EF12100016

  View details for PubMedID 2235486

• HUMAN-IMMUNODEFICIENCY-VIRUS INTEGRATION IN A CELL-FREE SYSTEM JOURNAL OF VIROLOGY Ellison, V., Abrams, H., ROE, T. Y., Lifson, J., BROWN, P. 1990; 64 (6): 2711-2715

  Abstract

  Integration of the viral genome into the nuclear DNA of a host cell plays a pivotal role in the replication of retroviruses. We have developed an in vitro method for studying the biochemistry of human immunodeficiency virus (HIV) integration by using extracts from HIV-infected cells. Analysis of the reaction products showed that HIV integration in vitro accurately reproduces the in vivo process. Integration occurred without apparent specificity for the target sequence, and the integrated provirus was directly flanked by a 5-base-pair duplication of DNA from the target site. HIV integration did not require a high-energy cofactor, and the enzymatic activities required for integration were recovered with the viral DNA when cell extracts were fractionated by gel exclusion chromatography.

  View details for Web of Science ID A1990DD73500031

  View details for PubMedID 2335814

• INTEGRATION OF RETROVIRAL DNA CURRENT TOPICS IN MICROBIOLOGY AND IMMUNOLOGY Brown, P. O. 1990; 157: 19-48

  View details for Web of Science ID A1990EE44400002

  View details for PubMedID 2203610

• RETROVIRAL INTEGRATION - STRUCTURE OF THE INITIAL COVALENT PRODUCT AND ITS PRECURSOR, AND A ROLE FOR THE VIRAL IN PROTEIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brown, P. O., Bowerman, B., Varmus, H. E., BISHOP, J. M. 1989; 86 (8): 2525-2529

  Abstract

  An essential step in the life cycle of a retrovirus is the integration of a DNA copy of the viral genome into a host cell chromosome. We have analyzed the structure of the initial covalent product of an in vitro retroviral integration reaction and determined the structure of the ends of the unintegrated linear viral DNA molecules present in vivo in cells infected with murine leukemia virus (MLV). Our results lead to the following conclusions: (i) Circularization of viral DNA plays no role in integration. The direct precursor to the integrated MLV provirus is a linear molecule. (ii) The initial step in the integration reaction is probably a cleavage that removes the terminal 2 bases from each 3' end of the viral DNA. This cleavage depends on a virally encoded protein, IN, that has previously been shown genetically to be required for integration. (iii) The resulting viral 3' ends are joined to target DNA to form the initial recombination intermediate.

  View details for Web of Science ID A1989U231400002

  View details for PubMedID 2539592

• A NUCLEOPROTEIN COMPLEX MEDIATES THE INTEGRATION OF RETROVIRAL DNA GENES & DEVELOPMENT Bowerman, B., Brown, P. O., BISHOP, J. M., Varmus, H. E. 1989; 3 (4): 469-478

  Abstract

  The integration of viral DNA into the host genome is an essential step in the retrovirus life cycle. To understand this process better, we have examined the native state of viral DNA in cells acutely infected by murine leukemia virus (MLV), using both a physical assay for viral DNA and a functional assay for integration activity (Brown et al. 1987). The viral DNA and integration activity copurify during velocity sedimentation, gel filtration, and density equilibrium centrifugation, indicating that viral DNA is in a large (approximately 160S) nucleoprotein complex that includes all functions required for integration activity in vitro. Analysis by immunoprecipitation shows that the viral capsid protein is part of the active nucleoprotein complex, but recognition of the complex by only a subset of anti-capsid sera implies that the protein is constrained conformationally. The viral DNA within this structure is accessible to nucleases; the effects of nucleases on the integrity of the complex suggest that the integration-competent particle is derived from and similar to the core of extracellular virions.

  View details for Web of Science ID A1989U742400004

  View details for PubMedID 2721960

• CORRECT INTEGRATION OF RETROVIRAL DNA INVITRO CELL Brown, P. O., Bowerman, B., Varmus, H. E., BISHOP, J. M. 1987; 49 (3): 347-356

  Abstract

  We have developed a cell-free system for studying the integration of retroviral DNA. In our assay, amber mutations in a bacteriophage lambda genome that serves as the target for integration are suppressed by integration of an MLV derivative that carries the E. coli supF gene. The structure of the reaction products is that expected from an authentic MLV integration reaction. Linear viral DNA from the cytoplasm of infected cells serves as a precursor, though not necessarily the immediate precursor, to the provirus integrated in vitro. The viral DNA in the infected cell appears to be tightly associated with the enzymatic machinery required for its integration. Supercoiling, chromatin structure, transcription, and replication are not required of the target DNA. Since no high-energy cofactor is necessary, the DNA breakage and joining steps in the integration reaction are probably coupled.

  View details for Web of Science ID A1987H297100010

  View details for PubMedID 3032450

• CATENATION AND KNOTTING OF DUPLEX DNA BY TYPE-1 TOPOISOMERASES - A MECHANISTIC PARALLEL WITH TYPE-2 TOPOISOMERASES PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES Brown, P. O., Cozzarelli, N. R. 1981; 78 (2): 843-847

  Abstract

  Escherichia coli omega protein, a type 1 topoisomerase, can catenate and knot duplex DNA circles. Previously, these activities were thought to be limited to type 2 topoisomerases. Catenation by omega requires a nick in one of the participating molecules, but it is not necessary that both be nicked. Catenation does not depend on sequence homology and is greatly stimulated by DNA-condensing agents such as spermidine. A eukaryotic type 1 topoisomerase can also interlock duplex DNA circles. These activities cannot easily be explained by the widely held topoisomerase model in which a reversible nick in DNA allows free rotation about the unbroken strand. We suggest instead passage of a DNA segment though a transient enzyme-bridged break in a single DNA strand. This is analogous to the sign inversion mechanism of the type 2 topoisomerases, and thus expresses an essential mechanistic unity among topoisomerases. As expected for relaxation by a single-strand passage, omega changes the linking number of DNA in steps of 1.

  View details for Web of Science ID A1981LF67000029

  View details for PubMedID 6262776

• STRUCTURE AND ACTIVITIES OF ESCHERICHIA-COLI DNA-GYRASE COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY Peebles, C. L., Higgins, N. P., Kreuzer, K. N., Morrison, A., Brown, P. O., Sugino, A., Cozzarelli, N. R. 1979; 43: 41-52

  View details for Web of Science ID A1978HF85900006

  View details for PubMedID 225110

• TOPOISOMERASE FROM ESCHERICHIA-COLI RELATED TO DNA GYRASE PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brown, P. O., Peebles, C. L., Cozzarelli, N. R. 1979; 76 (12): 6110-6114

  Abstract

  We have identified a topoisomerase activity from Escherichia coli related to DNA gyrase (topoisomerase II): we designate it topoisomerase II'. It was constructed of two subunits, which were purified separately. One is the product of the gyrA (formerly nalA) gene and is identical to subunit A of DNA gyrase. The other is a 50,000-dalton protein, which we have purified to homogeneity and call v. v may be a processed form of the much larger gyrase subunit B or may be derived from a transcript of part of the subunit B structural gene, because preliminary peptide maps of the two subunits are similar. Topoisomerase II' relaxes negatively supercoiled DNA and, uniquely among E. coli topoisomerases, relaxes positive supercoils efficiently. It is the only topoisomerase that can introduce positive supercoils; these are stoichiometric with enzyme molecules. Topoisomerase II' resembles gyrase in its sensitivity to oxolinic acid, the wrapping of DNA in an apparent positive supercoil around the enzyme, and the introduction in an aborted reaction of site-specific double-strand breaks in the DNA with concomitant covalent attachment of protein to both newly created 5' ends. Unlike DNA gyrase, topoisomerase II' has no negative supercoiling activity. Functional chimeric topoisomerases were constructed with the alpha subunit of the Micrococcus luteus gyrase and v or gyrase subunit B from E. coli. We discuss the implications of the dual of the gyrA gene product.

  View details for Web of Science ID A1979JA38200022

  View details for PubMedID 230498

• SIGN INVERSION MECHANISM FOR ENZYMATIC SUPERCOILING OF DNA SCIENCE Brown, P. O., Cozzarelli, N. R. 1979; 206 (4422): 1081-1083

  Abstract

  Both the introduction and the removal of supertwists by DNA gyrase change the linking number of DNA in steps of two. This surprising finding provides strong evidence that gyrase acts by a mechanism, called sign inversion, whereby a positive supercoil is directly inverted into a negative one via a transient double-strand break.

  View details for Web of Science ID A1979HU66700028

  View details for PubMedID 227059

• ENERGY COUPLING IN DNA GYRASE AND MECHANISM OF ACTION OF NOVOBIOCIN PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sugino, A., Higgins, N. P., Brown, P. O., Peebles, C. L., Cozzarelli, N. R. 1978; 75 (10): 4838-4842

  Abstract

  Escherichia coli DNA gyrase catalyzes negative supercoiling of closed duplex DNA at the expense of ATP. Two additional activities of the enzyme that have illuminated the energy coupling component of the supercoiling reaction are the DNA-dependent hydrolysis of ATP to ADP and P(i) and the alteration by ATP of the DNA site specificity of the gyrase cleavage reaction. This cleavage of both DNA strands results from treatment with sodium dodecyl sulfate of the stable gyrase-DNA complex that is trapped by the inhibitor oxolinic acid. Either ATP or a nonhydrolyzable analogue, adenyl-5'-yl-imidodiphosphate (App[NH]p), shifts the primary cleavage site on ColE1 DNA. The prevention by novobiocin and coumermycin A(1) of this cleavage rearrangement places the site of action of the antibiotics at a reaction step prior to ATP hydrolysis. The step blocked is the binding of ATP because coumermycin A(1) and novobiocin interact competitively with ATP in the ATPase and supercoiling assays; the K(i) values are more than four orders of magnitude less than the K(m) for ATP. This simple mechanism accounts for all effects of the drugs on DNA gyrase. Studies with App[NH]p, another potent competitive inhibitor of reactions catalyzed by gyrase, show that cleavage of a high energy bond is not required for driving DNA into the higher energy supercoiled form. With substrate levels of gyrase, App[NH]p induces supercoiling that is proportional to the amount of enzyme; a -0.3 superhelical turn was introduced per gyrase protomer A. We postulate that ATP and App[NH]p are allosteric effectors of a conformational change of gyrase that leads to one round of supercoiling. Nucleotide dissociation favored by hydrolysis of ATP returns gyrase to its original conformation and thereby permits enzyme turnover. Such cyclic conformational changes accompanying alteration in nucleotide affinity also seem to be a common feature of energy transduction in other diverse processes including muscle contraction, protein synthesis, and oxidative phosphorylation.

  View details for Web of Science ID A1978FU61400048

  View details for PubMedID 368801