Biomedical subjects

D Botstein

Publications and source records attributed to D Botstein.

At least 37 records · Page 2Linked to original sources

PubMed2001

Dominant-lethal alpha-tubulin mutants defective in microtubule depolymerization in yeast.

The dynamic instability of microtubules has long been understood to depend on the hydrolysis of GTP bound to beta-tubulin, an event stimulated by polymerization and necessary for depolymerization. Crystallographic studies of tubulin show that GTP is bound by beta-tubulin at the longitudinal dimer-dimer interface and contacts particular alpha-tubulin residues in the next dimer along the protofilament. This structural arrangement suggests that these contacts could account for assembly-stimulated GTP hydrolysis. As a test of this hypothesis, we examined, in yeast cells, the effect of mutating the alpha-tubulin residues predicted, on structural grounds, to be involved in GTPase activation. Mutation of these residues to alanine (i.e., D252A and E255A) created poisonous alpha-tubulins that caused lethality even as minor components of the alpha-tubulin pool. When the mutant alpha-tubulins were expressed from the galactose-inducible promoter of GAL1, cells rapidly acquired aberrant microtubule structures. Cytoplasmic microtubules were largely bundled, spindle assembly was inhibited, preexisting spindles failed to completely elongate, and occasional, stable microtubules were observed unattached to spindle pole bodies. Time-lapse microscopy showed that microtubule dynamics had ceased. Microtubules containing the mutant proteins did not depolymerize, even in the presence of nocodazole. These data support the view that alpha-tubulin is a GTPase-activating protein that acts, during microtubule polymerization, to stimulate GTP hydrolysis in beta-tubulin and thereby account for the dynamic instability of microtubules.

Cell Cycle↗

PubMed2001

Missing value estimation methods for DNA microarrays.

MOTIVATION: 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. RESULTS: 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.

Algorithms↗

PubMed2001

High-throughput genotyping with single nucleotide polymorphisms.

To make large-scale association studies a reality, automated high-throughput methods for genotyping with single-nucleotide polymorphisms (SNPs) are needed. We describe PCR conditions that permit the use of the TaqMan or 5' nuclease allelic discrimination assay for typing large numbers of individuals with any SNP and computational methods that allow genotypes to be assigned automatically. To demonstrate the utility of these methods, we typed >1600 individuals for a G-to-T transversion that results in a glutamate-to-aspartate substitution at position 298 in the endothelial nitric oxide synthase gene, and a G/C polymorphism (newly identified in our laboratory) in intron 8 of the 11-beta hydroxylase gene. The genotyping method is accurate-we estimate an error rate of fewer than 1 in 2000 genotypes, rapid-with five 96-well PCR machines, one fluorescent reader, and no automated pipetting, over one thousand genotypes can be generated by one person in one day, and flexible-a new SNP can be tested for association in less than one week. Indeed, large-scale genotyping has been accomplished for 23 other SNPs in 13 different genes using this method. In addition, we identified three "pseudo-SNPs" (WIAF1161, WIAF2566, and WIAF335) that are probably a result of duplication.

Alleles↗

PubMed2001

Microarrays in primary breast cancer--lessons from chemotherapy studies.

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.

Breast Neoplasms↗

PubMed2000

DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli.

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.

Escherichia coli↗

PubMed2000

Ongoing immunoglobulin somatic mutation in germinal center B cell-like but not in activated B cell-like diffuse large cell lymphomas.

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.

Biopsy↗

PubMed2000

Singular value decomposition for genome-wide expression data processing and modeling.

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.

Cell Cycle↗

PubMed2000

Molecular portraits of human breast tumours.

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.

Breast Neoplasms↗

PubMed2000

Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays.

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.

DNA Replication↗

PubMed2000

'Gene shaving' as a method for identifying distinct sets of genes with similar expression patterns.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Algorithms↗

PubMed2000

Two yeast forkhead genes regulate the cell cycle and pseudohyphal growth.

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.

Binding Sites↗

PubMed2000

Genome-wide characterization of the Zap1p zinc-responsive regulon in yeast.

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.

Gene Expression Profiling↗

PubMed2000

Desferrioxamine-mediated iron uptake in Saccharomyces cerevisiae. Evidence for two pathways of iron uptake.

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.

Amino Acid Sequence↗

PubMed2000

Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.

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.

Adult↗

PubMed2000

Integrating functional genomic information into the Saccharomyces genome database.

The Saccharomyces Genome Database (SGD) stores and organizes information about the nearly 6200 genes in the yeast genome. The information is organized around the 'locus page' and directs users to the detailed information they seek. SGD is endeavoring to integrate the existing information about yeast genes with the large volume of data generated by functional analyses that are beginning to appear in the literature and on web sites. New features will include searches of systematic analyses and Gene Summary Paragraphs that succinctly review the literature for each gene. In addition to current information, such as gene product and phenotype descriptions, the new locus page will also describe a gene product's cellular process, function and localization using a controlled vocabulary developed in collaboration with two other model organism databases. We describe these developments in SGD through the newly reorganized locus page. The SGD is accessible via the WWW at http://genome-www.stanford.edu/Saccharomyces/

Database Management Systems↗

PubMed2000

Lack of evidence for an association between alpha-adducin and blood pressure regulation in Asian populations.

Recent studies have found the tryptophan allele of a glycine to tryptophan polymorphism at position 460 (G460W) of the alpha-adducin protein to be associated with essential hypertension in European populations. We examined whether the tryptophan allele is associated with hypertension in a different population, comprised of subjects of Chinese origin from Taiwan, and Chinese and Japanese origin from the San Francisco Bay area and Hawaii. We adapted the 5' allelic discrimination assay or TaqMan to type individuals for the G460W polymorphism, and using this method we typed more than 1000 individuals. The frequency of the W allele was slightly increased in the treated subjects in the Chinese population (0.458 v 0.423) but not the Japanese population (0.549 v 0.558). We considered dominant, recessive, and additive models in our analysis. There was a significant result for a recessive model for systolic blood pressure in the Chinese population (chi2 6.84, df = 2, P < .05), but only suggestive evidence for diastolic blood pressure (chi2 3.30). In contrast, in the Japanese population, there was no evidence for a positive association under any model. For the combined Chinese and Japanese samples, the evidence for association with alpha-adducin was not significant.

Adult↗

PubMed2000

Systematic variation in gene expression patterns in human cancer cell lines.

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.

Breast↗

PubMed2000

A gene expression database for the molecular pharmacology of cancer.

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.

Antineoplastic Agents↗