Deval Lashkari

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 Institutes 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.

Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar–coding strategy

A quantitative and highly parallel method for analysing deletion mutants has been developed to aid in determining the biological function of thousands of newly identified open reading frames (ORFs) in Saccharomyces cerevisiae. This approach uses a PCR targeting strategy to generate large numbers of deletion strains. Each deletion strain is labelled with a unique 20–base tag sequence that can be detected by hybridization to a high–density oligonucleotide array. The tags serve as unique identifiers (molecular bar codes) that allow analysis of large numbers of deletion strains simultaneously through selective growth conditions. Hybridization experiments show that the arrays are specific, sensitive and quantitative. A pilot study with 11 known yeastgenes suggests that the method can be extended to include all of the ORFs in the yeast genome, allowing whole genome analysis with a single selective growth condition and a single hybridization.

Genome-wide mapping with biallelic markers in Arabidopsis thaliana

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

A radiation hybrid map of the zebrafish genome

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Functional Analysis of the Genes of Yeast Chromosome V by Genetic Footprinting

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.

A cDNA microarray gene expression database for cancer drug discovery

81 regulated during adverse drug reactions. Such information provides us with a more detailed understanding of the molecular mechanisms underlying toxic effects. Additionally, microarray technology can be used to identify gene expression profiles for new therapeutic compounds, thus providing an early screening method for potential toxic effects. An important step in using microarray analysis in toxicity screening is to construct a library of genes that are regulated positively or negatively during hepatotoxicity, as these genes may not be represented in libraries prepared from normal tissue. In order to facilitate this, we have treated rats with hepatotoxins representing diverse structural and therapeutic classes. These compounds induce a variety of hepatotoxic responses including peroxisomal proliferation, hepatic necrosis and apoptosis, cholestasis, DNA damage, protein synthesis inhibition, oxidative stress, mitochondrial damage, P-450 induction and phospholipidosis. The livers were harvested from the treated rats and mRNA was isolated. The mRNA was pooled and gene expression analysis of treated versus untreated rats was assayed using microarray analysis. The data gathered from microarray and sequence analysis allows us to identify signature sets of genes regulated during toxicity. These gene responses are then used to identify mechanisms of toxicity. Knowledge of specific toxicologic pathways can help determine potential human liabilities, and the identification of signature gene sets can be used to evaluate drug candidates for potential adverse effects. In a follow-up case study, using microarrays, we evaluated changes in gene expression in rat liver associated with hypertrophy, hyperplasia and single cell necrosis (apoptosis) following administration of a drug candidate.