Gene expression informatics--it's all in your mine.

Technologies for whole-genome RNA expression studies are becoming increasingly reliable and accessible. However, universal standards to make the data more suitable for comparative analysis and for inter-operability with other information resources have yet to emerge. Improved access to large electronic data sets, reliable and consistent annotation and effective tools for 'data mining' are critical. Analysis methods that exploit large data warehouses of gene expression experiments will be necessary to realize the full potential of this technology.

Drug target validation and identification of secondary drug target effects using DNA microarrays.

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.

Positionally cloned human disease genes: patterns of evolutionary conservation and functional motifs.

Positional cloning has already produced the sequences of more than 70 human genes associated with specific diseases. In addition to their medical importance, these genes are of interest as a set of human genes isolated solely on the basis of the phenotypic effect of the respective mutations. We analyzed the protein sequences encoded by the positionally cloned disease genes using an iterative strategy combining several sensitive computer methods. Comparisons to complete sequence databases and to separate databases of nematode, yeast, and bacterial proteins showed that for most of the disease gene products, statistically significant sequence similarities are detectable in each of the model organisms. Only the nematode genome encodes apparent orthologs with conserved domain architecture for the majority of the disease genes. In yeast and bacterial homologs, domain organization is typically not conserved, and sequence similarity is limited to individual domains. Generally, human genes complement mutations only in orthologous yeast genes. Most of the positionally cloned genes encode large proteins with several globular and nonglobular domains, the functions of some or all of which are not known. We detected conserved domains and motifs not described previously in a number of proteins encoded by disease genes and predicted functions for some of them. These predictions include an ATP-binding domain in the product of hereditary nonpolyposis colon cancer gene (a MutL homolog), which is conserved in the HS90 family of chaperone proteins, type II DNA topoisomerases, and histidine kinases, and a nuclease domain homologous to bacterial RNase D and the 3'-5' exonuclease domain of DNA polymerase I in the Werner syndrome gene product.

PDZ domain of neuronal nitric oxide synthase recognizes novel C-terminal peptide sequences.

PDZ domains are multifunctional protein-interaction motifs that often bind to the C-terminus of protein targets. Nitric oxide (NO), an endogenous signaling molecule, plays critical roles in nervous, immune, and cardiovascular function. Although there are numerous physiological functions for neuron-derived NO, produced primarily by the neuronal NO synthase (nNOS), excess nNOS activity mediates brain injury in cerebral ischemia and in animal models of Parkinson's disease. Subcellular localization of nNOS activity must therefore be tightly regulated. To determine ligands for the PDZ domain of nNOS, we screened 13 billion distinct peptides and found that the nNOS-PDZ domain binds tightly to peptides ending Asp-X-Val. This differs from the only known (Thr/Ser)-X-Val consensus that interacts with PDZ domains from PSD-95. Preference for Asp at the -2 peptide position is mediated by Tyr-77 of nNOS. A Y77D78 to H77E78 substitution changes the binding specificity from Asp-X-Val to Thr-X-Val. Guided by the Asp-X-Val consensus, candidate nNOS interacting proteins have been identified including glutamate and melatonin receptors. Our results demonstrate that PDZ domains have distinct peptide binding specificity.

Genome cross-referencing and XREFdb: implications for the identification and analysis of genes mutated in human disease.

Comparative genomics approaches and multi-organismal biology are valuable tools for genetic analysis. Cross-species connections between genes mutated in human disease states and homologues in model organisms can be particularly powerful, as model-organism gene function data and experimental approaches can shed light on the molecular mechanisms defective in the disease. We describe a project that is systematically identifying novel expressed sequence tag (EST) sequences that are highly related to genes in model organisms and mapping them to positions on the mouse and human maps. This process effectively cross-references model organism genes with mapped mammalian phenotypes, facilitating the identification of genes mutated in human disease states via the positional candidate approach. A public database, XREFdb (http:@www.ncbi.nlm.nih.gov/XREFdb/), disseminates similarity search, mapping and mammalian phenotype information and increases the rate at which these cross-species connections are established.

Characterization of the yeast transcriptome.

We have analyzed the set of genes expressed from the yeast genome, herein called the transcriptome, using serial analysis of gene expression. Analysis of 60,633 transcripts revealed 4,665 genes, with expression levels ranging from 0.3 to over 200 transcripts per cell. Of these genes, 1981 had known functions, while 2684 were previously uncharacterized. The integration of positional information with gene expression data allowed for the generation of chromosomal expression maps identifying physical regions of transcriptional activity and identified genes that had not been predicted by sequence information alone. These studies provide insight into global patterns of gene expression in yeast and demonstrate the feasibility of genome-wide expression studies in eukaryotes.

Exploiting the complete yeast genome sequence.

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.

Genes conserved in yeast and humans.

Evolutionary conservation of homologous gene products from distantly related organisms provides an information resource of great value for elucidating protein structure and function. Sequence similarities also serve as molecular cross-references between diverse organisms that offer different, or complementary, experimental approaches for analyzing gene expression and biochemistry in normal and abnormal states. There are now countless examples of information about a protein from one species contributing to the understanding of biological phenomena or disease in another species. Such connections are often unanticipated and surprising, but there is an opportunity to make them more systematically as concerted genome sequencing projects progress. In the present review we focus on connections between yeast and human proteins and their functional implications. We present several 'case studies' as well as survey results derived from comprehensive sequence comparisons among all yeast and human proteins currently present in the public databases.

Expression of an exogenous eukaryotic DNA methyltransferase gene induces transformation of NIH 3T3 cells.

Abnormal regional increases in DNA methylation, which have potential for causing gene inactivation and chromosomal instability, are consistently found in immortalized and tumorigenic cells. Increased DNA methyltransferase activity, which is also a characteristic of such cells, is a candidate to mediate these abnormal DNA methylation patterns. We now show that, in NIH 3T3 mouse fibroblasts, constitutive overexpression of an exogenous mouse DNA methyltransferase gene results in a marked increase in overall DNA methylation which is accompanied by tumorigenic transformation. These transformation changes can also be elicited by dexamethasone-inducible expression of an exogenous DNA methyltransferase gene. Our findings provide strong evidence that the increase in DNA methyltransferase activity associated with tumor progression could be a key step in carcinogenesis and provide a model system that can be used to further study this possibility.