Ronald W. Davis
Web: http://sequence-www.stanford.edu
Our laboratory is focused on the development and application of new molecular biology and manipulative genetic techniques to a variety of problems. As model organisms, we use Saccharomyces cerevisiae and Arabidopsis thaliana.
We are also developing new tools for rapid DNA sequencing and whole genome analysis. This project involves the development of automation and robotic approaches to increase the speed and reduce the labor. Because it is now possible to sequence whole genomes, it is now possible to conduct analysis on all genes of a genome. We are using Saccharomyces cerevisiae to conduct these initial whole genome analysis projects. The yeast genome sequence has approximately 6,000 genes. We are currently making a set of strains containing a complete deletion of each gene. In order to facilitate whole genome analysis each deletion is molecularly tagged with a unique 20 mer DNA sequence. This sequence acts as a molecular bar code and makes it easy to identify the presence of each deletion. The mixture of all such tag strains then allows for the analysis of the entire genome with the manipulation of a single sample. During growth under a variety of conditions the loss of a tag indicates the loss of a deletion from the population. The concentration of each tag is determined by PCR amplification of the tags and hybridization to an Affymetrix DNA chip that contains the complement to all of the DNA sequence tags. This approach can also be applied to other microorganisms.
In a second model study with Saccharomyces cerevisiae all of the open reading frames are being amplified by PCR. The extensive use of oligonucleotides is being made possible by a custom automated multiplex oligonucleotide synthesizer that makes 96 oligos at a time at much reduced cost. The amplified open reading frames are covalently attached to glass in a microarray. These microarrays can then be interrogated with fluorescently labeled probes made from messenger RNA. This allows the simultaneous evaluation of expression level of all genes from the genome. We have identified a number of wild isolates of yeast that grow at much higher temperatures than is typical for Saccharomyces cerevisiae and have also discovered that some of these strains show pathogenic traits and can be maintained in a live mouse. These microarrays can also be used to map complex genetic traits such as virulence traits in pathogenic Saccharomyces cerevisiae using genome mismatch scanning. They are also suitable for conducting two hybrid analysis that would allow one to generate a map of all interacting proteins coded for by the yeast genome. Also we are conducting studies with Arabidopsis thaliana involved in conducting whole genome sequence of this model plant. We are also generating microarrays of all currently known gene sequences using a newly developed HPLC method we can identify single base pair changes in the genome. This has allowed us to develop a bi-allelic map of Arabidopsis thaliana, and we are developing a method that will allow us to conduct whole genome genetic mapping using an Affymetrix DNA chip. Also using the denaturing HPLC method, we are identifying bi-allelic markers for the human Y chromosome. Because the Y chromosome does not recombine, a large set of such markers will allow one to trace the lineage of the Y chromosome through the human population.
Preuss, D., Lemieux, B., Yen, G., and Davis, R.W. (1993) A conditional sterile mutation eliminates surface components from Arabidopsis pollen and disrupts cell signaling during fertilization. Genes & Dev. 7: 974-985. (Medline)
McCusker, J.H., Clemons, K.V., Stevens, D.A., and Davis, R.D. (1994) Genetic Characterization of Pathogenic Saccharomyces cerevisiae Isolates. Genetics 136: 1261-1269. (Medline)
Lashkari, D.A., Hunicke-Smith, S.P., Norgren, R.M., Davis, R.W. and Brennan, T. (1995) An automated multiplex oligonucleotide synthesizer: Development of high-throughput, low-cost DNA synthesis. Proc. Natl. Acad. Sci. USA, 92: 7912-7915. (Medline)
Schena, M., Shalon, D., Davis, R.W., and Brown, P.O. (1995) Quantitative monitoring of gene expression pattern with a complementing DNA microarray. Science 270: 467-470. (Medline)
Schena, M. Shalon, D., Heller, R., Chai, A. Brown, P.O. and Davis, R. W. (1996) Parallel Human Genome Analysis: Microarray-Based Expression Monitoring of 1,000 Genes. Proc. Natl. Acad. Sci. USA, 93: 10614-10619. (Medline)
Shoemaker, D. D., Lashkari, D. A., Morris, D., Mittmann, M., and Davis, R.W. (1996) "Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy" Nature Genetics 14:450-456. (Medline)
