Patrick O. Brown
Web: http://cmgm.stanford.edu/pbrown
Several of the projects in Dr. Brown's laboratory are devoted to developing and applying new experimental methods for studying genes on a genomic scale.
Genetic footprinting is a "genomic" approach to determining the contribution made by each gene to the fitness of an organism. In a large-scale test of the feasibility of this approach, postdoctoral fellow Victoria Smith used genetic footprinting to analyze the biological roles of each gene on an entire chromosome, Chromosome V of Baker's yeast, Saccharomyces cerevisiae. With the recent completion of the sequence of the entire yeast genome, Dr. Brown's group now plans to use genetic footprinting to carry out a comprehensive analysis of the functions of each of the 6200 genes in this organism over the next two years.
Genomic mismatch scanning (GMS) is a method for genetic linkage mapping that has been under development in Dr. Brown's laboratory for several years. In this method, specialized enzymes, which can recognize the presence of differences between two DNA sequences, are used in a biochemical procedure that isolates all the regions of identity-by-descent between two relatives in a single procedure. The genetic locations of these regions are determined by labelling the isolated DNA with a fluorescent tag, and hybridizing it to a microscopic array of DNA sequences representing segments of the genome. In collaboration with Ron Davis's group, Dr. Brown's group has prepared microarrays containing approximately 3,000 individual yeast genes, soon to be expanded to include all 6200 yeast genes. These microarrays allow GMS to be used to map genes that underlie complex genetic traits in yeast, and to carry out detailed studies of the control and organization of meiotic recombination. Parallel work is directed at adapting the GMS methodology to the human genome. In the coming year, Dr. Brown's group expects to be able to genotype approximately 1,000 human loci, distributed throughout the genetic map, in a single hybridization.
In the course of their efforts to develop methods for making and using microscopic arrays of DNA molecules for GMS, Dr. Brown's group found that they could use microarrays of gene sequences as a tool for monitoring the expression patterns of many genes at once. Over the past year, they have made progress in developing and testing the use of cDNA microarrays for monitoring human gene expression patterns. Microarrays of several thousand genes have been assembled, and shown to be able to detect messages present at levels as low as 10-5 of total mRNA mass, and to distinguish 2-fold or smaller differences in RNA expression levels between different cell samples.
In parallel, over the past several years, large-scale human cDNA sequencing projects have deposited into public databases sequences representing at least 20,000 and perhaps as many as 50,000 different human genes. This number will certainly continue to grow, and may reach over 100,000 in the next year or two.
These developments create a wonderful opportunity to define, all at once, the RNA expression patterns of essentially every known human gene. This will be an extremely valuable source of information regarding the behavior and possible roles of each gene in health and disease. In the next year, Dr. Brown and his colleagues intend to develop and test a cDNA array that will allow the expression of 10,000 human genes to be monitored simultaneously. The expression of each of these genes will be determined for a set of hundreds of different cell and tissue types, developmental stages, disease states, and temporal programs of gene expression.
When a retrovirus infects a cell, it transcribes its RNA genome into a double-stranded DNA molecule, then inserts the viral DNA into the nuclear DNA of its host, establishing a provirus. Insertion of a provirus is essential for retroviral reproduction. How does a retrovirus get its DNA into the nucleus and integrate it into the host genome? Dr. Brown's group has developed methods for reproducing the integration reaction in vitro. The key enzyme in HIV integration, integrase, has been purified and its biochemical properties and molecular mechanism are a major focus of current work. One practical objective of the work with HIV is to identify inhibitors of integration, which could be useful as antiviral agents. The group is also investigating aspects of the cell-biology of retroviral infection, including how cellular components participate in the progression of the viral life cycle and how viral replication intermediates move to the correct cellular compartment.
Nelson, S. F., McCusker, J. H., Sander, M. A., Kee, Y., Modrich, P. and Brown, P. O. (1993) Genomic mismatch scanning: a new approach to genetic linkage mapping. Nat Genet 4, (1): 11-18. (Medline)
Roe, T., Reynolds, T. C., Yu, G. and Brown, P. O. (1993) Integration of murine leukemia virus DNA depends on mitosis. EMBO J 12, (5): 2099-2108. (Medline)
Brown, P. O. (1994) Genome Scanning Methods. Curr. Opinion in Genetics and Devel. 3, 366-373. (Medline)
Ellison, V., Gerton, J., Vincent, K. and Brown, P.O. (1995). An essential interaction between distinct domains of HIV-1 integrase mediates assembly of the active multimer. J. Biol. Chem. 270:3320-3326. (Medline)
Schena M; Shalon D; Davis RW; Brown PO. (1995) Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science, 270(5235):467-70. (Medline)
Smith V; Botstein D; Brown PO. (1995) Genetic footprinting: a genomic strategy for determining a gene's function given its sequence. Proc. Natl. Acad. Sci. USA, 92(14):6479-83. (Medline)
Shalon, D., Smith, S. J. and Brown, P.O. (1996). A DNA micro-array system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Research 6: 639-645. (Medline)
Smith, V., Chou, K., Lashkari, D., Botstein, D. and Brown, P.O. (1996). Functional analysis of the genes of yeast Chromosome V by genetic footprinting. Science 274: 2069-2074. (Medline)
