My research interests are centered on the mechanisms directing gene expression and chromatin domain formation in the simple eukaryote, Saccharomyces cerevisiae. Studies have shown that transcription can be aided or repressed by DNA elements residing thousands of base pairs away
from the target gene. These elements have been termed "enhancers" or "silencers" and seem to be a widespread phenomena in eukaryotic gene expression.
One example of a silencer is found in the regulation of mating type genes in yeast. Haploid yeast cells can be either a or a, depending on which genes are expressed at the MAT locus. Copies of the a and a genes are found on the left and right arms of chromosome III at HMLa and HMRa,
respectively. These genes are never expressed due to the action of silencers. Three known components make up the silencer at HMR E. One of these is an autonomously replicating sequence, thought to be an origin of replication. The other two components, RAP1 and ABF1, work elsewhere
in the yeast genome as gene activators. Several trans-acting proteins are also required for silencing, including SIR1, 2, 3, and 4. The exact function of these proteins within the context of silencing is unknown. Curiously, histone H4 is also required, leading to the assumption that
silencing involves a change in the regional chromatin structure of the gene.
Current work in my laboratory is directed toward exploring the relationship between replication and transcriptional silencing. My team of undergraduates are constructing plasmids and yeast strains to analyze how gene repression affects replication. Students have transformed
yeast with different plasmids, each having a unique silencer configuration. By removing the normal origin of replication from the plasmid, the mitotic stability of the plasmid can be measured. The plasmid will only be propagated in the yeast if the origin contained
within the silencer is working as an origin of replication. The ability of these constructs to repress transcription can be measured by simple b-galactosidase activities. These experiments will allow us to determine if the two activities, replication and repression, are dependent upon
each other. Preliminary studies indicate that strong silencers are also strong origins of replication.
Experiments done in Saccharomyces cerevisiae have an additional advantage in that yeast is a relatively inexpensive experimental organism. As a eukaryote, however, knowledge gained in this system is often true for higher life forms. The techniques used are general
molecular biology methods, and students will likely encounter many of them later on in their careers.
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