Fredric C. Volkert, PhD
Mechanisms of mitotic partitioning of yeast nuclear plasmids; control of type 1 protein phosphatase activity; using yeast cells as a model for human folate transport; activities of human Fragile X mental retardation protein.
The major focus of research in this laboratory is the basic biology of yeast plasmids, and yeast recombinant DNA technology. Basic studies on yeast plasmid biology provide insights into mechanisms of chromosome inheritance as well as fundamental knowledge necessary for technology development. Recent basic studies have focused on the interactions between yeast plasmids and the nuclear architecture which determine their strongly nonrandom pattern of mitotic inheritance; these studies have shown that two mechanisms previously proposed to explain this phenomenon - catenation of sister plasmid dimers and tethering of replication origins to the nuclear matrix - are, respectively, wrong, and quantitatively inadequate. Ongoing studies are aimed at identifying plasmid sequences other than replication origins that contribute to nonrandom inheritance. We are also interested in a non-centromeric plasmid partitioning system encoded by a native yeast episome, the 2-m circle DNA molecule. Our recent studies have refined the definition of a site on the plasmid necessary in cis for its activity, have ruled out previously proposed involvement of the replication origin in this system, and have established that the proteins of this system have an uneven distribution in the yeast nucleus. Recent applied yeast recombinant DNA studies have included construction for a system for variable-level gene expression which uses a 2-m circle encoded site-specific recombinase to excise copy number limiting sequences from cloning vectors in vivo. Using this system we have identified a set of High Copy Lethal genes that include key cell cycle regulatory genes, and have further studied regulation of one of these genes, GLC7, encoding type 1 phosphoprotein phosphatase. Our studies of GLC7 have shown that it is a cell cycle regulator and have identified upstream regulation of its catalytic activity by a subunit of a membrane fusion ATPase. Current studies are aimed at elucidating the mechanism of this regulation. Other applied studies are being carried out in collaboration with three other HSCB laboratories. In collaboration with Dr. Sheldon Rothenberg of the Department of Medicine, we are using yeast expression to study the function of human proteins involved in folate transport and to identify other proteins with which they interact in human cells. In collaboration with Dr.W. Ted Brown of our department and the Department of Medicine, we are modeling in yeast the action of the RNA-binding protein encoded by the human Fragile X Mental Retardation Syndrome gene. Finally, in a collaborative effort not involving yeast recombinant DNA, we are applying Pulsed Field Gel Electrophoresis (a technology formerly much used by yeast laboratories for mapping chromosomes) to tracking individual bacterial clones in outbreaks of nosocomial infection and in chronically/recurrently infected patients. This is being done in collaboration with the Vaccine Studies Center, which is directed by Dr. Kenneth Bromberg of the Department of Pediatrics.