[KIL-d]: A Novel Epigenetic Regulator of Viral Gene Expression in Yeast.

Rutgers, The State University Of New Jersey-Rbhs-Robert Wood, Piscataway NJ

Investigators

Abstract

This project aims to determine the genetic locus encoding [KIL-d], a cytoplasmically-inherited genetic element in yeast that exerts epigenetic regulatory effects on the M double-stranded (ds) RNA genomic segment of the cytoplasmic killer virus of yeast. In wild-type yeast cells, M dsRNA confers on infected cells the phenotypes of toxin production and toxin resistance, both activities of processed products of the virus-encoded preprotoxin protein. In the presence of [KIL-d], the expression of M dsRNA is normal in diploid cells, but upon meiosis each haploid progeny clone shows a different mitotically stable (variegated) defect in toxin production and/or resistance function. Upon mating of the defective haploids, wild-type virus expression is restored (healing), and upon meiosis variegated defective phenotypic expression in each haploid progeny reappears (resetting). The investigator has previously shown that the effects of [KIL-d] are exerted on M dsRNA but not on a cDNA clone derived from it. [KIL-d] is transmissible to a new strain by cytoplasmic transfer, but it does not exert its epigenetic effects on M dsRNA expression until the recipient haploid cells have gone through a cycle of mating and meiosis, indicating that nuclear functions are involved in the epigenetic regulation. [KIL-d] does not map on the viral M or L-A dsRNA segments, mitochondrial DNA or the 2-micron DNA plasmid. [KIL-d] has a relatively high rate of loss (like many plasmids), and an even higher rate of reappearance with variegated phenotypic expression in clones that have lost it. This behavior resembles the known prions of yeast, but not any known nucleic acid plasmid. This research will quantify the epigenetic effects of [KIL-d] on M dsRNA by determining whether it alters the size or quantity of viral genomic or transcript RNA or of the protein products of the virus. In order to determine the molecular nature of [KIL-d], a genomic library from a primary isolate in which this element has appeared after mutagenesis (available in this laboratory) will be used to generate a genomic DNA library (or a cDNA library), that will be screened for genes that can generate [KIL-d] when transferred to wild-type recipients. If [KIL-d] is a prion, then this screen should identify the gene(s) encoding the protein that forms the prion. If the prion model is incorrect, this screen should identify the genetic elements involved in this virus-specific epigenetic regulatory process, which appears to be the first such process to be subjected to molecular genetic analysis. Further studies using the genetic methods available in yeast will allow characterization of the genes identified in this library screening procedure. This research will provide a training opportunity for a postdoctoral fellow, graduate student and several undergraduate students, who will be trained in the methods of yeast molecular genetics. It will also include a new group summer research experience for a group of summer undergraduate students participating in two programs run on this campus with support from the NSF and an Initiative for Minority Student Development Award from the NIH. Misfolded proteins that can cause normal proteins of the same type to misfold are termed prions. In yeast, prions are cytoplasmically-inherited non-nucleic acid genetic elements. [KIL-d] is an unusual genetic element in yeast that alters expression of the genes of a viral RNA. This research will characterize this genetic element and determine whether it is a prion.