Worm Breeder's Gazette 12(3): 61 (June 15, 1992)

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Cloning of the Yeast rad6 Homolouge ( UBC-1 )in C. elegans

David Leggett, Don Jones, E. Peter, M. Candido

Department of Biochemistry, University of British Columbia, Vancouver B.C. Canada V6 T1Z3

The yeast RAD6 gene encodes a protein that is essential for DNA repair, induced mutagenesis and sporulation. It has recently been shown to encode a ubiquitin conjugating activity. This enzyme specifically ubiquitinates histones in vitro. and short-lived proteins targeted for degradation in vitro. Ubiquitin can be found in the cell either free or covalently bound to proteins. Ubiquitin bound to proteins serves as a tag to mark the protein for subsequent recognition by enzyme systems serving a variety of functions, e.g. selective protein degradation, DNA repair and receptor structure. In order to better understand the role of ubiquitin in higher eukaryotes, we have begun to study the RAD6 homologue in C elegans.

PCR using degenerate oligonucleotides primers designed from the RAD6 and corresponding human protein sequences was performed. This resulted in a 300 nucleotide fragment which corresponds to the C-terminal half of the coding region of the RAD6 homologue in C. elegans ( UBC-1 ).Sequencing of the PCR product has revealed a 58% identity to the human protein, and 52% identity to the yeast protein over the region characterized. Hybridization of this fragment to a YAC grid mapped the UBC-1 gene to Chromosome IV. This was subsequently refined to a 3.5 Kb Dral fragment of cosmid C04E2 .A cDNA clone of UBC-1 was also isolated by Bob Waterston's lab during the random sequencing of cDNAs as part of the genome project. Both the cDNA and genomic DNA are presently being sequenced. Northern analysis using the cDNA as a probe reveals two major transcripts of 3.0Kb and 1.7Kb. The level of these transcripts do not increase after heat shock.

Further work will involve examining UBC-1 's tissue and developmental expression, regulatory elements, target specificity as well as its biological role in the cell.

Supported by M.R.C. of Canada and the B.C. Health Research Foundation