This project characterizes the ribonucleotide reductase genes of the large DNA virus, vaccinia, and of variant viruses selected for the ability to grow in high concentrations of hydroxyurea. The variant viruses overproduce ribonucleotide reductase activity and a 34K early protein, and their DNA reveals restriction pattern aberration which are suggestive of gene amplification. Initially, the gene encoding the small subunit of ribonucleotide reductase will be mapped using an oliginucleotide probe, and its sequence determined. The 34K protein gene will also be mapped using hybrid-selected translation to confirm the identity of this protein and the ribonucleotide reductase small subunit. A transcriptional map of the reductase-encoding gene region will be determined, using Northern blots and S1 nuclease techniques. The necleotide sequence of the putative amplified DNA present in hydroxyurea- resistant virus will be determined to characterize the genomic rearrangements which have occurred and to form hypotheses about the mechanisms involved. The large subunit of viral ribonucleotide reductase will be identified by immunoprecipitation of holoenzyme by antibodies prepared to the overproduced 34K protein, and its gene mapped and sequenced. The temporal pattern of ribonucleotide reductase gene transcription throughout the infective cycle and its relation to viral DNA synthesis will be determined. Cloned constructs will be used in an in vitro transcription system to identify factors which may regulate early gene transcription in general, and expression of ribonucleotide reductase in particular. Insertion vectors designed to inactivate or replace the endogenous viral reductase genes will be constructed for use in evaluating whether this enzyme functions directly in initiation and termination of DNA synthesis. These studies are designed to evaluate the role of ribonucleotide reductase in replicating systems, and ultimately to ask whether this enzyme has a role in tumorigenesis.
