Expression of RAD4 gene of Saccharomyces cerevisiae that can be propagated in Escherichia coli without inactivation.

The RAD4 gene of Saccharomyces cerevisiae, which is essential for the nucleotide excision repair, was isolated from a yeast genomic library and the expression of this gene has been investigated. RAD4 mRNA was approximately 2.3 kb and di not contain intervening sequence, as determined by S1 nuclease mapping and Northern blot analysis. Transcription start site was located at 48 bp upstream of the ATG initiation codon. RAD4 gene is not induced by UV light damages as indicated by the absence of change in mRNA level after UV exposure in wild type yeast cells. The size of Rad4 protein overexpressed in Escherichia coli was found to be 89 kDa by SDS-PAGE. This is consistent with the size of the gene's ORF, which encodes 730 amino acids with the calculated molecular weight of 84456. The RAD4 protein contains many potential kinase dependent phosphorylation sites and its C-terminus is highly acidic like other DNA repair proteins of S. cerevisiae.