Isolation of the yeast gene encoding elongation factor 3 for protein synthesis

Abstract

The gene YEF-3 encoding the elongation factor 3 (EF-3) for peptide chain elongation in Saccharomyces cerevisiae has been isolated by immunoscreening of a yeast genomic library in the phage lambda gt11. The identity of the EF-3 gene was confirmed by several methods. First, a clone-encoded protein could affinity purify the antibody that specifically reacted with EF-3. Second, a recombinant fusion protein, which reacted with anti-beta-galactosidase antibody as well as with anti-EF-3 antibody, was found in the lysate of a positive clone lysogen. Third, the function of EF-3 in a yeast mutant in which the EF-3 activity is temperature-sensitive in an in vitro assay could be complemented by transformations. EF-3 protein was overproduced in a transformant which contained the EF-3 gene on a multicopy plasmid YEp-13. Southern blot analysis shows that YEF-3 is a single copy gene. The transcript unit as mapped by S1 nuclease mapping, is consistent with the size of the message determined by Northern blot analysis and shows no evidence of introns.