Expression of the HIS3 gene of Saccharomyces cerevisiae in polynucleotide phosphorylase-deficient strains of Escherichia coli K-12.
The PstI and BamHI fragments, containing the HIS3 (imidazoleglycerol phosphate dehydratase) gene of yeast obtained from pYehis2, and the ColE1-derived plasmid pBR322 were ligated in vitro and used to transform hisB463 strains of Escherichia coli K-12. Expression of the cloned HIS3 gene from yeast was markedly enhanced (3--5-fold) in polynucleotide phosphorylase (pnp)-deficient strains of E. coli. The levels of both HIS3 and plasmid-encoded mRNAs were increased in pnp- strains carrying the chimeric plasmids, whereas there was little difference in the levels of pBR322-specific mRNAs in pnp+ and pnp- strains. This increase in HIS3 mRNA appeared to be related to specific stabilization of the eukaryotic message due to its unique structural features, since the half-life of the HIS3 mRNA increased from 1.5 to 18.7 min, whereas no increase in the half-lives of pBR322 vehicle mRNAs was observed. A physical map of the plasmid pYehis2 was constructed using restriction endonuclease and molecular cloning techniques.
D002874:Chromosome Mapping; D004274:DNA, Recombinant / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D006836:Hydro-Lyases / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D012333:RNA, Messenger; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase