Selection by genetic complementation and characterization of the gene coding for the yeast porphobilinogen deaminase.
The porphobilinogen deaminase (PBG-D) gene of Saccharomyces cerevisiae has been isolated by genetic complementation of a mutant GL7 (alpha hem 3) strain, previously shown to be defective in this haembiosynthetic enzyme [Gollub, Liu, Dayan, Adlersberg & Sprinson (1977) J. Biol. Chem. 252, 2846-2854]. The gene was selected from a yeast wild-type genomic DNA library ligated into the shuttle vector YEp13. The complementing gene restored growth of the hem 3 (PBG-D) mutant strain on media in the absence of exogeneous haem or fatty acid and sterol supplements. The recombinant plasmid was retained in the Hem+ transformant provided that selective pressure for plasmid-dependent growth was maintained. Transformation of the mutant strain (hem 3) restored the PBG-D activity to levels up to 10-fold those of the parental strain. The mutant strain GL7 does not show any measurable enzymic activity. Analysis of the plasmid designated YEpPBG-D (containing the PBG-D gene) by hybrid-selected translation revealed that it contained the coding information for a single protein of apparent Mr 43,000. The coding region was localized on an 1.5 kb endonuclease-EcoRI fragment (E4), within the 5.5 kb genomic insert in YEpPBG-D.
D000642:Ammonia-Lyases / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D005815:Genetic Code; D005816:Genetic Complementation Test; D011163:Hydroxymethylbilane Synthase / Q000235:genetics*; D010957:Plasmids; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic
D005656:Fungal Proteins; D011163:Hydroxymethylbilane Synthase; D000642:Ammonia-Lyases