Selection by genetic complementation and characterization of the gene coding for the yeast porphobilinogen deaminase

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Abstract
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 and 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 exogenous haem or fatty acid and sterol supplements. The recombinant plasmid was reatined 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 hydrid-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.