Cloning by genetic complementation and restriction mapping of the yeast HEM1 gene coding for 5-aminolevulinate synthase

Abstract

We have cloned the structural gene HEM1 for 5-aminolevulinate (ALA) synthase from Saccharomyces cerevisiae by transformation and complementation of a yeast hem1-5 mutant which was previously shown to lack ALA synthase activity (Urban-Grimal and Labbe Bois 1981) and had no immunodetectable ALA synthase protein when tested with yeast ALA synthase antiserum. The gene was selected from a recombinant cosmid pool which contained wild-type yeast genomic DNA fragments of an average size of 40 kb. The cloned gene was identified by the restauration.of growth on a non fermentable carbon source without addition of exogenous ALA. Sub cloning of partial Sau3A digests and functional analysis by transformation allowed us to isolate three independent plasmids, each carrying a 6 kb yeast DNA fragment inserted in either orientation into the single BamHI site of the vector pHCG3 and able to complement hem1-5 mutation. Analysis of the three plasmids by restriction endonucleases showed that HEM1 is contained within a 2.9 kb fragment. The three corresponding yeast trans formants present a 1, 2.5 and 16 fold increase in ALA synthase activity as compared to the wild-type strain. The gene product immunodetected in the transformant yeast cells has identical size as the wild-type yeast ALA synthase and its amount correlates well with the increase in ALA synthase activity.