Cloning and disruption of a gene required for growth on acetate but not on ethanol: the acetyl-coenzyme A synthetase gene of Saccharomyces cerevisiae.
A DNA fragment of Saccharomyces cerevisiae with high homology to the acetyl-coenzyme A (acetyl-CoA) synthetase genes of Aspergillus nidulans and Neurospora crassa has been cloned, sequenced and mapped to chromosome I. It contains an open reading frame of 2139 nucleotides, encoding a predicted gene product of 79.2 kDa. In contrast to its ascomycete homologs, there are no introns in the coding sequence. The first ATG codon of the open reading frame is in an unusual context for a translational start site, while the next ATG, 24 codons downstream, is in a more conventional context. Possible implications of two alternative translational start sites for the cellular localization of the enzyme are discussed. A stable mutant of this gene, obtained by the gene disruption technique, had the same low basal activity of acetyl-CoA synthetase as wild-type cells when grown on glucose but completely lacked the strong increase in activity upon entering the stationary phase, providing direct proof that the gene encodes an inducible acetyl-CoA synthetase (ACS1) of yeast. As expected, the mutant was unable to grow on acetate as sole carbon source. Nevertheless, it showed normal induction of isocitrate lyase on acetate media, indicating that activity of acetyl-CoA synthetase is dispensable for induction of the glyoxylate cycle in S. cerevisiae. Surprisingly, disruption of the ACS1 gene did not affect growth on media containing ethanol as the sole carbon source, demonstrating that there are alternative pathways leading to acetyl-CoA under these conditions.
Department of Botany, University of Basel, Switzerland.
C
C
De Virgilio
N
N
Bürckert
G
G
Barth
J M
JM
Neuhaus
T
T
Boller
A
A
Wiemken
D000106:Acetate-CoA Ligase / Q000235:genetics*; D000085:Acetates / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D000431:Ethanol / Q000378:metabolism; D005800:Genes, Fungal / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D000085:Acetates; D000431:Ethanol; D000106:Acetate-CoA Ligase