Isolation of the yeast INO1 gene: located on an autonomously replicating plasmid, the gene is fully regulated.

AUTOR(ES)

Klig, L S

RESUMO

The Saccharomyces cerevisiae gene, INO1 , encoding the highly regulated enzyme, myo-inositol-1-phosphate synthase [1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent IN01 alleles ( ino1 -5 and ino1 -13). One of these mutants ( ino1 -5) fails to make any material that is crossreactive with antibody to the wild-type inositol-1-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-1-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO1 locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-1-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells ( ino1 -) containing the cloned INO1 gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INO1 . These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-1-phosphate synthase.

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