Abstract
The wild-type yeast nuclear gene, PIS, encodes phosphatidylinositol synthase (CDPdiacylglycerol-inositol 3-phosphatidyltransferase, EC 2.7.8.11) (Nikawa, J., and Yamashita, S. (1984) Eur. J. Biochem. 143, 251-256). We now report the sequence of the cloned 2, 129-base pair DNA and the location of the PIS coding region within the sequence. The PIS coding frame is capable of encoding 220 amino acid residues with a calculated molecular weight of 24,823. On Northern blot analysis, an RNA species that hybridized with the coding region was detected in the total poly(A)+ RNA of the wild-type yeast. The primary translation product contains a region showing local sequence homology with yeast phosphatidylserine synthase (EC 2.7.8.8) and Escherichia coli 3-phosphatidyl-1'-glycerol-3'-phosphate synthase (EC 2.7.8.5), suggesting that these three enzymes are evolutionarily related. The PIS gene was disrupted in vitro through insertion of the yeast HIS3 gene into the coding region. A heterozygous diploid, PIS/pis::HIS3, constructed from a PIS/PIS his3/his3 diploid by replacing one of the wild-type PIS genes with the disrupted PIS gene, showed no segregation of viable His+ spores on tetrad analysis, indicating that disruption of the PIS gene is lethal. The nonviable spores were in an arrested state with a characteristic terminal phenotype, suggesting that the function of the PIS gene is essential for progression of the yeast cell cycle.
Highlights
256).We report the sequence of the clo2n,e1d29- that the second messengers, derived from inositol phosphobasepairDNAandthelocationofthe
Subcloning experiments showed that the PIS gene is located in the 2.2-kbp insert of plasmidpPI514 (Fig. 1) [15].we determined the nucleotide sequence of the insertof plasmid pPI514 by the dideoxy method [22] using derivatives of the M13 phage [21]
Disruption of the Genomic PIS Locus-In previous studies [14],we demonstrated that analteration in the phosphatidylinositol synthase enzyme with the elevation of the apparent K, for myo-inositol leads to the requirement of myo-inositol by cells for both phosphatidylinositol synthesis and growth [14]
Summary
256).We report the sequence of the clo2n,e1d29- that the second messengers, derived from inositol phosphobasepairDNAandthelocationofthe. The PIS gene was disrupted in vitrothrough inser- membrane functions as a result of the inositol phospholipid tion of the yeast HIS3 gene into the coding region.A deficiency. Using this mutant we cloned the structural gene that encodes phosphatidylinositol synthase ( P I S )from a wild-type yeast DNA library by means of genetic complementation [15].
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