GENETIC AND MOLECULAR REGULATION OF INOSITOL BIOSYNTHESIS AND ITS COORDINATION WITH PHOSPHOLIPID BIOSYNTHESIS IN SACCHAROMYCES CEREVISIAE
KLIG, LISA SHARON
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The regulation of membrane biogenesis in a program of balanced cell growth is comprised of numerous complex interactions. Two components of this process are: (1) the regulation of the synthesis of phospholipid precursors, (2) the coordination of the synthesis of phospholipid precursors and the synthesis of phospholipids. Physically these events translate into the regulation of cytoplasmic enzyme activities and the coordination with membrane associated processes.;It has been shown that the enzyme inositol-1-phosphate synthase which catalyzes a reaction ultimately yielding the phospholipid precursor inositol-1-phosphate, is repressed 50-fold in wild type yeast grown in media containing 50(mu)M inositol. In order to study the mechanism(s) which regulates this enzyme, the gene encoding this enzyme (INO1) was cloned. The INO1 clone was used to analyze the homologous cellular mRNA. These studies identified two RNA species homologous to the INO1 locus. The smaller RNA (2.1 kilobases) was shown to be the mature messenger RNA. Data was obtained (by S1 nuclease mapping, utilization of yeast mutant strain rna2 and electron microscopy) which suggests that the larger RNA (2.8 kilobases) is the precursor out of which 0.7 kilobases is spliced generating the smaller mature mRNA. This splicing process (post-transcriptional) appears to be one of the levels at which the synthesis of this enzyme is regulated.;The regulation of the INO1 locus, coordinating the synthesis of inositol with the synthesis of phospholipids, was shown to occur at the transcriptional level. To further study this coordinated regulation the INO4 gene was cloned by complementation. The ino4 mutants map genetically to one locus, render the yeast inositol requiring and, simultaneously, repress the overall percentage of methylated phospholipids. Northern blot analysis revealed that this locus encodes a single very small mRNA. The quantity of mRNA homologous to the INO4 locus appears to increase in yeast grown under inositol-1-phosphate synthase repressing conditions. Furthermore, the amount of INO4 transcript present in different inositol mutants (auxotrophs and secretors) varies dramatically. This finding suggests that a locus involved in the coordinate regulation of phospholipid biosynthesis and phospholipid precursor biosynthesis is itself regulated.