1. The structural gene for cholinephosphate cytidylyltransferase (CCT) was isolated from a Sacchuromyces cerevisiae genomic library by means of complementation in a mutant of the yeast defective in the enzyme. The cloned DNA restored both the growth and cholinephosphate cytidylyltransferase activity of the mutant. Whereas the enzyme of the mutant was thermolabile, the enzyme produced by the transformant was indistinguishable in heat stability from that produced by the wild type.2. Strains carrying a multicopy recombinant plasmid overproduced cholinephosphate cytidylyltransferase. The overproduction of the enzyme brought about an increase in the synthesis of CDPcholine in the transformant, but there was no increase in the overall rate of phosphatidylcholine synthesis.3. The cloned DNA was subcloned into a 2.5-kb DNA fragment. The nucleotide sequence which contained CCT was determined by the dideoxy chain-termination method. The sequence contained an open reading frame capable of encoding a protein of 424 amino acid residues with a calculated relative molecular mass of 49 379.31. Northern blot analysis showed that this DNA segment is transcribed in yeast cells and the length of the transcript is consistent with the putative translation product.4. Hydropathy analysis according to Kyte and Doolittle indicated that the primary translation product contains extended hydrophilic stretches in its N-and C-terminal regions.5. The primary translation product contains a region showing local sequence homology with nucleotidyltransfer enzymes such as DNA polymerase (Escherichiu coli), CDPdiacylglycerol pyrophosphatase (E. coli), 3-deoxy-manno-octulosonate cytidylyltransferase (E. coli) and DNA ligase (T4 phage), suggesting that these five enzymes are evolutionarily related. Statistically significant sequence homology was also noted between the human c-fos gene product and the enzyme.Phosphatidylcholine is a major structural component of the cellular membranes of eukaryotes. In the yeast, Sacchuromyces cerevisiue, phosphatidylcholine comprises about 40% of the total phospholipids. Several lines of evidence indicate that phosphatidylcholine is absolutely required for yeast growth [l -31. S. cerevisiae possesses two pathways for the synthesis of phosphatidylcholine, the CDPcholine pathway and the phosphatidylethanolamine methylation pathway [4-61. Due to the feasibility of genetic manipulation of the yeast, the phosphatidylcholine-synthesizing system of S. cerevisiue has been the subject of extensive genetic studies. Mutants for all of the enzymes involved in the CDPcholine pathway and the phosphatidylethanolamine methylation pathway have been isolated [l, 2, 7-91. Characterization of these mutants has provided an insight into the genetic and
1. A yeast chromosomal DNA which contains the structural gene for phosphatidylserine synthase (PSS) was isolated by genetic complementation from a wild-type yeast genomic library. The PSS gene was subcloned into a 1.1-kb fragment of the yeast DNA on the YEp13 vector.2. The PSS gene on the multicopy plasmid caused the fourfold over-production of the enzyme and fully restored the phosphatidylserine content of the transformant. The phospholipid composition of the transformant was similar to that of the wild type.3. Sequence analysis showed that this DNA fragment contains an open reading frame capable of encoding 276 amino acid residues with a calculated relative molecular mass of 30804. Northern blot analysis of poly(A)-rich RNA of the wild-type yeast indicated that this DNA segment is transcribed into a single mRNA species.4. The DNA sequence contained two putative transcriptional initiation signals, each followed by the ATG initiator codon. Deletion experiments indicated that the 5'-proximal ATG codon is essential for the synthesis of the functional phosphatidylserine synthase.Phosphatidylserine synthase catalyzes the formation of phosphatidylserine from CDPdiacylglycerol and serine [l]. This enzyme is found in prokaryotes and lower eukaryotes, and corresponds to the base-exchange enzyme in higher eukaryotes which synthesizes phosphatidylserine utilizing preexisting phospholipid as the phosphatidyl donor instead of CDPdiacylglycerol [2]. In the yeast, phosphatidylserine comprises about 5% of the total phospholipids [3] and appears to play a role in the control of the net charge of the membrane. Furthermore, phosphatidylserine serves as a precursor for two major phospholipids, phosphatidylethanolamine and phosphatidylcholine. Decarboxylation of phosphatidylserine yields phosphatidylethanolamine [2] and subsequent methylation yields phosphatidylcholine [4]. The phosphatidylserine synthase of Escherichia coli was purified to homogeneity [5] and extensively characterized [6 -81. A eukaryotic enzyme has also been obtained in a highly purified form from the yeast, Saccharomyces cerevisiae [9]. Both the prokaryotic and eukaryotic type phosphatidylserine synthase genes have been cloned [lo, 111, but their nucleotide sequences remain unknown.Several lines of evidence indicate that yeast phosphatidylserine synthase is a regulatory enzyme. Inclusion of choline in the culture medium caused a significant reduction in phosphatidylserine synthase activity [12]. The enzyme level is dependent on the growth phase of the cells, being high in the Correspondence to S. Yamashita,
By genetic complementation in a yeast choline transport mutant from a yeast gene library, we isolated plasmids encoding choline transport. The cloned plasmids contained a common 4.0-kilobase DNA fragment and also complemented an ethanolamine transport defect. The cloned sequence present in the yeast genome was possibly unique.
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