| Budget Amount *help |
¥13,900,000 (Direct Cost: ¥13,900,000)
Fiscal Year 2005: ¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 2004: ¥7,200,000 (Direct Cost: ¥7,200,000)
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| Research Abstract |
(1)PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone ; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells t
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ransfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.
(2)Sindbis virus replicon-mediated gene expression involves RNA synthesis by viral replicase on cytoplasmic membranes. Here we examined the involvement of a membrane lipid, phosphatidylserine, in the replicon-mediated gene expression by using Chinese hamster ovary cell mutants defective in phosphatidylserine biosynthesis. When the mutant cells were transfected with a replicon RNA carrying a viral replicase gene followed by a subgenomic promoter-driven lacZ gene, expression of β-galactosidase from the replicon subgenomic RNA was inhibited under phosphatidylserine-deficient conditions. In contrast, expression of a replicase protein, nsP1, from the replicon genomic RNA and accumulation of the subgenomic RNA were not inhibited by the phosphatidylserine deficiency, indicating that inhibition of the reporter expression was not due to defects in production and function of the replicase. The reporter expression from an SV40 promoter-driven construct was found to be normal under similar conditions, implying the phosphatidylserine deficiency does not affect general translation and protein degradation. These results indicate that phosphatidylserine is specifically involved in a posttranscriptional event of Sindbis virus subgenomic promoter-driven gene expression. Less
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