| Project/Area Number |
12680642
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | Kansai Medical University |
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Principal Investigator |
FUJII Shigeru Kansai Medical University, Professor, 医学部, 教授 (60144482)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAGAWA Manabu Kansai Medical University, Assistant, 医学部, 助手 (50261053)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2000: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | Dictyostelium / differentiation factor / glycoprotein / protein structure / sugar chains / 細胞性粘菌 |
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| Research Abstract |
In the previous study, we have purified a factor inducing prespore cell differentiation with an apparent molecular mass of 106 kDa on SDS-PAGE, which we named psi-factor (prespore-cell-inducing factor). We analysed four partial amino acid sequences of the purified psi-factor, and synthesized an oligonucleotide corresponding to one partial amino acid sequence. By using it as a probe, we isolated cDNA clones encoding psi-factor. They contained the full-length psi-factor open reading frame with 1674 mucleotides, which encoded a protein of 557 amino acid residues. The amino acid sequence shows that the factor is a novel protein containing both proline- and cysteine- rich regions. The analysis of the N-terminal sequence of the purified psi-factor indicates that the first 19 amino acid residues are severed and the matured secreted psi-factor consists of 538 amino acid residues with a molecular mass of 60 kDa. This value is much smaller than the apparent molecular mass estimated by SDS-PAGE.. The difference may come from addition of sugar chains to the protein. The amino acid sequence shows six possible Asn-glycosylation sites, Asn-Xaa-Ser/Thr motifs. When psi-factor was treated with endoglycosidase H, its apparent molecular mass decreased on SDS-PAGE. It was strongly bound with ConA-agarose. These results indicated that psi-factor is a glycoprotein with Asn-linked carbohydrates. Knockout mutant Ax2 strains of the psi-factor gene were prepared by targeted integration. Rescuing the mutant strains by transforming actin 15-psi-factor expression vector caused overproduction of psi-factor protein and higher prespore-cell inducing activity. These results show that psi-factor is one of important differentiation factors in Dictyosteliium discoideum.
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