| Project/Area Number |
05650792
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
生物・生体工学
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
NAKAMURA Satoshi Tokyo Inst.Tech., Fac.Biosci.& Biotechnol.Associate Professor, 生命理工学部, 助教授 (50227899)
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| Co-Investigator(Kenkyū-buntansha) |
NAKAJINA Harushi Tokyo Inst.Tech.Fac.Biosci.& Biotechnol.Research Associate, 生命理工学部, 助手 (10217721)
AONO Rikizo Tokyo Inst.Tech.Fac.Biosci.& Biotechnol.Associate Professor, 生命理工学部, 助教授 (30126643)
HORIKOSHI Koki Tokyo Univ., Fac.Eng.Professor, 工学部, 教授 (80087551)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1994: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
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| Keywords | Haloohilic archaeon / Cell surface glycoprotein / Gene cloning / Gene expression / PCR |
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| Research Abstract |
Haloarcula japonica strain TR-1 is a predominantly triangular disc-shaped halophilic archaeon.Taxonomic characteristics, mode of cell division and ultrastructure of cell envelope have been studied for the strain to date. However, the mechanisms maintaing the triangular shape have been still unclear. We demonstrated occurrence of a glycoprotein on its cell surface (cell surface glycoprotein : CSG), which seemed to be important in maintenance of the characteristic shape of Ha.japonica. The Ha.japonica CSG was purified and characterized. It had an apparent molecular mass of 170 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)and the carbohydrate content was about 12%(w/w). The polypeptide portion contained a large proportion of acidic amino acids. Oligonucleotides, directed against conserved regions between the CSGs from Halobacterium halobium and Haloferax volcanii, were synthesized and used as PCR primers. CSG-related sequences were amplified from the chromosom
… More
al DNA of Ha.japonica by PCR.The PCR-generated DNA fragment of about 890 bp, presumed to be a part of the CSG gene of Ha.japonica, was cloned in Escherichia coli and sequenced. According to the predicted amino acid sequence, the CSG from Ha.japonica showed apparent homology with those from Hb, halobium and Hf.volcanii.
Chromosomal DNA from Ha.japonica was digested with EcoRI,ligated to a vactor plasmid, and then transformed into E.coli to construct a genomic library. Transformants were screened by colony hybridization using the PCR product as a probe. Positive clones were found to contain recombinant plasmids with an identical 3.5-kb EcoRI insert. The partial nucleotide sequence indicated that the 5'end of the CSG gene and upstream region. Therefore, the KpnI fragment of 2.5 kb was cloned from another genomic library of Ha.japonica by gene walding. The 3.5-kb and 2.5-kb fragments overlapped by 1.4 kb. The 4.6-kb KpnI-EcoRI fragment was obtained by recombining the above 3.5-kb and 2.5-kb fragments at a common SacI site, and then inserted in pUC119 vector to construct plasmid pJAG16.SDS-PAGE of cell extract of E.coli harboring pJAG16 revealed that the Ha.japonica CSG gene was expressid in E.coli. Less
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