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Development of mutant generation system of Streptococcus mutans, and analysis of the functional domain of glucosyltransferases using the mutant.

Research Project

Project/Area Number 09672104
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field 矯正・小児・社会系歯学
Research InstitutionOsaka University

Principal Investigator

FUJIWARA Taku  Dept.Pedodontics Fac.Dentistry, 歯学部・附属病院, 講師 (00228975)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000)
KeywordsStreptococcus mutans / erythromycin resistant gene / kanamycin resistant gene / テトラサイクリン耐性遺伝子 / エリスロマイシン耐性 / PCR
Research Abstract

The erythromycin resistant gene (erm) which works in Streptococcus mutans was cloned from Streptococcus-E.coli shuttle vector pVA838. pVA838 was digested with Hind III and Cla I, then inserted into a cloning vector pUC19.The DNA sequence of the insert was revealed that the essential part of the erm was app. 800 bp.Then PCR primers were designed to add restriction enzyme sites at the end of the essential gene.A smaller (830 bp) erythromycin resistant cassette was generated by PCR.The kanamycin resistant gene (aphA) was cloned from streptococcal transposon Tn 1545 by the similar procedure.DNA sequence of the 1.6 kb insert containing the aphA revealed that an open reading frame of the aphA was app. 1000 bp.From the sequence of the aphA gene, PCR primers were also synthesized.A smaller (1070 bp) kanamycin resistant cassette was produced by PCR.Furthermore, the tetracycline resistant gene was cloned from Tn 1545.
On other hand, randomly mutagenesis was performed to investigate the localization mechanism of the cell-associated glucosyltransferase (CA-GTase).The chromosomal DNA gene banks from S.mutans strains MT8148 and GS-5 were generated with pVA891.The gene bank was randomly transformed into S.mutans.The erythromycin resistant transformants were screened from Mitis-Salivarius agar.Then expression of CA-GTase was examined by the Western blot analysis.
Although over 1000 mutants were tested, the localization of the CA-GTase was not altered.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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