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

开发变形链球菌突变体产生系统，并利用该突变体分析葡萄糖基转移酶的功能域。

基本信息

批准号：
09672104
负责人：
FUJIWARA Taku
金额：
$ 0.51万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

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.

从链球菌突变体中起作用的红霉素耐药基因（ERM）是从链球菌 - e.coli shuttle shuttle vaector pva838克隆的。 PVA838用Hind III和CLA I消化，然后插入克隆向量PUC19。插入物的DNA序列显示出ERM的基本部分是App。设计了800 bp。然后设计了PCR引物来增加基本基因结束时的限制酶位点。较小（830 bp）耐红霉素的耐药盒是由PCR产生的。卡纳米霉素耐药基因（APHA）由链球菌TN 1545序列的均一序列（APHA）固定在1. tn kb中。揭示了APHA的开放式阅读框架是应用程序。 1000 bp。从APHA基因的序列，PCR引物也合成。较小（1070 bp）卡纳米霉素耐药盒由PCR.furthermore产生，四环素基因抗四环素的抗性基因是从TN 1545. tn 1545。（CA-GTase）。由pVA891产生了来自S.Mutans菌株MT8148和GS-5的染色体DNA基因库。将基因库随机转化为S.Mutans。抗红霉素的抗红霉素的抗性转化因子是从Mitis-Salivarius agar的筛查中筛选了CaR的均一表达，该二千群是由Ca-gtase的表达进行了测试。 CA-GTase的定位没有改变。