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。

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09672104/
• 关键词: Streptococcus 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

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.

在变形链球菌中起作用的红霉素抗性基因（erm）是从链球菌-大肠杆菌穿梭载体pVA838中克隆的。 pVA838用Hind III和Cla I双酶切，然后插入克隆载体pUC19中。插入片段的DNA序列显示，erm的主要部分是app。 800 bp。然后设计PCR引物，在必需基因末端添加限制性酶切位点。通过PCR生成更小的（830 bp）红霉素抗性盒。通过类似的程序从链球菌转座子Tn 1545克隆卡那霉素抗性基因（aphA）。含有aphA的1.6 kb插入片段的DNA序列 揭示了 aphA 的开放阅读框是 app。 1000 bp。根据aphA基因序列，还合成了PCR引物。通过PCR产生了较小的（1070 bp）卡那霉素抗性盒。此外，从Tn 1545克隆了四环素抗性基因。另一方面，进行随机诱变以研究细胞相关葡萄糖基转移酶的定位机制 (CA-GTase)。用pVA891生成S.mutans菌株MT8148和GS-5的染色体DNA基因库。将基因库随机转化到S.mutans中。从Mitis-Salivarius琼脂中筛选红霉素抗性转化子。然后通过Western blot分析检测CA-GTase的表达。虽然测试了1000多个突变体，但 CA-GTase 的定位没有改变。