Cloning of the gene encoding glucosyltransferase from Streptococcus sobrinus

Research Project

Project/Area Number	63570871
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	Functional basic dentistry
Research Institution	Okayama University
Principal Investigator	FUKUI Kazuhiro Okayama University Dental School, Associate Professor, 歯学部, 助教授 (70034171)
Co-Investigator(Kenkyū-buntansha)	KOKEGUCHI Susumu Okayama University Dental School, Assistant, 歯学部, 助手 (10144776) OHTA Hiroyuki Okayama University Dental School, Assistant, 歯学部, 助手 (80168947) KODAMA Takao Okayama University Dental School, Associate Professor, 歯学部, 助教授 (30034200) KATO Keijiro Okayama University Dental School, Professor, 歯学部, 教授 (50028718)
Project Period (FY)	1988 – 1989
Project Status	Completed (Fiscal Year 1989)
Budget Amount *help	¥2,100,000 (Direct Cost: ¥2,100,000) Fiscal Year 1989: ¥600,000 (Direct Cost: ¥600,000) Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Keywords	Streptococcus sobrinus / glucosyltransferase gene / cloning / water-insoluble glucan / dextran binding domain / Streptococcus mutans / 不溶性グネカン合成酵素 / グルコシ-ルトランスフェレ-ス / クロ-ニング / ショ糖 / スクラ-ゼ / 不溶性グルカン合成酵素 / グルコシールトランスフェレース遺伝子のクローニング / 蔗糖 / スクラーゼ
Research Abstract	The gene encoding glucosyltransferase responsible for water-insoluble glucansynthesis (GTF-I) of streptococcus sobrinus(formerly Streptococcus mutans 6715) was cloned, expressed, and sequenced. A gene bank from S.sobrinus 6715 DNA was contructed in vector pUC18 and screened with anti-GTF-I antibody to detect clones producing GTF-I peptide. Five immunopositive clones were isolated, all of which produced peptides that bound alpha-1,6 glucan. GTF-I activity was found in only two large peptides : one stretching over the full length of the GTF-I peptide and composed of about 1,600 amino acid residues (AB1 clone) and the other lacking about 80 N-terminal residues and about 260 C-terminal residues (AB2 clone). A deletion study of the AB2 clone indicated that specific glucan binding, which is essential for water-insoluble glucan synthesis, was lost prior to sucrase activity with an increase in deletion from the 3'end of the GTF-I gene. These results suggest that the GTF-I peptide consists of three segments : that for sucrose splitting (-1,100 residues), that for glucan binding (-240 residues), and that unknown function (-260 residues), in order from the N terminus. The primary structure of GTF-I peptide, deduced by DNA sequencing of the AB1 clone, was found to be very similar to that of the homologus protein from another strain of S.sobrinus.