Structural analysis of the movable genetic element that regulates G6PD gene expression and its transposition mechanism.

Research Project

Project/Area Number	61540458
Research Category	Grant-in-Aid for General Scientific Research (C)
Allocation Type	Single-year Grants
Research Field	遺伝学
Research Institution	HOKKAIDO UNIVERSITY
Principal Investigator	HORI Hiroshi HOKKAIDO UNIVERSITY, PROFESSOR fac. of sci., 理学部, 教授 (40000814)
Co-Investigator(Kenkyū-buntansha)	KIMURA Masahito HOKKAIDO UNIVERSITY, ASSOCIATE Fac. of Sci., 理学部, 助手 (30091440)
Project Period (FY)	1986 – 1987
Project Status	Completed (Fiscal Year 1987)
Budget Amount *help	¥1,400,000 (Direct Cost: ¥1,400,000) Fiscal Year 1987: ¥300,000 (Direct Cost: ¥300,000) Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
Keywords	Drosophila melanogaster / Movable genetic element / Gene expression / G6PD / P因子
Research Abstract	Our previous studies have suggested that a major genetic factor affecting G6PD gene expression in Drosophila melanogaster may be a movable genetic element which can insert in front of the G6PD coding region, but is easily excised therefrom upon outcrossing. In order to obtain physical evidence for this notion, we have first analyzed the DNA structure around the G6PD coding region by Southern blot using a cloned G6PD gene as a probe. As a result, two sorts of insertion sequences were found; one (IS1) in front of exon I, and the other within the intron (IS2), in three mutant strains which show overproduction of G6PD protein. Similar analyses with revertants derived the high G6PD-activity mutants showed that IS1 sequence was partially or totally excised out of the revertant genome with the IS2 sequence intact. It was thus demonstrated that it would be the IS1 sequence that is responsible for positive regulation of G6PD gene expression. We have next attempted to clone the IS1 sequence. Sinc … More e the sequence is highly unstable, it was very hard to clone it in its perfect form, but we finally succeeded to obtain a single clone among a million of plaques which carries the IS1 sequence. Sequencing data showed that the IS sequence is composed of four defective P elements tandemly arranged in such a way: P_L-P_<cL>-P_<cR>--P_R. The host repeats of P_L andP_<cL> are CTTGGGCG and those of P_<cR> and P_R are CTGCGGCG. P_L and P_R are both devoid of the sequence, nos.809-2560, and the 32nd base A is replaced by G in P_L and Tin P_R, respectively P_<cL> and P_<cR> are both devoid of the sequence, nos.206-2504, and the 32nd base is replaced by G. There is a connecting host sequence between P_<cL> and P_<cR>;TTTGTTTG.The sites of insertion are before this TTTGTTTG in the case of P_L and P_<cL> and after the TTTGTTTG in the case of P_<cR> and P_R. In the wild-type genome, there is a GC-rich sequence between the TTTGTTTG and the transcription start site which is highly homologous to the consensus sequence in front of the start site in several vertebrate house-keeping genes. We concluded therefore that the IS1 has inserted into the promoter region, thus affection G6PD gene expression. Less