Studies on the transposition of IS1 in Escherichia coli with two plasmid systems

• Main Authors: Hwang, Jiing-Luen, 黃景倫
• Other Authors: Chen, Jiann-Hwa
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/28248067402191739128

碩士 === 國立中興大學 === 分子生物學研究所 === 86 === 　　Two plasmid systems were used successfully for entrapping and analyzing IS1 plasmid insertion mutants in E. coli. Plasmid pS177 carries the sucrose-sensitive sacR/B genes and cells harboring the plasmid are lethal on 5% sucrose plates. Plasmid DNAs of the survivor colonies were extracted and screend for insertions of IS1 by restriction mapping and Southern hybridization. Ninety-one IS1 plasmid insertion mutants were collected and their insertion junction sequences determined. The results indicated that there were total of 24 insertions sistes for the 91 plasmid IS1 insertion mutants. The insertions were distributed fairly random in the target sacR/B genes. 　　Plasmids pBLT and pBLA were originally constructed by cloning the immunity region of bacteriophage lambda into the EcoRI site of pBR322 in both orientations. They carry the temperature-sensitive cI857 gene and part of the lambda left operon containing the left promoter/operator, N gene, tL1, and ral, cIII, ssb and kil genes. Cells harboring the plasmid are lethal at 40℃ because that the temperature-sensitive cI857 repressor is non-functional at 40℃ and the Kil protein is expressed. Plasmid DNAs of the six-hundred-and-ninety 40℃ survivor colonies were extracted and again screened for insertions of IS1 by restriction mapping and Southern hybridization. The restriction mapping results indicated that of the 323 IS1 insertions isolated, 226 had insertions in the N gene, 6 in the ssb gene and 91 in the kil gene. Nineteen IS1 insertion mutant plasmids, of which 5 had insertions in the N gene, 6 had insertions in the ssb gene and 8 had insertions in the kil gene, were randomly picked for insertion junction sequence determination. and the results confirmed the mapping results. Insertions in the N or kil gene were detected in both orientations at about equal frequency; but insertions in the ssb gene were detected only in one orientation in which transcription of the IS1 transposase is in the same direction as the oL/pL transcription. Several pBLT derivatives were constructed in which a copy of IS1 was cloned either in the ssb gene but in the reverse orientation, or in the region between the kil and the ssb genes in both orientations. Cells harboring these pBLT derivatives were plated out at both 40℃ and 30℃ and their ratios of cfu at 30℃were measured. The results showed that those cells were unable to grow at 40℃unless deletions had occurred in the containing plasmids so that the left promoters were deleted. This implied that the insertion of IS1 in one orientation, but not in the other orientation, generated a transcriptional terminator to block expression of the downstream kil gene, and that SSB and possibly N proteins were capable of antiterminating the termination. There were many IS1-mediated pBLT deletion mutants generated during the course of the study. Twenty such mutants were randomly chosen for restriction mapping of the deletion endpoints. While their one endpoints were located in the region between oL/pL and kil in several positions, their other endpoints, with one exception, were all located in the vicinity of the reported pBR332 IS1 insertion hotspot.