| Summary: | 博士 === 國立陽明大學 === 微生物及免疫學研究所 === 94 === Abstract
Insertion sequence IS629, initially isolated from the chromosome of Shigella sonnei, is a member of the IS3 family of bacterial transposable elements. IS629 is 1,310 base pairs long with a pair of 25-bp imperfect inverted repeats at its termini. Two partially overlapping open reading frames, orfA and orfB, are present in IS629, and two putative translational frameshift signals, T4G and A4T, are located near the 3’ end of orfA. With the lacZ gene as the reporter, both T4G and A4T motifs are determined to be a -1 frameshift signal. To verify that the -1 frameshifting occurred at these two motifs, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) approach is developed. The approach consists of three steps: (1) LC-MS/MS analysis of the protein digests, (2) primary data analysis using the known mRNA sequence, and (3) advanced data analysis using a new database containing distinct mRNA sequences with single insertion at particular positions. Two peptides representing the two transframe products designated OrfAB’ and OrfAB, are identified by the LC-MS/MS approach. Results of transposition assays show that OrfAB’ is the transposase and that OrfAB aids in the transposition of IS629. Pulse-chase experiments and E. coli two-hybrid assays demonstrate that OrfAB binds to and stabilizes OrfAB’, thus increasing the transposition activity of IS629. This is the first transposable element in the IS3 family shown to have two functional frameshifted products involved in transposition and to use a transframe product to regulate transposition.
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