One-ended Transposition of Tn1/3

Nimmo, Elaine Robertson (1986) One-ended Transposition of Tn1/3. MSc(R) thesis, University of Glasgow.

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The plasmids pBR322 and pPAK100 each contain one half of a transposon. pBR322 contains the right half of Tn3 while pPAK100 contains the left half of Tn1. These half transposons were assayed for the ability to transpose and in both cases transposition was detected at frequencies about 1000 times lower than wild type. The same half transposons were found to confer transposition immunity to a plasmid, and the level of immunity was found to be the same whether the whole transposon or only half of it was present. A series of deletion mutants was constructed, using restriction enzymes and the exonuclease BAL 31. These were assayed for the ability to transpose and to confer immunity to the host plasmid. All the deletion mutants except one were able to transpose and to cause transposition immunity. The exception which did not transpose was sequenced and found to have lost most of its transposon sequences, including all of the inverted repeat. The others all still contained their inverted repeat sequences. One-ended transposition resulted in the formation of cointegrates between the donor plasmid and the recipient, R388. All experiments were performed in recA strains of E. coli and in the absence of resolvase, therefore no break down of cointegrates was possible and the transposition event was stopped at the cointegrate stage. Restriction enzyme analysis of these cointegrates revealed that the donor plasmid was inserted at many places into the recipient plasmid and the two plasmids were joined at the junction of the inverted repeat and plasmid sequences. All the cointegrates analysed had the transposon inserted in the same orientation, but the relatively small number analysed means that the possibility of finding inserts in the opposite orientation cannot be excluded. No duplication of transposon sequences was detected using restriction enzymes, however a duplication of about 100bp. or less would not have been detected using this system. Sequencing work on three examples done in this laboratory (A. Arthur, pers. comm. ) has shown that there has been a small duplication of the inverted repeat sequences. In the three examples studied the duplication was 13bp., 32bp. and 100bp. Sequencing was also done to discover whether there had been any duplication of target sequences. It was found that, as in "normal" transposition, a 5bp. duplication had occurred. The data infers that transposons having only one inverted repeat are able to transpose, and that transposition can occur using either of the inverted repeats. It is still unclear whether transposition occurs by a symmetric or asymmetric mechanism, although the evidence of Craigie and Mizuuchi, (1985), strongly suggests that at least in the case of Mu, transposition occurs in a symmetric fashion.

Item Type: Thesis (MSc(R))
Qualification Level: Masters
Keywords: Genetics
Date of Award: 1986
Depositing User: Enlighten Team
Unique ID: glathesis:1986-76584
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:06
Last Modified: 19 Nov 2019 14:06

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