Expression of Tn1/3 Transposase

Hettle, Simon John Herald (1985) Expression of Tn1/3 Transposase. PhD thesis, University of Glasgow.

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It has previously been shown that the only sequences near the termini of Tn1/3 essential for the first step of the transposition process of these elements are the two 38bp. perfect inverted repeats at the extreme ends of the elements. This finding has been confirmed and it has also been shown that both of these sequences are equally good substrates for transposition. Also, it was shown that Tn1/3 transposition normally acts to replicate the DNA sequences that correspond to the entire transposon sequences. Further indirect evidence for the binding of Tn1 transposase to the inverted repeat sequences of Tn1/3 was obtained, supporting the hypothesis that it is to and upon these sequences that transposase binds and acts. The location of the HincII site present in Tn1 but absent in Tn3 has , also been determined by DNA sequencing and found to be at a position in Tn1 equivalent to co-ordinates 3037-3042 in Tn3, with cleavage occurring between base pairs 3039 and 3040. Thus, this site lies just within the tnpA gene coding region, near its N-terminal end. The sequence of 114bp. of Tn1 lying to the left of this site (i.e. extending towards the C-terminus of the tnpA gene) was determined and was found to have 97/114 bp. (=85 1%) homology at the DNA level and 37/38 residue (=97.4%) homology at the amino acid level with Tn3. This confirmed the prediction of a high degree of homology between the two elements, which had been made on the basis of their very similar restriction profiles. Using this information, a plamid (pSN015) was constructed that produced a fusion protein consisting of the whole Tn1 transposase protein with its three N-terminal amino acid residues removed and replaced by the first eleven N-terminal amino acid residues of B-galactosidase. As judged by the analysis of plasmid encoded proteins expressed in E. coli K-12 minicells, pSN015 did produce more protein than the derepressed Tn1 derivative Tn103 carried on a plasmid (pMB9) with a copy number c. 1/4 of that of pSN015. Yet the transposition of Tn3651 mediated in trans by each of these plasmids occurred at very similar frequencies at both 30

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Genetics
Date of Award: 1985
Depositing User: Enlighten Team
Unique ID: glathesis:1985-76572
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:07
Last Modified: 19 Nov 2019 14:07

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