The nucleotide sequence of IS402, from Pseudomonas cepacia, was determined. This transposable element was isolated previously from P. cepacia on the basis of its ability to increase expression of the Tn1 bla gene on plasmid pRP1. The IS402-containing region of pTGL52 (pRP1::IS402) was cloned into the Escherichia coli vector pBLUEKSP. Recombinant derivatives of pBLUEKSP carrying different regions of IS402 were constructed and were utilized for dideoxynucleotide sequence analysis of the element and flanking Tn1 DNA. IS402 (914-bp) had 17-bp terminal inverted repeat sequences with one mismatch, and three major open-reading-frames (ORFs) of 633, 441, and 264 nucleotides. It inserted into Tn1 170-bp upstream of its bla gene causing the direct duplication of 3-bp of target DNA. Comparison of IS402 with sequences in the GenBank and EMBL databases indicated that is was not closely related to previously described IS-elements. However the predicted amino acid sequence of the 441-nt ORF of IS402 exhibited 54% similarity with the putative transposase of IS427 from Agrobacterium tumefaciens. An effort was made to construct marked derivatives of IS402 suitable for testing whether its transposition might be triggered in response to nutrient starvation. For this purpose I cloned the fol gene of plasmid pR388 which confers resistance to trimethoprim (Tp) into the DraII site of IS402 adjacent to IR-L. The resulting element, IS402.2, was cloned into pTGL133, a broad-host-range plasmid which confers resistance to tetracycline and is temperature-sensitive with respect to its replication. The resulting plasmid, pTGL154, was transferred into P. cepacia and the transconjugants were propagated at 47$\sp\circ$C to isolate Tp$\sp{\rm R}$ transposants carrying IS402.2 in their chromosomes. Comparison of the extent of transposition of IS402.2 and of Tn5-751, a derivative of transposon Tn5 carrying a Tp$\sp{\rm R}$ marker, indicated that IS402.2 transposed at a relatively high rate. The orientation of the fol gene of IS402.2 was the same as the major ORFs of IS402 raising the possibility that the high frequency of transposition was due to increased transposase production caused by read-through transcription from the fol gene promoter. The high frequency of transposition of IS402.2 under normal conditions of growth makes it unsuitable for analysis of the influence of nutrient starvation on transposition. However, the results demonstrate the feasibility of constructing marked elements which may be suitable for this purpose.
| Identifer | oai:union.ndltd.org:UMASS/oai:scholarworks.umass.edu:dissertations-8381 |
| Date | 01 January 1992 |
| Creators | Ferrante, Anthony Adrian |
| Publisher | ScholarWorks@UMass Amherst |
| Source Sets | University of Massachusetts, Amherst |
| Language | English |
| Detected Language | English |
| Type | text |
| Source | Doctoral Dissertations Available from Proquest |
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