Six IS<I>61200</I>-transposants, obtained by intermolecular transposition of IS<I>6100</I> into the 'right-hand' chromosome end of <I>S. lividans</I>, and carrying amplifications and deletions were examined to investigate transposition-induced genome alterations. For this, cosmid clones containing representative regions of the chromosome ends, from two individuals genomic cosmid libraries of <I>S. lividans</I> and <I>S. coelicolor</I> were used as Southern hybridization probes. The sizes of the chromosomal amplifications, produced as a result of transposition of IS<I>6100</I>, were found to vary from 65.5kb to 350 kb. The nature of the telomeric sequences was further investigated, indicating that to a large extent sequences from the chromosome ends were retained in the mutants. Low-level amplifications in the 'right-hand' end were mapped to a large region extending between the terminal inverted repeat and the AUD Type 1 locus, but no concomitant large deletions were found. In parallel, isolation, cloning, and sequencing the putative terminal-DNA from <I>S. avermitilis </I>was attempted. Preserving the covalently attached terminal protein at the 5' end of the linear chromosome of <I>S. avermitilis, </I>total DNA was isolated. Restriction fragments obtained from this preparation were used for cloning. Four candidate clones obtained independently more than once were examined to check if any of them carry the chromosome end. Comparison of the DNA sequences from the four clones with another seven terminal DNA sequences revealed no similarity. However one clone, D showed 95% homology to the <I>rho</I> gene of <I>S. lividans</I> ZX7, indicating that the <I>S. avermitilis</I> <I>rho</I> gene had been cloned. The analysis of an oligonucleotide probe designed from the consensus sequence present in seven other terminal sequences. The probe generated a signal with two controls, DNA from <I>S. lividans</I> 1326 and ZX1 strains, whereas no signal was detected with DNA from <I>S. avermitilis</I> suggesting that the terminal sequence of <I>S. avermitilis </I>is not similar to the consensus sequence.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:637187 |
| Date | January 1998 |
| Creators | Gunes, G. |
| Publisher | Swansea University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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