Nucleotide sequence analysis of the junctions of the terminal inverted repeats of the Streptomyces lividans linear chromosome

Abstract

The junctions of the Streptomyces lividans chromosomal terminal inverted repeats (TIRs) were isolated from cosmid clones as 6.2 kb PstI and 2.9 kb BamHI fragments, respectively. The fragments were completely sequenced. In each of the fragments just one open reading frame could be identified. One putative gene product showed significant similarities to a sensor and the other to a transcriptional regulator protein of prokaryotic two-component signalling systems. Next to one TIR numerous long direct repeats were found within a region of about 400 bp.

Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans

“…Loss of one or both chromosomal ends, by transposition, other illegitimate or homologous recombination events (using for example multiple copies of IS elements or small direct repeats like those observed at the end of one TIR; Volff et al 1997) may lead to circularized or inverted duplicated chromosomes which, at high frequency, undergo deletions and amplifications by overreplication of DNA due to the lack of replication terminator sequences. Studies on DNA replication and termination should provide further evidence for this model.…”

“…This temperature-sensitive vector carries the minimal replicon and kanamycin resistance gene (Kan r ) isolated from the Streptomyces plasmid pGM11 (Muth et al 1989) as BamHI-HindIII fragment and inserted into the BamHI/ HindIII-cut E. coli vector pUC19 (Yaniseh-Perron et al 1984). A 4 kb BglII fragment from pJOE2527 (see accompaning paper, Volff et al 1997) was inserted into the BglII site of pJOE915, which was constructed by inserting a 1.05 kb BclI fragment bearing the thiostrepton resistance gene tsr (Thompson et al 1980) into the EcoRV site of pIC20H (Volff et al 1996). A 2.8 kb EcoRI-BamHI fragment from pJOE2518 (accompaning paper; Volff et al) was inserted on the other side of tsr and the tsr gene together with the two TIR junction fragments was then inserted as a HindIII fragment into pJOE1082 to give pJOE2701.…”

Genetic instability associated with insertion of IS6100 into one end of the Streptomyces lividans chromosome

“…3). This amplification overlaps the junction between the TIR and flanking unique sequence DNA, which, from sequence analysis, contains numerous long direct repeats (Volff & Altenbuchner, 1997). The other amplification end point was concluded to lie in the region spanned by cosmid 66, which detected novel amplified and single-copy fragments of 25 kb and 13 kb, respectively, in addition to several wild-type single-copy fragments.…”

“…Due to the duplication of TIR sequences, the question of whether both TIRs are present is complicated. To investigate this further in the IS6100-amplified mutants, we looked for the presence of a unique 4n5 kb XhoI fragment which overlaps the innermost portion of the TIR in question Volff & Altenbuchner, 1997). XhoI chromosomal digests were probed with cosmid 68\7, which contains a region overlapping the TIR together with adjacent unique sequences.…”

Influence of disruption of the recA gene on genetic instability and genome rearrangement in Streptomyces lividans

“…The chloramphenicol resistance gene cmlR was amplified by PCR (16) from the genomic DNA of strain TK64 by using primers derived from the sequence previously published (4). Plasmid pJOE2424 reveals the junctions of the terminal inverted repeats of the chromosome (25). The absence of a line or an arrow indicates the presumed deletion.…”