| dc.description | The viomycin-resistance derivative, Tn4560, of S. fradiae transposon Tn4556 can insert at various sites in Streptomyces chromosomes. To introduce Tn4560 into S. venezuelae (a producer of chloramphenicol) in which linkage can be easily analysed by generalized transduction, a Cys$\sp-$ strain of S. lividans in which Tn4560 had transposed from plasmid pUC1169 into the host chromosome was first isolated. In that strain Tn4560 transposed from the chromosome into a high-copy-number temperature-sensitive plasmid pGM160 and a low copy-number plasmid pDQ101. The Tn4560-containing plasmid pDQ201, derived from pDQ101, was transferred into S. venezuelae by transformation to generate the Vio$\sp{\rm R}$Thio$\sp{\rm R}$ strain, LG206. In LG206, pDQ201 was unstable at 43$\sp\circ$C. About 40,000 colonies were screened for the Thio$\sp{\rm S}$ phenotype after growth at 43$\sp\circ$C in the presence of viomycin. Southern hybridization analysis of genomic DNA fragments of 14 Vio$\sp{\rm R}$ Thio$\sp{\rm S}$ isolates showed that Tn4560 had transposed to at least four different chromosomal sites. Conjugation mapping located $\Omega$-1 between hisA6 and pdx-4, and $\Omega$-3 between pdx-4 and lysA; SV1-mediated generalized transduction located $\Omega$-2 very close to pdx-4, and $\Omega$-4 within the chloramphenicol biosynthesis (cml) gene cluster. A junction fragment containing the $\Omega$-4 insertion site was cloned for future use as a probe to identify chloramphenicol biosynthesis genes. To demonstrate transposition into a previously mapped gene in S. venezuelae, pDQ201 was transferred into a homoserine-sensitive thrC mutant and plasmids were subsequently eliminated at 43$\sp\circ$C. Selection for homoserine resistance gave thrB thrC double mutants. Cotransduction of vph, $thr\sp-$ and $lysA\sp+$ in a lysA recipient confirmed the location of Tn4560 ($\Omega$-5) in or very close to the thrB gene. | en_US |
