Insertional mutagenesis using a temperature-sensitive replicon.

<p>A. Northern blot analysis. Total RNAs were extracted from <i>S. coelicolor</i> strains with marker only, wild type <i>ssrA</i> or <i>ssrA-DD</i> inserted at the <i>attB</i> locus and analysed by Northern blot using a probe that does not distinguish between wild type and mutant tmRNA. Ethidium bromide stained ribosomal RNA (rRNA) is shown as a loading control. B. Schematic diagrams illustrating the anticipated genomic maps of the <i>ssrA</i> locus, the <i>attB</i> locus following integration of <i>ssrA</i> or <i>ssrA-DD</i>, and the <i>ssrA</i> locus following correct integration of the HygR cassette. The expected sizes of <i>Nco</i>I restriction fragments are indicated in each case. Lower case letters to the right of each fragment indicates fragment labeled in panel C. C. Southern blot analysis of <i>Nco</i>I-digested genomic DNA isolated from “positive” strains that were resistant to hygromycin and sensitive to thiostrepton. Ten isolates from each group were analyzed, five of which are illustrated. The top Southern blot was probed with an <i>ssrA</i> probe while the bottom blot was hybridized with a probe for the HygR cassette after the same blot was stripped. Control lane (Con) is genomic DNA from an untransformed wild type strain. Arrows and letters indicate genomic fragments illustrated in Panel B. D. Two of the ten positives from the <i>ssrA</i>/- group with an unusual Southern blot result (one of these is indicated by asterisk in panel B) were further analyzed by genomic PCR of the <i>ssrA</i> region with genomic DNA template from wild type cells used as a control. An ethidium bromide-stained agarose gel is illustrated.