Figure 2
Confirmation of gene disruption in MG_207 by Southern and immunoblot analyses. A. Southern analysis of M. genitalium DNA from wild type G37 and TIM207 strains. Membranes were probed with radiolabeled MG_207 and gentamicin gene sequences. G37 and TIM207 represent M. genitalium wild type and MG_207 mutant strains. Sizes of DNA fragments are indicated in kilo bases (kb). B. Immunoblot analysis of wild type G37 and TIM207 strains. SDS-PAGE separated proteins were transferred to nitrocellulose membrane and probed with anti-His10MG207 rabbit antiserum (1:500). After treating with peroxidase labeled second antibody (1:10,000 dilution), blots were developed with chemiluminiscent method (ECL) and the signals autoradiographed. G37 and TIM207 represent M. genitalium wild type and MG_207 mutant strains, respectively. The size (kDa) of the marker protein is given on the left. C. Schematics showing the organization of MG_207 in the genome of M. genitalium. I. Organization of genes around MG_207. Arrows represent genes and their direction of transcriptions. Numbers above the arrows indicate the assigned number of each gene. II. Restriction sites around MG_207 gene. Open boxes represent regions adjacent to MG_207: Black box represents the gene MG_207. Arrow within the black box indicates the direction of transcription of MG_207. SpeI indicates the locations of SpeI restriction site around MG_207. TIS indicates the site of transposon insertion.