ISSN:
1617-4623
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Summary We have developed and tested a general method for the construction of tandem genetic duplications of defined regions of the Myxococcus xanthus chromosome, using transposon Tn5. A strain is constructed with a copy of Tn5-wt, coding for resistance to kanamycin, inserted at one end of the region to be duplicated, and a copy of Tn5-132, coding resistance to tetracycline, at the other end. The desired duplication forms by unequal recombination between the two transposons. The duplication is then isolated by transduction into a new strain by selecting for the copy of Tn5 at the novel joint. In order to test the method we constructed a duplication of the region between the previously described insertions Ω2224 and Ω925. This duplication was shown to have the correct restriction map in the vicinity of both copies of Tn5 and to produce antibiotic-sensitive segregants by recombinational loss of the Tn5 at the novel joint. Ω925 and Ω2224 are too far apart to cotransduce. Nevertheless, the duplication of the chromosomal segment between the two insertions can be transduced, as expected for a tandem duplication. To demonstrate the potential of constructed tandem duplications as genetic tools, three markers in the duplicated region were studied: cglB, a stimulatable A-motility gene, tgl, a stimulatable S-motility gene, and rif, the site of spontaneous rifampicin resistance mutations. Using the constructed tandem duplication, a cglB + cglB2 heterozygote was made. Since this strain was motile, cglB + is dominant. The cglB2 allele was mapped by measuring the ratio of non-motile to motile segregants obtained when the heterozygote lost the duplication and the results compared with transductional mapping of the same locus. tgl +tgl-1 heterozygotes could also be constructed, establishing the map order as Ω2224-tgl-Ω925. These heterozygotes were S-motile, indicating that tgl-1 is recessive. cglB2 rif +cglB +rif rdouble heterozygotes were also constructed. These were RfS, showing that rif + is dominant as in E. coli. Three-point analysis of segregants from the double heterozygote strains allowed determination of gene order. The order of genes in the rif region was determined to be Ω2224-cglB2-rif-tgl-1-Ω925.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00330897
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