Biogenesis of vaccinia: analysis by three-factor crosses reveals mutual influence on stability of drug resistance and temperature sensitivity when both markers occur in some recombinant virus isolates.
Recombination analysis with 5 temperature-sensitive (ts) mutants of vaccinia virus, relegated to the E category phenotype and mimicking closely the effects produced by the antibiotic rifampicin, was undertaken to determine whether the genetic loci determining this phenotype were clustered on the genetic map. Optimum conditions for analysis were established with respect to the MOI and duration of incubation. At an MOI of 5-10, consistent values of recombination frequency (RF) were obtained, but became significantly lower when the MOI was 1 or less. Constant RF values were recorded when the duration of the infection was 8-24 h. These values were increased 3-fold by extending the incubation to 36 h. Under the standard conditions for analysis adopted, each mutant partner was added at an MOI of 10 and incubation was for 18 h. The map of group E mutants drawn from RF values obtained by 2-factor crosses was confirmed by 3-factor cross analysis, in which recombinants carried both the ts mutations and the rifampicin-resistance (R) marker. Both 2- and 3-factor cross data revealed that the 5 group E mutations were not clustered on the vaccinia virus genome. Surprisingly, after recombining ts 7743 with rifampicin resistance (R), different isolates of ts 7743 R could be mapped at two loci to the right or left of another isolate, ts 9383. Characterization of five independent plaque isolates of the progeny arising from a ts 7743 R parent showed that one isolate possessed a cold-sensitive phenotype (33 degrees/40 degrees = 1:26), two isolates retained their original ts character, and two became phenotypically like wild type (wt) with respect to thermosensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)
D004279:DNA, Viral / Q000302:isolation & purification; D004351:Drug Resistance; D010641:Phenotype; D011995:Recombination, Genetic; D013696:Temperature; D014616:Vaccinia virus / Q000235:genetics*
D004279:DNA, Viral