Genetic instability of vaccinia virus containing artificially duplicated genome regions

A double recombinant of vaccinia virus (W-lacZ/J-tk/F) was obtained, which contains two inverted copies of the virus tk gene, separated by 45 kb: (i) the native copy located in the HindIII J fragment of the virus genome was inactivated due to insertion of E. coli lacZ gene; (ii) the second active copy was artificially inserted into the HindIII F fragment. The virus expressing both thymidine kinase and beta-galactosidase (tk+lac+ phenotype) was cloned. Due to the presence of duplicated inverted sequences of the tk gene in the virus genome extensive recombination was observed leading to genetic heterogeneity of the virus population. The population consisted mainly of the virions with the tk+lac- (77%) and tk+lac+ (23%) phenotypes. Passages in the presence of BUdR revealed minor fractions of the tk-lac+ and tk-lac- phenotypes. Structural analysis of DNA isolated from virions confirmed the genetic heterogeneity of the virus population. Nine different HindIII fragments were detected containing HindIII F, J and (or) lacZ sequences. The structure of these fragments indicates that predominantly two types of recombination events occur in the population: (i) translocation of the lacZ gene between duplicated sequences of the tk gene or displacement of lacZ by tk via intergenome and intragenome double crossing over; (ii) inversion of a 45 kb sequence in the conserved region of the genome between duplicated sequences of the tk gene due to a intragenome single crossing over.