GENETIC INSTABILITY OF VACCINIA VIRUS CONTAINING ARTIFICIALLY DUPLICATED GENOME REGIONS

A double recombinant of vaccinia virus (W-lacZ/J-tk/F) was obtained, w hich contains two inverted copies of the virus tk gene, separated by 4 5 kb: (i) the native copy located in the HindIII J fragment of the vir us genome was inactivated due to insertion of E. coli lacZ gene; (ii) the second active copy was artificially inserted into the HindIII F fr agment. The virus expressing both thymidine kinase and P-galactosidase (tk(+)lac(+) phenotype) was cloned. Due to the presence of duplicated inverted sequences of the tk gene in the virus genome extensive recom bination was observed leading to genetic heterogeneity of the virus po pulation. The population consisted mainly of the virions with the tk()lac(-) (77%) and tk(+)lac(+) (23%) phenotypes. Passages in the presen ce of BUdR revealed minor fractions of the tk(-)lac(+) and tk(-)lac(-) phenotypes. Structural analysis of DNA isolated from virions confirme d the genetic heterogeneity of the virus population. Nine different Hi ndIII fragments were detected containing HindIII F, J and (or) lacZ se quences. The structure of these fragments indicates that predominantly two types of recombination events occur in the population: (i) transl ocation of the lacZ gene between duplicated sequences of the tk gene o r displacement of lacZ by tk via intergenome and intragenome double cr ossing over; (ii) inversion of a 45 kb sequence in the conserved regio n of the genome between duplicated sequences of the tk gene due to a i ntragenome single crossing over.