A genetic investigation of the mechanism of adenovirus marker rescue

Abstract

We have developed quantitative and segregational methods for investigating the mechanism of genetic exchange in adenovirus marker rescue. Estimates of “marker rescue frequency” (m.r.f.) were used to show that marker rescue increases linearly with increasing dose of fragment up to equimolarity with the full-length genome. The m.r.f. is also affected by the size of the rescuing fragment and the position of the wild-type allele within it, regardless of whether the fragment is terminal or internal. This is compatible with marker rescue being based on homologous exchange between the recombining partners. Examination of individually transfected cells showed that there is very wide variability in the values of the m.r.f.'s. This suggests that marker transfer can occur after replication of the full-length genome has begun, and can occur late into the infectious cycle. Unselected markers on the rescuing fragment were shown to be co-inherited frequently. This suggests that physical linkage is accompanied by genetic linkage. To examine this more closely, a multifactorial marker rescue was performed. The data show unequivocally that markers resident on the same fragment as the selected allele are inherited at high frequency, with a gradient of transfer in which markers closest to the selected marker are transferred most frequently. Markers up to 13 and perhaps as many as 17 kb apart can be inherited together. There are very few examples of the inheritance of distal markers in the absence of proximal ones. These data suggest that large pieces of DNA are transferred in a concerted reaction during marker rescue.