8 Supplement

Abstract

During the last year a number of new techniques and approaches have begun to be applied to the genetic analysis of adenoviruses, and although still in their infancy, these probably indicate important future directions within the field. Of prime importance has been the utilization of recombinant DNA technologies. Many laboratories have cloned defined segments of the adenovirus genome in plasmid vectors, and these have served as convenient substrates for mutagenesis. The effects of various mutations can be studied either following their transfer back into viral genomes or using in vivo or in vitro assays that allow the expression of viral genes contained within plasmids to be monitored.

Stow (1981) used a GC-tailing procedure to clone the HpaI E fragment from the left end of the adenovirus-2 genome (0–4.5 map units) into the PstI site of pBR322. Sequences between 0 and 3.8 map units were transferred back into progeny virus by excising this fragment from the plasmid, ligating it to a fragment spanning from 3.8 to 100 map units (isolated from the virion DNA of the adenovirus-5 variant dl309) (Jones and Shenk 1979), and transfecting tissue-culture cells with the resulting unit-length molecules. Because the cloned fragment retained its biological activity, it was possible to begin introducing mutations within it. Small deletions were introduced at position 2.8 by linearizing the plasmid with XmaI and then treating it sequentially with nuclease S1 and DNA ligase; these sequences were transferred to progeny virus as before. The resulting mutants were unable to replicate in...