ABSTRACT
Two full-length contiguous HpaI fragments of the 0 to 18.2% region of T7 H DNA (HpF-H and HpG) were inserted into plasmids pHV14 or pC194 using oligo(dG . dC) connectors or synthetic HindIII adaptors. Amplification of the two early T7 fragments was achieved by transforming lysostaphin-treated S. aureus W57 with the hybrid plasmids. Experimental evidence is presented suggesting that neither of these T7 segments can be cloned in an intact form in E. coli. One of the hybrids, pHV14-HpF-H, proved to be unstable even in B. subtilis 168. The supercoiled recombinant plasmids were tested for their capacity to support RNA synthesis by purified E. coli or T7 RNA polymerases and to serve as templates in a cell-free T7 DNA replication system. The results of these in vitro studies indicate the presence of active "early" promoters in the cloned fragment HpF-H and active "late" promoters, as well as a functional origin of replication in the cloned fragment HpG.
Subject(s)
DNA Replication , DNA, Recombinant , DNA, Viral/genetics , Genes, Viral , T-Phages/genetics , Cloning, Molecular , DNA, Circular/genetics , DNA, Superhelical/genetics , Operon , Plasmids , Transcription, GeneticABSTRACT
A defective genotype of herpes simplex virus strain Angelotti (HSV ANG) accumulates in the course of controlled serial high MOI virus passages representing 50-60% of the total number of mature virions in the seventh of these passages. Defective HSV ANG significantly differs from other defective HSV genotypes described so far: the DNA of the defective particles has the same buoyant density as nondefective DNA. In contrast to non-defective HSV ANG DNA, it is not attacked by the restriction endonucleases Eco R I, Hpa I and Hind III. Defective virions strongly suppress the formation of progeny virus. They do not interfere, however, with the synthesis of HSV-specified thymidine (TdR) kinase.
