Recombinant plasmids carrying promoters, genes and the origin of DNA replication of the early region of bacteriophage T7

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.