Cloning of bacteriophage T5 promoters.

Abstract

Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonucleases PstI and HindIII and the resulting fragments were inserted into the plasmid pBR322. Selection of transformants for Aps-Tcr-phenotype made it possible to screen the hybrid plasmids that contained promoter sequences in the cloned fragments. Two PstI/HindIII fragments, 720 bp (51% of the T5 DNA length) and 1,200 bp (70%) were cloned in this study. Tcr levels for these plasmids were as high as 18 micrograms/ml and 75 micrograms/ml, respectively. The presence of Escherichia coli RNA polymerase binding sites on both fragments was shown using the nitrocellulose filter assay. These binding sites are situated between 35 bp and 95 bp from the HindIII cleavage site on the 1,200 bp fragment; and within 420 bp from the HindIII site on the 720 bp fragment.