Cloning of bacteriophage T5 promoters |
| |
Authors: | Vladimir N Ksenzenko Tatjana P Kamynina Nina M Pustoshilova Valentine M Kryukov and A A Bayev |
| |
Institution: | (1) Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, 142292 Pushchino, Moscow region, USSR |
| |
Abstract: | Summary Bacteriophage T5 was subjected to combined hydrolysis with the restriction endonuclease 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 fragment, 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 g/ml and 75 g/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.Abbreviations Ap
ampicillin
- Tc
tetracycline
- bp
base pairs
- NTPs
nucleoside triphosphates
- PBB
polymerase binding buffer |
| |
Keywords: | |
本文献已被 SpringerLink 等数据库收录! |
|