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T4 polynucleotide kinase; cloning of the gene (pseT) and amplification of its product.
Author(s) -
Midgley C.A.,
Murray N.E.
Publication year - 1985
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1985.tb03989.x
Subject(s) - biology , gene , microbiology and biotechnology , phosphatase , nucleic acid sequence , polynucleotide , genetics , biochemistry , enzyme
The T4 gene (pseT) for polynucleotide kinase (pnk) has been cloned in lambda. Induction of a lambda E‐W‐S‐cI857 prophage in which the pseT gene can be transcribed from the late lambda promoter, PR1, leads to greater than 100‐fold amplification of pnk activity; pnk comprises approximately 7% of the total soluble cell protein. The purified enzyme, as expected, is both a 5′‐kinase and a 3′‐phosphatase. The amino acid sequence deduced from an open reading frame identified as the pseT gene contains a sequence which corresponds particularly well with that part of the adenine nucleotide binding site of adenylate kinase shown to form a flexible loop. A deletion mutant that lacks 5′‐kinase activity, and possibly also 3′‐phosphatase activity, has lost two amino acids from within the proposed loop structure. A second region of the pnk sequence shares homology with phosphoglycerate kinase, yeast inorganic pyrophosphatase and histone 2b from various organisms.