Cloning, overexpression and nucleotide sequence of a thermostable DNA ligase-encoding gene.
Thermostable DNA ligase has been harnessed for the detection of single-base genetic diseases using the ligase chain reaction [Barany, Proc. Natl. Acad. Sci. USA 88 (1991) 189-193]. The Thermus thermophilus (Tth) DNA ligase-encoding gene (ligT) was cloned in Escherichia coli by genetic complementation of a ligts 7 defect in an E. coli host. Nucleotide sequence analysis of the gene revealed a single chain of 676 amino acid residues with 47% identity to the E. coli ligase. Under phoA promoter control, Tth ligase was overproduced to greater than 10% of E. coli cellular proteins. Adenylated and deadenylated forms of the purified enzyme were distinguished by apparent molecular weights of 81 kDa and 78 kDa, respectively, after separation via sodium dodecyl sulfate-polyacrylamide-gel electrophoresis.
Barany
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Department of Microbiology, Cornell University Medical College, New York, NY 10021.
Gelfand
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D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D011088:DNA Ligases / Q000235:genetics*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics*
D003062:Codon; D011088:DNA Ligases