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AssigneeUniversity of Maryland
InventorsDaniel C. Stein, Andrejez S. Piekarowicz, Robert T. Yuan
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E. coli bacterial strains encoding a restriction gene that degrades methylated DNA, and prevents cloning of genes expressing the methyltransferase responsible for methylation, are mutated by a chemical or physical mutagen, so as to make the restriction enzyme temperature sensitive. Mutant cells are rendered competent, and plasmids expected or known to contain genes encoding methyltransferase enzymes are introduced. The transformants grow at the permissive temperature, where the restriction enzyme system is inactivated due to the mutated gene. Successful clones, expressing a methyltransferase, can be quickly identified by those which grow at the permissive temperature, but not at the non-permissive temperature. The valuable methyltransferases, as well as restriction enzymes associated therewith, can accordingly be recovered in large quantity.
A method for detecting E. coli bacterial cells containing a cloned methyltransferase gene, said cells expressing a methyltransferase enzyme, comprising:
A method for producing methyltransferase, comprising:
A method of producing mutant, temperature sensitive E. coli bacteria capable of expressing a methyltransferase enzyme, comprising:
An E. coli novel bacterial cell, having a genome comprising a plasmid which encodes a methyltransferase, said genome further comprising a gene encoding a temperature sensitive restriction enzyme reactive with methylated DNA produced by said methyltransferase, the restriction enzyme being active at a first, non-permissive temperature range, and inactive at a second, permissive temperature range, wherein-said bacterial cell can grow at said permissive temperature range, but not at said non-permissive temperature range.
Dependencies are inferred from the claim text.