Escherichia coli hydride prodn. with enhanced mutation rates - by incorporation of a palindromic DNA sequence into plasmid or bacteriophage DNA

摘要

In the prodn. of Escherichia coli mutants in which plasmid or bacteriophage DNA is produced contg. a transposone, (A) (i) the non-symmetrical region of the tranposone is completely exercised from the resulting tranposone-contg. DNA by means of a restriction endonuclease and (ii) the transposone ends of the resulting DNA are linked with a ligase and DNA is produced having a mirror-image symmetrical region, or (B) (i) by means of one or more restriction endonucleases, the non-symmetrical region of the transposone is completely excised from the transposone-contg. DNA and, in addition, either one DNA fragment with one of the two transposone ends is excised, the other transposone and remaining on the shortened plasmid or bacteriophage DNA, or two DNA fragments each comprising one of the transposone ends are excised and (ii) the resulting DNA fragment(s) comprising transposone ends are linked with a plasmid or bacteriophage DNA having two transposone ends with the aid of one or more ligases to produce DNA having a mirror-image symmetrical region (the plasmid or bacteriophage DNA with transposone ends having been produced by the procedure of step B(i); (C) the resulting DNA is transformed or transduced into Escherichia coli to form E.coli hybrids; and (D) the E. coli mutants formed as a consequence of the unstable DNA mirror-image symmetry in the E.coli hybrids are cultivated and selectively tested, individual mutants opt. being isolated. The procedure gives enhanced mutation rates (increasing the probability of obtaining useful mutants) because not only the mirror-image symmetrical region of the DNA but also neighbouring regions are rendered unstable by the mirror-image symmetry.