Saturation mutagenesis of the inside end of insertion sequence IS50

Gene. 1989 Dec 21;85(1):75-81. doi: 10.1016/0378-1119(89)90466-6.

Abstract

A 19-bp segment at the inside (I) end of IS50 (Tn5) is needed for efficient transposition. The importance of each position was assayed by making at least one base substitution at each position by either chemical-or oligodeoxyribonucleotide-directed mutagenesis. Mutant I ends were paired with a wild-type (wt) segment from the outside (O) end of IS50 and the transposase (tnp) gene was placed either between the ends or 1200 bp from the O end. The frequency of transposition of the resultant elements to bacteriophage lambda was measured. At least one substitution at each of the 19 I-end positions decreased transposition activity to less than 25% of wt, and most substitutions (25 of 28) decreased it to less than 5% of wt from one or both donor plasmids. These results show that each position in the I end is important during transposition.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • DNA Transposable Elements*
  • Escherichia coli / genetics*
  • Molecular Sequence Data
  • Mutation*
  • Nucleic Acid Hybridization
  • Oligonucleotide Probes
  • Plasmids

Substances

  • DNA Transposable Elements
  • Oligonucleotide Probes