N-terminal domain-deleted mu transposase exhibits increased transposition activity with low target site preference in modified buffers.
BACKGROUND
Transposition using MuA and the Mu-transposon is frequently used for insertional and deletional mutagenesis and to introduce primer sequences into DNA of unknown sequence. However, mutagenesis studies are often limited by the number of transposition events taking place within a small target sequence. Although the intrinsic target site preference of MuA is low, it nevertheless may limit the recovery of desired events.


METHODS
In the current study, we have compared the transposition activity of wild-type, C-terminally deleted, N-terminally deleted and both C- and N-terminally deleted MuA.


RESULTS
We found that N-terminally deleted MuA was most active with an about sevenfold increase in transposition efficiency compared to wild-type. In addition, we identified a novel reaction condition in which the transposition activity of N-terminally deleted MuA was increased about 45-fold.


CONCLUSIONS
The use of the N-terminally deleted MuA with the newly established condition enhanced the relative transposition activity about 280-fold compared to that of wild-type MuA with the conventional buffer. In addition, the optimized buffer decreased the target site preference of N-terminally deleted MuA resulting in a more even distribution of integration events throughout the target gene.
Kim
Yun Cheol
YC
Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA 90095, USA. kyc8402@ucla.edu
Morrison
Sherie L
SL
D004251:DNA Transposable Elements; D016254:Mutagenesis, Insertional / Q000379:methods*; D011995:Recombination, Genetic; D017384:Sequence Deletion; D019895:Transposases / Q000235:genetics* / Q000378:metabolism*
D004251:DNA Transposable Elements; C114697:mu transposase; D019895:Transposases