Artificial transposable elements in the study of the ends of IS1.
We have constructed artificial IS1-based transposons by attaching synthetic oligodeoxynucleotides, corresponding to the sequence of the ends of IS1, to a selectable DNA segment ['omega' fragment; Prentki and Krisch, Gene 29 (1984) 303-313]. These transposons were used to examine the sequence requirements at the ends for IS1 transposition. We show here that a 24- to 28-bp sequence from the left or right ends of IS1 is capable of transposition when present at both ends of the omega fragment in the correct orientation. Transposition activity requires the presence of an intact IS1 in cis on the same plasmid molecule. In trans, however, neither resident genomic copies of IS1, nor copies carried by a compatible, high-copy-number plasmid present in the same cell, complement the artificial transposons efficiently. Transposition frequencies in the presence of a cis-complementing IS1 are, however, similar to those of the naturally occurring IS1-based transposon, Tn9. In addition, transposition results in a 9-bp duplication in the target DNA molecule as is usually the case for insertion of the intact IS1. Using this system, we have obtained evidence indicating that the activity of a synthetic IS1 end is not determined exclusively by its sequence, but can be strongly enhanced by a second, wild-type end used in the transposition event. The data also show that single base pair mutations can exhibit a cumulative effect in reducing transposition activity.
Prentki
P
P
Molecular Biology, University of Southern California, Los Angeles 90089-1481.
Pham
M H
MH
Gamas
P
P
Chandler
M
M
Galas
D J
DJ
D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D009154:Mutation; D011815:R Factors
D004251:DNA Transposable Elements; D004269:DNA, Bacterial