Vectors for constructing kan gene fusions: direct selection of mutations affecting IS10 gene expression.
We describe several vectors for constructing translational fusions to the kan gene of Tn5. Fusions are constructed in vitro using multi-copy vectors containing unique cloning sites situated between upstream transcriptional terminators and a downstream kan gene lacking transcriptional and translational start signals. Multi-copy fusions can be converted to single-copy chromosomal fusions by in vivo recombination with specific phage lambda vectors and vice versa. We find that kan fusions are often more suitable than lacZ fusions for the direct selection of mutations that increase fusion expression. These vectors were developed for isolating mutations that increase IS10 transposase expression; we describe strategies used to isolate such mutations, which map to IS10 or the Escherichia coli himA, himD(hip), dam or infC genes.
Sussman
J K
JK
Department of Microbiology, University of California, Los Angeles 90024.
Masada-Pepe
C
C
Simons
E L
EL
Simons
R W
RW
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D007612:Kanamycin; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics; D010957:Plasmids; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins; D019895:Transposases
D004251:DNA Transposable Elements; D011993:Recombinant Fusion Proteins; D007612:Kanamycin; D009713:Nucleotidyltransferases; D019895:Transposases