TnphoA and TnphoA' elements for making and switching fusions for study of transcription, translation, and cell surface localization.
We describe a set of elements based on the transposon TnphoA for making transcriptional fusions to the lacZ gene and for making translational fusions to the phoA or lacZ structural gene. Each element can be switched, one for another, by homologous recombination, thereby allowing testing for transcription, translation, or cell surface localization determinants at the same site within a gene. We describe three kinds of elements for making each fusion type. Two kinds are transposition proficient (Tnp+): one encodes kanamycin resistance, and the other encodes tetracycline resistance. The third kind is transposition defective (Tnp-) and encodes kanamycin resistance. In addition, we describe one Tnp- element that has no reporter gene and encodes chloramphenicol resistance; this element is used primarily as a tool to aid in switching fusions. Switching is efficient because each element has in common 254 bp of DNA at the phoA end and 187 bp (or more) of DNA at the IS50R end of TnphoA, and switching is straightforward because individual elements encode different drug resistances. Thus, switched recombinants can be selected as drug-resistant transductants, and they can be recognized as ones that have lost the parental drug resistance and fusion phenotype. Further, switching Tnp+ elements to Tnp- elements reduces problems due to transposition that can arise in P1 crosses or cloning experiments. Some TnphoA and TnphoA' elements cause polar mutations, while others provide an outward promoter for downstream transcription. This feature is especially useful in the determination of operon structures. Strategies for the use of TnphoA and TnphoA' elements in gene analysis are also described.
Wilmes-Riesenberg
M R
MR
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907.
Wanner
B L
BL
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D004251:DNA Transposable Elements / Q000235:genetics*; D004274:DNA, Recombinant / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015321:Gene Rearrangement / Q000235:genetics; D007613:Kanamycin Resistance / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic; D014161:Transduction, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; C039919:RNA, recombinant; D011993:Recombinant Fusion Proteins; D012313:RNA; D001616:beta-Galactosidase