PMID:8335256
| Citation |
Metcalf, WW and Wanner, BL (1993) Construction of new beta-glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacement. Gene 129:17-25 |
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| Abstract |
Five cassettes carrying uidA, encoding beta-glucuronidase, were made for the construction of insertion mutants with transcriptional fusions to uidA. Three uidA cassettes contain antibiotic-resistance genes, for chloramphenicol (Cm), for kanamycin (Km) and neomycin (Nm), or for streptomycin (Sm) and spectinomycin (Sp). Some cause polar insertions while others provide a promoter for downstream gene expression. The expression of these uidA cassettes was compared to the expression of lacZ at the same site in phnD, a phosphate-regulated gene for phosphonate use. Several phn::uidA or phn::lacZ insertions were recombined onto the chromosome to test mutational effects and to measure gene expression in single copy. This was done using one of three methods for allele replacement. A new method involved recombination of mutations in M13 onto the chromosome by infection of an Escherichia coli rep mutant that fails to propagate single-stranded DNA phages. Merodiploid recombinants were selected using a resistance marker carried by the M13 phage; segregants lacking M13 sequences were then selected as deoxycholate-resistant (DocR) ones. An improved method for recombination of mutations in pir-dependent, oriR6K vectors involved the use of plasmids containing genes for tetracycline resistance (TcR). Merodiploid recombinants were selected by conjugative transfer of such plasmids into a recipient lacking pir (encoding the pi protein of the R6K plasmid); segregants lacking vector sequences were subsequently selected as Tc-sensitive ones. Both procedures are efficient and allow for recombining marked as well as unmarked mutations onto the chromosome. In addition, some insertions with an antibiotic-resistance marker were directly recombined onto the chromosome by transformation of a recD mutant with linear DNA.(ABSTRACT TRUNCATED AT 250 WORDS) |
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| Keywords |
Alleles; Amino Acid Sequence; Base Sequence; Escherichia coli/genetics; Gene Expression; Glucuronidase/genetics; Molecular Sequence Data; Mutagenesis, Insertional/methods; Plasmids; Transcription, Genetic; beta-Galactosidase/genetics |
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