CONSTRUCTION OF NEW BETA-GLUCURONIDASE CASSETTES FOR MAKING TRANSCRIPTIONAL FUSIONS AND THEIR USE WITH NEW METHODS FOR ALLELE REPLACEMENT
CONSTRUCTION OF NEW BETA-GLUCURONIDASE CASSETTES FOR MAKING TRANSCRIPTIONAL FUSIONS AND THEIR USE WITH NEW METHODS FOR ALLELE REPLACEMENT
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DOI：
10.1016/0378-1119(93)90691-u
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发表时间：
1993-07-15
期刊：
影响因子：
3.5
通讯作者：
WANNER, BL
中科院分区：
文献类型：
--
作者：
METCALF, WW;WANNER, BL
制作了五个携带编码β-葡萄糖醛酸酶的uidA的盒，用于构建与uidA转录融合的插入突变体。三个uidA盒包含抗生素抗性基因，氯霉素(Cm)、卡那霉素(Km)和新霉素(Nm)、或链霉素(Sm)和壮观霉素(Sp)。有些引起极性插入，而另一些则提供下游基因表达的启动子。将这些uidA盒的表达与phnD（一种用于磷酸盐使用的磷酸盐调节基因）中相同位点的lacZ的表达进行比较。将多个 phn::uidA 或 phn::lacZ 插入重组到染色体上，以测试突变效应并测量单拷贝中的基因表达。这是使用三种等位基因替换方法之一完成的。一种新方法涉及通过感染无法繁殖单链 DNA 噬菌体的大肠杆菌 rep 突变体，将 M13 中的突变重组到染色体上。使用M13噬菌体携带的抗性标记来选择分二倍体重组体；然后选择缺乏M13序列的分离子作为脱氧胆酸抗性(Doc(R))分离子。一种在pir依赖性ori(R6K)载体中重组突变的改进方法涉及使用含有四环素抗性(Tc(R))基因的质粒。通过将此类质粒接合转移至缺乏 pir（编码 R6K 质粒的 PI 蛋白）的受体中来选择部分二倍体重组体；随后选择缺乏载体序列的分离子作为Tc敏感的分离子。这两种方法都很有效，并且允许将标记和未标记的突变重组到染色体上。此外，通过用线性DNA转化recD突变体，一些带有抗生素抗性标记的插入片段被直接重组到染色体上。还描述了删除了 uidA、lacZ 和 phoA 的新大肠杆菌突变体，包括允许同时使用多个报告基因的突变体。
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 (Doc(R)) ones. An improved method for recombination of mutations in pir-dependent, ori(R6K) vectors involved the use of plasmids containing genes for tetracycline resistance (Tc(R)) . 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. New E. coli mutants deleted for uidA, lacZ and phoA are also described, including ones that allow for the simultaneous use of multiple reporter genes.