Recombination-deficient strains

A Bacterial Artificial Chromosome (BAC) is a vector that allows the propagation of larger exogenous DNA fragments, up to several hundred kb. BACs are propagated in recombination-deficient strains of E. coli. They are more stable and easier to handle than yeast artificial chromosomes (YACs). 

A particularly interesting feature of the ATP-dependent DNase is its possible involvement in genetic recombination. Thus, certain recombination deficient strains of E. coli with the recB and recC phenotypes (15) appear to lack this enzyme. 

DNA repair. Polymerase-deficient, and thus DNA repair-deficient, E. coli has provided the basis for a test that depends on the fact that the growth of a deficient strain is inhibited more by a DNA-damaging agent than is that of a repair-competent strain. The recombinant assay using Bacillus subtilis is conducted in much the same way because recombinant deficient strains are more sensitive to DNA-damaging agents. 

Bacillus sub tilts (gram-positive) is useful in this assay because its cellular membrane is more permeable to chemicals than that of some gram-negative enteric bacteria. A recombination-deficient strain that has the highest sensitivity and the widest spectra to typical mutagens has been selected from a... 

Stable Strontium. In mutagenicity assays using the Rec" (recombination-repair-deficient) strain of Bacillus subtilis, strontium chloride had a negative effect in vitro (Kanematsu et al. 1980). Furthermore, in a survey of the effect of metal salts, strontium was found to have no adverse effect on the fidelity of DNA synthesis in vitro, which was thought to be consistent with its reported lack of mutagenicity and carcinogenicity (Loeb et al. 1977)... 

The typical results shown in Table 3 indicate the minimum concentrations (jLig/ml) necessary to inhibit growth of either wild-type or the recombination-deficient rec45) strain in liquid broth at 37°C. The ratio MIC (Rec )/MIC (Rec ) indicates the degree of DNA damage that was nonreparable in the repair-deficient strain in either the presence or the absence of S-9 metabolic activation. 

F hsdR17 r m endAl gyrA96 EizV) recAl relAl supE44 thi-1. A recombination-deficient suppressing strain generally useful for plasmid amplification. 

Metabolic pathways containing dioxygenases in wild-type strains are usually related to detoxification processes upon conversion of aromatic xenobiotics to phenols and catechols, which are more readily excreted. Within such pathways, the intermediate chiral cis-diol is rearomatized by a dihydrodiol-dehydrogenase. While this mild route to catechols is also exploited synthetically [221], the chirality is lost. In the context of asymmetric synthesis, such further biotransformations have to be prevented, which was initially realized by using mutant strains deficient in enzymes responsible for the rearomatization. Today, several dioxygenases with complementary substrate profiles are available, as outlined in Table 9.6. Considering the delicate architecture of these enzyme complexes, recombinant whole-cell-mediated biotransformations are the only option for such conversions. E. coli is preferably used as host and fermentation protocols have been optimized [222,223]. 

This microbial sensor system is based on the inhibitory action of the mutagens on the respiration of B subtilis Rec . B subtilis M45 (Rec") is genetically deficient in the DNA recombination enzyme system, whereas B subtilis H17 (Rec+) is a wild strain which has the ability to repair damaged DNA. The subsequent death of Rec bacteria is preceded by the decrease of respiration. As a result, the number of Rec cells on the surface of the oxygen electrode decreased and the current of the Rec electrode increased. On the other hand, the damaged DNA of Rec+ bacteria is repaired with the recombination system. Therefore, the number of Rec+ cells did not change and the current of the Rec electrode did not increase. 

S. avermitilis and the biosynthesis of avermectins constitute an interesting example where traditional techniques such as chemical mutagenesis and protoplast fusion combined with recombinant DNA technology have been successfully applied in mutant isolation and strain improvement. In addition, this system offered the first opportunity to apply mutasynthesis to the production of better analogs, an application that had never before been exploited commercially. An intense doramectin development effort was therefore initiated with the Mrf-deficient mutants of S. avermitilis. The first step in this process involved the isolation of mutants deficient in 5-Omethyltransferase activity to maximize levels of the more bioactive class B avermectins [13], Thereafter, a combination of strain im-... 

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