Plasmids comprising transgene insertions in four lines of transgenic mice have been retrieved by plasmid rescue into a set of Escherichia coli strains with mutations in different members of the methylation-dependent restriction system (MDRS). Statistical analysis of plasmid rescue frequencies has revealed that the MDRS loci detect differential modifications of the transgene insertions among mouse lines that show distinctive patterns of transgene expression. Plasmids in mice that express hybrid insulin transgenes during development can be readily cloned into E. coli strains carrying mutations in two of the MDRS loci, merA and mcrB. In mice in which transgene expression is inappropriately delayed into adulthood, plasmids can only be cloned into E. coli that carry mutations in all known MDRS activities. Differential cloning frequencies in the presence or absence of the various methylation-dependent restriction genes represent a further way to distinguish regions of mammalian chromosomes. These multiply deficient E. coli strains will also facilitate the molecular cloning of modified chromosomal DNA.
Regulation of the expression of the histidase coded by hutK of Klebsiella aerogenes in Salmonella typhimurium and in Escherichia coli and of the expression of the histidase coded by huts of S. typhimurium in E. coli was investigated. The hutK histidase was found to be sensitive to catabolite repression in K. aerogenes and in E. coli, but insensitive to catabolite repression in S. typhimurium; huts histidase has previously been shown to be catabolite sensitive in all three organisms. The expression of both hutK and huts histidase in E. coli was activated by nitrogen starvation. Apparently, the glutamine synthetase ofE. coli may activate the formation of some glutamate-and ammonia-producing enzymes. Salmonella typhimurium and Klebsiella aerogenes are able to degrade histidine to glutamate, ammonia, and formamide; in both organisms the structural genes for the first two enzymes of the pathway, histidase (EC 4.3.13) and urocanase (EC 4.2.1.49), are in one hut operon, and the structural genes for the third and fourth enzymes of the pathway, imidazolonepropionase (EC 3.5.2.9) and formiminoglutamase (EC 3.5.3.8), are in the second, closely linked hut operon (7, 9, 17). Neidhardt and Magasanik observed that in K. aerogenes the formation of histidase was subject to strong catabolite repression when the organism was grown in a medium containing glucose, histidine, and ammonium sulfate (11); they observed little repression of histidase when the cells were grown in a medium containing glucose and histidine but no ammonium salt. This observation has recently been
A lambda phage (lambdaNK55) carrying the translocatable element Tn10, conferring tetracycline resistance (Tetr), has been utilized to isolate glutamine auxotrophs of Escherichia coli K-12. Such strains lack uridylyltransferase as a result of an insertion of the TN10 element in the glnD gene. The glnD::Tn10 insertion has been mapped at min 4 on the E. coli chromosome and 98% contransducible by phage P1 with dapD. A lambda transducing phage carrying the glnD gene has been identified. A glnD::Tn10 strain synthesizes highly adenylylated glutamine synthetase under all conditions of growth and fails to accumulate high levels of glutamine synthetase in response to nitrogen limitation. However, this strain, under nitrogen-limiting conditions, allows synthesis of 10 to 20 milliunits of biosynthetically active glutamine synthetase per mg of protein, which is sufficient to allow slow growth in the absence of glutamine. The GlnD phenotype in E. coli can be suppressed by the presence of mutations which increase the quantity of biosynthetically active glutamine synthetase.
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