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Cloning of multiple genes involved with cobalamin (Vitamin B12) biosynthesis in Bacillus megaterium. 总被引:7,自引:6,他引:1
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An effective shotgun cloning procedure was developed for Bacillus megaterium by amplifying gene libraries in Bacillus subtilis. This technique was useful in isolating at least 11 genes from B. megaterium which are involved with cobalamin (vitamin B12) biosynthesis. Amplified plasmid banks were transformed into protoplasts of both a series of Cob mutants blocked before the biosynthesis of cobinamide and Cbl mutants blocked in the conversion of cobinamide into cobalamin. Amplification of gene libraries overcame the cloning barriers inherent in the relatively low protoplast transformation frequency of B. megaterium. A family of plasmids was isolated by complementation of seven different Cob and Cbl mutants. Each plasmid capable of complementing a Cob or Cbl mutant was transformed into each one of the series of Cob and Cbl mutants; many of the plasmids isolated by complementation of one mutation carried genetic activity for complementation of other mutations. By these criteria, four different complementation groups were resolved. At least six genes involved in the biosynthesis of cobinamide are carried on a fragment of DNA approximately 2.7 kilobase pairs in length; other genes involved in the biosynthesis of cobinamide were located in two other complementation groups. The physical and genetic data permitted an ordering of genes within several of the complementation groups. The presence of complementing plasmids in mutants blocked in cobalamin synthesis resulted in restoration of cobalamin biosynthesis. 相似文献
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N. Cohen G. Weber B.L. Banner A.F. Welton W.C. Hope H. Crowley W.A. Anderson B.A. Simko M. O'Donnell J.W. Coffey C. Fiedler-Nagy C. Batula-Bernardo 《Prostaglandins & other lipid mediators》1984,27(4):553-562
The syntheses and biological activity of (all
)-7,7-dimethyl-5-8,- 11,14-eicosatetraenoic acid, (all
)-7,7,-dimethyl-5,8,11-eicosatrienoic acid, (
,
-7,7-dimethyl-5,8-eicosadienoic acid, (all
)-10,10-dimetyl- 5,8,11,14-eicosatetraenoic acid, (all
-10,10-dimethyl-5,8,11-eicosatrienoic acid, and
.-(
,
-15-hydroxy-7,7-dimethyl-5,8-eicosadienoic acid are described. These arachidonic acid analogs are all inhibitors of ionophore-induced SRS-A biosynthesis in rat peritoneal cells. Their mode of action may involve inhibition of phospholipase A2 rather than Δ5-lipoxygenase. These compounds failed to exhibit significant activity in an
model designed to detect inhibitors of antigen-induced, leukotriene-mediated bronchoconstriction is sensitized guinea pigs. 相似文献
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On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase 总被引:13,自引:0,他引:13
T Alber D W Banner A C Bloomer G A Petsko D Phillips P S Rivers I A Wilson 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1981,293(1063):159-171
Triose phosphate isomerase is a dimeric enzyme of molecular mass 56 000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel beta-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanisms for catalysis involving polarization of the substrate carbonyl group. 相似文献
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A series of N-alkylmaleimides has been synthesized and used to investigate the thiol groups that are essential for the activity of rat liver microsomal glucose 6-phosphatase. All of the N-alkylmaleimides inactivated glucose 6-phosphatase when preincubated with microsomes (microsomal fractions) at pH 6.5 and 30 degrees C. When enzyme activity was assayed in intact microsomes, the inactivation was non-linear with respect time, showing an initial rapid phase followed by a slower secondary phase. During the initial rapid phase the inactivation may apparently be completely reversed by disrupting the microsomal membrane with detergent. However, after longer exposure to N-alkylmaleimides the reversal is no longer complete. This observation was explained by the results obtained from studying the inactivation in detergent-disrupted microsomes. In this case glucose 6-phosphatase was also completely inactivated, but much more slowly than was seen in intact microsomes, and the process was linear with respect to time. When assayed in both intact and detergent-disrupted microsomes, glucose 6-phosphatase inactivation was dependent on the number of carbon atoms in the alkyl side chain of the N-alkylmaleimides; this dependence was much more marked in disrupted microsomes. Analysis of the data showed that in neither case was there a saturating effect at high concentrations of maleimide. The data have been interpreted to suggest that there are are least two thiol groups essential for activity located in two separate non-polar regions of the membrane-enzyme system. The conclusions are discussed in the light of the current model for the microsomal glucose 6-phosphatase system. 相似文献
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Structure of the ColE1 rop protein at 1.7 A resolution 总被引:14,自引:0,他引:14
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Alberto J. León David Banner Luoling Xu Longsi Ran Zhiyu Peng Kang Yi Chao Chen Fengping Xu Jinrong Huang Zhen Zhao Zhen Lin Stephen H. S. Huang Yuan Fang Alyson A. Kelvin Ted M. Ross Amber Farooqui David J. Kelvin 《Journal of virology》2013,87(4):1957-1966
Ferrets have become an indispensable tool in the understanding of influenza virus virulence and pathogenesis. Furthermore, ferrets are the preferred preclinical model for influenza vaccine and therapeutic testing. Here we characterized the influenza infectome during the different stages of the infectious process in ferrets with and without prior specific immunity to influenza. RNA from lung tissue and lymph nodes from infected and naïve animals was subjected to next-generation sequencing, followed by de novo data assembly and annotation of the resulting sequences; this process generated a library comprising 13,202 ferret mRNAs. Gene expression profiles during pandemic H1N1 (pdmH1N1) influenza virus infection were analyzed by digital gene expression and solid support microarrays. As expected during primary infection, innate immune responses were triggered in the lung tissue; meanwhile, in the lymphoid tissue, genes encoding antigen presentation and maturation of effector cells of adaptive immunity increased dramatically. After 5 days postinfection, the innate immune gene expression was replaced by the adaptive immune response, which correlates with viral clearance. Reinfection with homologous pandemic influenza virus resulted in a diminished innate immune response, early adaptive immune gene regulation, and a reduction in clinical severity. The fully annotated ferret infectome will be a critical aid to the understanding of the molecular events that regulate disease severity and host-influenza virus interactions among seasonal, pandemic, and highly pathogenic avian influenzas. 相似文献
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