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71.
Recombinant Escherichia coli fadR atoC(Con) mutants containing the polyhydroxyalkanoate (PHA) biosynthesis genes from Alcaligenes eutrophus are able to incorporate significant levels of 3-hydroxyvalerate (3HV) into the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]. We have used E. coli fadR (FadR is a negative regulator of fatty acid oxidation) and E. coli atoC(Con) (AtoC is a positive regulator of fatty acid uptake) mutants to demonstrate that either one of these mutations alone can facilitate copolymer synthesis but that 3HV levels in single mutant strains are much lower than in the fadR atoC(Con) strain. E. coli atoC(Con) mutants were used alone and in conjunction with atoA and atoD mutants to determine that the function of the atoC(Con) mutation is to increase the uptake of propionate and that this uptake is mediated, at least in part, by atoD+. Similarly, E. coli fadR mutants were used alone and in conjunction with fadA, fadB, and fadL mutants to show that the effect of the fadR mutation is dependent on fadB+ and fadA+ gene products. Strains that were mutant in the fadB or fadA locus were unable to complement a PHA biosynthesis pathway that was mutant at the phaA locus (thiolase), but a strain containing a fadR mutation and which was fadA+ fadB+ was able to complement the phaA mutation and incorporated 3HV into P(3HB-co-3HV) to a level of 29 mol%.  相似文献   
72.
We developed a biotin–streptavidin-based sandwich ELISA for the sensitive and specific detection of Yersinia pestis. In this assay, the F1 capsular protein and Y. pestis were captured by anti-F1 mouse monoclonal antibody followed by detection with biotinylated-anti-F1 rabbit polyclonal antibody and HRP-conjugated streptavidin. The developed F1 ELISA could detect not only the F1 protein up to 29 and 17 pg/ml but also Y. pestis up to 177.8 and 129.2 CFU/ml in PBS buffer and human serum, respectively. In addition, the F1 ELISA did not show any cross-reactivity with various proteins and bacterial strains.  相似文献   
73.
Both ThyA and ThyX proteins catalyze the transfer of the methyl group from methylenetetrahydrofolate (CH(2) H(4) -folate) to dUMP, forming dTMP. To estimate the relative steady state expression levels of ThyA and ThyX, Western blot analysis was performed using ThyA or ThyX antiserum on total protein from the wild-type, ΔthyX, and thyX-complemented strains of Corynebacterium glutamicum. The level of ThyA decreased gradually during the stationary growth phase but that of ThyX was maintained steadily. Whereas the expression level of ThyA in a ΔsigB strain was comparable to that of the wild-type, the level of ThyX was significantly diminished in the deletion mutant and was restored to that of the wild-type in the complemented strain, indicating that the level of ThyX was regulated by SigB. Growth of the C.?glutamicum ΔsigB strain was dependent upon coupling activity of dihydrofolate reductase (DHFR) with ThyA for the synthesis of thymidine, and thus showed sensitivity to the inhibition of DHFR by the experimental inhibitor, WR99210-HCl. These results suggested that the relative levels of ThyA and ThyX differ in response to different growth phases and that SigB is necessary for maintenance of the level of ThyX during transition into the stationary growth phase.  相似文献   
74.
D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size. In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected. The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.  相似文献   
75.
The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.  相似文献   
76.

Background  

One of the big remaining challenges in evolutionary biology is to understand the evolution and maintenance of meiotic recombination. As recombination breaks down successful genotypes, it should be selected for only under very limited conditions. Yet, recombination is very common and phylogenetically widespread. The Red Queen Hypothesis is one of the most prominent hypotheses for the adaptive value of recombination and sexual reproduction. The Red Queen Hypothesis predicts an advantage of recombination for hosts that are coevolving with their parasites. We tested predictions of the hypothesis with experimental coevolution using the red flour beetle, Tribolium castaneum, and its microsporidian parasite, Nosema whitei.  相似文献   
77.
An enrichment semi-nested PCR procedure was developed for detection of Clostridium botulinum types A, B, E, and F. It was applied to sediment samples to examine the prevalence of C. botulinum in the Korean environment. The first pair of primers for the semi-nested PCR was designed using a region shared by the types A, B, E, and F neurotoxin gene sequences, and the second round employed four nested primers complementary to the BoNT/A, /B, /E, and /F encoding genes for simultaneous detection of the four serotypes. Positive results were obtained from the PCR analysis of five of 44 sediments (11%) collected from Yeong-am Lake in Korea; all were identified as deriving from type B neurotoxin (bontb) genes. Two of the C. botulinum type B organisms were isolated, and their bontb genes sequenced. The deduced amino acid sequences of BoNT/B showed 99.5 and 99.8% identity with the amino acid sequence of accession no. AB084152. Our data suggest that semi-nested PCR is a useful tool for detecting C. botulinum in sediments, and renders it practicable to conduct environmental surveys.  相似文献   
78.
The nucleotide sequences corresponding to bovine alpha S2- and beta- casein mRNAs have been determined by cDNA analysis. Both sequences appear to be complete at their 5' ends. The nucleotide sequence of alpha S2-casein, when compared with the corresponding cavine A sequence, helps to define the boundaries of a large amino acid repeat (approximately 80 residues) whereas comparisons with the nucleotide sequences of rat gamma- and mouse epsilon-casein mRNAs also reveal extensive sequence similarities. An alignment of these four sequences shows that the divergence of their translated regions has been characterized by the duplication and deletion of discrete segments of sequence that probably correspond to exons. A high degree of nucleotide substitution is also found when the four sequences are compared, except for well-conserved leader-peptide and phosphorylation-site sequences and, to a lesser extent, the 5'-untranslated regions. Similar comparison of the bovine and rat beta-caseins shows that their divergence has involved a high rate of nucleotide substitution but that no major insertions or deletions of sequence have occurred. The several splice sites that have veen defined in the rat beta-casein gene are likely to have been conserved in the bovine. The contrasting evolutionary histories of the alpha- and beta-casein coding sequences correlate with the distinctive functions of these proteins in the casein micelle system in milk.   相似文献   
79.
Anthrax is caused by the spore‐forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA‐acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent‐spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.  相似文献   
80.
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