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181.
182.
Twin-arginine targeting (Tat) protein secretion systems consist of two protein types, members of the TatA and TatC families. Homologues of these proteins are found in many archaea, bacteria, chloroplasts and mitochondria. Every prokaryotic organism with a fully sequenced genome exhibits either neither family member, or between one and three paralogues of these two family members. The Arabidopsis thaliana genome encodes three of each. Although many mitochondrially encoded TatC homologues have been identified, corresponding TatA homologues have not been found in this organelle. Phylogenetic analyses reveal that most prokaryotic Tat systems consist of one TatC homologue and two sequence-divergent TatA homologues (TatA and TatB). When only one TatA homologue is present, TatB is missing, and when three TatA homologues are present, the third one arose by duplication of TatA, not TatB. Further, homologues most resembling TatB are more sequence-divergent than those more closely resembling TatA. In contrast to the TatA family, the TatC family shows phylogenetic clustering in strict accordance with organismal type. These results are discussed in terms of their probable structural, functional and evolutionary significance. 相似文献
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Edgley M D'Souza A Moulder G McKay S Shen B Gilchrist E Moerman D Barstead R 《Nucleic acids research》2002,30(12):e52
About 40% of the genes in the nematode Caenorhabditis elegans have homologs in humans. Based on the history of this model system, it is clear that the application of genetic methods to the study of this set of genes would provide important clues to their function in humans. To facilitate such genetic studies, we are engaged in a project to derive deletion alleles in every gene in this set. Our standard methods make use of nested PCR to hunt for animals in mutagenized populations that carry deletions at a given locus. The deletion bearing animals exist initially in mixed populations where the majority of the animals are wild type at the target. Therefore, the production of the PCR fragment representing the deletion allele competes with the production of the wild type fragment. The size of the deletion fragment relative to wild type determines whether it can compete to a level where it can be detected above the background. Using our standard conditions, we have found that when the deletion is <600 bp, the deletion fragment does not compete effectively with the production of the wild type fragment in PCR. Therefore, although our standard methods work well to detect mutants with deletions >600 bp, they do not work well to detect mutants with smaller deletions. Here we report a new strategy to detect small deletion alleles in complex DNA pools. Our new strategy is a modification of our standard PCR based screens. In the first round of the nested PCR, we include a third PCR primer between the two external primers. The presence of this third primer leads to the production of three fragments from wild type DNA. We configure the system so that two of these three fragments cannot serve as a template in the second round of the nested PCR. The addition of this third primer, therefore, handicaps the amplification from wild type template. On the other hand, the amplification of mutant fragments where the binding site for the third primer is deleted is unabated. Overall, we see at least a 500-fold increase in the sensitivity for small deletion fragments using our new method. Using this new method, we report the recovery of new deletion alleles within 12 C.elegans genes. 相似文献
185.
Slipped-strand DNAs formed by long (CAG)*(CTG) repeats: slipped-out repeats and slip-out junctions
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Pearson CE Tam M Wang YH Montgomery SE Dar AC Cleary JD Nichol K 《Nucleic acids research》2002,30(20):4534-4547
The disease-associated expansion of (CTG)·(CAG) repeats is likely to involve slipped-strand DNAs. There are two types of slipped DNAs (S-DNAs): slipped homoduplex S-DNAs are formed between two strands having the same number of repeats; and heteroduplex slipped intermediates (SI-DNAs) are formed between two strands having different numbers of repeats. We present the first characterization of S-DNAs formed by disease-relevant lengths of (CTG)·(CAG) repeats which contained all predicted components including slipped-out repeats and slip-out junctions, where two arms of the three-way junction were composed of complementary paired repeats. In S-DNAs multiple short slip-outs of CTG or CAG repeats occurred throughout the repeat tract. Strikingly, in SI-DNAs most of the excess repeats slipped-out at preferred locations along the fully base-paired Watson–Crick duplex, forming defined three-way slip-out junctions. Unexpectedly, slipped-out CAG and slipped-out CTG repeats were predominantly in the random-coil and hairpin conformations, respectively. Both the junctions and the slip-outs could be recognized by DNA metabolizing proteins: only the strand with the excess repeats was hypersensitive to cleavage by the junction-specific T7 endonuclease I, while slipped-out CAG was preferentially bound by single-strand binding protein. An excellent correlation was observed for the size of the slip-outs in S-DNAs and SI-DNAs with the size of the tract length changes observed in quiescent and proliferating tissues of affected patients—suggesting that S-DNAs and SI-DNAs are mutagenic intermediates in those tissues, occurring during error-prone DNA metabolism and replication fork errors. 相似文献
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Mitochondria,nitric oxide,and cardiovascular dysfunction 总被引:6,自引:0,他引:6
Ramachandran A Levonen AL Brookes PS Ceaser E Shiva S Barone MC Darley-Usmar V 《Free radical biology & medicine》2002,33(11):1465-1474
Cardiovascular diseases encompass a wide spectrum of abnormalities with diverse etiologies. The molecular mechanisms underlying these disorders include a variety of responses such as changes in nitric oxide- (NO) dependent cell signaling and increased apoptosis. An interesting aspect that has received little or no attention is the role mitochondria may play in the vascular changes that occur in both atherosclerosis and hypertension. With the changing perspective of the organelle from simply a role in metabolism to a contributor to signal transduction pathways, the role of mitochondria in cells with relatively low energy demands such as the endothelium has become important to understand. In this context, the definition of the NO-cytochrome c oxidase signaling pathway and the influence this has on cytochrome c release is particularly important in understanding apoptotic mechanisms involving the mitochondrion. This review examines the role of compromised mitochondrial function in a variety of vascular pathologies and the modulation of these effects by NO. The interaction of NO with the various mitochondrial respiratory complexes and the role NO plays in modulating mitochondrial-mediated apoptosis in these systems will be discussed. 相似文献
188.
The human erythrocyte glucose transport protein (GluT1) is an adenine nucleotide binding protein. When complexed with cytosolic ATP, GluT1 exhibits increased affinity for the sugar export site ligand cytochalasin B, prolonged substrate occlusion, reduced net sugar import capacity, and diminished reactivity with carboxyl terminal peptide-directed antibodies. The present study examines the kinetics of nucleotide interaction with GluT1. When incorporated into resealed human red blood cell ghosts, (2,3)-trinitrophenyl-adenosine-triphosphate (TNP-ATP) mimics the ability of cytosolic ATP to promote high-affinity 3-O-methylglucose uptake. TNP-ATP fluorescence increases upon interaction with purified human red cell GluT1. TNP-ATP binding to GluT1 is rapid (t(1/2) approximately 0.5 s at 50 microM TNP-ATP), cooperative, and pH-sensitive and is stimulated by ATP and by the exit site ligand cytochalasin B. Dithiothreitol inhibits TNP-ATP binding to GluT1. GluT1 preirradiation with saturating, unlabeled azidoATP enhances subsequent GluT1 photoincorporation of [gamma-32P]azidoATP. Reduced pH enhances azidoATP photoincorporation into isolated red cell GluT1 but inhibits ATP modulation of sugar transport in resealed red cell ghosts and in GluT1 proteoliposomes. We propose that cooperative nucleotide binding to reductant-sensitive, oligomeric GluT1 is modulated by a proton-sensitive saltbridge. The effects of ATP on GluT1-mediated sugar transport may be determined by the number of ATP molecules complexed with the transporter. 相似文献
189.
Zhao L Cuff CA Moss E Wille U Cyrus T Klein EA Praticò D Rader DJ Hunter CA Puré E Funk CD 《The Journal of biological chemistry》2002,277(38):35350-35356
Targeted gene disruption or overexpression of 12/15-lipoxygenase in mice on the genetic background of apolipoprotein E or low density lipoprotein-receptor (LDL-R) deficiency has implicated 12/15-lipoxygenase in atherogenesis. The data support indirectly a role for 12/15-lipoxygenase in the oxidative modification of low density lipoprotein. In this study we set out to explore other potential mechanisms for 12/15-lipoxygenase in atherosclerosis using apolipoprotein B mRNA editing catalytic polypeptide-1/LDL-R double-deficient mice, a model highly related to the human condition of familial hypercholesterolemia. 12/15-Lipoxygenase deficiency in this strain led to approximately 50% decrease in aortic lesions in male and female mice at 8 months on a chow diet in the absence of cholesterol differences. While studying 12/15-lipoxygenase-deficient macrophages in culture, we discovered a remarkable selective defect (75-90% decrease) in interleukin-12 production but not in tumor necrosis factor-alpha or nitric oxide release, in response to lipopolysaccharide in the presence or absence of interferon-gamma priming. The lipopolysaccharide/interferon-gamma response was associated with a 33-50% decrease in nuclear interferon consensus sequence-binding protein, which is consistent with interferon consensus sequence-binding protein containing protein complex-dependent regulation of the interleukin-12 p40 gene. The decrease in interleukin-12 production was recapitulated in vivo in mouse aortas of the triple knockout group and was reflected in a marked decrease in interferon-gamma expression. The data provide support for a novel mechanism linking the 12/15-lipoxygenase pathway to a known immunomodulatory Th1 cytokine in atherogenesis. 相似文献
190.
Talledo M Rivera IN Lipp EK Neale A Karaolis D Huq A Colwell RR 《Environmental microbiology》2003,5(5):350-354
A Vibrio cholerae bacteriophage, family Myoviridae, was isolated from seawater collected from the coastal water of Lima, Peru. Genome size was estimated to be 29 kbp. The temperate phage was specific to V. cholerae and infected 12/13 V. cholerae O1 strains and half of the four non-O1/non-O139 strains tested in this study. Vibrio cholerae O139 strains were resistant to infection and highest infection rates were obtained in low nutrient media amended with NaCl or prepared using seawater as diluent. 相似文献