The power of association studies to detect the contribution of candidate genetic loci to variation in complex traits.
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The statistical power of five association study test statistics (two haplotype-based tests, two marker-based tests, and the Transmission Disequilibrium Test-Q5) to detect single nucleotide polymorphism (SNP)/phenotype associations in a linkage-disequilibrium-based candidate gene scan employing a number of SNPs is examined. Power is estimated as a function of realistic parameters expected to affect the likelihood of detecting a significant association: the number of SNPs examined, the scaled recombination size of the region examined, the proportion of variance in the trait attributable to a hidden causative polymorphism within the region, and the number of individuals or families examined. For the different combinations of parameter values, power is estimated from a large number of realizations of a simulated coalescent describing a single random mating population with mutation, random genetic drift, and recombination. This explicit population genetics model results in a distribution of DNA marker heterozygosities and linkage disequilibria that are likely to resemble those expected in actual population samples. The study concludes that (1) marker-based permutation tests are more powerful than simple haplotype-based tests, (2) there is sufficient power to detect the presence of causative polymorphisms of small effect if on the order of 500 individuals are sampled, (3) greater power is achieved by increasing the sample size than by increasing the number of polymorphisms, (4) association studies are generally more powerful than transmission disequilibrium-based tests, and (5) for the range of parameters considered association studies have a low repeatability unless sample sizes are on the order of 500 individuals. Estimates of 4Nc for a number of gene regions and human populations will be of use in determining the density of SNPs that are likely to be required for successful association studies. (+info)
The transmembrane domains of the human multidrug resistance P-glycoprotein are sufficient to mediate drug binding and trafficking to the cell surface.
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The human multidrug resistance P-glycoprotein (P-gp) is organized in two tandem repeats with each repeat consisting of an N-terminal hydrophobic domain containing six potential transmembrane segments followed by a hydrophilic domain containing a nucleotide-binding fold. A series of deletion mutants together with an in vivo drug-binding assay were used to test whether the deletion mutants interacted with substrates or were transported to the cell surface. We found that a deletion mutant consisting of only the transmembrane domains (residues 1-379 plus 681-1025) retained the ability to interact with drug substrates. In the absence of drug substrates, the deletion mutant was sensitive to trypsin and endoglycosidase H. Expression in the presence of verapamil, vinblastine, capsaicin, or cyclosporin A, however, resulted in a mutant protein that was resistant to trypsin and endoglycosidase H. The mutant was then detected at the cell surface and was sensitive to digestion by endoglycosidase F. By contrast, the N-terminal transmembrane domain (residues 1-379) alone did not interact with drug substrates, since it was sensitive to only endoglycosidase H and was not detected at the cell surface. These results show that the nucleotide-binding domains are not required for interaction of P-gp with substrate or for trafficking of P-gp to the cell surface. (+info)
Control of apical membrane chloride permeability in the renal A6 cell line by nucleotides.
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1. The effect of extracellular nucleotides applied on the apical side of polarised A6 cells grown on permeant filters was investigated by measuring the changes in (i) the 36Cl efflux through the apical membranes, (ii) the intracellular chloride concentrations (aCli, measured with N-(6-methoxyquinolyl) acetoethyl ester, MQAE), (iii) ICl, the short-circuit current in the absence of Na+ transport and (iv) the characteristics of the apical chloride channels using a patch-clamp approach. 2. ATP or UTP (0.1-500 microM) transiently stimulated ICl. The sequence of purinergic agonist potencies was UTP = ATP > ADP >> the P2X-selective agonist beta,gamma-methylene ATP = the P2Y-selective agonist 2-methylthioATP. Suramin (100 microM) as the P2Y antagonist Reactive Blue 2 (10 microM) had no effect on the UTP (or ATP)-stimulated current. These findings are consistent with the presence of P2Y2-like receptors located on the apical membranes of A6 cells. Apical application of adenosine also transiently increased ICl. This effect was blocked by theophylline while the UTP-stimulated ICl was not. The existence of a second receptor, of the P1 type is proposed. 3. ATP (or UTP)-stimulated ICl was blocked by apical application of 200 microM N-phenylanthranilic acid (DPC) or 100 microM niflumic acid while 100 microM glibenclamide was ineffective. 4. Ionomycin and thapsigargin both transiently stimulated ICl; the nucleotide stimulation of ICl was not suppressed by pre-treatment with these agents. Chlorpromazin (50 microM), a Ca2+-calmodulin inhibitor strongly inhibited the stimulation of ICl induced either by apical UTP or by ionomycin application. BAPTA-AM pre-treatment of A6 cells blocked the UTP-stimulated ICl. Niflumic acid also blocked the ionomycin stimulated ICl. 5. A fourfold increase in 36Cl effluxes through the apical membranes was observed after ATP or UTP application. These increases of the apical chloride permeability could also be observed when following aCli changes. Apical application of DPC (1 mM) or 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB; 500 microM) produced an incomplete inhibition of 36Cl effluxes through the apical membranes in ATP-stimulated and in untreated monolayers. 6. In single channel patch-clamp experiments, an apical chloride channel with a unitary single channel conductance of 7.3 +/- 0.6 pS (n = 12) was usually observed. ATP application induced the activation of one or more of these channels within a few minutes. 7. These results indicate that multiple purinergic receptor subtypes are present in the apical membranes of A6 cells and that nucleotides can act as modulators of Cl- secretion in renal cells. (+info)
Mcm2, but not RPA, is a component of the mammalian early G1-phase prereplication complex.
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Previous experiments in Xenopus egg extracts identified what appeared to be two independently assembled prereplication complexes (pre-RCs) for DNA replication: the stepwise assembly of ORC, Cdc6, and Mcm onto chromatin, and the FFA-1-mediated recruitment of RPA into foci on chromatin. We have investigated whether both of these pre-RCs can be detected in Chinese hamster ovary (CHO) cells. Early- and late-replicating chromosomal domains were pulse-labeled with halogenated nucleotides and prelabeled cells were synchronized at various times during the following G1-phase. The recruitment of Mcm2 and RPA to these domains was examined in relation to the formation of a nuclear envelope, specification of the dihydrofolate reductase (DHFR) replication origin and entry into S-phase. Mcm2 was loaded gradually and cumulatively onto both early- and late-replicating chromatin from late telophase throughout G1-phase. During S-phase, detectable Mcm2 was rapidly excluded from PCNA-containing active replication forks. By contrast, detergent-resistant RPA foci were undetectable until the onset of S-phase, when RPA joined only the earliest-firing replicons. During S-phase, RPA was present with PCNA specifically at active replication forks. Together, our data are consistent with a role for Mcm proteins, but not RPA, in the formation of mammalian pre-RCs during early G1-phase. (+info)
Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow.
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Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy. Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH. In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium. Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1. When C. cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition. In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited. These data indicate that C. cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose. This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C. cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing. (+info)
Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase.
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The nucleotide binding properties of mutants with alterations to Asp(351) and four of the other residues in the conserved phosphorylation loop, (351)DKTGTLT(357), of sarcoplasmic reticulum Ca(2+)-ATPase were investigated using an assay based on the 2', 3'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP) photolabeling of Lys(492) and competition with ATP. In selected cases where the competition assay showed extremely high affinity, ATP binding was also measured by a direct filtration assay. At pH 8.5 in the absence of Ca(2+), mutations removing the negative charge of Asp(351) (D351N, D351A, and D351T) produced pumps that bound MgTNP-8N(3)-ATP and MgATP with affinities 20-156-fold higher than wild type (K(D) as low as 0.006 microM), whereas the affinity of mutant D351E was comparable with wild type. Mutations K352R, K352Q, T355A, and T357A lowered the affinity for MgATP and MgTNP-8N(3)-ATP 2-1000- and 1-6-fold, respectively, and mutation L356T completely prevented photolabeling of Lys(492). In the absence of Ca(2+), mutants D351N and D351A exhibited the highest nucleotide affinities in the presence of Mg(2+) and at alkaline pH (E1 state). The affinity of mutant D351A for MgATP was extraordinarily high in the presence of Ca(2+) (K(D) = 0.001 microM), suggesting a transition state like configuration at the active site under these conditions. The mutants with reduced ATP affinity, as well as mutants D351N and D351A, exhibited reduced or zero CrATP-induced Ca(2+) occlusion due to defective CrATP binding. (+info)
Differential rates of NTP hydrolysis by the mutant [S69G]RecA protein. Evidence for a coupling of NTP turnover to DNA strand exchange.
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The x-ray crystal structure of the Escherichia coli RecA protein indicates that the phosphate groups of the nucleotide cofactor are bound by a loop whose amino acid sequence ((66)GPESSGKT(73)) corresponds to a consensus phosphate binding loop sequence (GXXXXGK[T/S]) found in many NTP-binding proteins. As part of an investigation of the role of the P-loop in ATP hydrolysis, we prepared a mutant RecA protein in which serine 69 was replaced by a glycine residue. We have found that the [S69G]RecA mutation has a differential effect on the hydrolysis of various nucleoside triphosphates. The [S69G]RecA protein catalyzes the single-stranded DNA-dependent hydrolysis of rATP, ddATP, and dATP with turnover numbers of 10, 20, and 36 min(-1), respectively. The wild type RecA protein, in contrast, hydrolyzes each of these nucleoside triphosphates with similar turnover numbers of 20-24 min(-1). Significantly, the [S69G]RecA protein promotes strand exchange with all three nucleoside triphosphates, and the rate of strand exchange is directly proportional to the rate of hydrolysis of each of the nucleotide cofactors. These findings with the [S69G]RecA protein provide support for the existence of a mechanistic coupling between NTP hydrolysis and DNA strand exchange. (+info)
Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y(2) receptor (-/-) mice.
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To test for the role of the P2Y(2) receptor (P2Y(2)-R) in the regulation of nucleotide-promoted Ca(2+) signaling in the lung, we generated P2Y(2)-R-deficient (P2Y(2)-R(-/-)) mice and measured intracellular Ca(2+)(i) responses (DeltaCa(2+)(i)) to nucleotides in cultured lung fibroblasts and nasal and tracheal epithelial cells from wild type and P2Y(2)-R(-/-) mice. In the wild type fibroblasts, the rank order of potencies for nucleotide-induced DeltaCa(2+)(i) was as follows: UTP >/= ATP >> ADP > UDP. The responses induced by these agonists were completely absent in the P2Y(2)-R(-/-) fibroblasts. Inositol phosphate responses paralleled those of DeltaCa(2+)(i) in both groups. ATP and UTP also induced Ca(2+)(i) responses in wild type airway epithelial cells. In the P2Y(2)-R(-/-) airway epithelial cells, UTP was ineffective. A small fraction (25%) of the ATP response persisted. Adenosine and alpha,beta-methylene ATP were ineffective, and ATP responses were not affected by adenosine deaminase or by removal of extracellular Ca(2+), indicating that neither P1 nor P2X receptors mediated this residual ATP response. In contrast, 2-methylthio-ADP promoted a substantial Ca(2+)(i) response in P2Y(2)-R(-/-) cells, which was inhibited by the P2Y(1) receptor antagonist adenosine 3'-5'-diphosphate. These studies demonstrate that P2Y(2)-R is the dominant purinoceptor in airway epithelial cells, which also express a P2Y(1) receptor, and that the P2Y(2)-R is the sole purinergic receptor subtype mediating nucleotide-induced inositol lipid hydrolysis and Ca(2+) mobilization in mouse lung fibroblasts. (+info)