A haplotype-based algorithm for multilocus linkage disequilibrium mapping of quantitative trait loci with epistasis

For tightly linked loci, cosegregation may lead to nonrandom associations between alleles in a population. Because of its evolutionary relationship with linkage, this phenomenon is called linkage disequilibrium. Today, linkage disequilibrium-based mapping has become a major focus of recent genome research into mapping complex traits. In this article, we present a new statistical method for mapping quantitative trait loci (QTL) of additive, dominant, and epistatic effects in equilibrium natural populations. Our method is based on haplotype analysis of multilocus linkage disequilibrium and exhibits two significant advantages over current disequilibrium mapping methods. First, we have derived closed-form solutions for estimating the marker-QTL haplotype frequencies within the maximum-likelihood framework implemented by the EM algorithm. The allele frequencies of putative QTL and their linkage disequilibria with the markers are estimated by solving a system of regular equations. This procedure has significantly improved the computational efficiency and the precision of parameter estimation. Second, our method can detect marker-QTL disequilibria of different orders and QTL epistatic interactions of various kinds on the basis of a multilocus analysis. This can not only enhance the precision of parameter estimation, but also make it possible to perform whole-genome association studies. We carried out extensive simulation studies to examine the robustness and statistical performance of our method. The application of the new method was validated using a case study from humans, in which we successfully detected significant QTL affecting human body heights. Finally, we discuss the implications of our method for genome projects and its extension to a broader circumstance. The computer program for the method proposed in this article is available at the webpage http://www.ifasstat.ufl.edu/genome/~LD.     

Zinc-induced decrease of the thermal stability and regeneration of rhodopsin

Zinc is present at high concentrations in the photoreceptor cells of the retina where it has been proposed to play a role in the visual phototransduction process. In order to obtain more information about this role, the study of the effect of zinc on several properties of the visual photoreceptor rhodopsin has been investigated. A specific effect of Zn(2+) on the thermal stability of rhodopsin, obtained from bovine retinas and solubilized in dodecyl maltoside detergent, in the dark is reported. The thermal stability of rhodopsin in its ground state (dark state) is clearly reduced with increasing Zn(2+) concentrations (0-50 microm Zn(2+)). The thermal bleaching process is accelerated in the presence of Zn(2+) with k rate constants, at 55 degrees C, of 0.028 +/- 0.002 min(-1) (0 microm Zn(2+)) and 0.056 +/- 0.003 min(-1) (50 microm Zn(2+)), corresponding to t(12) values of 24.4 +/- 1.6 min and 11.8 +/- 0.1 min, respectively. Thermodynamic parameters derived from Arrhenius plots show a significant E(a) increase at 50 microm Zn(2+) for the process, with deltaG++ decrease and increase in deltaH++ and deltaS++ possibly reflecting conformational rearrangements and reordering of water molecules. The stability of the metarhodopsin II intermediate is also decreased and changes in the metarhodopsin II decay pathway are also detected. The extent of rhodopsin regeneration in vitro is also reduced by zinc. These effects, specific for zinc, are also seen for rhodopsin in native disc membranes, and may be relevant to the suggested role of Zn(2+) in normal and pathological retinal function.     

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor,target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.     

Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay

Cryptosporidium parvum oocysts were stored in 1-ml aliquots of filtered river water at -20, 4, 10, and 21-23 C in the dark. Oocysts were also added to filter-sterilized river water samples and stored at 21-23 C. The infectivity of oocysts stored under different conditions was assayed at weekly intervals through infection of human adenocarcinoma ileocecal (HCT-8) cell monolayers. Wells containing between 10 and 100 foci of infection were enumerated by immunofluorescent microscopy, and the number of infective oocysts was calculated. No infectious oocysts were detected after 1 wk at -20 C. The number of infective oocysts stored at 4 C decreased 5-fold, and the number of those stored at 10 C decreased 2.5-fold after 14 wk. The infectivity of oocysts stored in potassium dichromate (positive control) at 4 C decreased 2-fold over 14 wk. The number of infective oocysts in filter-sterilized and non-filter-sterilized river water stored at 21-23 C decreased by 3.3 and 2.6 log units, respectively, over 12 wk, and no foci of infection were detected at 14 wk. The results show that as temperature increased from 4 to 23 C, the duration of oocyst infectivity decreased.     

Ashworthius Patriciapilittae n. sp. (Trichostrongyloidea: Haemonchinae), an abomasal nematode in Odocoileus virginianus from Costa Rica,and a new record for species of the genus in the Western Hemisphere

A species of Ashworthius is reported for the first time in the Western Hemisphere, and A. patriciapilittae n. sp. is described on the basis of specimens in white-tailed deer Odocoileus virginianus from Costa Rica. Among 8 known species, A. patriciapilittae is morphologically similar to A. tuyenquangi in red muntjac Muntjacas muntjak from northern Vietnam. The synlophe in A. patriciapilittae is composed of 26 ridges in the cervical zone and is continuous to the caudal extremity in males and females. Males are characterized by a complex dorsal ray and narrow trifurcate spicules (351-356 microm long) lacking an "eyelet." with dissimilar ventral and dorsal processes; the gubernaculum is 45-48% of the spicule length. Females have a prominent linguiform flap at the vulva and large eggs (108-142 microm long). The presence of A. patriciapilittae in Costa Rica is examined in the context of competing hypotheses for cospeciation or contemporary host-switching in cervids: either A. patriciapilittae is a component of an endemic Central and South American fauna that has diversified through coevolution of Ashworthius and cervid hosts or it has been introduced. Among haemonchines in the Western Hemisphere, specimens of A. patriciapilittae may be confused with 3 species of Haemonchus, including H. contortus, H. placei, and H. similis, that occur in both domestic and wild ruminants. Discovery of A. patriciapilittae emphasizes the continued need for survey and inventory to define the structure and distribution of parasite faunas in wild and domestic ruminants from the Nearctic and Neotropical regions.     

Kinetic model of BiP- and PDI-mediated protein folding and assembly

A mechanism for heavy chain binding protein (BiP)- and protein disulfide isomerase (PDI)- mediated protein folding and assembly has been proposed. It considers BiP chaperoning action and PDI catalytic activity. A kinetic model has been developed based on the proposed mechanism. The model was used for quantifying the influence of polypeptide concentration and ratio, and the effect of BiP and PDI concentration on the kinetics of folding and assembly. An optimum value for polypeptide concentration that minimizes assembly times was found, and different kinetic behaviors were identified for polypeptide concentrations higher or lower than the optimum. Pulse-chase experiments and the dependence of assembly time on unassembled polypeptides ratio predicted by the model are similar to those found during in vitro and in vivo folding and assembly of antibodies and human chorionic gonadotropin (hCG), as well as bovine pancreatic trypsin inhibitor (BPTI) folding. The model also explains the increase in folding and assembly rates during overexpression of BiP and PDI.     

Neither agouti-related protein nor neuropeptide Y is critically required for the regulation of energy homeostasis in mice

Agouti-related protein (AgRP), a neuropeptide abundantly expressed in the arcuate nucleus of the hypothalamus, potently stimulates feeding and body weight gain in rodents. AgRP is believed to exert its effects through the blockade of signaling by alpha-melanocyte-stimulating hormone at central nervous system (CNS) melanocortin-3 receptor (Mc3r) and Mc4r. We generated AgRP-deficient (Agrp(-/-)) mice to examine the physiological role of AgRP. Agrp(-/-) mice are viable and exhibit normal locomotor activity, growth rates, body composition, and food intake. Additionally, Agrp(-/-) mice display normal responses to starvation, diet-induced obesity, and the administration of exogenous leptin or neuropeptide Y (NPY). In situ hybridization failed to detect altered CNS expression levels for proopiomelanocortin, Mc3r, Mc4r, or NPY mRNAs in Agrp(-/-) mice. As AgRP and the orexigenic peptide NPY are coexpressed in neurons of the arcuate nucleus, we generated AgRP and NPY double-knockout (Agrp(-/-);Npy(-/-)) mice to determine whether NPY or AgRP plays a compensatory role in Agrp(-/-) or NPY-deficient (Npy(-/-)) mice, respectively. Similarly to mice deficient in either AgRP or NPY, Agrp(-/-);Npy(-/-) mice suffer no obvious feeding or body weight deficits and maintain a normal response to starvation. Our results demonstrate that neither AgRP nor NPY is a critically required orexigenic factor, suggesting that other pathways capable of regulating energy homeostasis can compensate for the loss of both AgRP and NPY.     

Properties of triple helices formed by parallel-stranded hairpins containing 8-aminopurines

Parallel-stranded hairpins with a polypyrimidine sequence linked to a complementary purine carrying 8-aminopurines such as 8-aminoadenine, 8-aminoguanine and 8-aminohypoxanthine bind polypyrimidine sequences complementary (in an antiparallel sense) to the purine part by a triple helix. The relative stabilities of triplexes were assessed by UV-absorption melting experiments as a function of pH and salt concentration. Hairpins carrying 8-aminopurines give very stable triple helical structures even at neutral pH, as confirmed by gel-shift experiments, circular dichroism and nuclear magnetic resonance spectroscopy. The modified hairpins may be redesigned to cope with small interruptions in the polypyrimidine target sequence.