Model of ribosomal translocation coupled with intra- and inter-subunit rotations

The ribosomal translocation involves both intersubunit rotations between the small 30S and large 50S subunits and the intrasubunit rotations of the 30S head relative to the 30S body. However, the detailed molecular mechanism on how the intersubunit and intrasubunit rotations are related to the translocation remains unclear. Here, based on available structural data a model is proposed for the ribosomal translocation, into which both the intersubunit and intrasubunit rotations are incorporated. With the model, we provide quantitative explanations of in vitro experimental data showing the biphasic character in the fluorescence change associated with the mRNA translocation and the character of a rapid increase that is followed by a slow single-exponential decrease in the fluorescence change associated with the 30S head rotation. The calculated translation rate is also consistent with the in vitro single-molecule experimental data.     

A simple network thermodynamic method for series-parallel coupled flows III. Application to coupled solute and volume flows through epithelial membranes

Using the network thermodynamic methods presented in the first two papers in this series (Mikulecky, Wiegand & Shiner, 1977; Mikulecky, 1977), an application to the coupled flows of salt and volume through epithelial membranes is developed. In another paper, the kidney proximal tubule is treated as a specific example (Thomas & Mikulecky,1978; Thomas, 1977). Two different experimental situations are examined in detail. The first is the case of transport across an epithelium with no external driving forces and a pump; the second is the case with transepithelial driving forces for both volume flow and solute flow but with no pump. For the linear network, the result for the most general case with a pump and transepithelial driving forces is obtained by superposition.The results obtained for the linear model lead to a number of interesting conclusions. The composition of transported fluid is independent of pump rate but does depend on the characteristics of the various membranes in the epithelium. The link between the genetic determination of epithelial membranes and the tonicity of blood is discussed. The absence of a need for a “standing gradient” in the interstitial space is clearly demonstrated, although the actual tonicity of that space can play a significant role, as is shown in detail in the work on the kidney proximal tubule. The topological aspects of the experimental design affect the manifestation of Onsager reciprocity in different parts of the system. Also, much of the epithelial function can be seen in terms of simple network concepts, such as the current divider properties of parallel pathways. The changes in topology brought about by different experimental design can be seen to alter these current-division patterns.     

多重等位基因特异性扩增—— 微流控芯片电泳法同时测定多个单核苷酸多态性位点

文章以微流控芯片电泳为检测平台, 建立了一种基于DNA适配器连接介导的多重等位基因特异性扩增同时测定多个单核苷酸多态性(SNP)位点的方法。以白细胞介素1β(IL1B)基因中的7个SNP位点(794C>T、1274C>T、2143T>C、2766T>del、3298G>A、5200G>A和5277C>T)为检测对象, 通过PCR预扩增得一段含该7个待测SNP位点的长片段; 用限制性内切酶MboⅠ将其消化成短片段, 再与DNA适配器(adapter)相连; 以连接产物为模板, 在两管中分别用7条等位基因特异性引物和一条公用引物进行7重等位基因特异性扩增; 最后用微流控芯片电泳法分离等位基因特异性扩增产物, 根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。采用本法成功测定了48名健康中国人的IL1B基因上的7个SNP位点, 与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)和测序法测定结果完全一致。本法结果准确, 可用于同时测定多个SNP位点; 以微流控芯片电泳作为检测平台, 分析速度快, 样品需要量少; 借助于自制筛分凝胶和重复使用芯片, 使得SNP分析成本大大降低。     

Analysis of mouse embryonic gene library for the frequency of single and multiple copy genes

A gene library was constructed from embryonic mouse DNA by ligating DNA fragments generated by partialEco RI digestion with Charon 4A vector andin vitro packaging. A special consideration was given to randomization of target DNA. The general applicability of a gene library prepared in this manner was assessed through cloning a variety of genes of known reiteration frequency in the mouse genome. The survey included a single copy gene — C region of the immunoglobulin heavy chain, and genes that appear in more than one copy — V region of the immunoglobulin light chain genes and the endogenous retrovirus related genes. In all cases tested the frequency of clone isolation was in good agreement with the expected incidence based on the number of genome equivalents screened and the reiteration frequency of that particular gene. Moreover, we found no preference with regard to the clonability of genes contained in fragments of a wide-size range.Abbreviations kb kilobases - pfu plaque forming unit - poly(A) polyadenilic acid - Mo-MuL V Moloney strain of murine leukemia virus     

Bayesian inference for kappa from single and multiple studies

Cohen's kappa coefficient is a widely popular measure for chance-corrected nominal scale agreement between two raters. This article describes Bayesian analysis for kappa that can be routinely implemented using Markov chain Monte Carlo (MCMC) methodology. We consider the case of m > or = 2 independent samples of measured agreement, where in each sample a given subject is rated by two rating protocols on a binary scale. A major focus here is on testing the homogeneity of the kappa coefficient across the different samples. The existing frequentist tests for this case assume exchangeability of rating protocols, whereas our proposed Bayesian test does not make any such assumption. Extensive simulation is carried out to compare the performances of the Bayesian and the frequentist tests. The developed methodology is illustrated using data from a clinical trial in ophthalmology.     

Biochemical and functional evidence for the cosecretion of multiple messengers from single and multiple compartments

Examination of the secretory profile and subcellular localization of some of the multiple export products of the adrenal medullary chromaffin cells indicates that several compartments (chromaffin vesicle, lysosomes, endoplasmic reticulum) are coupled to specific receptors and to cell depolarization through Ca2+-dependent mechanism(s). The activation of the release process results in the concerted cosecretion of endogenous catecholamines, newly incorporated catecholamines, adenine nucleotides, chromogranins, dopamine beta-hydroxylase (EC 1.14.17.1), enkephalins and related opioid peptides, stored ascorbate and newly incorporated ascorbate, lysosomal hydrolases, and soluble acetylcholinesterase. This complex organization for the coexistence of these multiple putative messengers and their cosecretion may be relevant to other endocrine cells and neurons where coexistence of transmitters has been found. This coexistence in multiple secretory compartments may provide the subcellular basis for independent regulation of the synthesis, packaging, and secretion of individual transmitters within the multiplicity of putative messengers secreted by a particular endocrine cell or nerve terminal.     

Comparative informativeness for linkage of multiple SNPs and single microsatellites

Single nucleotide polymorphisms (SNPs), or biallelic markers, are popular in genetic linkage studies due to their abundance in the genome, stability, and ease of scoring. We determined the 'information ratio' (IR) of closely spaced SNPs in simulated nuclear families and affected sib pairs (ASPs). (The IR is the ratio of actual average maximum lod score to the maximum lod score attainable if the marker were fully informative.) The nuclear families included parental information, whereas the ASPs did not. We analyzed these SNPs in two ways: (1) using multipoint analysis, and (2) treating the SNPs as 'composite markers' (i.e., haplotypes, as assigned by GENEHUNTER). (3) We also calculated the IR of a single microsatellite marker with multiple alleles and compared with the IR from the SNPs. For each set of input conditions, we simulated 1000 nuclear families, of 2, 3, 4, or 5 children each, as well as 1000 ASPs. We generated SNP marker data for strings of k = 1, 2, 3, 5, 7, and 10 SNP loci, with no recombination (theta = 0) and no linkage disequilibrium among the SNPs. The MAF (minor allele frequency) was either 0.5 or 0.25, and allele frequencies were the same for all k loci in any analysis. We also generated marker data for one single-locus microsatellite marker, with m = 3, 4, 5, 6, 7, and 9 equally frequent alleles. In all simulations, the disease was fully penetrant dominant, and there was no recombination or linkage disequilibrium among markers or between marker and disease. When multipoint analysis was used, we found that 5-7 closely spaced SNPs were usually enough to yield an IR of approximately 100%, for nuclear families of any size. However, for the ASPs, even 7-10 SNPs yielded an IR of only 70-80%. A microsatellite with 9 equally frequent alleles yielded about the same IR (86-88%) as a string of 4-5 SNPs, in nuclear families. SNPs analyzed as 'composite markers' analyses performed worse, due to the inherent ambiguity of SNP haplotyping.     

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

We describe solid-phase cloning (SPC) for high-throughput assembly of expression plasmids. Our method allows PCR products to be put directly into a liquid handler for capture and purification using paramagnetic streptavidin beads and conversion into constructs by subsequent cloning reactions. We present a robust automated protocol for restriction enzyme based SPC and its performance for the cloning of >60 000 unique human gene fragments into expression vectors. In addition, we report on SPC-based single-strand assembly for applications where exact control of the sequence between fragments is needed or where multiple inserts are to be assembled. In this approach, the solid support allows for head-to-tail assembly of DNA fragments based on hybridization and polymerase fill-in. The usefulness of head-to-tail SPC was demonstrated by assembly of >150 constructs with up to four DNA parts at an average success rate above 80%. We report on several applications for SPC and we suggest it to be particularly suitable for high-throughput efforts using laboratory workstations.     

Estimating haplotype frequencies and standard errors for multiple single nucleotide polymorphisms

Estimating haplotype frequencies becomes increasingly important in the mapping of complex disease genes, as millions of single nucleotide polymorphisms (SNPs) are being identified and genotyped. When genotypes at multiple SNP loci are gathered from unrelated individuals, haplotype frequencies can be accurately estimated using expectation-maximization (EM) algorithms (Excoffier and Slatkin, 1995; Hawley and Kidd, 1995; Long et al., 1995), with standard errors estimated using bootstraps. However, because the number of possible haplotypes increases exponentially with the number of SNPs, handling data with a large number of SNPs poses a computational challenge for the EM methods and for other haplotype inference methods. To solve this problem, Niu and colleagues, in their Bayesian haplotype inference paper (Niu et al., 2002), introduced a computational algorithm called progressive ligation (PL). But their Bayesian method has a limitation on the number of subjects (no more than 100 subjects in the current implementation of the method). In this paper, we propose a new method in which we use the same likelihood formulation as in Excoffier and Slatkin's EM algorithm and apply the estimating equation idea and the PL computational algorithm with some modifications. Our proposed method can handle data sets with large number of SNPs as well as large numbers of subjects. Simultaneously, our method estimates standard errors efficiently, using the sandwich-estimate from the estimating equation, rather than the bootstrap method. Additionally, our method admits missing data and produces valid estimates of parameters and their standard errors under the assumption that the missing genotypes are missing at random in the sense defined by Rubin (1976).     

Evidence for an intra- and extraplastidic pre-chorismate pathway

Pea plants grown under different conditions of cultivation, and eight different plant species with variegated leaves were used to study the intracellular localization of shikimate oxidoreductase (EC 1.1.1.25), the marker enzyme of the pre-chorismate pathway. The two series of experiments indicated an intra-and an extraplastidic compartimentalization of the enzyme, and both enzyme activities are regulated differentially. While the extraplastidic activity is permanently demonstrable, the intraplastidic activity is subject to the plants' developmental state and also depends on both illumination and fertilization.     

APOLLO: a quality assessment service for single and multiple protein models

SUMMARY: We built a web server named APOLLO, which can evaluate the absolute global and local qualities of a single protein model using machine learning methods or the global and local qualities of a pool of models using a pair-wise comparison approach. Based on our evaluations on 107 CASP9 (Critical Assessment of Techniques for Protein Structure Prediction) targets, the predicted quality scores generated from our machine learning and pair-wise methods have an average per-target correlation of 0.671 and 0.917, respectively, with the true model quality scores. Based on our test on 92 CASP9 targets, our predicted absolute local qualities have an average difference of 2.60 ? with the actual distances to native structure. AVAILABILITY: http://sysbio.rnet.missouri.edu/apollo/. Single and pair-wise global quality assessment software is also available at the site.     

A simple network thermodynamic method for modeling series-parallel coupled flows. I. The linear case

Photosynthetic CO₂ uptake in leaf mesophyll can be modelled in a general way using a second-order partial differential equation, which is derived and discussed and compared with existing models using finite series-parallel combinations of a few resistance elements.When mesophyll porosity is constant and when local CO₂ uptake is proportional to intercellular CO₂ concentration C, the equation takes the form Δ²C = α²C. Analytic solutions are given for this special case, with the boundary condition being equivalence of internal CO₂ uptake with that moving through the stomates. These solutions show the importance of accounting for the three-dimensional nature of CO₂ transport in the mesophyll, which most existing models do not do.The model is useful for exploring optimal geometry of leaves. Its implications for leaf thickness, stomatal size and spacing, and mesophyll porosity are discussed. In particular, it yields a prediction of optimal leaf thickness very close to values commonly occurring in nature.     

Measures of transinformation for multiple input/single output neuronal systems

This paper presents a method for the calculation of the information transfer, or transinformation, in multiple input/single output neuronal systems. Our approach is an extension of an approach introduced by Eckhorn and Poepel in 1974. These authors computed the transinformation in single input/single output neuronal channels by regarding the spike (or stimulus) trains involved as finite Markov chains. The expressions for multiple input systems presented here are derived in close analogy to formulae in linear systems theory which show explicity the correlations between the different input channels. A number of equivalent forms for the transinformation are discussed.     

Genome-wide identification of allele-specific effects on gene expression for single and multiple individuals

The analysis of allele-specific gene expression (ASE) is essential for the mapping of genetic variants that affect gene regulation, and for the identification of alleles that modify disease risk. Although RNA sequencing offers the opportunity to measure expression at allele levels, the availability of powerful statistical methods for mapping ASE in single or multiple individuals is limited. We developed a maximum likelihood model to characterize ASE in the human genome. Approximately 17% of genes displayed an allele-specific effect on gene expression in a single individual. Simulations using our model gave a better performance and improved robustness when compared with the binomial test, with different coverage levels, allelic expression fractions and random noise. In addition, our method can identify ASE in multiple individuals, with enhanced performance. This is helpful in understanding the mechanism of genetic regulation leading to expression changes, alternative splicing variants and even disease susceptibility.     

Host genetic architecture in single and multiple infections

Hosts are often target to multiple simultaneous infections by genetically diverse parasite strains. The interaction among these strains and the interaction of each strain with the host was shown to have profound effects on the evolution of parasite traits. Host factors like genetic architecture of resistance have so far been largely neglected. To see whether genetic architecture differs between different kinds of infections we used joint scaling analysis to compare the genetic components of resistance in the red flour beetle Tribolium castaneum exposed to single and multiple strains of the microsporidian Nosema whitei. Our results indicate that additive, dominance and epistatic components were more important in single infections whereas maternal components play a decisive role in multiple infections. In detail, parameter estimates of additive, dominance and epistatic components correlated positively between single and multiple infections, whereas maternal components correlated negatively. These findings may suggest that specificity of host–parasite interactions are mediated by genetic and especially epistatic components whereas maternal effects constitute a more general form of resistance.