Biochemical and immunological studies of three genetic variants of 3-phosphoglycerate kinase 2 from the mouse

Three electrophoretic variants of 3-phosphoglycerate kinase 2 (PGK-2A, PGK-2B, and PGK-2C) were purified from DBA/2J, C3H/HeJ, and C57L/J mice, respectively. PGK-2C exhibits only 2% of the specific activity of PGK-2A and PGK-2B in the reaction leading to the formation of 1,3-diphosphoglycerate. Compared to PGK-2A and PGK-2B, PGK-2C exhibits broader coenzyme specificity and lower K_ms for substrate and coenzymes. Incubation at 45C revealed that PGK-2B is more heat stable than either PGK-2A or PGK-2C. Enzyme immunoinactivation and double immunodiffusion studies showed that mice carrying any one of these three PGK-2 alleles have similar amounts of proteins for PGK-1 and PGK-2 in testes. The results of these studies suggest that low PGK-2C activity in C57L/J mice is a result of a structural rather than a regulatory gene mutation.     

Fine Mapping Causal Variants with an Approximate Bayesian Method Using Marginal Test Statistics

Two recently developed fine-mapping methods, CAVIAR and PAINTOR, demonstrate better performance over other fine-mapping methods. They also have the advantage of using only the marginal test statistics and the correlation among SNPs. Both methods leverage the fact that the marginal test statistics asymptotically follow a multivariate normal distribution and are likelihood based. However, their relationship with Bayesian fine mapping, such as BIMBAM, is not clear. In this study, we first show that CAVIAR and BIMBAM are actually approximately equivalent to each other. This leads to a fine-mapping method using marginal test statistics in the Bayesian framework, which we call CAVIAR Bayes factor (CAVIARBF). Another advantage of the Bayesian framework is that it can answer both association and fine-mapping questions. We also used simulations to compare CAVIARBF with other methods under different numbers of causal variants. The results showed that both CAVIARBF and BIMBAM have better performance than PAINTOR and other methods. Compared to BIMBAM, CAVIARBF has the advantage of using only marginal test statistics and takes about one-quarter to one-fifth of the running time. We applied different methods on two independent cohorts of the same phenotype. Results showed that CAVIARBF, BIMBAM, and PAINTOR selected the same top 3 SNPs; however, CAVIARBF and BIMBAM had better consistency in selecting the top 10 ranked SNPs between the two cohorts. Software is available at https://bitbucket.org/Wenan/caviarbf.     

Proteome-Scale Analysis of Biochemical Activity

ABSTRACT

During the last 10 years, there has been a large increase in the number of genome sequences available for study, altering the way that the biology of organisms is studied. In particular, scientific attention has increasingly focused on the proteome, and specifically on the role of all the proteins encoded by the genome. We focus here on several aspects of this problem. We describe several technologies in widespread use to clone genes on a genome-wide scale, and to express and purify the proteins encoded by these genes. We also describe a number of methods that have been developed to analyze various biochemical properties of the proteins, with attention to the methodology and the limitations of the approaches, followed by a look at possible developments in the next decade.     

Biochemical and immunological characterization of genetic variants of phosphoglucose isomerase from mouse

Two electrophoretic variants of phosphoglucose isomerase (PGI) were purified from whole body extracts of DBA/2J and C57BL/6J mice by a substrate-affinity elution from an 8-(6-aminohexyl) amino-ATP-Sepharose column followed by preparative isoelectric focusing. Both PGI variants were shown to be dimers of the same molecular weight, sedimentation coefficient, and K _m for fructose-6-phosphate. The isoelectric points were found to be 8.4 and 8.7 for variants from DBA/2J and C57BL/6J mice, respectively. Differential thermal stability was observed for the two variants in 0.1 m tris-HCl buffer, pH 8.0, at 54 C; the half-lives of the purified PGI from DBA/2J and C57BL/6J mice were shown to be 3.4 and 1.8 min, respectively, under those conditions. Similar differences were observed for the enzyme variants in the crude homogenates. Antisera against PGI from DBA/2J mice were raised in rabbits. The variants from DBA/2J and C57BL/6J mice showed no significant differences in their respective inactivation curves by the antisera. Results of amino acid composition analyses and peptide mappings of the two PGI variants indicate that the genetic variation of this enzyme might result from a single charged amino acid substitution.D. J. C. is a National Institutes of Health Visiting Fellow.     

Phenotypic and genetic integration of personality and growth under competition in the sheepshead swordtail,Xiphophorus birchmanni

Competition for resources including food, physical space, and potential mates is a fundamental ecological process shaping variation in individual phenotype and fitness. The evolution of competitive ability, in particular social dominance, depends on genetic (co)variation among traits causal (e.g., behavior) or consequent (e.g., growth) to competitive outcomes. If dominance is heritable, it will generate both direct and indirect genetic effects (IGE) on resource‐dependent traits. The latter are expected to impose evolutionary constraint because winners necessarily gain resources at the expense of losers. We varied competition in a population of sheepshead swordtails, Xiphophorus birchmanni, to investigate effects on behavior, size, growth, and survival. We then applied quantitative genetic analyses to determine (i) whether competition leads to phenotypic and/or genetic integration of behavior with life history and (ii) the potential for IGE to constrain life history evolution. Size, growth, and survival were reduced at high competition. Male dominance was repeatable and dominant individuals show higher growth and survival. Additive genetic contributions to phenotypic covariance were significant, with the G matrix largely recapitulating phenotypic relationships. Social dominance has a low but significant heritability and is strongly genetically correlated with size and growth. Assuming causal dependence of growth on dominance, hidden IGE will therefore reduce evolutionary potential.     

Genome-wide functional screen of 3′UTR variants uncovers causal variants for human disease and evolution

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Now and future of mouse mutagenesis for human disease models

One of the major objectives of the Human Genome Project is to understand the biological function of the gene and genome as well as to develop clinical applications for human diseases. For this purpose, the experimental validations and preclinical trails by using animal models are indispensable. The mouse (Mus musculus) is one of the best animal models because genetics is well established in the mouse and embryonic manipulation technologies are also well developed. Large-scale mouse mutagenesis projects have been conducted to de-velop various mouse models since 1997. Originally, the phenotype-driven mutagenesis with N-ethyl-N-nitrosourea (ENU) has been the major efforts internationally then knockout/conditional mouse projects and gene-driven mutagenesis have been following. At the beginning, simple monogenic traits in the experimental condition have been elucidated. Then, more complex traits with variety of environmental interactions and gene-to-gene interactions (epistasis) have been challenged with mutant mice. In addition, chromosomal substitution swains and collaborative cross strains are also available to elucidate the complex Waits in the mouse. Altogether, mouse models with mutagenesis and various laboratory strains will accelerate the studies of functional genomics in the mouse as well as in human.     

Genetic Analysis and Mapping of Biochemical Markers in an F2 Intercross of Two Inbred Strains of the Rabbit (Oryctolagus cuniculus)

A total of 40 biochemical and four immunological markers found to be polymorphic in the rabbit in previous studies were screened in the AX/JU and IIIVO/JU inbred strains. Although the strains are considered unrelated, only eight (biochemical) markers were found to be polymorphic between the two strains. These eight markers were analyzed in an F₂ intercross population. Linkage was found for Est-5 and C on chromosome 1 and for Es-1, Est-2, Est-4, Est-6 and HP on linkage group VI. Two polymorphic markers, Es-3 and Mhr-1 could not be linked to any of the other markers.     

Phenotypic variation and covariation indicate high evolvability of acoustic communication in crickets

Studying the genetic architecture of sexual traits provides insight into the rate and direction at which traits can respond to selection. Traits associated with few loci and limited genetic and phenotypic constraints tend to evolve at high rates typically observed for secondary sexual characters. Here, we examined the genetic architecture of song traits and female song preferences in the field crickets Gryllus rubens and Gryllus texensis. Song and preference data were collected from both species and interspecific F1 and F2 hybrids. We first analysed phenotypic variation to examine interspecific differentiation and trait distributions in parental and hybrid generations. Then, the relative contribution of additive and additive‐dominance variation was estimated. Finally, phenotypic variance–covariance ( P ) matrices were estimated to evaluate the multivariate phenotype available for selection. Song traits and preferences had unimodal trait distributions, and hybrid offspring were intermediate with respect to the parents. We uncovered additive and dominance variation in song traits and preferences. For two song traits, we found evidence for X‐linked inheritance. On the one hand, the observed genetic architecture does not suggest rapid divergence, although sex linkage may have allowed for somewhat higher evolutionary rates. On the other hand, P matrices revealed that multivariate variation in song traits aligned with major dimensions in song preferences, suggesting a strong selection response. We also found strong covariance between the main traits that are sexually selected and traits that are not directly selected by females, providing an explanation for the striking multivariate divergence in male calling songs despite limited divergence in female preferences.     

Understanding the Origins of Loss of Protein Function by Analyzing the Effects of Thousands of Variants on Activity and Abundance

Understanding and predicting how amino acid substitutions affect proteins are keys to our basic understanding of protein function and evolution. Amino acid changes may affect protein function in a number of ways including direct perturbations of activity or indirect effects on protein folding and stability. We have analyzed 6,749 experimentally determined variant effects from multiplexed assays on abundance and activity in two proteins (NUDT15 and PTEN) to quantify these effects and find that a third of the variants cause loss of function, and about half of loss-of-function variants also have low cellular abundance. We analyze the structural and mechanistic origins of loss of function and use the experimental data to find residues important for enzymatic activity. We performed computational analyses of protein stability and evolutionary conservation and show how we may predict positions where variants cause loss of activity or abundance. In this way, our results link thermodynamic stability and evolutionary conservation to experimental studies of different properties of protein fitness landscapes.     

High-Resolution Mapping of Complex Traits with a Four-Parent Advanced Intercross Yeast Population

A large fraction of human complex trait heritability is due to a high number of variants with small marginal effects and their interactions with genotype and environment. Such alleles are more easily studied in model organisms, where environment, genetic makeup, and allele frequencies can be controlled. Here, we examine the effect of natural genetic variation on heritable traits in a very large pool of baker’s yeast from a multiparent 12th generation intercross. We selected four representative founder strains to produce the Saccharomyces Genome Resequencing Project (SGRP)-4X mapping population and sequenced 192 segregants to generate an accurate genetic map. Using these individuals, we mapped 25 loci linked to growth traits under heat stress, arsenite, and paraquat, the majority of which were best explained by a diverging phenotype caused by a single allele in one condition. By sequencing pooled DNA from millions of segregants grown under heat stress, we further identified 34 and 39 regions selected in haploid and diploid pools, respectively, with most of the selection against a single allele. While the most parsimonious model for the majority of loci mapped using either approach was the effect of an allele private to one founder, we could validate examples of pleiotropic effects and complex allelic series at a locus. SGRP-4X is a deeply characterized resource that provides a framework for powerful and high-resolution genetic analysis of yeast phenotypes and serves as a test bed for testing avenues to attack human complex traits.     

Extent of QTL Reuse During Repeated Phenotypic Divergence of Sympatric Threespine Stickleback

How predictable is the genetic basis of phenotypic adaptation? Answering this question begins by estimating the repeatability of adaptation at the genetic level. Here, we provide a comprehensive estimate of the repeatability of the genetic basis of adaptive phenotypic evolution in a natural system. We used quantitative trait locus (QTL) mapping to discover genomic regions controlling a large number of morphological traits that have diverged in parallel between pairs of threespine stickleback (Gasterosteus aculeatus species complex) in Paxton and Priest lakes, British Columbia. We found that nearly half of QTL affected the same traits in the same direction in both species pairs. Another 40% influenced a parallel phenotypic trait in one lake but not the other. The remaining 10% of QTL had phenotypic effects in opposite directions in the two species pairs. Similarity in the proportional contributions of all QTL to parallel trait differences was about 0.4. Surprisingly, QTL reuse was unrelated to phenotypic effect size. Our results indicate that repeated use of the same genomic regions is a pervasive feature of parallel phenotypic adaptation, at least in sticklebacks. Identifying the causes of this pattern would aid prediction of the genetic basis of phenotypic evolution.     

Coevolutionary genetics of hosts and parasites with quantitative inheritance

Summary A model of host—parasite coevolution is analysed. A host resistance trait and a parasite virulence trait interact to determine the outcome of a parasitic attack, where each trait is determined by quantitative genetic variation. The resistance and virulence traits are assumed to have a fitness cost. Each host and parasite genotype is treated as a separate species in a multidimensional Lotka—Volterra system in which the numerical abundance of each genotype is free to change. Thus, the epidemiological effects of fluctuating population sizes are analysed jointly with changes in genotype frequencies. Population sizes fluctuate increasingly as the parasites' reproductive capacity increases and as resistance and virulence benefits per unit cost decline. The patterns of genetic variability depend mainly on the stability of population sizes and on the shape of the relationship between the costs and benefits of a trait.     

Phenotypic plasticity and the evolution of trade-offs: the quantitative genetics of resource allocation in the wing dimorphic cricket, Gryllus firmus

In the wing dimorphic sand cricket, Gryllus firmus, there is a pronounced trade-off between flight capability and fecundity. This trade-off is found both between morphs and within the macropterous morph, in which fecundity is negatively correlated with the mass of the principle flight muscles, the dorso-longitudinal muscles (DLM). In this paper, we examine how this trade-off is affected by a reduction in food and its genetic basis. We find that the relative fitness of the two wing morphs is not changed although both fecundity and DLM mass are decreased. A quantitative genetic analysis shows that the trade-off function is genetically variable but that most of the variation occurs in the intercept rather than the slope of the function. Analysis further indicates a very high genetic correlation between environments (food ration) supporting the hypothesis of a strong functional constraint between reproduction and flight capability.     

Phenotypic integration in flowers of neotropical lianas: diversification of form with stasis of underlying patterns

Phenotypic integration is essential to the understanding of organismal evolution as a whole. In this study, a phylogenetic framework is used to assess phenotypic integration among the floral parts of a group of Neotropical lianas. Flowers consist of plant reproductive organs (carpels and stamens), usually surrounded by attractive whorls (petals and sepals). Thus, flower parts might be involved in different functions and developmental constraints, leading to conflicting selective forces. We found that Bignonieae flowers have very similar patterns of variance/covariance among traits and that such patterns are uncorrelated with the phylogenetic relationships between species. However, in spite of pattern stasis, our results also indicate that diversification of floral morphology in this group has occurred throughout the evolution of magnitudes of correlation among traits. Thus, we suggest that stabilizing selection has played an important role in phenotypic integration, resulting in the long‐term stasis of covariance patterns underlying flower diversification during the ca. 50 Myr of evolution of Bignonieae. This is the first report of long‐term stasis in the phenotypic integration of angiosperms, suggesting that patterns of floral morphology can be recognizable as specific attributes of distinct botanical families.     

短季棉早熟不早衰生化性状的遗传分析

用5个早熟不早衰的短季棉品种和5个早衰的短季棉品种进行双列杂交,并对亲本、F1和F2代于2001年和2002年田间试验研究与短季棉早熟不早衰有关的抗氧化系统保护酶(SOD、POD和CAT)、叶绿素及激素(生长素和脱落酸)的遗传特性。结果表明：抗氧化系统保护酶CAT、POD和SOD存在着不同的遗传特性,CAT酶以加性上位性效应为主,其次为显性效应;POD酶以加性效应为主,其次为加性上位性效应;SOD酶活以显性效应为主,其次为加性上位性效应;IAA以显性效应为主,其次为加性效应;ABA以加性效应为主,其次为显性效应。且这些生化性状的遗传率较高,在后代能稳定遗传;同时棉株在不同发育时期体内生化性状表达不同,在花铃期CAT、POD和SOD酶以显性效应为主,其次为加性上位性效应,加性效应表达量很小;棉株体内生化性状的表达也是相互联系、相互制约,CAT酶与POD酶存在着遗传和表型负相关,与SOD酶存在着遗传和表型正相关;POD酶与SOD酶存在着遗传和表型负相关;抗氧化系统保护酶与激素之间存在着复杂的遗传关系。因此,研究生化性状的遗传特性和表达特征,为选育短季棉早熟不早衰新品种和生化性状的QTLs定位提供理论依据。     

Differential intolerance to loss of function and missense mutations in genes that encode human matricellular proteins

Targeted gene disruption in mice has provided valuable insights into the functions of matricellular proteins. Apart from missense and loss of function mutations that have been associated with inherited diseases, however, their functions in humans remain unclear. The availability of deep exome sequencing data from over 140,000 individuals in the Genome Aggregation Database provided an opportunity to examine intolerance to loss of function and missense mutations in human matricellular genes. The probability of loss-of-function intolerance (pLI) differed widely within members of the thrombospondin, CYR61/CTGF/NOV (CCN), tenascin, small integrin-binding ligand N-linked glycoproteins (SIBLING), and secreted protein, acidic and rich in cysteine (SPARC) gene families. Notably, pLI values in humans had limited correlation with viability of the corresponding homozygous null mice. Among the thrombospondins, only THBS1 was highly loss-intolerant (pLI = 1). In contrast, Thbs1 is not essential for viability in mice. Several known thrombospondin-1 receptors were similarly loss-intolerant, although thrombospondin-1 is not the exclusive ligand for some of these receptors. The frequencies of missense mutations in THBS1 and the gene encoding its signaling receptor CD47 indicated conservation of some residues implicated in specific receptor binding. Deficits in missense mutations were also observed for other thrombospondin genes and for SPARC, SPOCK1, SPOCK2, TNR, and DSPP. The intolerance of THBS1 to loss of function in humans and elevated pLI values for THBS2, SPARC, SPOCK1, TNR, and CCN1 support important functions for these matricellular protein genes in humans, some of which may relate to functions in reproduction or responding to environmental stresses.