Association of the heart fatty acid-binding protein (FABP3) gene with milk traits in Manchega breed sheep

The ovine fatty acid-binding protein type 3 gene has been chosen as a functional candidate gene for milk traits. Two different single nucleotide polymorphisms (SNPs) of ovine FABP3 gene have been tested in a daughter design comprising 13 families. No association was found between estimated breeding values for milk yield, protein and fat contents (FC) and genotypes across families using anova and transmission disequilibrium test (TDT). In within-family analysis, one family showed a significant effect for FC. These results could indicate linkage disequilibrium between the FABP3 gene and a quantitative trait loci (QTL) for FC, with the heterozygous genotype associated with a positive effect in this trait.     

Phenotypic effect of the functioning of nucleolar-organizing regions of human chromosomes

Effects of functioning of nucleolar-organizing regions (NOR) of human chromosomes on a number of quantitative morphophysiological traits were studied. Sexual dimorphism of correlation of chromosomal functional polymorphism with some morphophysiological traits was revealed. Statistically significant positive correlations of ribosomal genes total activity with height, weight, chest circumference and diastolic blood pressure have been found in females but not in males. The data obtained suggest that functional activity of rRNA gene clusters in individuals of different sexes may exert different modifying effect on the level of expression of great number of genes responsible for some quantitative traits in man.     

Conformational effects of selected cancer-related amino acid substitutions in the p53 protein.

The tumor suppressor gene p53 has been identified as the most frequent target of genetic alterations in human cancers. Cancer-related mutations in the human p53 protein tend to cluster in four of the five highly conserved domains of the protein, and, in particular, in the central region of domain IV from residues 241 to 253. Using conformational energy analysis based on ECEPP (Empirical Conformational Energies for Polypeptides Program), we have determined the preferred three dimensional structures for this tridecapeptide sequence for the human wild-type p53 protein and four cancer-related mutant p53 proteins (Ala 245, Ile 246, Trp 248, Ser 249). The results show that the mutant peptides adopt conformations that are distinctly different from that of the wild-type peptide. These results are consistent with experimental conformational studies demonstrating altered detectability of antigenic epitopes in murine wild-type and mutant p53 proteins. These results suggest that the oncogenic effects of human mutant p53 proteins may be mediated by distinct local conformational changes in the protein.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

The frequency of the perfect genotype in a population subject to pleiotropic mutation

We consider a large population of asexual organisms characterised by a number of quantitative traits that are subject to stabilising selection. Mutation is taken to act pleiotropically, with every mutation generally changing all of the traits under selection. We focus on the equilibrium distribution of the population, where mutation and selection are in balance. It has been previously established that the equilibrium distribution of genotypic effects may be anomalous, as it may contain a singular spike--a Dirac delta function--corresponding to a non-zero proportion of the population having exactly optimal genotypic values. In the present work, we present exact results for the case where three traits are under selection. These results give the equilibrium genetic variance of the population, and the proportion of the population that have the optimal genotype. This is achieved for two different spherically symmetric distributions of mutant effects. Additionally, a simple and robust numerical approach is also presented that allows the treatment of some other mutation distributions, where there are an arbitrary number of selected traits.     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins

asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo-polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.     

Alanine-Scanning Mutagenesis of Protein Phosphatase Type 1 in the Yeast Saccharomyces Cerevisiae

Protein phosphatase type 1, encoded by GLC7 in Saccharomyces cerevisiae, is an essential serine/threonine phosphatase implicated in the regulation of a diverse array of physiological functions. We constructed and examined 20 mutant alleles of GLC7 in which codons encoding clusters of charged residues were changed to alanine codons. Three of 20 mutant alleles alter residues in the active site of the phosphatase and are unable to rescue the lethality of a glc7::LEU2 disruption. The 17 alleles that support growth confer a range of mutant traits including cell cycle arrest, 2-deoxyglucose resistance, altered levels of glycogen, sensitivity to high salt, and sporulation defects. For some traits, such as 2-deoxyglucose resistance and cell cycle arrest, the mutated residues map to specific regions of the protein whereas the mutated residues in glycogen-deficient mutants and sporulation-defective mutants are more widely distributed over the protein surface. Many mutants have complex phenotypes, each displaying a diverse range of defects. The wide range of phenotypes identified from the collection of mutant alleles is consistent with the hypothesis that Glc7p-binding proteins, which are thought to regulate the specificity of Glc7p, have overlapping binding sites on the surface of Glc7p. This could account for the high level of sequence conservation found among type 1 protein phosphatases from different species.     

Chemical- and Irradiation-induced Mutants of Indica Rice IR64 for Forward and Reverse Genetics

IR64, the most widely grown indicarice in South and Southeast Asia, possesses many positive agronomic characteristics (e.g., wide adaptability, high yield potential, tolerance to multiple diseases and pests, and good eating quality,) that make it an ideal genotype for identifying mutational changes in traits of agronomic importance. We have produced a large collection of chemical and irradiation-induced IR64 mutants with different genetic lesions that are amenable to both forward and reverse genetics. About 60,000 IR64 mutants have been generated by mutagenesis using chemicals (diepoxybutane and ethylmethanesulfonate) and irradiation (fast neutron and gamma ray). More than 38,000 independent lines have been advanced to M₄ generation enabling evaluation of quantitative traits by replicated trials. Morphological variations at vegetative and reproductive stages, including plant architecture, growth habit, pigmentation and various physiological characters, are commonly observed in the four mutagenized populations. Conditional mutants such as gain or loss of resistance to blast, bacterial blight, and tungro disease have been identified at frequencies ranging from 0.01% to 0.1%. Results from pilot experiments indicate that the mutant collections are suitable for reverse genetics through PCR-detection of deletions and TILLING. Furthermore, deletions can be detected using oligomer chips suggesting a general technique to pinpoint deletions when genome-wide oligomer chips are broadly available. M₄ mutant seeds are available for users for screening of altered response to multiple stresses. So far, more than 15,000 mutant lines have been distributed. To facilitate broad usage of the mutants, a mutant database has been constructed in the International Rice Information System (IRIS; http: //www.iris.irri.org) to document the phenotypes and gene function discovered by users.     

Quantitative variability of 342 plasma proteins in a human twin population

The degree and the origins of quantitative variability of most human plasma proteins are largely unknown. Because the twin study design provides a natural opportunity to estimate the relative contribution of heritability and environment to different traits in human population, we applied here the highly accurate and reproducible SWATH mass spectrometry technique to quantify 1,904 peptides defining 342 unique plasma proteins in 232 plasma samples collected longitudinally from pairs of monozygotic and dizygotic twins at intervals of 2–7 years, and proportioned the observed total quantitative variability to its root causes, genes, and environmental and longitudinal factors. The data indicate that different proteins show vastly different patterns of abundance variability among humans and that genetic control and longitudinal variation affect protein levels and biological processes to different degrees. The data further strongly suggest that the plasma concentrations of clinical biomarkers need to be calibrated against genetic and temporal factors. Moreover, we identified 13 cis‐SNPs significantly influencing the level of specific plasma proteins. These results therefore have immediate implications for the effective design of blood‐based biomarker studies.     

Alteration of an amino acid residue outside the active site of the ricin A chain reduces its toxicity towards yeast ribosomes

Summary Yeast transformants containing integrated copies of a galactose-regulated, ricin toxin A chain (RTA) expression plasmid were constructed and used in an attempt to isolate RTA-resistant yeast mutants. Analysis of RNA from mutant strains demonstrated that approximately half contained ribosomes that had been partially modified by RTA, although all the strains analysed transcribed full-length RTA RNA. The mutant strains could have mutations in yeast genes giving rise to RTA-resistant ribosomes or they could contain alterations within the RTA-encoding DNA causing production of mutant toxin. Ribosomes isolated from mutant strains were shown to be susceptible to RTA modification in vitro suggesting that the strains contain alterations in RTA. This paper describes the detailed analysis of one mutant strain which has a point mutation that changes serine 203 to asparagine in RTA protein. Although serine 203 lies outside the proposed active site of RTA its alteration leads to the production of RTA protein with a greatly reduced level of ribosome modifying activity. This decrease in activity apparently allows yeast cells to survive expression of RTA as only a proportion of the ribosomes become modified. We demonstrate that the mutant RTA preferentially modifies 26S rRNA in free 60S subunits and has lower catalytic activity compared with native RTA when produced in Escherichia coli. Such mutations provide a valuable means of identifying residues important in RTA catalysis and of further understanding the precise mechanism of action of RTA.     

Pervasive genetic integration directs the evolution of human skull shape

It has long been unclear whether the different derived cranial traits of modern humans evolved independently in response to separate selection pressures or whether they resulted from the inherent morphological integration throughout the skull. In a novel approach to this issue, we combine evolutionary quantitative genetics and geometric morphometrics to analyze genetic and phenotypic integration in human skull shape. We measured human skulls in the ossuary of Hallstatt (Austria), which offer a unique opportunity because they are associated with genealogical data. Our results indicate pronounced covariation of traits throughout the skull. Separate simulations of selection for localized shape changes corresponding to some of the principal derived characters of modern human skulls produced outcomes that were similar to each other and involved a joint response in all of these traits. The data for both genetic and phenotypic shape variation were not consistent with the hypothesis that the face, cranial base, and cranial vault are completely independent modules but relatively strongly integrated structures. These results indicate pervasive integration in the human skull and suggest a reinterpretation of the selective scenario for human evolution where the origin of any one of the derived characters may have facilitated the evolution of the others.     

Convergent analysis of cDNA and short oligomer microarrays, mouse null mutants and bioinformatics resources to study complex traits

Gene expression data sets have recently been exploited to study genetic factors that modulate complex traits. However, it has been challenging to establish a direct link between variation in patterns of gene expression and variation in higher order traits such as neuropharmacological responses and patterns of behavior. Here we illustrate an approach that combines gene expression data with new bioinformatics resources to discover genes that potentially modulate behavior. We have exploited three complementary genetic models to obtain convergent evidence that differential expression of a subset of genes and molecular pathways influences ethanol-induced conditioned taste aversion (CTA). As a first step, cDNA microarrays were used to compare gene expression profiles of two null mutant mouse lines with difference in ethanol-induced aversion. Mice lacking a functional copy of G protein-gated potassium channel subunit 2 (Girk2) show a decrease in the aversive effects of ethanol, whereas preproenkephalin (Penk) null mutant mice show the opposite response. We hypothesize that these behavioral differences are generated in part by alterations in expression downstream of the null alleles. We then exploited the WebQTL databases to examine the genetic covariance between mRNA expression levels and measurements of ethanol-induced CTA in BXD recombinant inbred (RI) strains. Finally, we identified a subset of genes and functional groups associated with ethanol-induced CTA in both null mutant lines and BXD RI strains. Collectively, these approaches highlight the phosphatidylinositol signaling pathway and identify several genes including protein kinase C beta isoform and preproenkephalin in regulation of ethanol- induced conditioned taste aversion. Our results point to the increasing potential of the convergent approach and biological databases to investigate genetic mechanisms of complex traits.     

Alterations of DNA and chromatin structures at telomeres and genetic instability in mouse cells defective in DNA polymerase alpha

Telomere length is controlled by a homeostatic mechanism that involves telomerase, telomere-associated proteins, and conventional replication machinery. Specifically, the coordinated actions of the lagging strand synthesis and telomerase have been argued. Although DNA polymerase alpha, an enzyme important for the lagging strand synthesis, has been indicated to function in telomere metabolism in yeasts and ciliates, it has not been characterized in higher eukaryotes. Here, we investigated the impact of compromised polymerase alpha activity on telomeres, using tsFT20 mouse mutant cells harboring a temperature-sensitive polymerase alpha mutant allele. When polymerase alpha was temperature-inducibly inactivated, we observed sequential events that included an initial extension of the G-tail followed by a marked increase in the overall telomere length occurring in telomerase-independent and -dependent manners, respectively. These alterations of telomeric DNA were accompanied by alterations of telomeric chromatin structures as revealed by quantitative chromatin immunoprecipitation and immunofluorescence analyses of TRF1 and POT1. Unexpectedly, polymerase alpha inhibition resulted in a significantly high incidence of Robertsonian chromosome fusions without noticeable increases in other types of chromosomal aberrations. These results indicate that although DNA polymerase alpha is essential for genome-wide DNA replication, hypomorphic activity leads to a rather specific spectrum of chromosomal abnormality.     

Design of the helix-turn-helix motif: nonlocal effects of quaternary structure in DNA recognition investigated by laser Raman spectroscopy

The operator-binding domain of phage lambda repressor provides a model for DNA recognition by the helix-turn-helix (HTH) motif. In the wild-type protein, dimerization is mediated by hydrophobic packing (of the dyad-related helix 5), which serves as an indirect determinant of operator affinity. The mutant repressor, Tyr88----Cys, forms an intersubunit disulfide linkage and exhibits enhancement of both structural stability and operator affinity. Yet the dimer-specific operator affinity of the mutant is 10-fold weaker than that of the wild-type (noncovalent) dimer, suggesting nonlocal effects of the intersubunit disulfide bond on HTH recognition (Sauer et al., 1986). To explore such nonlocal effects, we describe laser Raman studies of the Cys88 mutant repressor and its interaction with operator sites OL1 and OR3. The following results have been obtained: (i) Wild-type and mutant dimers exhibit similar secondary structures, indicated by quantitative comparison of Raman amide I and amide III bands. (ii) The engineered disulfide of the mutant lacks rigorous symmetry; we observe mainly the gauche/gauche/trans CC-S-S-CC rotamer. (iii) Remarkably, distinctive local and nonlocal differences are observed in the mechanisms of DNA recognition by wild-type and mutant repressors. These differences involve specific hydrogen-bonding interactions between the protein and DNA, including guanine N7 sites in the major groove of DNA, and alterations in DNA phosphodiester conformation induced by protein binding. We analyze these differences in relation to crystal structures of the wild-type dimer with and without bound DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ligand binding, reactivity and biological activity of a distal pocket mutant of neuroglobin

We have generated the Lys67Glu mutant form of neuroglobin. Experimental spectral studies are consistent with a six coordinate heme in which the distal histidine bond is stretched compared to the wild type protein. Carbon monoxide binding to the ferrous form of the mutant follows a hyperbolic concentration dependence limiting at the histidine dissociation rate of 0.7 s(-1). Further analysis indicates a significantly lowered histidine binding constant. Oxygen binding kinetic studies confirm the higher heme ligand dissociation level and indicate a p50 value for oxygen binding<1 mmHg. The ferrous form of the protein yields an oxygenated intermediate on reaction with oxygen. The rate of oxidation, by oxygen, follows a complex concentration dependence, consistent with the presence of two distinct oxidation mechanisms. A quantitative model for the two oxidation processes has been developed, which is consistent with a lowered distal histidine binding constant in the mutant form of the protein. These data suggest that the protein structure surrounding the heme site in neuroglobin limits access to external ligands and provides an energy barrier to the structural changes following ligand binding in this protein. However, the mutation does not appear to affect reactivity with cytochrome c and the anti-apoptotic activity of the mutant in human cells of neuronal origin is increased as compared to the wild type protein.     

The carboxyl-terminal extension of the precursor D1 protein of photosystem II is required for optimal photosynthetic performance of the cyanobacterium Synechocystis sp. PCC 6803

The D1 protein is an integral component of the photosystem II reaction center complex. In the cyanobacterium Synechocystis sp. PCC 6803, D1 is synthesized with a short 16-amino acids-long carboxyl-terminal extension. Removal of this extension is necessary to form active oxygen-evolving photosystem II centers. Our earlier studies have shown that this extension is cleaved by CtpA, a specific carboxyl-terminal processing protease. The amino acid sequence of the carboxyl-terminal extension is conserved among D1 proteins from different organisms, although at a level lower than that of the mature protein. In the present study we have analyzed a mutant strain of Synechocystis sp. PCC 6803 with a duplicated extension, and a second mutant that lacks the extension, to investigate the effects of these alterations on the function of the D1 protein in vivo. No significant difference in the growth rates, photosynthetic pigment composition, fluorescence induction, and oxygen evolution rates was observed between the mutants and the control strain. However, using long-term mixed culture growth analysis, we detected significant decreases in the fitness of these mutant strains. The presented data demonstrate that the carboxyl-terminal extension of the precursor D1 protein is required for optimal photosynthetic performance.     

Marker Effects in the Genetic Transduction of Tryptophan Mutants of E. COLI

Recombination frequencies have been determined in crosses involving 28 mutant strains for 20 of which the site of the alteration is known from studies of amino-acid substitutions in the protein products. Three of these mutants showed especially high frequencies of recombination when crossed to other single mutants or when crossed to a strain carrying two alterations at opposite ends of the trpA gene. There is no obvious molecular explanation of the high recombination of these three mutants. They include one missense mutant, one amber and one ochre. The low-frequency recombination mutants include all these same classes as well as frameshift mutants. There is nothing unique about the intragenic location of the high-recombination mutants; in each case there is at least one low-recombination mutant in the same codon.-Crosses involving mutants which were isolated in an altered wild type have shown that the behavior of a high-recombination mutant does not result from its molecular configuration alone, but from its combination with the homologous wild-type sequence from the other parent.-Several lines of evidence indicate that recombination in this system frequently involves closely-spaced double exchanges (about 40 codons apart).