Mapping economic trait loci for somatic cell score in Holstein cattle using microsatellite markers and selective genotyping

Marker-assisted selection (MAS) uses genetic marker genotypes to predict an animal's production potential and will provide additional selection information for progeny testing. With the discovery of highly polymorphic microsatellite markers, the tools now exist to begin the search for economic trait loci (ETL), which is the first step toward MAS. The objective of this study was to identify ETL for somatic cell score in an existing Holstein population. Using the granddaughter design, sons from seven grandsire families were genotyped with 20 autosomal microsatellites from five chromosomes (4, 8, 13, 17, 23), with an emphasis on chromosome 23, which is the location of the bovine major histocompatibility complex (BoLA). Selective genotyping was used to reduce the number of genotypes required, in which the 10 highest and 10 lowest sons from the phenotypic distribution curve were tested (140 sons in seven families). One marker (513), located near BoLA, showed evidence of an ETL in three of five polymorphic families. Additional sons were genotyped from the five families to estimate the effect and to compare selective and ‘complete’ genotyping. Both methods detected an ETL at marker 513, but in different families. This study provides evidence of the usefulness of microsatellite markers and the granddaughter design in the detection of ETL; however, additional markers need to be evaluated to determine the usefulness of selective genotyping. Based on the results from the 20 studied markers, the most likely position of a somatic cell score ETL lies near marker 513, located on chromosome 23.     

Signal transduction by the epidermal growth factor receptor is attenuated by a COOH-terminal domain serine phosphorylation site.

It has been proposed that the acute desensitization of epidermal growth factor receptor (EGF-R) function can be accounted for, in part, by the effect of EGF to increase phosphorylation of the receptor at Ser1046/7 (Countaway, J.L., Nairn, A.C., and Davis, R.J. (1992) J. Biol. Chem. 267, 1129-1140). Here, we show that the mutational removal of this phosphorylation site causes an activation of EGF-R function and a potentiation of signal transduction. The mechanism of potentiation results from 1) defective down-regulation of the EGF-R when cells are incubated with high concentrations of EGF; and 2) increased EGF-stimulated tyrosine phosphorylation. The increased EGF-stimulated phosphorylation is associated with an alteration of the apparent specificity of tyrosine phosphorylation and is independent of the down-regulation defect. Together, these data strongly support the hypothesis that Ser1046/7 is a biologically significant site of regulatory phosphorylation of the EGF-R.     

A top-down approach to mechanistic biological modeling: application to the single-chain antibody folding pathway

A top-down approach to mechanistic modeling of biological systems is presented and exemplified with the development of a hypothesis-driven mathematical model for single-chain antibody fragment (scFv) folding in Saccharomyces cerevisiae by mediators BiP and PDI. In this approach, model development starts with construction of the most basic mathematical model—typically consisting of predetermined or newly-elucidated biological behavior motifs—capable of reproducing desired biological behaviors. From this point, mechanistic detail is added incrementally and systematically, and the effects of each addition are evaluated. This approach follows the typical progression of experimental data availability in that higher-order, lumped measurements are often more prevalent initially than specific, mechanistic ones. It also necessarily provides the modeler with insight into the structural requirements and performance capabilities of the resulting detailed mechanistic model, which facilitates further analysis. The top-down approach to mechanistic modeling identified three such requirements and a branched dependency-degradation competition motif critical for the scFv folding model to reproduce experimentally observed scFv folding dependencies on BiP and PDI and increased production when both species are overexpressed and promoted straightforward prediction of parameter dependencies. It also prescribed modification of the guiding hypothesis to capture BiP and PDI synergy.     

Progress toward heterologous expression of active G‐protein‐coupled receptors in Saccharomyces cerevisiae: Linking cellular stress response with translocation and trafficking

High-level expression of mammalian G-protein-coupled receptors (GPCRs) is a necessary step toward biophysical characterization and high-resolution structure determination. Even though many heterologous expression systems have been used to express mammalian GPCRs at high levels, many receptors are improperly trafficked or are inactive in these systems. En route to engineering a robust microbial host for GPCR expression, we have investigated the expression of 12 GPCRs in the yeast Saccharomyces cerevisiae, where all receptors are expressed at the mg/L scale. However, only the human adenosine A₂a (hA₂aR) receptor is active for ligand-binding and located primarily at the plasma membrane, whereas other tested GPCRs are mainly retained within the cell. Selective receptors associate with BiP, an ER-resident chaperone, and activated the unfolded protein response (UPR) pathway, which suggests that a pool of receptors may be folded incorrectly. Leader sequence cleavage of the expressed receptors was complete for the hA₂aR, as expected, and partially cleaved for hA₂bR, hCCR5R, and hD_2LR. Ligand-binding assays conducted on the adenosine family (hA₁R, hA₂aR, hA₂bR, and hA₃R) of receptors show that hA₂aR and hA₂bR, the only adenosine receptors that demonstrate leader sequence processing, display activity. Taken together, these studies point to translocation as a critical limiting step in the production of active mammalian GPCRs in S. cerevisiae.     

Transgressing Wallace's Line brings hyperdiverse weevils down to earth

Wallace's Line, located in the heart of the Indo-Australian archipelago, has historically been hypothesized to strongly inhibit dispersal. Taxa crossing this barrier are confronted with different biota of Asian or Australian origin, respectively, but the extent to which these conditions have affected the evolution of the colonizing lineages remains largely unknown. We examined the potential correlations of body size, lifestyle and biogeographical distribution in the weevil genus Trigonopterus. These beetles are highly diverse both on foliage and in litter east of Wallace's Line but occur exclusively in leaf litter in the west. Based on a comprehensive, dated phylogeny of 303 species, we inferred nine crossing events of Wallace's Line, all from east to west. Five previously foliage-dwelling lineages changed their lifestyle to leaf litter habitats after crossing this barrier. Our results indicate that dispersal is not more likely in edaphic lineages, but rather that abiotic and/or biotic factors may be responsible for the exclusive leaf litter habitat of Trigonopterus in Sundaland. This includes differences in climate, and the different predatory faunas of Australia-New Guinea, Wallacea and Sundaland. A mimicry complex in New Guinea with Trigonopterus species as presumable model may be of relevance in this context.     

Signal Recognition Particle-dependent Targeting of Ribosomes to the Rough Endoplasmic Reticulum in the Absence and Presence of the Nascent Polypeptide-associated Complex

Proteins with RER-specific signal sequences are cotranslationally translocated across the rough endoplasmic reticulum through a proteinaceous channel composed of oligomers of the Sec61 complex. The Sec61 complex also binds ribosomes with high affinity. The dual function of the Sec61 complex necessitates a mechanism to prevent signal sequence-independent binding of ribosomes to the translocation channel. We have examined the hypothesis that the signal recognition particle (SRP) and the nascent polypeptide-associated complex (NAC), respectively, act as positive and negative regulatory factors to mediate the signal sequence-specific attachment of the ribosome-nascent chain complex (RNC) to the translocation channel. Here, SRP-independent translocation of a nascent secretory polypeptide was shown to occur in the presence of endogenous wheat germ or rabbit reticulocyte NAC. Furthermore, SRP markedly enhanced RNC binding to the translocation channel irrespective of the presence of NAC. Binding of RNCs, but not SRP-RNCs, to the Sec61 complex is competitively inhibited by 80S ribosomes. Thus, the SRP-dependent targeting pathway provides a mechanism for delivery of RNCs to the translocation channel that is not inhibited by the nonselective interaction between the ribosome and the Sec61 complex.     

Antinociceptive, anti-inflammatory and antipyretic properties of Melastoma malabathricum leaves aqueous extract in experimental animals

The present study was carried out to establish the antinociceptive, anti-inflammatory, and antipyretic properties of the aqueous extract of Melastoma malabathricum leaves in experimental animals. The antinociceptive activity was measured using abdominal constriction, hot-plate, and formalin tests, whereas the anti-inflammatory and antipyretic activities were measured using carrageenan-induced paw edema and brewer's yeast-induced pyrexia tests, respectively. The extract, which was obtained after soaking the air-dried leaves in distilled water for 72 h and then preparing in concentrations of 10%, 50%, and 100% (v/v), was administered subcutaneously 30 min prior to subjection to the above mentioned assays. At all concentrations tested, the extract was found to exhibit significant (P < 0.05) antinociceptive, anti-inflammatory, and antipyretic activities in a concentration-independent manner. Our findings that the aqueous extract of M. malabathricum possesses antinociceptive, anti-inflammatory, and antipyretic activities supports previous claims on its traditional uses to treat various ailments.     

Role of the cytoplasmic segments of Sec61alpha in the ribosome-binding and translocation-promoting activities of the Sec61 complex

The Sec61 complex performs a dual function in protein translocation across the RER, serving as both the high affinity ribosome receptor and the translocation channel. To define regions of the Sec61 complex that are involved in ribosome binding and translocation promotion, ribosome-stripped microsomes were subjected to limited digestions using proteases with different cleavage specificities. Protein immunoblot analysis using antibodies specific for the NH(2) and COOH terminus of Sec61alpha was used to map the location of proteolysis cleavage sites. We observed a striking correlation between the loss of binding activity for nontranslating ribosomes and the digestion of the COOH- terminal tail or cytoplasmic loop 8 of Sec61alpha. The proteolyzed microsomes were assayed for SRP-independent translocation activity to determine whether high affinity binding of the ribosome to the Sec61 complex is a prerequisite for nascent chain transport. Microsomes that do not bind nontranslating ribosomes at physiological ionic strength remain active in SRP-independent translocation, indicating that the ribosome binding and translocation promotion activities of the Sec61 complex do not strictly correlate. Translocation-promoting activity was most severely inhibited by cleavage of cytosolic loop 6, indicating that this segment is a critical determinant for this function of the Sec61 complex.     

The Saccharomyces cerevisiae YFR041C/ERJ5 gene encoding a type I membrane protein with a J domain is required to preserve the folding capacity of the endoplasmic reticulum

YFR041C/ERJ5 was identified in Saccharomyces cerevisiae as a gene regulated by the unfolded protein response pathway (UPR). The open reading frame of the gene has a J domain characteristic of the DnaJ chaperone family of proteins that regulate the activity of Hsp70 chaperones. We determined the expression and topology of Erj5p, a type I membrane protein with a J domain in the lumen of the endoplasmic reticulum (ER) that colocalizes with Kar2p, the major Hsp70 in the yeast ER. We identified synthetic interactions of Deltaerj5 with mutations in genes involved in protein folding in the ER (kar2-159, Deltascj1Deltajem1) and in the induction of the unfolded protein response (Deltaire1). Loss of Erj5p in yeast cells with impaired ER protein folding capacity increased sensitivity to agents that cause ER stress. We identified the ERJ5 mRNA and confirmed that agents that promote accumulation of misfolded proteins in the ER regulate its abundance. We found that loss of the non-essential ERJ5 gene leads to a constitutively induced UPR, indicating that ERJ5 is required for maintenance of an optimal folding environment in the yeast ER.     

Identification of substrate recognition determinants for human ERK1 and ERK2 protein kinases

Two epidermal growth factor-stimulated protein kinases that correspond to ERK1 and ERK2 have been purified from human epidermoid carcinoma cells (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). A consensus primary sequence for substrates of ERK1 has been identified as -Pro-Leu-Ser/Thr-Pro- (Alvarez, E., Northwood, I. C., Gonzalez, F. A., Latour, D. A., Seth, A., Abate, C., Curran, T., and Davis, R. J. (1991) J. Biol. Chem. 266, 15277-15285). However, the structural determinants for substrate recognition are not understood. We performed a systematic analysis of the effect of point mutations in the primary sequence of peptide substrates on the rate of phosphorylation by ERK1 and ERK2. The results of this investigation demonstrate that the substrate specificities of the ERK1 and ERK2 protein kinases are very similar. We propose that the primary sequence of substrates for ERK1 and ERK2 protein kinases can be generalized as -Pro-Xaan-Ser/Thr-Pro- (where Xaa is a neutral or basic amino acid and n = 1 or 2).