Single amino acid substitutions in procollagen VII affect early stages of assembly of anchoring fibrils

Procollagen VII is a homotrimer of 350-kDa pro-alpha1(VII) chains, each consisting of a central collagenous domain flanked by the noncollagenous N-terminal NC1 domain and the C-terminal NC2 domain. After secretion from cells, procollagen VII molecules form anti-parallel dimers with a C-terminal 60-nm overlap. Characteristic alignment of procollagen VII monomers forming a dimer depends on site-specific binding between the NC2 domain and the triple-helical region adjacent to Cys-2634 of the interacting procollagen VII molecules. Formation of the intermolecular disulfide bonds between Cys-2634 and either Cys-2802 or Cys-2804 is promoted by the cleavage of the NC2 domain by procollagen C-proteinase. By employing recombinant procollagen VII variants harboring G2575R, R2622Q, or G2623C substitutions previously disclosed in patients with dystrophic epidermolysis bullosa, we studied how these amino acid substitutions affect intermolecular interactions. Binding assays utilizing an optical biosensor demonstrated that the G2575R substitution increased affinity between mutant molecules. In contrast, homotypic binding between the R2622Q or G2623C molecules was not detected. In addition, kinetics of heterotypic binding of all analyzed mutants to wild type collagen VII were different from those for binding between wild type molecules. Moreover, solid-state binding assays demonstrated that R2622Q and G2623C substitutions prevent formation of stable assemblies of procollagen C-proteinase-processed mutants. These results indicate that single amino acid substitutions in procollagen VII alter its self-assembly and provide a basis for understanding the pathomechanisms leading from mutations in the COL7A1 gene to fragility of the dermal-epidermal junction seen in patients with dystrophic forms of epidermolysis bullosa.     

Egg-laying preference of female fungus gnat Bradysia sp. nr. coprophila (Diptera: Sciaridae) on three different soilless substrates

Fungus gnats, Bradysia spp., are major insect pests in greenhouses. Adult female fungus gnats prefer to lay eggs in growing medium that is microbially active or that contains high amounts of peat moss or hardwood bark. However, egg-laying preference has not been demonstrated quantitatively. This study was designed to determine whether fungus gnat Bradysia sp. nr. coprophila females prefer any of the three soilless growing media provided. The three soilless growing media tested were Metro-Mix 560 with Scott's Coir, Sunshine LC1 Mix, and Universal SB 300 Mix. Initially, the egg-laying potential of the fungus gnat species used in this study was assessed by dissecting mated females after 24, 48, and 72 h. For the egg-laying preference experiment, adults that emerged from pupae were aspirated into a plastic vial, sexed, and then allowed to mate for 24 h. Individual mated females were released into an experimental chamber (15 by 15 by 5-cm plastic container) consisting of four 6-cm petri dishes, three of which contained soilless growing media and one with filter paper (control). In total, there were 50 experimental chambers, with each chamber representing a replication. Females remained in the experimental chambers for 48 h after which the growing media were processed using a flotation/extraction method. The number of eggs laid by female fungus gnats ranged from 21 to 217 with most eggs recovered after 48 h (141.0 +/- 9.3). There were no significant differences among the three soilless growing media in terms of number of eggs laid, although all three growing media were significantly different from the filter paper with higher numbers of eggs laid in the soilless growing media than the filter paper. Despite no significant difference among the growing media in the number of eggs laid, fungus gnat females tended to lay eggs more often, based on the number of petri dishes in which at least one egg was laid, in Metro-Mix 560 (86%) than Sunshine LC1 (66%), Universal SB 300 (52%), or filter paper (18%). Based on the results of this study, female fungus gnats may not prefer a specific growing medium for oviposition. However, fungus gnat females may rely on other factors not tested in this study such as moisture content and volatiles emitted from growing media in their decision where to lay eggs.     

Endothelial stress induces the release of vitamin D-binding protein, a novel growth factor

Endothelial cells (EC) under stress release paracrine mediators that facilitate accumulation of vascular smooth muscle cells (VSCM) at sites of vascular injury. We found that medium conditioned by serum-starved EC increase proliferation and migration of VSCM in vitro. Fractionation of the conditioned medium followed by mass spectral analysis identified one bioactive component as vitamin D-binding protein (DBP). DBP induced both proliferation and migration of VSMC in vitro in association with increased phosphorylation of ERK 1/2. PD 98059, a biochemical inhibitor of ERK 1/2, abrogated these proliferative and migratory responses in VSMC. DBP is an important carrier for the vitamin-D sterols, 25-hydroxyvitamin-D, and 1alpha,25-dihydroxyvitamin-D. Both sterols inhibited the activity of DBP on VSMC, suggesting that vitamin D binding sites are important for initiating the activities of DBP on VSMC. Release of DBP at sites of endothelial injury represents a novel pathway favoring accumulation of VSMC at sites of vascular injury.     

Polyamine sensing during antizyme mRNA programmed frameshifting

A key regulator of cellular polyamine levels from yeasts to mammals is the protein antizyme. The antizyme gene consists of two overlapping reading frames with ORF2 in the +1 frame relative to ORF1. A programmed +1 ribosomal frameshift occurs at the last codon of ORF1 and results in the production of full-length antizyme protein. The efficiency of frameshifting is proportional to the concentration of polyamines, thus creating an autoregulatory circuit for controlling polyamine levels. The mRNA recoding signals for frameshifting include an element 5' and a pseudoknot 3' of the shift site. The present work illustrates that the ORF1 stop codon and the 5' element are critical for polyamine sensing, whereas the 3' pseudoknot acts to stimulate frameshifting in a polyamine independent manner. We also demonstrate that polyamines are required to stimulate stop codon readthrough at the MuLV redefinition site required for normal expression of the GagPol precursor protein.     

Evaluation of Candidate Gene Effects for Beef Backfat via Bayesian Model Selection

Candidate gene approaches provide tools for exploring and localizing causative genes affecting quantitative traits and the underlying variation may be better understood by determining the relative magnitudes of effects of their polymorphisms. Diacyglycerol O-acyltransferase 1 (DGAT1), fatty acid binding protein (heart) 3 (FABP3), growth hormone 1 (GH1), leptin (LEP) and thyroglobulin (TG) have been previously identified as genes contributing to genetic control of subcutaneous fat thickness (SFT) in beef cattle. In the present research, Bayesian model selection was used to evaluate effects of these five candidate genes by comparing competing non-nested models and treating candidate gene effects as either random or fixed. The analyses were implemented in SAS to simplify the programming and computation. Phenotypic data were gathered from a F₂ population of Wagyu × Limousin cattle. The five candidate genes had significant but varied effects on SFT in this population. Bayesian model selection identified the DGAT1 model as the one with the greatest model probability, whether candidate gene effects were considered random or fixed, and DGAT1 had the greatest additive effect on SFT. The SAS codes developed in the study are freely available and can be downloaded at: http://www.ansci.wsu.edu/programs/.Mention of a proprietary product does not constitute a guarantee or warranty of the product by USDA, Montana AES, or the authors and does not imply its approval to the exclusion of other products that may also be suitable.     

Impairment of cytotype regulation of P-element activity in Drosophila melanogaster by mutations in the Su(var)205 gene

Cytotype regulation of transposable P elements in the germ line of Drosophila melanogaster is associated with maternal transmission of P elements inserted at the left telomere of the X chromosome. This regulation is impaired in long-term stocks heterozygous for mutations in Suppressor of variegation 205 [Su(var)205], a gene implicated in the control of telomere length. Regulation by TP5, a structurally incomplete P element at the X telomere, is more profoundly impaired than regulation by TP6, a different incomplete P element inserted at the same site in a TAS repeat at the X telomere. Genetic analysis with the TP5 element indicates that its regulatory ability is not impaired in flies whose fathers came directly from a stock heterozygous for a Su(var)205 mutation, even when the flies themselves carry this mutation. However, it is impaired in flies whose grandfathers came from such a stock. Furthermore, this impairment occurs even when the Su(var)205 mutation is not present in the flies themselves or in their mothers. The impaired regulatory ability of TP5 persists for at least several generations after TP5 X chromosomes extracted from a long-term mutant Su(var)205 stock are made homozygous in the absence of the Su(var)205 mutation. Impairment of TP5-mediated regulation is therefore not directly dependent on the Su(var)205 mutation. However, it is characteristic of the six mutant Su(var)205 stocks that were tested and may be related to the elongated telomeres that develop in these stocks. Impairment of regulation by TP5 is also seen in a stock derived from Gaiano, a wild-type strain that has elongated telomeres due to a dominant mutation in the Telomere elongation (Tel) gene. Regulation by TP6 is not impaired in the Gaiano genetic background. The regulatory abilities of the TP5 and TP6 elements are therefore not equally susceptible to the effects of elongated telomeres in the mutant Su(var)205 and Gaiano stocks.     

Genetics of species differences in the wild annual sunflowers, Helianthus annuus and H. petiolaris

Much of our knowledge of speciation genetics stems from quantitative trait locus (QTL) studies. However, interpretations of the size and distribution of QTL underlying species differences are complicated by differences in the way QTL magnitudes are estimated. Also, many studies fail to exploit information about QTL directions or to compare inter- and intraspecific QTL variation. Here, we comprehensively analyze an extensive QTL data set for an interspecific backcross between two wild annual sunflowers, Helianthus annuus and H. petiolaris, interpret different estimates of QTL magnitudes, identify trait groups that have diverged through selection, and compare inter- and intraspecific QTL magnitudes. Our results indicate that even minor QTL (in terms of backcross variance) may be surprisingly large compared to levels of standing variation in the parental species or phenotypic differences between them. Morphological traits, particularly flower morphology, were more strongly or consistently selected than life history or physiological traits. Also, intraspecific QTL were generally smaller than interspecific ones, consistent with the prediction that larger QTL are more likely to spread to fixation across a subdivided population. Our results inform the genetics of species differences in Helianthus and suggest an approach for the simultaneous mapping of inter- and intraspecific QTL.     

Single amino acid substitutions in the C-terminus of collagen II alter its affinity for collagen IX

The structural integrity of cartilage depends on the presence of extracellular matrices (ECM) formed by heterotypic fibrils composed of collagen II, collagen IX, and collagen XI. The formation of these fibrils depends on the site-specific binding between relatively small regions of interacting collagen molecules. Single amino acid substitutions in collagen II change the physicochemical and structural characteristics of those sites, thereby leading to an alteration of intermolecular collagen II/collagen IX interaction. Employing a biosensor to study interactions between R75C, R789C or G853E collagen II mutants and collagen IX, we demonstrated significant changes in the binding affinities. Moreover, analyses of computer models representing mutation sites defined exact changes in physicochemical characteristics of collagen II mutants. Our study shows that changes in collagen II/collagen IX affinity could represent one of the steps in a cascade of changes occurring in the ECM of cartilage as a result of single amino acid substitutions in collagen II.