Genome‐wide association analysis for growth,muscularity and meat quality in Piétrain pigs

Improvement in growth and meat quality is one of the main objectives in sire line pig breeding programmes. Mapping quantitative trait loci for these traits using experimental crosses and a linkage‐based approach has been performed frequently in the past. The Piétrain breed often was involved as a founder breed to establish the experimental crosses. This breed was selected for muscularity and leanness but shows relatively poor meat quality. It is frequently used as a sire line breed. With the advent of genome‐wide and dense SNP chips in pig genomic research, it is possible to also conduct genome‐wide association studies within the Piétrain breed. In this study, around 500 progeny‐tested sires were genotyped with 60k SNPs. Data filtering showed that around 48k SNPs were useable in this sample. These SNPs were used to conduct a genome‐wide association study for growth, muscularity and meat quality traits. Because it is known that a mutation in the RYR1 gene located on chromosome 6 shows a major effect on meat quality, this mutation was included in the models. Single‐marker and multimarker association analyses were performed. The results revealed between zero and eight significant associations per trait with P < 5 × 10^?5. Of special interest are SNPs located on SSC6, SSC10 and SSC15.     

Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.     

Stimulation of poliovirus synthesis in a HeLa cell-free in vitro translation-RNA replication system by viral protein 3CDpro

The plus-strand RNA genome of poliovirus serves three distinct functions in the life cycle of the virus. The RNA is translated and then replicated, and finally the progeny RNAs are encapsidated. These processes can be faithfully reproduced in a HeLa cell-free in vitro translation-RNA replication system that produces viable poliovirus. We have previously observed a stimulation of virus synthesis when an mRNA, encoding protein 3CD(pro), is added to the translation-RNA replication reactions of poliovirus RNA. Our aim in these experiments was to further define the factors that affect the stimulatory activity of 3CD(pro) in virus synthesis. We observed that purified 3CD(pro) protein also enhances virus synthesis by about 100-fold but has no effect on the translation of the polyprotein. Optimal stimulation is observed only when 3CD(pro) is present early in the incubation period. The stimulation, however, is abolished by a mutation either in the RNA binding domain of 3CD(pro), 3C(pro)R84S/I86A, or by each of two groups of complementary mutations R455A/R456A and D339A/S341A/D349A at interface I in the 3D(pol) domain of 3CD(pro). Surprisingly, virus synthesis is strongly inhibited by the addition of both 3C(pro) and 3CD(pro) at the beginning of incubation. We also examined the effect of other viral or cellular proteins on virus synthesis in the in vitro system. No enhancement of virus synthesis occurred with viral proteins 3BC, 3ABC, 3BCD, 3D(pol), and 3C(pro) or with cellular protein PCBP2. These results suggest that 3CD(pro) has to be present in the reaction at the time the replication complexes are assembled and that both the 3C(pro) and 3D(pol) domains of the protein are required for its activity that stimulates virus production.     

An Interaction between Glutathione and the Capsid Is Required for the Morphogenesis of C-Cluster Enteroviruses

Glutathione (GSH) is the most abundant cellular thiol playing an essential role in preserving a reduced cellular environment. Cellular GSH levels can be efficiently reduced by the GSH biosynthesis inhibitor, L-buthionine sulfoximine (BSO). The aim of our study was to determine the role of GSH in the growth of two C-cluster enteroviruses, poliovirus type 1 (PV1) and coxsackievirus A20 (CAV20). Our results show that the growth of both PV1 and CAV20 is strongly inhibited by BSO and can be partially reversed by the addition of GSH. BSO has no effect on viral protein synthesis or RNA replication but it strikingly reduces the accumulation of 14S pentamers in infected cells. GSH-pull down assays show that GSH directly interacts with capsid precursors and mature virus made in the absence of BSO whereas capsid precursors produced under GSH-depletion do not bind to GSH. In particular, the loss of binding of GSH may debilitate the stability of 14S pentamers, resulting in their failure to assemble into mature virus. Immunofluorescence cell imaging demonstrated that GSH-depletion did not affect the localization of viral capsid proteins to the replication complex. PV1 BSO resistant (BSOr) mutants evolved readily during passaging of the virus in the presence of BSO. Structural analyses revealed that the BSOr mutations, mapping to VP1 and VP3 capsid proteins, are primarily located at protomer/protomer interfaces. BSOr mutations might, in place of GSH, aid the stability of 14S particles that is required for virion maturation. Our observation that BSOr mutants are more heat resistant and need less GSH than wt virus to be protected from heat inactivation suggests that they possess a more stable capsid. We propose that the role of GSH during enterovirus morphogenesis is to stabilize capsid structures by direct interaction with capsid proteins both during and after the formation of mature virus particles.     

Nectin couples cell-cell adhesion and the actin scaffold at heterotypic testicular junctions

Actin-based cell-cell adherens junctions (AJs) are crucial not only for mechanical adhesion but also for cell morphogenesis and differentiation. While organization of homotypic AJs is attributed mostly to classic cadherins, the adhesive mechanism of heterotypic AJs in more complex tissues remains to be clarified. Nectin, a member of a family of immunoglobulin-like adhesion molecules at various AJs, is a possible organizer of heterotypic AJs because of its unique heterophilic trans-interaction property. Recently, nectin-2 (-/-) mice have been shown to exhibit the defective sperm morphogenesis and the male-specific infertility, but the role of nectin in testicular AJs has not been investigated. We show here the heterotypic trans-interaction between nectin-2 in Sertoli cells and nectin-3 in spermatids at Sertoli-spermatid junctions (SspJs), heterotypic AJs in testes. Moreover, each nectin-based adhesive membrane domain exhibits one-to-one colocalization with each actin bundle underlying SspJs. Inactivation of the mouse nectin-2 gene causes not only impaired adhesion but also loss of the junctional actin scaffold at SspJs, resulting in aberrant morphogenesis and positioning of spermatids. Localization of afadin, an adaptor protein of nectin with the actin cytoskeleton, is also nectin-2 dependent at SspJs. These results indicate that the nectin-afadin system plays essential roles in coupling cell-cell adhesion and the cortical actin scaffold at SspJs and in subsequent sperm morphogenesis.     

Dual Stem Loops within the Poliovirus Internal Ribosomal Entry Site Control Neurovirulence

In the human central nervous system, susceptibility to poliovirus (PV) infection is largely confined to a specific subpopulation of neuronal cells. PV tropism is likely to be determined by cell-external components such as the PV receptor CD155, as well as cell-internal constraints such as the availability of a suitable microenvironment for virus propagation. We reported previously that the exchange of the cognate internal ribosomal entry site (IRES) within the 5′ nontranslated region of PV with its counterpart from human rhinovirus type 2 (HRV2) can eliminate the neuropathogenic phenotype in a transgenic mouse model for poliomyelitis without diminishing the growth properties in HeLa cells. We now show that attenuation of neurovirulence of PV/HRV2 chimeras is not confined to CD155 transgenic mice but is evident also after intraspinal inoculation into Cynomolgus monkeys. We have dissected the PV and HRV2 IRES elements to determine those structures responsible for neurovirulence (or attenuation) of these chimeric viruses. We report that two adjacent stem loop structures within the IRES cooperatively determine neuropathogenicity.     

Flavonol synthase from Citrus unshiu is a bifunctional dioxygenase

Flavonol synthase was classified as a 2-oxoglutarate-dependent dioxygenase converting natural (2R,3R)-dihydroflavonols, i.e. dihydrokaempferol, to the corresponding flavonols (kaempferol). Flavonol synthase from Citrus unshiu (Satsuma mandarin), expressed in Escherichia coli and purified to homogeneity, was shown to accept also (2S)-naringenin as a substrate, producing kaempferol in high yield and assigning sequential flavanone 3beta-hydroxylase and flavonol synthase activities to the enzyme. In contrast, dihydrokaempferol was identified as the predominant product from assays performed with the unnatural (2R)-naringenin as substrate. The product which was not converted any further on repeated incubations was identified by 1H NMR and CD spectroscopies as (-)-trans-dihydrokaempferol. The data demonstrate that Citrus flavonol synthase encompasses an additional non-specific activity trans-hydroxylating the flavanones (2S)-naringenin as well as the unnatural (2R)-naringenin at C-3.     

UV-B Inhibition of Phytochrome-Mediated Anthocyanin Formation in Sinapis alba L. Cotyledons : Action Spectrum and the Role of Photoreactivation

An action spectrum was measured for ultraviolet (UV) radiation-induced damage to (inhibition of) phytochrome-induced anthocyanin formation in cotyledons of 40-hour-old Sinapis alba L. seedlings. The action spectrum showed maximum effectiveness in the 260 to 280 nanometer waveband with little effect above 295 nanometers. The damaging effect of UV could be photorepaired by subsequent exposure to sunlight or to long wavelength (360 nanometers) UV radiation. Because this form of damage is subject to photorepair (photoreactivation), it is probably due to the formation of pyrimidine dimers, and the results suggest that it would not be ecologically relevant even if there was an increase in solar UV due to a decrease in stratospheric ozone levels of about 30%. If a dark period of more than 1 hour is interspersed between the phytochrome induction and the UV irradiation, the inhibition of the phytochrome induction gradually decreases with increasing dark period.     

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O₂ in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe³⁺ (g = 4.3) and aqueous Mn²⁺ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe³⁺ hemes were observed, probably arising from cytochrome c peroxidase and the a₃:Cu_b site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe₂S₂]⁺ cluster of succinate dehydrogenase, the [Fe₂S₂]⁺ cluster of the Rieske protein of cytochrome bc ₁, and the [Fe₃S₄]⁺ cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g _ave = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe₄S₄ clusters (60–85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O₂, was a quadrupole doublet with ΔE _Q = 1.15 mm/s and δ = 0.45 mm/s, assigned to [Fe₄S₄]²⁺ clusters. Substantial high-spin non-heme Fe²⁺ (up to 20%) and Fe³⁺ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.