Development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene.

Milk fat percentage is an important production trait of dairy cattle and is one of the goals of breeding programs. Over 95% of the milk fat accounts for triacylglycerols. AGPAT6 (1-acylglycerol-3-phosphate O-acyltransferase 6) catalyzes an intermediary step of triglyceride synthesis in the mammary cells. Genome-wide association studies identified SNP rs211250281 (g27: 36520069 G/T) in the agpat6 gene associated with milk fat content and fat-to-protein ratio in dairy cattle. The article presents data on the development of TaqMan PCR assay for genotyping SNP rs211250281 of the bovine agpat6 gene. In this method, a primer pair, initiating amplification of 75-bp fragments of the agpat6 gene, and two allele-specific TaqMan probes are used. Identification of the G and T alleles is based on a comparison of the final fluorescence intensity of FAM and VIC dyes, respectively. The developed TaqMan PCR assay was validated by Sanger sequencing method. The results of both methods fully coincided, that confirmed high accuracy of the developed TaqMan PCR assay. This reliable, simple, rapid, and high-throughput method could be a suitable tool for studying the distribution of the SNP rs211250281 among different cattle breeds and its association with milk fat content.

Identification of novel integration sites for bovine leukemia virus proviral DNA in cancer driver genes in cattle with persistent lymphocytosis.

Enzootic bovine leukosis is one of the unsolved problems of cattle breeding in many countries. The etiological agent of the disease is the bovine leukemia virus (BLV) - an oncogenic retrovirus, that infects B-lymphocytes in cattle. The number and genetic content of BLV provirus integration sites in the bovine genome were reported to can be used as an early diagnostic sign of leukemogenesis in the infected cattle, but patterns of BLV provirus integration into the bovine genome and associations between genomic features of the integration sites and development of lymphocytosis and B-cell lymphomas remain poorly elucidated. Here we present data on five novel BLV provirus integration sites in the genome of cattle with persistent lymphocytosis. Two of these sites were located in introns of scfd2 and pgpep1 genes, which have been recognized as cancer driver genes. Three of the rest integration sites were found in the intergenic spaces between ctps1 and cited4, nampt and ccdc71, skp2 and lmbrd2 genes, from which cited4 and skp2 also possess oncogenic properties. These data support previous findings of the association between localization of BLV proviral DNA near cancer driver genes and leukemogenesis in the BLV-infected cattle.

Molecular characterization and phylogenetic study of Theileria sp. parasites detected in cattle from the Moscow region of Russia.

Theileriae are obligate intracellular protozoan parasites which are transmitted by ixodid ticks and infect both wild and domestic ruminants worldwide. Theileriosis causes significant economic losses to the livestock industry in many countries due to the high morbidity and mortality in cattle herds. In Russia, information concerning prevalence of Theileria spp. in cattle is very limited. This study reports on molecular characterization and phylogenetic analysis of Theileria spp. parasites detected in cattle from the Moscow region of Russia. Phylogenetic analysis based on the full length 18S rRNA gene revealed that the Russian Theileria parasites belong to the Theileria orientalis / Theileria buffeli / Theileria sergenti group and share a common genotype with T. buffeli Marula from Kenya, T. buffeli isolates from Japan and South Korea, T. orientalis isolate from Australia and T. sergenti isolate from Japan, which belong to the pathogenic Chitose genotype.

Development of TaqMan PCR assay for detection of A and B variants of the bovine ß-lactoglobulin.

ß-Lactoglobulin (BLG) is one of the prevalent whey protein in cattle. To date, several variants of bovine BLG have been found, but the most common are A and B, which differ from each other by SNPs rs109625649 and rs110066229. Numerous studies showed effects of A and B variants of BLG on milk yield, fat and protein content and cheese-making properties. To date, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), allele-specific polymerase chain reaction (ASPCR), PCR single-strand conformation polymorphism (PCR-SSCP) and high resolution melting (HRM) methods have been proposed for detection of A and B variants of bovine BLG. These methods involve multistep sample processing, which is an essential disadvantage in conducting large-scale cattle genotyping projects. This article describes a development of TaqMan PCR assay for detection of A and B variants (rs109625649) of bovine BLG. In this method a primer pair, initiating amplification of 101-bp fragment of BLG gene, and two allele-specific TaqMan probes are used. Identification of B and A variants of BLG is based on comparison of final fluorescence intensity of FAM and VIC dyes, respectively. The developed one-step method requires less time and is more suitable for large-scale genotyping of cattle compared to the commonly used PCR-RFLP.

Draft genome sequence data and comparative analysis of Erysipelothrix Rhusiopathiae vaccine strain VR-2.

Erysipelothrix rhusiopathiae VR-2 is a commercially available live attenuated vaccine strain widely used in Russia, Kazakhstan, and a number of European countries for immunization of pigs against swine erysipelas. The draft genome sequence of E. rhusiopathiae strain VR-2 reported in this paper is 1,704,727 bp in length, has CG content of 36.5%, and contains 1680 genes, including 51 tRNA, 3 rRNA, and 1408 protein-coding genes. Comparative sequence analysis between Fujisawa (serovar 1a), VR-2 and six other serovar N strains of E. rhusiopathiae revealed wide genetic variability of the chromosomal region essential for serovar-specific antigenicity and virulence of E. rhusiopathiae strains. We have performed a BLAST search and found 12 genomic loci potentially specific for the E. rhusiopathiae VR-2 strain. These data could be helpful for developing genetic assays for differentiation of field isolates and this live attenuated vaccine strain, which is especially important for epizootical monitoring of swine erysipelas in countries, where the live vaccine strain E. rhusiopathiae VR-2 is used for pig immunization, as well as for the design of recombinant vaccines against swine erysipelas. The genome of E. rhusiopathiae VR-2 has been submitted in GenBank under accession number RJTK00000000.1.

Real-time PCR assay with an endogenous internal amplification control for detection and quantification of Anaplasma marginale in bovine blood.

Bovine anaplasmosis is a tick-borne rickettsial disease, causing significant economic losses in many countries. The main causative agent of bovine anaplasmosis is Anaplasma marginale (Rickettsiales, Anaplasmataceae). To date, several PCR assays for A. marginale DNA detection were proposed, but most of them do not provide an internal amplification control, which allows to prevent false-negative results and is required for reliability of the results of pathogen DNA detection by PCR assay. In the present study, a real-time PCR assay based on the species-specific and highly conserved fragment of msp1α gene was developed for detection and quantification of A. marginale in bovine blood. The real-time PCR assay is able to detect as few as one copу of msp1α gene per reaction. To prevent false-negative results, simultaneous amplification and detection of the bovine genomic DNA fragment as an endogenous internal amplification control (IAC) was provided. The assay can be used as a highly specific and sensitive method for detection and quantification of A. marginale in infected cattle, and for the evaluation of the efficacy of anti-rickettsial drugs and anaplasmosis vaccines.

Bovine leukemia virus tax gene/Tax protein polymorphism and its relation to Enzootic Bovine Leukosis.

Bovine leukemia virus (BLV) is an oncogenic retrovirus of the Deltaretrovirus genus, which causes persistent infection in its natural hosts - cattle, zebu, and water buffalo with diverse clinical manifestations through the defeat of B-cells. The BLV proviral genome, along with structural genes (gag, pro, pol, and env), includes nonstructural ones (R3, G4, tax, rex, AS, pre-miRs (for miRNAs). We have shown in our previous data the association of some pre-miRs-B' (for BLV miRNA) alleles with leukocyte (WBC - white blood cell) number in BLV-infected cows. Multifunctional properties of Tax protein have led us to an assumption that tax gene/Tax protein could have too population variations related to WBC counts. Here we report about several tax alleles/Tax protein variants, which have a highly significant association with an increase or a decrease of WBC number in BLV-infected cows. We have provided evidence that Tax A, H variants (tax b, c, d, f, e alleles) are correlated with reduced WBC counts at the level of BLV-negative groups of animals and thus could be the feature of the aleukemic (AL) form of BLV infection. We suggest this finding could be used in BLV testing for the presence of Tax A, H in the proviral DNA consider such strains of BLV as AL ones, and because of this, minimize the clinical losses due to BLV infection in cattle.

Co-infection with tick-borne disease agents in cattle in Russia.

Tick-borne diseases cause significant livestock losses worldwide. In Russia, information concerning single or mixed infections with different Anaplasma, Theileria and Babesia species in cattle is very limited. This study was conducted to determine the level of co-infection with protozoan pathogens (Theileria spp. and Babesia spp.) and rickettsial pathogens (A. marginale and A. phagocytophilum) in cattle in central Russia. Blood samples were examined with real time polymerase chain reaction (RT-PCR) for A. marginale and A. phagocytophilum, and by amplifying the V4 hypervariable region of the 18S rRNA gene, followed by cloning, DNA sequencing, and phylogenetic analyses, for Babesia and Theileria species. In total 67% of examined blood samples were positive for Theileria spp. or A. marginale, and 19% of the animals were co-infected with Theileria spp. and A. marginale. Seasonal variation in prevalence was found for Theileria spp. Phylogenetic analysis based on 18S rRNA gene sequences revealed the presence of five Theileria species: T. annulata, T. orientalis, T. buffeli, T. sergenti, and T. sinensis. No samples were positive for Babesia spp. or A. phagocytophilum. The data obtained for prevalence of bovine theileriosis and anaplasmosis in the central part of Russia underscore the need for improved surveillance and control programs to reduce tick-borne diseases in cattle.

Molecular survey and genetic characterization of Anaplasma marginale isolates in cattle from two regions of Russia.

Anaplasma marginale is an intraerythrocytic tick-borne rickettsial pathogen that causes bovine anaplasmosis, an economically important disease of cattle worldwide. Major surface protein MSP1α has been used as a stable marker in identifying geographical strains of A. marginale. The genetic diversity of A. marginale based on MSP1α has been reported in several countries all over the world. Only a few molecular surveys of A. marginale strains have been conducted in Russia. The aim of this study was molecular detection and characterization of A. marginale isolates in cattle from two regions of Russia. Blood samples from 62 cattle were collected and screened for the presence of A. marginale by real-time PCR targeting the msp4 gene. Anaplasma marginale DNA was detected in 26 cattle (42%). The partial msp1α gene containing tandem repeat sequences and msp4 gene were amplified from msp4-positive samples, cloned and sequenced. Sequence analysis revealed that two msp4 genotypes were found. The genetic diversity of A. marginale strains was analyzed based on the MSP1α tandem repeats structure and 5'-UTR microsatellite. Sixteen new genotypes of A. marginale were found in 17 animals. Seven animals (41%) were infected by more than one genotype. Eight new tandem repeats are described for the first time. The number of repeats differed between 1 and 6 across the isolates. The msp1α microsatellite analysis revealed that six genotypes were identified; one of them was not previously described. Phylogenetic analysis revealed that Russian isolates formed four separate clades. The tandem repeat and microsatellite analyses of the msp1α gene showed a high genetic diversity among the isolates. The present study provided the first evidence of genetic diversity of A. marginale in cattle in Russia.

Mutagenesis Studies and Structure-function Relationships for GalNAc/Gal-Specific Lectin from the Sea Mussel Crenomytilus grayanus.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) with anticancer activity represents а novel lectin family with ß-trefoil fold. Earlier, the crystal structures of CGL complexes with globotriose, galactose and galactosamine, and mutagenesis studies have revealed that the lectin contained three carbohydrate-binding sites. The ability of CGL to recognize globotriose (Gb3) on the surface of breast cancer cells and bind mucin-type glycoproteins, which are often associated with oncogenic transformation, makes this compound to be perspective as a biosensor for cancer diagnostics. In this study, we describe results on in silico analysis of binding mechanisms of CGL to ligands (galactose, globotriose and mucin) and evaluate the individual contribution of the amino acid residues from carbohydrate-binding sites to CGL activity by site-directed mutagenesis. The alanine substitutions of His37, His129, Glu75, Asp127, His85, Asn27 and Asn119 affect the CGL mucin-binding activity, indicating their importance in the manifestation of lectin activity. It has been found that CGL affinity to ligands depends on their structure, which is determined by the number of hydrogen bonds in the CGL-ligand complexes. The obtained results should be helpful for understanding molecular machinery of CGL functioning and designing a synthetic analog of CGL with enhanced carbohydrate-binding properties.

A new recombinant endo-1,3-ß-D-glucanase from the marine bacterium Formosa algae KMM 3553: enzyme characteristics and transglycosylation products analysis.

A specific endo-1,3-ß-D-glucanase (GFA) gene was found in genome of marine bacterium Formosa algae KMM 3553. For today this is the only characterized endo-1,3-ß-D-glucanase (EC 3.2.1.39) in Formosa genus and the only bacterial EC 3.2.1.39 GH16 endo-1,3-ß-D-glucanase with described transglycosylation activity. It was expressed in E. coli and isolated in homogeneous state. Investigating the products of polysaccharides digestion with GFA allowed to establish it's substrate specificity and classify this enzyme as glucan endo-1,3-ß-D-glucosidase (EC 3.2.1.39). The amino-acid sequence of GFA consists of 556 residues and shows sequence similarity of 45-85% to ß-1,3-glucanases of bacteria belonging to the CAZy 16th structural family of glycoside hydrolases GH16. Enzyme has molecular weight 61 kDa, exhibits maximum of catalytic activity at 45 °C, pH 5.5. Half-life period at 45 °Ð¡ is 20 min, complete inactivation happens at 55 °C within 10 min. Km for hydrolysis of laminarin is 0.388 mM. GFA glucanase from marine bacteria F. algae is one of rare enzymes capable to catalyze reactions of transglycosylation. It catalyzed transfer of glyconic part of substrate molecule on methyl-ß-D-xylopyranoside, glycerol and methyl-α-D-glucopyranoside. The enzyme can be used in structure determination of ß-1,3-glucans (or mixed 1,3;1,4- and 1,3;1,6-ß-D-glucans) and enzymatic synthesis of new carbohydrate-containing compounds.

А new Gal/GalNAc-specific lectin from the mussel Mytilus trossulus: Structure, tissue specificity, antimicrobial and antifungal activity.

In the present study, a new Gal/GalNAc specific lectin from the mussel Mytilus trossulus (designated as MTL) was identified, and its expression levels, both in tissues and toward pathogen stimulation, were then characterized. The MTL primary structure was determined via cDNA sequencing. Deduced sequence of 150 amino acid residues showed 89% similarity to lectins from the mussels Crenomytilus grayanus and Mytilus galloprovincialis that were the first members of a new family of zoolectins. The results indicated that the MTL might be involved in immune response toward pathogen infection, and it might perform different recognition specificity toward bacteria or fungi.

Bioinspired enzymatic synthesis of silica nanocrystals provided by recombinant silicatein from the marine sponge Latrunculia oparinae.

The process of silica formation in marine sponges is thought to be mediated by a family of catalytically active structure-directing enzymes called silicateins. It has been demonstrated in biomimicking syntheses that silicateins facilitated the formation of amorphous SiO2. Here, we present evidence that the silicatein LoSiLA1 from the marine sponge Latrunculia oparinae catalyzes the in vitro synthesis of hexa-tetrahedral SiO2 crystals of 200­300 nm. This was possible in the presence of the silica precursor tetrakis-(2-hydroxyethyl)-orthosilicate that is completely soluble in water and biocompatible, experiences hydrolysis­condensation at neutral pH and ambient conditions.

Carbohydrate-binding motifs in a novel type lectin from the sea mussel Crenomytilus grayanus: Homology modeling study and site-specific mutagenesis.

The GalNAc/Gal-specific lectin from the sea mussel Crenomytilus grayanus (CGL) was shown to represent a novel family of lectins and to be characterized by three amino acid tandem repeats with high (up to 73%) sequence similarities to each other. We have used homology modeling approach to predict CGL sugar-binding sites. In silico analysis of CGL-GalNAc complexes showed that CGL contained three binding sites, each of which included conserved HPY(K)G motif. In silico substitutions of histidine, proline and glycine residues by alanine in the HPY(K)G motifs of the Sites 1-3 was shown to lead to loss of hydrogen bonds between His and GalNAc and to the increasing the calculated CGL-GalNAc binding energies. We have obtained recombinant CGL and used site-specific mutagenesis to experimentally examine the role of HPK(Y)G motifs in hemagglutinating and carbohydrate binding activities of CGL. Substitutions of histidine, proline and glycine residues by alanine in the HPYG motif of Site 1 and Site 2 was found to led to complete loss of CGL hemagglutinating and mucin-binding activities. The same mutations in HPKG motif of the Site 3 resulted in decreasing the mucin-binding activity in 6-folds in comparison with the wild type lectin. The mutagenesis and in silico analysis indicates the importance of the all three HPY(K)G motifs in the carbohydrate-binding and hemagglutinating activities of CGL.

cDNA cloning and structural characterization of a lectin from the mussel Crenomytilus grayanus with a unique amino acid sequence and antibacterial activity.

An amino acid sequence of GalNAc/Gal-specific lectin from the mussel Crenomytilus grayanus (CGL) was determined by cDNA sequencing. CGL consists of 150 amino acid residues, contains three tandem repeats with high sequence similarities to each other (up to 73%) and does not belong to any known lectins family. According to circular dichroism results CGL is a ß/α-protein with the predominance of ß-structure. CGL was predicted to adopt a ß-trefoil fold. The lectin exhibits antibacterial activity and might be involved in the recognition and clearance of bacterial pathogens in the shellfish.

Mannan-binding lectin of the sea urchin Strongylocentrotus nudus.

A novel lectin specific to low-branched mannans (MBL-SN) was isolated from coelomic plasma of the sea urchin Strongylocentrotus nudus by combining anion-exchange liquid chromatography on DEAE Toyopearl 650 M, affinity chromatography on mannan-Sepharose and gel filtration on the Sephacryl S-200. The molecular mass of MBL-SN was estimated by sodium dodecyl sulphate polyacrylamide gel electrophoresis under non-reducing conditions to be about 34 kDa. MBL-SN was shown to be a dimer with two identical subunits of about 17 kDa. The native MBL-SN exists as a tetramer. The physico-chemical properties of MBL-SN indicate that it belongs to C-type mannan-binding lectins. The cDNA encoding MBL-SN was cloned from the total cDNA of S. nudus coelomocytes and encodes a 17-kDa protein of 144 amino acid residues that contains a single carbohydrate-recognition domain of C-type lectins. Prediction of the MBL-SN tertiary structure using comparative modelling revealed that MBL-SN is an α/ß-protein with eight ß-strands and two α-helices. Comparison of the MBL-SN model with available three-dimensional structures of C-type lectins revealed that they share a common fold pattern.

Enzymatic and molecular characterization of an endo-1,3-ß-d-glucanase from the crystalline styles of the mussel Perna viridis.

The retaining endo-1,3-ß-d-glucanase (EC 3.2.1.39) was isolated from the crystalline styles of the commercially available Vietnamese edible mussel Perna viridis. It catalyzes hydrolysis of ß-1,3-bonds in glucans and enables to catalyze a transglycosylation reaction. Resources of mass-spectrometry for analysis of enzymatic products were studied. cDNA sequence of endo-1,3-ß-d-glucanase was determined by RT-PCR in conjunction with the rapid amplification of cDNA ends (RACE) methods. The cDNA of 1380bp contains an open reading frame of 1332bp encoding a mature protein of 328 amino acids. On basis of amino acid sequence analysis endo-1,3-ß-d-glucanase was classified as a glycoside hydrolase of family 16.

Occurrence of a silicatein gene in glass sponges (Hexactinellida: Porifera).

Silicatein genes are involved in spicule formation in demosponges (Demospongiae: Porifera). However, numerous attempts to isolate silicatein genes from glass sponges (Hexactinellida: Porifera) resulted in a limited success. In the present investigation, we performed analysis of potential silicatein/cathepsin transcripts in three different species of glass sponges (Pheronema raphanus, Aulosaccus schulzei, and Bathydorus levis). In total, 472 clones of such transcripts have been analyzed. Most of them represent cathepsin transcripts and only three clones have been found to represent transcripts, which can be related to silicateins. Silicatein transcripts were identified in A. schulzei (Hexactinellida; Lyssacinosida; Rosselidae), and the corresponding gene was called AuSil-Hexa. Expression of AuSil-Hexa in A. schulzei was confirmed by real-time PCR. Comparative sequence analysis indicates high sequence identity of the A. schulzei silicatein with demosponge silicateins described previously. A phylogenetic analysis indicates that the AuSil-Hexa protein belongs to silicateins. However, the AuSil-Hexa protein contains a catalytic cysteine instead of the conventional serine.

Silicatein genes in spicule-forming and nonspicule-forming Pacific demosponges.

Silicatein genes are known to be involved in siliceous spicule formation in marine sponges. Proteins encoded by these genes, silicateins, were recently proposed for nanobiotechnological applications. We studied silicatein genes of marine sponges Latrunculia oparinae collected in the west Pacific region, shelf of Kuril Islands. Five silicatein genes, LoSilA1, LoSilA1a, LoSilA2, and LoSilA3 (silicatein-alpha group), LoSilB (silicatein-beta group), and one cathepsin gene, LoCath, were isolated from the sponge L. oparinae for the first time. The deduced amino acid sequence of L. oparinae silicateins showed high-sequence identity with silicateins described previously. LoCath contains the catalytic triad of amino acid residues Cys-His-Asn characteristic for cathepsins as well as motifs typical for silicateins. A phylogenetic analysis places LoCath between sponge silicateins-beta and L-cathepsins suggesting that the LoCath gene represents an intermediate form between silicatein and cathepsin genes. Additionally, we identified, for the first time, silicatein genes (AcSilA and AcSilB) in nonspicule-forming marine sponge, Acsmall a, Cyrillicnthodendrilla sp. The results suggest that silicateins could participate also in the function(s) unrelated to spiculogenesis.

An endo-(1-->3)-beta-D-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization.

An endo-(1-->3)-beta-d-glucanase (L(0)) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1-->3)-beta-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 degrees C. L(0) hydrolyzed laminaran with K(m) approximately 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl betad-glucoside as acceptor (K(m) approximately 2mg/mL for laminaran) and laminaran as donor and as acceptor (K(m) approximately 5mg/mL) yielding p-nitrophenyl betad-glucooligosaccharides (n=2-6) and high-molecular branching (1-->3),(1-->6)-beta-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of beta-(1-->6)-glycosidic bonds, and laminaran with 10% of beta-(1-->6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L(0) was characteristic for a protein with prevailing beta secondary-structural elements. Binding L(0) with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L(0) with glucose (K(a)=7.4 x 10(5)+/-1.1 x 10(5)M(-1)) and stoichiometry (n=13.3+/-0.7) was calculated. The cDNA encoding L(0) was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.