Isolation and sequence of sheep immunoglobulin E heavy-chain complementary DNA.

A complementary DNA (cDNA) corresponding to sheep immunoglobulin E heavy (IgE H) chain messenger RNA (mRNA) was isolated and sequenced. A fragment of sheep IgE constant H (epsilon)-chain was initially synthesized using the polymerase chain reaction (PCR) and single-stranded cDNA prepared from the mRNA of parasite-stimulated sheep lymph nodes. This fragment was then used to probe a cDNA library, again prepared from a parasite-stimulated lymph node. A recombinant clone containing cDNA encoding a sheep IgE H-chain of 1802 base pairs was isolated and the H-chain cDNA-sequenced. The nucleotide sequence was found to code for a complete sheep IgE H-chain, consisting of both variable (V) and constant (C) regions. When the amino acid sequence derived from the nucleotide sequence was compared to other species epsilon-chains, interesting homologies and differences between corresponding domains were found, including conservation in cysteine and tryptophan residues and variable glycosylation sites.     

Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone

OBJECTIVES: To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences. SAMPLE POPULATION: Parathyroid glands from 1 healthy cat. PROCEDURES: A cDNA library was constructed in lambda phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences. RESULTS: Screening of approximately 2 X 10(5) recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence. CONCLUSIONS AND CLINICAL RELEVANCE: The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats.     

V(D)J recombinational signal sequence DNA binding activities expressed by fetal bovine thymus

V(D)J recombination, or immunoglobulin gene rearrangement is a developmentally regulated, cell type specific, site directed recombination event that brings either immunoglobulin or T-cell receptor gene segments together to form mature, expressible Ig or TCT genes. This DNA recombination is directed by the recombinational signal sequences or RSS elements present adjacent to Ig and TCR gene segments. The RSS element is composed of a conserved nonamer element and a conserved heptamer element separated by a conserved length spacer region. In this report, we examine the expression of DNA binding proteins that interact with the RSS element in the bovine fetal thymus using EMSA assays. Our data indicates that the nonamer portion of the RSS element is the primary site of recognition for RSS binding proteins expressed in the bovine fetal thymus. We also show that these proteins are expressed from early stages of bovine fetal development through to full term development.     

Characterization and validation of bovine gonadotripin releasing hormone receptor (GNRHR) polymorphisms

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4 bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.     

Characterization and validation of bovine Gonadotripin releasing hormone receptor (GNRHR) polymorphisms

Gonadotropin releasing hormone and its receptor (GNRHR) play a critical role in sexual differentiation and reproduction. Available evidence shows a strong genetic component in the timing of puberty. In bovines, there are significant differences within and among beef breeds in the time when bulls reach puberty. Despite its economic importance, there are not many SNPs or genetic markers associated with this characteristic. The aims of the study were to identify DNA polymorphism in the bovine GNRHR by re-sequencing analysis, determine haplotype phases, and perform a population study in a selected tag SNP in six breeds. Eight SNPs were detected, including: one in the Upstream Regulatory Region (URR), five in the coding regions, and two in non-coding regions. This polymorphism level corresponds to one variant every 249.4 bp and a global nucleotide diversity of 0.385. Two haplogroups comprising nine haplotypes and two linkage blocks were detected. Despite 5 tag SNPs were required to capture all variability, just one SNP allowed to define both haplogroups, and only two SNPs were needed to differentiate the most common haplotypes. An additional taq SNP was necessary to identify both URR variants. Allele-frequency analysis of a selected taq SNP among breeds showed a geographical cline. European Bos taurus breeds had lower frequencies of the C allele than B. indicus type cattle, while Creole cattle and Wagyu breeds had intermediate frequency. There was a significant correlation between frequency profile and timing of puberty among the studied breeds, which seems to suggest that genetic variation within bovine GNRHR gene could explain at least part of the reported variability.     

The polymorphisms of bovine melanocortin-3 receptor pseudogene

Mammalian melanocortin-3 receptor (MC3R) plays an important role in the central control of energy homeostasis, and several functional polymorphisms of mc3r have been detected. Interestingly, the bovine mc3r was a pseudogene, and its polymorphisms and function remain to be investigated. Single-strand conformation polymorphism (SSCP) showed 5, 2 and 3 genotypes in fragment F1, F2 and F3 of mc3r in seven cattle breeds, respectively. All genotypes revealed novel sequences. Three SNPs 657G > T, 756C > T, 822T > C were detected in fragment F1, five SNPs 1091T > C, 1133T > C, 1144C > T, 1259T > C and 1319G > A were detected in fragment F2, and two SNPs 1687G > A, 1860C > T were detected in F3. The SNPs in fragment F1 and F2 were located at exon 2. The five SNPs in fragment F2 demonstrated a tight linkage disequilibrium status. Variation detected here might have an impact on the function of bovine mc3r pseudogene.     

牛FSHR基因第4外显子多态性检测及序列分析

以西门塔尔和夏洛莱种公牛为对象,采用PCR-SSCP技术检测了促卵泡素受体(FSHR)基因第4外显子在西门塔尔、夏洛莱种公牛45个个体中的遗传多态性,结果发现多态性,旨在为研究该基因多态性与西门塔尔和夏洛莱种公牛繁殖性状的相关性提供理论依据,为筛选繁殖性状分子标记奠定基础.结果表明,牛FSHR基因第4外显子存在2个等位基因A和B,3种基因型分别为AA型、AB型和BB型.对多态片段的测序分析表明,FSHR基因第4外显子第38位碱基处发生碱基C→G的颠换,使得FSHR基因编码的受体胞外域部分出现一个脯氨酸到丙氨酸的变化,结合FSHR蛋白空间结构分析发现该氨基酸变化不直接影响FSHR与FSH的结合及FSHR转导信号能力.此外,据牛、绵羊、猪、马、人和大鼠FSHR基因第4外显子序列同源性比较表明:牛与绵羊该部分序列的同源最高为100%,与大鼠同源性最低为83%.     

单核苷酸多态性(SNPs)——第三代DNA分子标记

单核苷酸多态性 (SNPs)是指染色体基因组水平上由单个核苷酸变异引起的DNA序列多态性 ,具有分布广、密度高、多态性丰富等特点 ,且易于实现自动化检测 ,被公认为是最新的第 3代DNA分子标记。其在基因作用机理、疾病的研究及动物育种等领域具有广泛的应用前景。本文简要介绍了 6种高通量的SNPs检测技术 :基因芯片、TaqMan、变性高效液相色谱、双色荧光偏振检测入侵、基质辅助激光解吸附电离飞行时间质谱及单碱基延伸标签阵列的原理及其优、缺点。     

Molecular cloning of complementary DNA from a pneumopathic strain of bovine viral diarrhea virus and its diagnostic application.

A pneumopathic strain of bovine viral diarrhea virus was grown in cell culture and purified. Genomic ribonucleic acid was extracted, polyadenylated at the 3' end, and copied into complementary DNA after oligo-dT priming. Complementary DNA was male double stranded and cloned into the pUC9 plasmid. Approximately 200 complementary DNA clones varying in length from 0.5 to 2.5 kilobases were obtained. Hybridization assays indicated that the sequences isolated were specific for bovine viral diarrhea virus and that at least 5.5 kilobases of bovine viral diarrhea virus genome was represented in the library of complementary DNA clones, the majority of which may have originated from the 3' end of the virus genome. One cloned complementary DNA sequence was used as a 32P-labelled hybridization probe for bovine viral diarrhea virus detection. The probe hybridized with all cytopathic and noncytopathic strains of bovine viral diarrhea virus tested and was 100 times more sensitive than infectivity assays for the detection of bovine viral diarrhea virus. Hybridization did not occur with nucleic acids from bovine coronavirus, bluetongue virus, bovine adenovirus or uninfected cell cultures. Native plasmid DNA sequences, labelled with 32P, did not hybridize with bovine viral diarrhea virus ribonucleic acid.     

中华拟德氏吸虫与日本血吸虫的RAPD遗传标志分析

中华拟德氏吸虫(Paradeontacylix sinensis)是冷血吸虫,日本血吸虫(Schistosoma japonicum)是温血吸虫的重要代表。分别对中华拟德氏吸虫和日本血吸虫成虫的全基因组进行随机扩增多态性研究,筛选出31种随机引物,对2种成虫扩增得到189条多态性基因片断,S6、S46对中华拟德氏吸虫全基因组有特异性,分别扩增出一条大小约399bp和695bp的特异性强条带;引物S2对日本血吸虫基因组有特异性,只扩增1条1670bp的特异性强条带。多数引物对中华拟德氏吸虫和日本血吸虫成虫全基因组扩增出大小相似的条带,推测中华拟德氏吸虫与日本血吸虫的亲缘关系密切。     

Search for bovine papilloma virus DNA in bovine ocular squamous cell carcinomas (BOSCC) and BOSCC-derived cell lines.

Although the cause of bovine ocular squamous cell carcinoma (BOSCC) is attributed to viruses in addition to cofactors (eg, UV light), to our knowledge, the final causative agent has not been described. Bovine papilloma virus (BPV)-like particles were detected in approximately 33% of various putative precursor lesions of BOSCC. In contrast, it was reported that, using BPV-specific antibodies, it was not possible to detect viral antigens in BOSCC. Fourteen established BOSCC and 9 BOSCC-derived cell lines were examined for BPV DNA. Probes of all 6 known BPV types were used in various hybridization assays. Neither Southern blot analysis, under high and low stringency conditions, nor in situ hybridization resulted in detection of BPV DNA. Papilloma viruses were not observed in electron microscopic studies. Results exclude direct association between BOSCC and BPV types 1 to 6, or as yet unknown closely related BPV types. However, BPV may contribute to induction of precursor lesions or events leading to carcinogenic transformation, without being relevant for maintenance of the tumor.     

A PCR-based assay for discriminating Cervus and Rangifer (Cervidae) antlers with mitochondrial DNA polymorphisms

This study describes a method for discriminating Rangifer antlers from true Cervus antlers using agarose gel electrophoresis, capillary electrophoresis, quantitative real-time PCR, and allelic discrimination. Specific primers labeled with fluorescent tags were designed to amplify fragments from the mitochondrial D-loop genes for various Cervus subspecies and Rangifer tarandus differentially. A 466-bp fragment that was observed for both Cervus and Rangifer antlers served as a positive control, while a 270-bp fragment was specifically amplified only from Rangifer antlers. Allelic discrimination was used to differentiate between Cervus and Rangifer antlers, based on the amplification of specific alleles for both types of antlers. These PCR-based assays can be used for forensic and quantitative analyses of Cervus and Rangifer antlers in a single step, without having to obtain any sequence information. In addition, multiple PCR-based assays are more accurate and reproducible than a single assay for species-specific analysis and are especially useful in this study for the identification of original Cervus deer products from fraudulent Rangifer antlers.     

DNA sequence analysis of glycoprotein G gene of bovine ephemeral fever virus and development of a double oil emulsion vaccine against bovine ephemeral fever

The surface glycoprotein G is considered as the major neutralizing and protective antigen of bovine ephemeral fever virus (BEFV). Comparison of the deduced amino acid sequence of G protein of BEFV isolates during the period 1984-2004 outbreaks in Taiwan showed amino acid substitutions in the neutralizing epitopes. All the isolates differ markedly in the neutralizing epitope at the same amino acid positions compared to the currently available killed vaccine strain (Tn73). Tn88128 strain isolated in 1999 showed the maximum variability of 12 amino acids, 5 amino acid in the neutralization epitope and 7 apart from, respectively. Combinations of both Tn88128 (1999) and commercially available vaccine strain (Tn73) were developed and its safety was evaluated in mice, guinea pigs, calves, and pregnant cows. None of the animals showed any adverse effect or clinical signs. Calves were immunized with commercial vaccine (Tn73) and, combined vaccine (Tn73 and Tn88128), respectively, with adjuvants such as Al-gel and water-in-oil-in-water (w/o/w) oil and PBS alone and challenged with Tn88128 strains. Except PBS administered animals, all the vaccinated animals showed protective immune response. However, animals immunized with combined vaccine plus w/o/w adjuvant elicited stronger neutralization antibodies and long lasting immunity compared to other vaccines.     

The enzymatic profile of urine and plasma in bovine urinary bladder cancer (enzootic bovine haematuria)

The enzymatic profile of urine and plasma in field cases of bovine bladder cancer was studied. Urinary lactate dehydrogenase activity was significantly altered along with the isoenzyme pattern. Activity of alkaline phosphatase and -glucuronidase was decreased in the affected animals. No significant changes were observed in acid phosphatase, or arylsulphatase A and B activity. In plasma, lactate dehydrogenase activity was elevated without any change in the isoenzyme pattern. No significant changes were observed in the other plasma enzymes studied or in the sialic acid concentration.     

Evaluation of safety and efficacy of DNA vaccines against bovine herpesvirus-1 (BoHV-1) in calves

Four DNA vaccines against BoHV-1 were evaluated for their efficacy in calves. Twelve animals were divided into four groups which were injected with four different DNA vaccines: pVAX-tgD (Vaccine A); pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B); pVAX-UbiLacI-tgD-L (Vaccine C); pVAX-UbiLacI-tgD-L co-immunised with pVAX-48CpG (Vaccine D). Three additional calves were given the plasmid vector and served as controls. Ninety days after the first vaccination all calves were challenge infected with BoHV-1.All animals developed a severe form of infections bovine rhinotracheitis. Only the calves given the pVAX-tgD co-immunised with pVAX-48CpG (Vaccine B) developed humoral antibodies against BoHV-1 between 56 and 90 days after the first vaccination, whereas in calves of other groups and in the controls, antibodies appeared only after the infection. In the calves vaccinated with either pVAX-tgD (Vaccine A) or pVAX-tgD combined with pVAX-48CpG (Vaccine B), BoHV-1-specific IFN-γ secreting cells were detected in PBMCs 90 days after the first vaccination and their number increased after challenge exposure. In the other groups the IFN-γ secreting cells were detected after virus infection and at low values.     

牛白细胞介素-2基因的克隆与序列分析

白细胞介素-2(interleukin-2,IL-2)是一种具有广泛生物学活性的细胞因子,主要由激活的T淋巴细胞产生,它能提高免疫细胞杀伤癌细胞、病毒或细菌感染细胞以及促进抗体生成和分泌的能力[1-2],在机体正常免疫过程中起着十分重要的作用.     

Characterisation of the fiber gene and partial sequence of the early region 4 of bovine adenovirus 2 (short communication)

The full sequence of the fiber gene and partial sequence of the putative 17 kD protein gene of bovine adenovirus-2 (BAdV-2) were determined. The size of the fiber gene of BAdV-2 proved to be 561 amino acids, of which the amino acids 37 to 385 form a typical shaft domain of 22 repetitive motifs. On the complementary strand, a gene homologous to the 17 kD protein coded in the E4 region of several human adenoviruses was found. The sequence analysis seems to confirm the presence of an intron in the sequenced part of the E4 region.