应用PCR检测隐孢子虫卵囊的研究

隐孢子虫病是一种重要的人畜共患原虫病。为了在临床样品中更准确、快速地检测隐孢子虫卵囊，从初步纯化的含有不同数量隐孢子虫卵囊的样品中和含有不同数量隐孢子虫卵囊的奶牛粪便中，直接提取DNA或用DNA纯化试剂盒对提取的奶牛粪便中卵囊DNA进行纯化之后用作PCR模板，用1对人工合成寡核苷酸作为PCR引物，扩增片段大小为452bp。优化了Mg^2 浓度、引物浓度和dNTP浓度，并进行了特异性检验。建立的PCR具有隐孢子虫属特异性，不仅扩增出新鲜样品DNA提取物中的目的片段，而且扩增出放置6年之久的DNA提取物中的目的片段。样品经过初步纯化之后，最低检测值100个卵囊／ml；从含有隐孢子虫卵囊的奶牛粪便中提取DNA，尔后经过DNA纯化试剂盒纯化，PCR最低检测值为10^5个卵囊/g粪便。     

Detection of γ-H2AX,a Biomarker for DNA Double-strand Breaks,in Urinary Bladders of N -Butyl- N -(4-Hydroxybutyl)-Nitrosamine-Treated Rats

To evaluate the potential role of DNA repair in bladder carcinogenesis, we performed an immunohistochemical analysis of expression of various DNA repair enzymes and γ-H2AX, a high-sensitivity marker of DNA double-strand breaks, in the urothelium of male F344 rats treated with N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN), a bladder-specific carcinogen. Our results clearly demonstrated that γ-H2AX aggregation was specifically generated in nuclei of bladder epithelial cells of BBN-treated rats, which was not found in untreated controls or mesenchymal cells. γ-H2AX-positive cells were detected not only in hyperplastic and neoplastic areas but also in the normal-like urothelium after BBN treatment. These data indicate that γ-H2AX has potential as a useful biomarker for early detection of genotoxicity in the rat urinary bladder. To the best of our knowledge, this is the first report demonstrating expression of γ-H2AX during bladder carcinogenesis.     

DNA testing for type III von Willebrand disease in Dutch Kooiker dogs

Von Willebrand disease type III is widespread in Dutch Kooiker dogs. To eradicate von Willebrand disease from the breed, affected dogs and nonsymptomatic carriers must be excluded from breeding. Previous efforts to detect carriers in Kooiker dogs by a von Willebrand factor antigen assay were not satisfactory because of considerable overlap of plasma concentrations in normal dogs and carriers. The aim of this study was to develop and apply a DNA test for the detection of von Willebrand disease carriers in the Kooiker breed. Two mutations in the von Willebrand factor gene in affected Kooiker dogs have been described previously, a splice site mutation at the border of intron 16 and exon 16 and a missense mutation in exon 3. We have developed polymerase chain reaction tests for both mutations in genomic DNA. The missense mutation most likely is a neutral variant and appears to be a polymorphism present in many breeds. The allele-specific oligonucleotide test for the splice site mutation was applied in the selection of animals cleared to breed by the Dutch breeding club. In a few years, the mutation has been eliminated from the breeding stock without apparent increase of inbreeding or preferential sire usage.     

安哥拉山羊与建昌黑山羊及其杂种后代的DNA指纹分析

以 (CA/GATA/TCC) 5探针 ,HinfI酶切 ,研究安哥拉山羊和建昌黑山羊及安哥拉山羊与建昌黑山羊级进杂交的F2 、F3的DNA指纹图谱。结果 ,在供试山羊中 ,个体平均检出 1 8.2± 0 .4条谱带 ,安哥拉山羊、F2 、F3的鉴别机率分别为 1 .38× 1 0 - 12 ,1 .1 8× 1 0 - 13,0 .50× 1 0 - 10 ;群体内相似系数安哥拉山羊为0 .4 539,F2 为 0 .4 0 77,F3为 0 .61 1 1 ;亲子鉴定的父权概率W =0 .9889;安哥拉山羊与建昌黑山羊比较 ,2 .3kb和 8.6kb谱带为安哥拉山羊的特异谱带     

基因组印记的研究进展

综述基因组印记的可能发生机制及人和鼠中常见的印记基因，论述了且印记作用的生物学意义。     

Study on Genetic Diversity and System Evolution of mtDNA D-Loop Region in Xianglushan Chicken

This study was aimed to analyze the genetic diversity and phylogenetic evolution in Xianglushan chicken.The full-length sequences of mitochondrial DNA D-Loop (mtDNA D-Loop) region of 121 Xianglushan chickens were analyzed by PCR amplification combined with bidirectional sequencing,and the composition,variation and maternal origin of mtDNA D-Loop region were discussed.The results showed that the total length of mtDNA D-Loop region in Xianglushan chicken was 1 231-1 233 bp.The contents of A,T,C and G were 26.62%,33.55%,26.49% and 13.34%,respectively.The content of T was the highest,the content of G was the lowest,and the content of A+T was significantly higher than that of G+C,it indicated that region might have certain base hobbies.Analysis of 121 full-length sequences were found to coexist in 11 haplotypes and 26 mutation sites,of which 2 were single polymorphic information sites,24 were simple information sites,4 bases were inserted and 2 bases were missing.The genetic diversity analysis results showed that the haplotype diversity was 0.814,the nucleotide diversity was 0.00447,the average nucleic acid difference was 5.494,which indicated that the genetic diversity of Xianglushan chicken was relatively rich,the effect of preservation was better,which had a certain breeding space.Tajima's D was 0.39378 and the test results were not significant (P>0.10),in line with neutral mutations.The cluster analysis results showed that Xianglushan chicken and Gallus gallus gathered as one,indicating that Xianglushan chicken originated from Gallus gallus, and there were 4 branches inside the branch,indicating that Xianglushan chicken had many matrilineal origins.The results could provide some reference data for the protection and exploitation of pheasant germplasm resources in Xianglushan chicken.     

Methylation of free-floating deoxyribonucleic acid fragments in the bronchoalveolar lavage fluid of dogs with chronic bronchitis exposed to environmental tobacco smoke

Background

The etiology of canine chronic bronchitis (CB) is not completely understood, although exposure to environmental tobacco smoke (ETS) affects the airway inflammatory responses in some dogs with CB. The mechanism by which this occurs is unknown.

Findings

We investigated the concentrations and methylation rates of free-floating DNA fragments in bronchoalveolar lavage fluid (BALF) from dogs with chronic bronchitis. Based on serum cotinine levels, dogs with CB were divided into 2 groups: dogs that either had or had not been exposed to ETS. Our results demonstrated that the total nucleated cell and macrophage numbers increased in BALF of ETS-exposed dogs with CB. There were no significant differences in DNA concentrations and methylation rates in BALF between the 2 groups. However, 3 out of 8 dogs exposed to ETS had high DNA methylation rates in their BALF samples.

Conclusion

Our results suggest that ETS exposure leads to epigenetic modifications of cellular components in BALF in dogs diagnosed with CB.     

线粒体DNA在鳞翅目昆虫系统学研究中的应用

线粒体DNA具有母性遗传与进化速率快等特点,已作为理想的分子标记广泛应用于昆虫的分类学、群体遗传学、系统发育和分子进化等研究。本文介绍了鳞翅目昆虫线粒体基因组的特征,对线粒体DNA应用于鳞翅目昆虫系统发生和分子进化等方面的研究进行了综述,并总结了线粒体基因或区段在鳞翅目昆虫不同分类阶元的适用性。     

Detection of papillomavirus-DNA in mesenchymal tumour cells and not in the hyperplastic epithelium of feline sarcoids

Abstract We examined 12 formalin-fixed paraffin-embedded feline skin tumours which had the histopathological features of fibropapillomas for the presence of papillomavirus (PV) DNA using touchdown polymerase chain recation (PCR), DNA sequencing and nonradioactive in situ hybridization. Nine of the tumours contained a 102-bp PCR product demonstrated using consensus PV primers that amplify a portion of the L1 gene. The nucleotide sequences are closely related, but not identical to that of ovine PV type 2, rabbit oral PV and reindeer PV. The deduced amino acid sequences had strong homologies with the major capsid protein L1 of deer PV, bovine papillomavirus (BPV) 1 and BPV 2, and European elk PV. Although PV antigens were not detected in any of the tumours by immunohistochemistry, PV DNA was demonstrated in individual mesenchymal cells or cell nests of 4/12 tumours by in situ hybridization. A nonproductive infection of mesenchymal fibroblast-like tumour cells with a papillomavirus would explain the lack of PV antigen expression and the absence of PV DNA in the hyperplastic epithelium. Because these tumours and their pathogenesis are similar to equine sarcoids, we suggest that they should be reclassified as 'feline sarcoids' instead of fibropapillomas.     

H19和IGF2R基因在体细胞克隆猪各组织中的甲基化状态

为了探求新生克隆猪可能的死亡原因以及是否存在不完全的DNA甲基化重编程,本试验运用亚硫酸氢盐测序法分别检测了H19基因和IGF2R基因差异甲基化区（DMR）在新生死亡克隆猪和同期正常猪心脏、肝脏、脾脏、肺脏和肾脏中的甲基化状态。结果发现,H19基因DMR在克隆猪肺脏中表现为超甲基化,极显著高于正常猪（95．20％VS46．80％P〈0．01）,且10个测序克隆中存在2处连续的全甲基化CpG位点（4-9位、12-S17位）,而在其他组织中甲基化差异不显著（P〉0．05）;IGF2R基因DMR在肝脏中处于超甲基化状态,显著高于正常猪（80．00％V839．41％P〈0．05）,而在肺脏中为去甲基化状态,板显著低于正常猪（14．71％VS66．47％P〈0．01）,在其他组织差异不显著（P〉0．05）。结果说明,在死亡克隆猪中,H19基因DMR在肺脏和IGF2R基因在肝脏与肺脏中存在不完全的DNA甲基化重编程,这可能是导致克隆动物死亡的因素之一。