Gene-conversion in rabbit B-cell ontogeny and during immune responses in splenic germinal centers

Combinatorial diversity is limited in rabbits because only a few V(H) genes rearrange. Most diversification of the primary repertoire is generated by somatic hypermutation and gene conversion-like changes of rearranged V(H) in B cells that migrate to appendix and other gut associated lymphoid tissues (GALT) of young rabbits. The changes are referred to as gene conversion-like because the non-reciprocal nature of the alterations introduced has not yet been demonstrated. There are many similarities between rabbits and chickens in how their B cells develop and diversify their repertoires. However, although the majority of rabbit B cells may have rearranged and diversified their V genes early in life, some B cells in adult rabbits have rearranged VH sequences that are identical or nearly identical to germline sequences. We found these cells in splenic germinal centers (GC) on days 7 and 10 after immunization of normal adult rabbits with DNP-BGG. By day 15, all rearranged V(H) sequences were diversified. We find an overall pattern of splenic precursor cells whose germline or near germline sequences change both by gene conversion and point mutations during early divisions and mainly by point mutations during later divisions. These events, in parallel with diversification of light chain sequences, may produce the diverse combining sites that serve as substrates for further affinity maturation by selection either within GC or later among emigrant cells in sites such as bone marrow. Some of the sequences altered by gene conversion in splenic germinal centers may also produce new members of the B-cell repertoire in adult rabbits comparable to those produced in GALT of neonatal rabbits.     

Developmental progression of immunoglobulin heavy chain diversity in sheep

In order to assess the respective impacts of combinatorial rearrangement, junctional diversification, somatic hypermutation and gene conversion in the generation of immunoglobulin heavy chain variable regions diversity, the sequences of 42 variable regions from late fetal, newborn and young sheep were determined and compared to those of adult animals. At earlier stages of development, the use of germline diversity segments appears restricted, junctional variability is already established, and somatic hypermutations are scarce. The sequence diversity in adults is much higher, which we suggest results from a higher hymermutation activity and possibly from the use of a variety of diversity segments. Altogether, this pattern is very reminiscent of the situation observed in cattle, except for the length of the third complementarity determining regions (CDR3) which are shorter in sheep than in bovine. Unlike the chicken and rabbit systems, it seems that new rearrangements continue to occur in sheep for at least several months after birth.     

Immunoglobulin genes and diversity: what we have learned from domestic animals

ABSTRACT: This review focuses on the diversity of immunoglobulin (Ig) genes and Ig isotypes that are expressed in domestic animals. Four livestock species--cattle, sheep, pigs, and horses--express a full range of Ig heavy chains (IgHs), including mu, delta, gamma, epsilon, and alpha. Two poultry species (chickens and ducks) express three IgH isotypes, mu, upsilon, and alpha, but not delta. The kappa and lambda light chains are both utilized in the four livestock species, but only the lambda chain is expressed in poultry. V(D)J recombination, somatic hypermutation (SHM) and gene conversion (GC) are three distinct mechanisms by which immunoglobulin variable region diversity is generated. Different domestic animals may use distinct means to diversify rearranged variable regions of Ig genes.     

Somatic hypermutation leads to diversification of the heavy chain immunoglobulin repertoire in cattle

The availability of unique variable (VH), diversity (D), and joining (JH) gene segments in the vertebrate germline determines the extent to which a primary immunoglobulin (Ig) repertoire can be generated through combinatorial rearrangement. Although bovine D segments possess unusual properties, the diversity of the primary Ig heavy chain (IgH) repertoire in cattle is restricted by the dominance of a single family of germline VH genes of limited number and diversity. Cattle therefore must employ other diversification strategies in order to generate a functional IgH repertoire, the main candidates being gene conversion and somatic hypermutation. In considering these possibilities, we predicted that if somatic hypermutation was active during B lymphocyte development, the process would introduce nucleotide substitutions to the VDJ exon and also non-coding region lying downstream of the rearranged JH segment. In contrast, our expectation was that gene conversion would show a greater tendency to confine modification to the IgH coding sequence, leaving intron regions substantially unmodified. An analysis of rearranged IgH sequences from cattle of different ages revealed that the diversification of germline sequences could be observed in very young calves and that substitution frequency increased with age. The age-dependent accumulation of mutations was particularly apparent in the second IgH complementarity-determining region (CDR2). Single base substitutions were found to predominate, with purines targeted more frequently than pyrimidines and transitions favoured over transversions. In non-coding regions, mutations were detected at a normalised frequency that was indistinguishable from that observed in CDR2. These data are consistent with a process of IgH diversification driven predominantly by somatic hypermutation.     

The immunoglobulin light chain locus of the turkey, Meleagris gallopavo

To date, most jawed vertebrate species encode more than one immunoglobulin light (IgL) chain isotypes. It has been shown that several bird species (chickens, white Pekin or domestic duck, and zebra finches) exclusively express lambda isotype. We analyze here the genomic organization of another bird species turkey IgL genes based on the recently released genome data. The turkey IgL locus located on chromosome 17 spans approximately 75.2kb and contains a single functional V(λ) gene, twenty V(λ) pseudogenes, and a single functional J(λ)-C(λ) block. These data suggest that the genomic organization of bird IgL chain genes seems to be conserved. Ten cDNA clones from turkey Igλ chain containing almost full-length V(λ), J(λ) and C(λ) segments were acquired. The comparison of V(λ) cDNA sequences to all the germline V(λ) segments suggests that turkey species may be generating IgL chain diversity by gene conversion and somatic hypermutation like the chicken. This study provides insights into the immunoglobulin light chain genes in another bird species.     

新疆地方绵羊品种PRNP基因多态性研究

从新疆采取了8个地方绵羊品种的血液样品171份,提取绵羊基因组DNA,用PCR方法扩增绵羊PRNP基因,通过序列测定,对它们的PRNP基因型进行研究,确定了PRNP基因136、154、171位密码子的多态性为136(A/A),154(H/R)和171(Q/R/H/K),结果发现所检测的新疆地方绵羊品种PRNP基因136位密码子均为A,其基因型均为A型痒病抵抗性基因型。     

Site-directed mutagenesis of the myostatin gene in ovine fetal myoblast cells in vitro

Myostatin is an important negative regulator of muscle growth and development. Natural mutations of the myostatin gene cause a double muscling phenotype in beef cattle, pigs and sheep. Therefore, it is feasible to produce a high growth domestic breed by generating a transgenic animal with a mutation, deletion or knockout of the myostatin gene. Our objective was to introduce a subtle mutation of G to A 281-bp upstream of the 3' untranslated region (3'UTR) end of the myostatin gene in Poll Dorset fetal myoblast cells in vitro. Fetal myoblast cells were isolated from fetuses at day 50 of gestation from Poll Dorset sheep and transfected with linear gene-targeting vector pMSTN-A using electroporation. We obtained seven gene-targeted cell colonies with homologous recombination, which were positive as confirmed by PCR, Southern blot. The Western blot analysis result demonstrated that the myostatin protein expression in positive colonies is lower than that of negative ones. These results strongly suggest that we successfully mutated the myostatin gene of Poll Dorset ovine fetal myoblast cells and the mutation can effectively downregulate the myostatin protein expression.     

Genetic characterization of Indian peste des petits ruminants virus (PPRV) by sequencing and phylogenetic analysis of fusion protein and nucleoprotein gene segments

Peste des petits ruminants (PPR) is an important viral disease of sheep and goats, endemic in India. The study was undertaken to characterize the local PPRV by sequencing fusion (F) protein and nucleoprotein (N) gene segments and phylogenetic analysis, so as to focus on genetic variation in the field viruses. Selected regions of PPRV genome were amplified from clinical samples collected from 32 sheep and goats by RT-PCR and the resulting amplicons were sequenced for phylogenetic analysis. The phylogenetic tree based on the 322bp F gene sequences of PPRV from five different locations clustered them into lineage 4 along with other Asian isolates. While the 425bp N gene sequences revealed a different pattern of branching, yielding three distinct clusters for Nigerian, Turkey and Indian isolates. Thus, classification of PPRV into lineages based on the N gene sequences appeared to yield better picture of molecular epidemiology for PPRV.     

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.     

Analysis of the genomes of bluetongue virus serotype 10 vaccines and a recent BTV-10 isolate from Washington

The 10 double-stranded RNA gene segments of 2 vaccinal strains of bluetongue virus (BTV) serotype 10 that are used in the United States (BTV CA8 California and BT-8 Colorado), and a BTV-10 isolate recently obtained from infected sheep in Washington (state) were characterized by oligonucleotide fingerprint analyses. It was determined that although the 2 BTV-10 vaccinal strains are genotypically distinct, they are closely related both to each other and to the United States prototype BTV-10 virus. The BTV-10 field isolate appears to be a naturally occurring reassortment virus with genome segments derived from both United States prototype BTV-10 and BTV-11 viruses. However, one RNA segment of the isolate was totally unlike the corresponding segments of United States prototype BTV-10, -11, -13 and -17 viruses.     

Amino acid polymorphisms of PrP gene in Mongolian sheep

To characterize amino acid polymorphisms in sheep prion protein (PrP), we analyzed the PrP genes from 271 sheep of 4 breeds (Khalkh, Yeroo, Orkhon and Khangai) raised in central Mongolia (Tuv, Uvurkhangai and Selenge prefectures). A total of 16 genotypes and 8 allelic variants of the PrP gene at codons 112, 136, 154 and 171 were found. At codon 171, 1.8% of the sheep had arginine/arginine (R/R) (resistant to scrapie) and 66.8% had glutamine/glutamine (Q/Q) (susceptible to scrapie). Several Yeroo and Orkhon sheep raised in Selenge prefecture had valine at codon 136 (136V) (highly susceptible to scrapie). Several Yeroo, Orkhon and Khangai sheep raised in Selenge prefecture had histidine at codon 154 (154H). Novel polymorphisms of valine (V) and serine (S) at codon 127, lysine (K) at codon 171, and leucine (L) and arginine (R) at codon 189 were also found in Khalkh, Yeroo and Orkhon sheep. It is not known whether these novel polymorphisms affect scrapie susceptibility.     

Polymorphism Analysis of Taste Receptor Family 1 Member Gene Exons in Sheep

This study was aimed to investigate the distribution of genetic polymorphism of taste receptor family 1 member (T1R) gene between Ujimqin sheep and Hu sheep.DNA pools direct sequencing method and MALDI-TOFMS method were used to analyze genetic variation of T1R genes in 172 sheep of two Chinese sheep strains of Mongolian,and bioinformatics software predicted what impact polymorphic loci had to mRNA and protein secondary structure of T1R gene.The results showed that 9 SNPs were screened in T1R gene of two groups.Chi-square test for independence was taken to find the genotypes of the 5 SNPs which were significantly different between two sheep population(P<0.05),SNP2 located in TAS1R1 gene,SNP4,SNP7 and SNP8 located in TAS1R2 gene,SNP10 located in TAS1R3 gene.SNP2,SNP7 and SNP10 were silent mutations.SNP2 and SNP10 lead to corresponding gene mRNA secondary structure and the minimum free energy change,while the SNP7 only lead to the minimum free energy changes.SNP4 and SNP8 were missense mutations,the two missense mutations respectively led to asparagine(Asn) into serine (Ser),threonine (Thr) into methionine (Met),and according to online software forecast,the protein secondary structure of TAS1R2 gene all changed in mutations before and after.     

杜寒绵羊多胎性能候选基因BMPR-1B和BMP15的研究

为了从分子水平探讨杜寒绵羊的多胎机制,本试验在山西省某养殖场采集了87只经产杜寒母绵羊的耳组织,选取骨形态发生蛋白15 （bone morphogenetic protein 15,BMP15）和骨形态发生蛋白受体1B （bone morphogenetic protein receptor 1B, BMPR-1B）为候选基因,采用PCR-SSCP、PCR-RFLP法,结合母羊产羔数与所产羊羔初生重,分析其与杜寒绵羊多胎性能的相关性。结果显示,杜寒绵羊的BMPR-1B基因在第746位碱基处发生了A→G突变,检测到3种基因型：AA、AG和GG,A等位基因频率（0.5230）略高于G等位基因（0.4770）,A为优势等位基因;AG基因型频率（0.5172）高于GG（0.2184）和AA（0.2644）基因型,AG为优势基因型。χ²适合性检验显示该位点处于Hardy-Weinberg平衡状态;BMPR-1B基因第864位碱基未发生突变。杜寒绵羊的BMP15基因不存在V31D和S300G位点突变。在该群体中,BMPR-1B基因A746G位点GG、AG基因型个体的产羔数极显著高于AA基因型个体（P<0.01）,羔羊初生重在3种基因型间差异不显著（P>0.05）。综上所述,BMPR-1B基因是影响杜寒绵羊繁殖性能的一个主效基因,可以作为分子标记对杜寒绵羊进行辅助育种,初步排除BMP15基因突变对杜寒绵羊多胎性能影响的可能性。     

绵羊味觉受体第一家族基因外显子多态性分析

为了探讨绵羊味觉受体第一家族（taste receptor family 1 member,T1R）基因外显子多态性及其基因型在乌珠穆沁羊和湖羊群体中分布的差异性,试验采用DNA池直接测序及飞行时间质谱（MALDI-TOFMS）法对中国蒙古系两个绵羊品种共172个个体T1Rs基因外显子的遗传变异情况进行分析,利用生物信息学软件预测多态位点对T1Rs基因mRNA二级结构和蛋白质二级结构的影响。结果表明,在两个群体的T1R家族基因中筛查到9个SNPs。独立性卡方检验显示有5个多态位点基因型的分布在两个绵羊群体中存在显著性差异（P<0.05）,分别为TAS1R1基因上的SNP2,TAS1R2基因上的SNP4、SNP7和SNP8,TAS1R3上的SNP10,其中,SNP2、SNP7和SNP10为同义突变;SNP2和SNP10导致相应基因mRNA二级结构和最小自由能的改变,而SNP7仅导致TAS1R2基因最小自由能发生改变;SNP4和SNP8为错义突变,分别导致TAS1R2蛋白质中第379位天冬酰胺变为丝氨酸和第701位苏氨酸变成蛋氨酸,且突变前后受体蛋白的二级结构均发生改变。     

抗口蹄疫病毒单克隆抗体1C7重轻链可变区基因的克隆及序列分析

应用分子生物学技术，从分泌抗O型口蹄疫病毒单克隆抗体的杂交瘤细胞1C7中提取总RNA，经反转录，PCR扩增及克隆，分别得到VH基因及VL基因。序列测定结果表明：1C7的VH基因为368bp,VL基因为323bp。用NCBI GenBank分析表明，VH和VL均符合小鼠抗体可变区特征，为功能性重排的抗体可变区基因。根据Kabat分类体系，1C7的VH基因中的VH基因片段隶属于抗体重链第7183家族，其VL基因中的VL基因片段隶属于抗体K轻链20家族，VH基因由VH76－1BG－DFL16．1－JH4重排而形成，VL基因由KVbw20-JK2重排而形成。1C7的VH和VL基因的克隆为抗FMDV scFv的构建与表达奠定了基础。     

Protective effect of the AT137RQ and ARQK176 PrP alleles against classical scrapie in Sarda breed sheep

The susceptibility of sheep to scrapie is under the control of the host’s prion protein (PrP) gene and is also influenced by the strain of the agent. PrP polymorphisms at codons 136 (A/V), 154 (R/H) and 171 (Q/R/H) are the main determinants of susceptibility/resistance of sheep to classical scrapie. They are combined in four main variants of the wild-type ARQ allele: VRQ, AHQ, ARH and ARR. Breeding programmes have been undertaken on this basis in the European Union and the USA to increase the frequency of the resistant ARR allele in sheep populations. Herein, we report the results of a multi-flock study showing the protective effect of polymorphisms other than those at codons 136, 154 and 171 in Sarda breed sheep. All ARQ/ARQ affected sheep (n = 154) and 378 negative ARQ/ARQ controls from four scrapie outbreaks were submitted to sequencing of the PrP gene. The distribution of variations other than those at the standard three codons, between scrapie cases and negative controls, was statistically different in all flocks. In particular, the AT₁₃₇RQ and ARQK₁₇₆ alleles showed a clear protective effect. This is the first study demonstrating a protective influence of alleles other than ARR under field conditions. If further investigations in other sheep breeds and with other scrapie sources confirm these findings, the availability of various protective alleles in breeding programmes of sheep for scrapie resistance could be useful in breeds with a low frequency of the ARR allele and would allow maintaining a wider variability of the PrP gene.     

绵羊高繁殖力候选基因BMPR-IA的研究

以骨形态发生蛋白受体IA（bone morphogenetic protein receptor IA，BMPR-IA）基因为候选基因，采用PCR—SSCP技术检测该基因在高繁殖力绵羊品种（小尾寒羊、湖羊）以及低繁殖力绵羊品种（多赛特羊、特克塞尔羊、德国肉用美利奴羊）中的单核苷酸多态性，同时研究该基因对小尾寒羊高繁殖力的影响。结果表明：在小尾寒羊中检测到AA、AB两种基因型，在湖羊中只检测到一种基因型BB，而在低繁殖力的3个绵羊品种中只检测到一种基因型AA。统计结果表明：小尾寒羊A等位基因频率为0．964，B等位基因频率为0．036。测序结果表明：BB型与AA型相比有6处核苷酸发生了突变。独立性检验表明：外尾寒羊与低繁殖力绵羊品种间基因型分布差异不显著（P〉0．05），而湖羊与小尾寒羊、低繁殖力绵羊品种间基因型分布差异极显著（P〈0．001）。AB基因型小尾寒羊平均产羔数比AA基因型多0．15只，但差异不显著（P〉0．05）。研究表明：BMPR-IA基因不是小尾寒羊和湖羊高繁殖力的主效基因。     

Congenital malformations in sheep resulting from in utero inoculation of Cache Valley virus

Serologic evidence indicated that an episode of congenital abnormalities in sheep was caused by Cache Valley virus (CVV), a bunyavirus indigenous to the United States. To determine the teratogenic potential of CVV in sheep, fetuses were infected in utero between 27 and 54 days of gestation with an isolate (CK-102) obtained in 1987 from a sentinel sheep in San Angelo, Texas. The dams of these fetuses were euthanatized between 28 and 75 days after inoculation, and the fetuses were examined for malformations. Twenty-eight of 34 fetuses had congenital abnormalities, including arthrogryposis, hydranencephaly, mummification, reabsorption, and oligohydroamnion. Virus was isolated from the allantoic fluid of 11 of 17 fetuses euthanatized at less than 70 days of gestation. The virus-positive fetuses, which were all negative for CVV-neutralizing antibody, had lesions ranging from none to severe arthrogryposis and hydranencephaly. Virus was not recovered from the allantoic fluid of fetuses after 76 days' gestation when CVV-specific antibody could be detected in 5 of 8 fetuses examined. The 2 fetuses infected on days 50 and 54 of gestation appeared normal and 1 had antibody to CVV.