Characterization of the bovine immunoglobulin lambda light chain constant IGLC genes

To characterize the bovine immunoglobulin lambda light chain constant region (IGLC) genes, we have isolated a bacterial artificial chromosome (BAC) clone by a PCR based approach from a bovine genomic DNA library, constructed using a genital ridge cell line derived from a male Holstein fetus. The positive BAC clone, containing the bovine IGLC genes, was fully sequenced and had a 138 kb insert. Sequence analysis revealed that the bovine immunoglobulin lambda light chain locus consisted of four joining-constant gene recombination units spanning approximately 20 kb DNA in length. A detailed examination of the recombination signal sequences, RNA splicing sites and coding sequences of the four joining-constant gene recombination units suggested that only two IGLC genes (IGLC2 and IGLC3) were functional while the IGLC1 and IGLC4 appeared to be pseudogenes. This conclusion was further confirmed by a series of RT-PCR amplifications, which also showed that among these four genes the IGLC3 was preferentially expressed in cattle. Phylogenetic analysis indicated that the bovine IGLC genes were more closely related to their equivalents in sheep and goats than that to other mammals.     

哺乳动物免疫球蛋白重链恒定区基因研究进展

免疫球蛋白分子是由2条相同的重链和2条相同的轻链所构成的多肽链结构,其中重链有5种,与之相应的免疫球蛋白分子为5类,整个免疫球蛋白分子可分为恒定区和可变区2部分。由于免疫球蛋白重链恒定区在免疫过程中起多种效应作用,目前已对多种动物的免疫球蛋白重链恒区基因进行了研究,如哺乳动物、鱼类、两栖类、爬行类、鸟类等。了解哺乳动物免疫球蛋白的结构、功能起到了重要的作用,也对了解免疫系统的起源和进化提供依据。     

Novel insight into antibody diversification from cattle

The bovine preimmune repertoire develops in the absence of maternal antibodies due to the placental barrier formed by syndesmochorial type of placenta. The limited germline sequence diversity, both at the heavy and light chain loci, imposes constraints on generation of combinatorial diversity in cattle. The cattle, thus, must employ other strategies for antibody diversification. Analysis of VDJ rearrangements in adult cattle have led identification of generation of large IgM antibody molecules that may have an exceptionally long CDR3H region (up to 61 amino acids). The IgM antibodies with an exceptionally long CDR3H are indeed functional as some of these recognize structurally dissimilar antigens. The antibody diversification in cattle involves generation of an exceptionally long CDR3H in addition to point somatic mutations.     

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.     

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.     

Nucleotide sequence and restriction fragment length polymorphisms of the equine Cvarepsilon gene

IgE is the dominant immunoglobulin isotype involved in type I hypersensitivities in mammals. The heavy chain constant region domains of equine IgE are encoded by a single gene, the Cvarepsilon gene. By restriction analysis of cDNA from 15 unrelated horses, we have now identified two Cvarepsilon alleles, characterised by a Sma I restriction fragment length polymorphism, which we designated Cvarepsilon(a) and Cvarepsilon(b). Sequence analysis of both, Cvarepsilon(a) and Cvarepsilon(b) cDNA, showed in addition two single base exchanges resulting in two amino acid substitutions. Both sequences have only 95.9% homology of the coding region sequence with the published equine Cvarepsilon sequence, which could represent a third haplotype. Polymorphism of the IgE heavy chain constant region gene, as described here, might well impose genetic variability on the effector functions of equine IgE predisposition to allergic diseases in horses.     

Cross-reactivity of monoclonal antibodies to bovine immunoglobulins with immunoglobulins of other species.

Monoclonal antibodies (Mabs) to bovine immunoglobulin heavy chain of the four major isotypes gamma 1, gamma 2, alpha, mu and the light chains (combined kappa and lambda) were produced and found to cross-react in enzyme-linked immunoassay (ELISA) with immunoglobulins of some other animal species despite the discrete specificity associated with an antibody derived from a single clone. This cross-reactivity, particularly amongst ruminants, could be utilized in serological testing for the diagnosis of disease in these species. For example, Mabs produced against bovine immunoglobulin light chain cross-react with bison immunoglobulin light chain and were used successfully in serological testing as the secondary detection antibody in an indirect ELISA for the diagnosis of Brucella abortus in bison herds in north-western Canada.     

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.     

Tritrichomonas foetus isolates from cats and cattle show minor genetic differences in unrelated loci ITS-2 and EF-1α

The protozoan parasite Tritrichomonas foetus is well known as an important causative agent of infertility and abortion in cattle (bovine trichomonosis). This World Organisation for Animal Health (O.I.E.) notifiable disease is thought to be under control in many countries including Switzerland. In recent studies, however, T. foetus has also been identified as an intestinal parasite that causes chronic large-bowel diarrhoea in cats. Since the feline isolates were considered indistinguishable from bovine isolates, the possibility and risk of parasite transmission from cats to cattle and vice versa has been intensively discussed in current literature. Therefore, we investigated if cat and cattle isolates are genetically distinct from each other or in fact represent identical genotypes. For this purpose, two independent genetic loci were selected that turned out to be well-suited for a PCR sequencing-based genotyping of trichomonad isolates: (i) previously published internal transcribed spacer region 2 (ITS-2) and (ii) a semi-conserved sequence stretch of the elongation factor-1 alpha (EF-1α) gene used for the first time in the present study. Respective comparative analyses revealed that both loci were sufficiently variable to allow unambiguous genetic discrimination between different trichomonad species. Comparison of both genetic loci confirmed that T. suis and T. mobilensis are phylogenetically very close to T. foetus. Moreover, these two genetic markers were suited to define host-specific genotypes of T. foetus. Both loci showed single base differences between cat and cattle isolates but showed full sequence identity within strains from either cat or cattle isolates. Furthermore, an additional PCR with a forward primer designed to specifically amplify the bovine sequence of EF-1α was able to discriminate bovine isolates of T. foetus from feline isolates and also from other trichomonads. The implications these minor genetic differences may have on the biological properties of the distinct isolates remain to be investigated.     

Identification of genes responsible for hereditary diseases in Japanese beef cattle

In the breeding of domestic animals, selection of economically desired traits has been the most important consideration for the improvement of animals, but excluding negative factors in animal production, such as causative genes for hereditary diseases, is also required for the genetic improvement of domestic animals. The incidence of various hereditary diseases has been reported in Japanese beef cattle and these diseases have caused serious problems in the breeding and raising of healthy beef cattle. This article reviews the identification of causative genes for the following three hereditary diseases in Japanese beef cattle: (i) Chediak–Higashi syndrome; (ii) renal tubular dysplasia; and (iii) bovine chondrodysplastic dwarfism. Chediak–Higashi syndrome is a hereditary bleeding disorder reported in Japanese black cattle. To identify the cause of this disease, we cloned and sequenced the bovine LYST gene, which has been found to be involved in Chediak–Higashi syndrome in humans, and found that an amino acid substitution of histidine to arginine at amino acid residue 2015 is the causative mutation in the cattle disease. Renal tubular dysplasia is a hereditary disease of Japanese black cattle showing renal failure and growth retardation. We mapped the locus for this disease to the 4 cM region of bovine chromosome 1 by linkage analysis and found a large deletion in this region. The deleted region contained the PCLN1 gene encoding a tight‐junction protein of renal epithelial cells, and we concluded that deletion of the PCLN1 gene is responsible for the disease. Bovine chondrodysplastic dwarfism is a hereditary disease of Japanese brown cattle, displayed by short limbs and joint abnormalities. We mapped the locus for the disease to a region of bovine chromosome 6 by linkage analysis. By constructing YAC and BAC contigs covering this region and sequence analysis, we identified a novel gene (LIMBIN), which plays an essential role in bone formation in this region, and found two mutations responsible for the disease. The identification of these mutations provided the basis for DNA‐based diagnostic systems for these three diseases, and after development of the diagnosis systems, the incidences of these hereditary diseases have dramatically decreased.     

牛Fas基因的组织表达分析及其功能预测

采用RT-PCR技术从中国西门塔尔牛睾丸组织总RNA中反转录FascDNA,将其克隆于pMD19-Tvector后进行测序分析,并对其表达的蛋白结构功能进行预测、分析。结果表明:牛的FascDNA序列为1109bp,编码323个氨基酸残基,在氨基酸序列上与羊、猪、人、小鼠的相似性分别为90.5%、65.3%、56.7%和48.6%。Fas蛋白的胞外区有2个糖基化位点和3个富含半胱氨酸残基的结构亚域,对Fas蛋白的定位及凋亡信号识别有重要作用。牛与羊、猪、人、小鼠Fas的死亡结构域(Death domain,DD)氨基酸序列的相似性分别为94.3%、68.5%、59.6%和70.8%,体现了该结构在各物种间较强的保守性及接受凋亡信号诱导靶细胞凋亡的重要性。组织表达谱发现,Fas不仅表达于牛的淋巴、脾等淋巴系统,而且高表达于牛生殖系统的睾丸中,这对于进一步阐明Fas在公牛精子发生过程中的调控作用奠定基础。     

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat (Felis domesticus) Ig epsilon constant region

A feline splenic cDNA library was screened with a (32)P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline C epsilon gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.     

Characterization of the immunoglobulin A heavy chain gene of the Atlantic bottlenose dolphin (Tursiops truncatus)

Immunoglobulin constant region heavy chain genes of the dolphin (Tursiops truncatus) have been described for IgM and IgG but not for IgA. Here, the heavy chain sequence of dolphin IgA has been cloned and sequenced as cDNA. RT-PCR amplification from blood peripheral lymphocytes was carried out using degenerate primers and a single sequence was detected. The inferred heavy chain structure shows conserved features typical of mammalian IgA heavy chains, including three constant (C) regions, a hinge region between constant region domain 1 (C1) and constant region domain 2 (C2), and conserved residues for interaction with the Fc alpha R1 and N-glycosylation sites. Comparisons of the deduced amino acid sequences of the IgA heavy chain for the dolphin and the evolutionarily related artiodactyl species showed high similarity. In cattle and sheep, as in dolphins, a single IgA subclass has been identified. Southern blot analysis as well as genomic PCR confirmed the presence of multiple IGHA sequences suggesting that IGHA pseudogenes may be present in the dolphin genome.     

STAT3基因对广西黄牛肌肉干细胞成肌诱导分化的调控研究

研究旨在检测信号转导与转录激活因子3（STAT3）的表达规律，克隆STAT3基因，构建真核表达载体，并探索STAT3基因对广西黄牛肌肉干细胞（MuSCs）分化的调控作用。采集6、12、18月龄广西黄牛肌肉组织以及生长期（GM）和分化期（DM）肌肉干细胞，分别提取RNA并反转录为cDNA，通过实时荧光定量PCR检测STAT3基因和成肌相关基因的表达规律；克隆黄牛STAT3基因完整编码区序列，构建过表达载体pCD-STAT3并检测其对黄牛肌肉干细胞分化的影响。实时荧光定量PCR结果表明，STAT3基因在6、12、18月龄黄牛肌肉组织中均有表达，且在18月龄中表达量最高，12月龄中表达量最低；STAT3和成肌调节因子6（MYF6）基因在分化期肌肉干细胞中表达量均极显著高于生长期（P<0.01）。广西黄牛STAT3基因编码区全长2 313 bp，与空载体pCDNA3.1相连成功构建过表达载体pCD-STAT3，将pCD-STAT3转入体外培养广西黄牛肌肉干细胞，肌肉干细胞中STAT3基因及成肌决定蛋白（MyoD1）、骨骼肌肌球蛋白重链（MyHC）基因表达量极显著升高（P<0.01），肌细胞生成素（MyoG）基因表达量显著升高（P<0.05），且肌管数量、大小均显著高于pCDNA3.1组（P<0.05）。本研究检测了转录因子STAT3在广西黄牛背最长肌中的表达规律；且过表达STAT3基因显著促进了广西黄牛肌肉干细胞的成肌分化，为深入研究STAT3基因在肌肉生长发育中的作用及其分子调控机制奠定基础。     

秦川牛Snail2基因扩增、序列特征及表达特性分析

试验旨在扩增秦川牛Snail2基因，构建Snail2基因在其各组织不同发育阶段及脂肪细胞不同分化时期的时空表达规律，为进一步研究Snail2基因在牛脂肪沉积中的功能及调控作用奠定基础。以秦川牛为研究对象，经PCR扩增得到Snail2基因CDS区序列，运用生物信息学软件对其功能结构进行预测，同时采用实时荧光定量PCR检测Snail2基因在新生牛和成年牛各组织及脂肪细胞成脂分化过程中的时空表达谱。结果显示，秦川牛Snail2基因编码序列全长为952 bp。不同物种系统进化树结果表明，牛Snail2蛋白在家牛、水牛、山羊、绵羊中高度保守。磷酸化位点分析发现，存在41个潜在磷酸化位点，其中周期蛋白依赖性激酶1（cyclin dependent kinase 1，CDK1）、CDK5、CKⅡ、CKⅠ等多个细胞周期相关激酶参与了Snail2蛋白的磷酸化修饰。蛋白结构域预测发现，牛、人和鼠等8个物种中有8个相似Motif。Snail2蛋白二级结构主要由不规则卷曲构成，二、三级结构预测结果一致。Snail2基因启动子区序列分析发现1个CpG岛及E2F、C/EBPα（CCAAT/enhancer binding proteins alpha）、AP2和Sp1等与脂肪生成相关的转录因子结合位点。实时荧光定量PCR结果显示，Snail2基因在牛脂肪组织中呈现较高表达水平，且随着脂肪细胞成脂分化和牛生长发育过程均呈现上升趋势，表明Snail2基因在牛脂肪沉积过程中发挥重要作用。研究结果为进一步揭示Snail2基因通过影响脂肪细胞增殖和分化进而影响肉牛脂肪沉积作用机制奠定基础。