日本牙鲆主要组织相容性复合体DAB等位基因的多态性

根据牙鲆(Paralichthys olivaceus)主要组织相容性复合体(MHC)DAB基因序列设计特异性引物,在日本牙鲆基因组中扩增了包括DAB基因完整外显子2和内含子1在内的长度为408 bp的DNA片段.对该片段克隆测序后,发现了37个MHC-DAB等位基因,各等位基因的频次以及各主型中亚型数目分布都不平衡.在第二外显子273 bp核苷酸序列中有50个位点发生变异,核苷酸多样性PI值为0.0618,在编码的氨基酸序列中存在多态变异位点30个,其中简约信息位点29个,单变异位点1个.非同义替代与同义替代的比率在抗原结合区(PBR)和非PBR编码区分别为10.0和1.62,分析表明正向选择机制可能是产生牙鲆MHC-DAB多态性的主要因素.估计的核苷酸的转换和颠换数随着遗传距离的增加而增加.各等位基因间的系统发生关系表明,19个主型分为两个群.对氨基酸组分和密码子的偏倚性以及分布频率的分析表明各同义密码子的使用频率不均衡.本研究为牙鲆MHC-DAB基因的遗传进化和分子标记辅助育种的研究提供了理论基础.     

藏鸡主要组织相容性复合体B-LBⅡ基因第二外显子序列遗传变异分析

根据鸡主要组织相容性复合体B-LBⅡ基因序列设计特异性引物,在藏鸡基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,并通过克隆和PCR直接测序获得了该片段的核苷酸序列。发现了15个B-LBⅡ新等位基因。对18个B-LBⅡ等位基因核苷酸序列和其所编码的MHCB-LBⅡ分子β1结构域的氨基酸序列分析显示,第二外显子核苷酸序列遗传多态性异常丰富,存在着62个多态变异位点(共包括80个突变),其中41个为简约性多态位点;衡量该序列遗传多样性的π值为0.0718;反映其群体内遗传变异度的平均遗传距离为0.056±0.008,低于在5个外来品种所估算的平均遗传距离。该编码区核苷酸相对异义替换率(15.61±2.69%)显著高于其同义替换率(3.25±0.94%),进一步分析表明,基因重组和平衡选择机制可能是引起B-LBⅡ基因序列变异的主要因素。在β1结构域氨基酸序列中,存在11个同义替换和27个异义替换;在24个肽结合位点中有12个变异位点;与其他6个中国地方鸡品种和一个外来品种比较发现,有11个异义氨基酸替换仅出现在藏鸡群体中,并被认为与藏鸡的免疫特异性有关,可为鸡的抗病力研究提供分子依据。     

中国部分地方鸡种MHC B-G基因第二外显子的遗传多态性

根据鸡主要组织相容性复合体BG基因序列设计特异性引物,在9个中国地方鸡种和1个国外引进鸡种基因组中扩增了包括其第一内含子和第二外显子在内、长度为401bp的DNA片段。经PCRSSCP分型筛选后,对该片段的核苷酸序列进行克隆测序和直接PCR测序及比对分析,发现了31个MHCBG新等位基因;各等位基因主型所含有的亚型数及其在不同品种间的分布极不均衡。第二外显子核苷酸序列和其所编码的MHCBG抗原类IgV结构域氨基酸序列比较表明,在中国地方鸡种BG基因第二外显子的207bp序列中有37个多态性变异位点,其中简约性信息位点29个,单个位点的变异8个;等位基因间的遗传变异范围0.0013-0.1433;各变异位点的核苷酸变异指数0.206-1.462。该编码区核苷酸的异义替换率为9.26%±1.92%,高于同义替换率2.34%±0.90%。所估计的核苷酸转换数和颠换数随着遗传距离的增加而逐渐增加,当核苷酸转换数和颠换数达到平衡后,该片段核苷酸转换数的增加幅度逐渐高于颠换数。在其所编码的类IgV结构域氨基酸序列中,多态变异位点有22个,其中简约性信息位点6个,单变异位点16个;所估测的等电点为8.45,疏水性氨基酸占40.3%,亲水性氨基酸占29.9%;该序列具有明显的疏水性特点。等位基因间的系统发生分析表明,31个BG等位基因分为两个群,相同主型的等位基因首先聚类。本研究为鸡BG基因的免疫功能和遗传进化研究提供了分子依据     

黄麂Mhc-DRB 基因多态性及其维持机制

利用牛DRB3 特异性引物(LA31 和LA32),通过聚合酶链式反应(PCR)、单链构象多态性(SSCP)以及克隆测序技术,从12 只黄麂个体中共获得20 个DRB 第二外显子等位基因,其中6 个个体具有3 ～ 4 个等位基因,提示利用该引物从黄麂中至少可以扩增出2 个DRB 位点。所有序列均无插入、缺失和终止密码子。基于序列比对(与牛DRB3 和鹿科DRB 基因同源性非常高),以及所检测到的氨基酸变异位点主要位于抗原结合区,推测本文所获得的黄麂序列为表达的、且具有重要功能的DRB 位点。抗原结合区氨基酸位点的非同义替换(d_N )显著大于同义替换(d_S )(P < 0.01),说明历史上黄麂DRB 基因经历过正选择作用。CODMEL 程序中的模型M7 和M8 似然比检测(Likelihood ratio test,LRT)结果同样支持上述推论。进一步利用经验贝叶斯法准确地检测出6 个受正选择作用的氨基酸位点(位点11、37、61、67、71、86),其中的5 个位点位于PBR 区。因此,正选择作用可能是维持黄麂DRB 基因多态性的主要机制之一。基于DRB 外显子2 序列利用邻接法(NJ)
构建了部分偶蹄动物系统发生关系,在NJ 树上,黄麂DRB 基因与其它鹿科动物DRB 基因呈镶嵌式分布,提示跨物种进化是维持黄麂DRB 基因多态性的另一重要机制。此外,黄麂两个等位基因(Mure-DRB1 和Mure-DRB11)和马鹿的两个等位基因(Ceel-DRB34 和Ceel-DRB46)与牛科的等位基因构成一个独立的进化枝,说明黄麂和马鹿的某些DRB 基因具有非常古老的谱系。     

乌龟MHCⅡ类B基因第二外显子的克隆及序列分析

利用一对兼并性引物扩增了乌龟MHCⅡ类分子B基因第二外显子的部分片段,并对PCR产物进行了克隆和测序,结果得到8种长度为166 bp的不同序列。经分析,序列中有84个变异位点,核苷酸的非同义替换(dN)多于同义替换(dS),造成39个位点氨基酸的改变。氨基酸的替换趋于集中在假定的抗原结合位点附近。利用MEGA、PAUP软件分别构建NJ树和MP树,两种树极为相似,均分为两支。同一个体中出现有多种序列,提示乌龟MHCⅡ类分子B基因可能存在着座位重复。研究表明:乌龟MHCⅡ类分子B基因第二外显子有较高的多态性,有利于乌龟野生种群的遗传保护。     

合作猪SLA-DQA基因外显子2多态性分析

旨在研究合作猪DQA基因外显子2多态性,确定其等位基因数、核苷酸多态位点、氨基酸多态位点及各个等位基因之间的遗传关系,分析其进化意义。选用PCR-SSCP对439只合作猪SLA-DQA基因外显子2的多态性进行检测;测序群体内因变异而产生的各等位基因序列,并分析序列数据。结果显示,在合作猪SLA-DQA外显子2中发现了7个新等位基因,共18个核苷酸多态位点,10个氨基酸多态位点。合作猪SLA-DQA外显子2具有较丰富的多态性,群体内可能蕴藏着更加丰富的遗传资源;合作猪SLA-DQA外显子2基因最初可能由一个等位基因突变分化成一大类基因;合作猪SLA-DQA外显子2序列与各个猪种的SLA-DQA外显子2序列具有较高的同源性,预示着这些猪种的SLA-DQA外显子2基因最早可能来源于其分歧之前的共同祖先原始序列;新发现的7个SLA-DQA外显子2等位基因,可能由遗传关系较近的两个等位基因突变产生。     

定位于5q31-5q32的DFNA52的20个候选基因的突变筛查

为了克隆定位于5号染色体微卫星标记D5S2056和D5S638之间约8.8 cM的区间内的非综合征性常染色体显性遗传性耳聋 DFNA52 (OMIM: 607683)的致病基因, 文章根据基因在耳蜗组织的表达情况, 筛选出20个候选基因, 设计合成了扩增20个基因外显子及外显子与内含子交界的引物, 用DNA直接测序法进行序列变异分析。结果显示, 在基因外显子及侧翼区共发现了45个单核苷酸多态, 其中42个变异在多态数据库已报道, 其余3个为新发现的单核苷酸多态, 序列变异与疾病表型无共分离现象, 排除了这些基因外显子突变导致遗传性耳聋的可能性。     

黑麂MHC - DQA2 基因多态性

利用牛特异性扩增DQA2 第2 外显子的嵌套引物,对黑麂基因组DNA 进行PCR 扩增和克隆测序,基于该
序列设计出黑麂DQA2 基因第2 外显子特异性引物。利用该引物,通过PCR - SSCP 以及克隆测序技术,从40 个
黑麂样品中获得4 个不同的DQA2 等位基因。没有一个个体同时具有2 个以上的等位基因,所有序列均不含插入
或缺失突变,不含终止密码子,因此,本研究所扩增的DQA2 基因可能是表达的单基因座位。抗原结合区(Peptide
binding region,PBR)非同义替换率(dn)显著大于同义替换率(ds) (P <0.05),暗示该座位曾经历过明
显的正选择作用;进一步利用CODEML 程序中的相关模型以及贝叶斯法检测出4 个受选择作用的氨基酸位点
(α11、α58、α62、α66),这4 个位点均位于PBR 区。基于NJ 法构建的部分偶蹄类DQA 外显子2 系统发生关系
显示,黑麂4 个DQA2 等位基因与牛、羊以及梅花鹿的DQA2 等位基因构成独立的进化枝,在该进化枝内,黑麂
DQA2 等位基因优先与牛DQA2 等位基因聚类,暗示黑麂DQA2 基因在进化过程中存在跨物种进化现象。上述结
果表明平衡选择是维持黑麂DQA2 基因多态性的主要机制。然而,本研究从40 个样品中仅检测出2 个杂合子,
黑麂DQA2 等位基因之间的频率存在显著差异,推测可能是所检测的样品来源于不同种群,由于华伦德效应
(The Whalund effect)导致杂合度降低,也不排除本文所设计的引物在PCR 扩增过程中存在无效等位基因。     

中国地方鸡种MHC B-LBⅡ新等位基因的遗传多态性研究

为研究鸡MHC B-LBⅡ基因的遗传多态性,首先在8个中国地方鸡种(藏鸡、仙居鸡、北京油鸡、固始鸡、斗鸡、丝羽乌骨鸡、白耳鸡和狼山鸡)B-LBⅡ基因第二外显子扩增了一长度为 175 bp 的 DNA 片段并进行 SSCP 基因型分析;在8 个地方鸡种共 467 个个体中检测到 37 个 PCR-SSCP 基因型;从被检样品中筛选出不同基因型的个体,并在其 B-LBⅡ基因组中扩增了一个包括其第二外显子和第二内含子在内长度为374 bp的片段,通过克隆和测序获得了该片段的核苷酸序列。经序列分析,在前述地方鸡种被筛选出的 30 个无血缘关系的个体中发现了 31 个 B-LBⅡ新等位基因,并参照哺乳动物 MHC II 类 B 等位基因命名规则进行了命名。对这 31 个 B-LBⅡ新等位基因长度为 374 bp 的 DNA 片段进行比对表明,在其第二外显子序列上共有 68 个多态性变异位点,其中简约性信息位点 51 个,单变异位点 17 个,具有丰富的遗传多态性。在这些多态性变异位点中,出现在遗传密码子第一和第二位上的碱基替换率分别为 36.76% 和 35.29%。等位基因序列间的相似性估测为 90.6%-99.5%;B-LBⅡ基因第二外显子的错义替换率和同义替换率分别为 14.64±2.67%和 2.92±0.94%。结果表明,B-LBⅡ基因的丰富遗传多态性主要是由基因重组和平衡选择效应所引起的。对 B-LBⅡ等位基因第二外显子所编码的 B-LBⅡ分子β1 结构域氨基酸序列比对发现,31 个 B-LBⅡ新等位基因属于 26 个等位基因主型;在β1结构域氨基酸序列的 33个变异位点上,存在 6 个同义替换和 27 个错义替换。分析认为,那些发生在多肽结合位点上的氨基酸错义替换与鸡 MHC B-LBⅡ分子的免疫特异性有关。该结果可为鸡的抗病育种研究提供分子生物学依据。     

合作猪SLA-DQA基因第4外显子多态性分析

合作猪的MHC-DQA基因的适应性变异,其抗原识别区域(即外显子4)通过PCR扩增和随后的单链构象多态性(SSCP)和序列分析,结果显示在439个合作猪个体,SLA-DQA第4外显子检出4个等位基因和6个基因型(AA、BB、DD、AB、AC和AD),其中A等位基因和AA基因型的频率最高,为优势基因和优势基因型。对不同型的PCR-SSCP条带测序分析,发现7个突变位点(5 068 bp T→C,5 109 bp和5 149 bp处缺失C,5 131 bp A→G导致丝氨酸变为甘氨酸,5 135 bp C→T,5 234 bp G→A,5 136 bp处插入A)。遗传学分析发现,合作猪多态信息含量(PIC)为0.240 1,属于低度多态,各种基因型的分布不显著。研究结果证实,合作猪SLA—DQA基因第4外显子为低度多态。     

Evolution of MHC class IIB in the genome of wild and ornamental guppies, Poecilia reticulata

This is the first study to quantify genomic sequence variation of the major histocompatibility complex (MHC) in wild and ornamental guppies, Poecilia reticulata. We sequenced 196-219 bp of exon 2 MHC class IIB (DAB) in 56 wild Trinidadian guppies and 14 ornamental strain guppies. Each of two natural populations possessed high allelic richness (15-16 alleles), whereas only three or fewer DAB alleles were amplified from ornamental guppies. The disparity in allelic richness between wild and ornamental fish cannot be fully explained by fixation of alleles by inbreeding, nor by the presence of non-amplified sequences (ie null alleles). Rather, we suggest that the same allele is fixed at duplicated MHC DAB loci owing to gene conversion. Alternatively, the number of loci in the ornamental strains has contracted during >100 generations in captivity, a hypothesis consistent with the accordion model of MHC evolution. We furthermore analysed the substitution patterns by making pairwise comparisons of sequence variation at the putative peptide binding region (PBR). The rate of non-synonymous substitutions (dN) only marginally exceeded synonymous substitutions (dS) in PBR codons. Highly diverged sequences showed no evidence for diversifying selection, possibly because synonymous substitutions have accumulated since their divergence. Also, the substitution pattern of similar alleles did not show evidence for diversifying selection, plausibly because advantageous non-synonymous substitutions have not yet accumulated. Intermediately diverged sequences showed the highest relative rate of non-synonymous substitutions, with dN/dS>14 in some pairwise comparisons. Consequently, a curvilinear relationship was observed between the dN/dS ratio and the level of sequence divergence.     

Evolution of introns and exons of class II major histocompatibility complex genes of vertebrates

 The phylogenetic relationships and patterns of nucleotide substitution were compared for introns and exons of class II major histocompatibility complex (MHC) genes in three datasets: human DRB1, human DQA1, and cyprinid fish DAB1. In both human DRB1 and cyprinid DAB1, there was strong evidence that recombination events between alleles have occurred in such a way that intron and exon sequences of a given allele do not necessarily share the same evolutionary history. In the case of human DRB1, recombination was found to have homogenized intron 1 and intron 2 sequences relative to exon 2 sequences within lineages of alleles but not between lineages. As a result, mean divergence times of intron sequences are much more recent than those of exonic sequences. Thus, the divergence time of DRB1 introns cannot be used to date that of exons in the same alleles, and the hypothesis that most human DRB1 polymorphism is of very recent origin is not supported. Received: 5 September 1999 / Revised: 30 December 1999     

凹耳蛙MHCII类B基因第二外显子多态性分析

两栖类正经历全球范围内的种群衰退,很多两栖动物集群灭绝事件与环境病原体(如壶菌(Batrachochytrium dendrobatidis)的侵扰有关。MHC基因的表达产物在有颌脊椎动物免疫应答过程中起关键作用,其多态性通常与动物对疾病的抗性或易感性密切相关,因而被认为是研究动物适应性进化的最佳候选基因之一。本文对中国特有的无尾两栖动物凹耳蛙(Odorrana tormota)MHC II类B基因多态性进行初步研究。首先,利用1对通用引物扩增出凹耳蛙MHC II类B基因exon2长约180bp的DNA片段。在此基础上,利用ligation-mediated PCR进一步获取侧翼未知序列,序列拼接后长2,030bp,包含exon2以及intron1和intron2的部分序列。基于上述序列设计出凹耳蛙B基因exon2特异性引物(IIQ1BU/IIQ1BD),对该物种黄山种群32个样品进行PCR扩增和克隆测序,共获得34个不同的等位基因,等位基因序列核苷酸和氨基酸变异位点的比例分别为16.17%(33/204)和26.87%(18/67),大多数氨基酸变异位点位于推测的抗原结合位点(antigen binding sites,ABS)。每个样品包含2-5个等位基因,结合等位基因序列特征以及cDNA表达分析结果,推测凹耳蛙至少拥有3个可表达的B基因座位。与文献报道的蛙科其他物种比较后发现,尽管凹耳蛙目前的分布区非常狭窄,但其MHC II类B基因多态性明显高于蛙科其他动物。等位基因碱基替换模式提示凹耳蛙MHC II类B基因曾经历过强烈的正选择作用,ABS区的dN值显著大于dS(P<0.05),PAML软件包CODEML程序中不同模型的似然比检测(likelihood rate test)结果同样支持上述推论,贝叶斯经验贝叶斯路径(Bayesian Em-pirical Bayes)共检测出5个显著受正选择作用的氨基酸位点。贝叶斯系统树的拓扑结构显示,无尾两栖类不同科的等位基因分别形成单系群,但蛙科不同属的等位基因未能形成单系群,蛙属绿池蛙(Rana clamitans)的1个等位基因与臭蛙属凹耳蛙的部分等位基因享有共同的谱系关系,提示蛙科不同属间的B基因存在跨种多态性。     

Two new polymorphisms in introns 2 and 3 of the human porphobilinogen deaminase gene

Sequencing of the polymerase chain reaction amplified exon 2–4 fragment of the human porphobilinogen deaminase gene revealed a G/T polymorphism (I2G and I2T) in intron 2, and a G/A polymorphism (I3G and I3A) in intron 3 of the gene. The frequencies of these alleles are presented.The nucleotide sequence data reported in this paper (the sequence of introns 2 and 3, and the polymorphic sites) will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Database with accession nos. D10608 and D12722     

Lineage‐specific sequence evolution and exon edge conservation partially explain the relationship between evolutionary rate and expression level in A. thaliana

Rapidly evolving proteins can aid the identification of genes underlying phenotypic adaptation across taxa, but functional and structural elements of genes can also affect evolutionary rates. In plants, the ‘edges’ of exons, flanking intron junctions, are known to contain splice enhancers and to have a higher degree of conservation compared to the remainder of the coding region. However, the extent to which these regions may be masking indicators of positive selection or account for the relationship between dN/dS and other genomic parameters is unclear. We investigate the effects of exon edge conservation on the relationship of dN/dS to various sequence characteristics and gene expression parameters in the model plant Arabidopsis thaliana. We also obtain lineage‐specific dN/dS estimates, making use of the recently sequenced genome of Thellungiella parvula, the second closest sequenced relative after the sister species Arabidopsis lyrata. Overall, we find that the effect of exon edge conservation, as well as the use of lineage‐specific substitution estimates, upon dN/dS ratios partly explains the relationship between the rates of protein evolution and expression level. Furthermore, the removal of exon edges shifts dN/dS estimates upwards, increasing the proportion of genes potentially under adaptive selection. We conclude that lineage‐specific substitutions and exon edge conservation have an important effect on dN/dS ratios and should be considered when assessing their relationship with other genomic parameters.     

Allelic variation, balancing selection and positive selected sites detected from MHC class Iα gene of olive flounder

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and pattern of evolution in MHC genes has been investigated in many mammals, however, much less is known in teleost. In the present study, we have investigated complete MHC Iα gene consists of 7 exons and 6 introns in Olive flounder (Paralichthys olivaceus). Genetic variation in the MHC class Iα gene was also tested in flounder. In 32 individuals, a total of 62 alleles were detected from exon 2 of MHC class Iα gene. The rate of non-synonymous substitutions (d ( N )) occurred at a significantly higher frequency than that of synonymous substitutions (d ( S )) in PBR and non-PBR, suggesting that balancing selection for maintaining polymorphisms at the MHC Iα locus. Many positive selection sites were found to act very intensively on antigen binding sites. Our founding suggests a snapshot in an evolutionary process of MHC Iα gene evolution of the P. olivaceus.     

The noncoding regions of HLA-DRB uncover interlineage recombinations as a mechanism of HLA diversification

The mechanisms generating new alleles at the MHC loci are still unknown in detail, and several proposals have been made to explain the extent of polymorphism. The patchwork pattern of polymorphism in the 2nd exon of HLA-DRB1 recommends this locus as a model for the study of the potential of interallelic gene conversion. In general, the inference of gene conversion-like events based exclusively on exon sequence comparisons may be misleading because the identity of the putative donor allele remains unknown. In this study, we describe five alleles of the HLA-DRB1 gene, which intron regions give evidence for interlineage recombination events either strictly located at the 2nd exon or involving the adjacent introns. Furthermore, we show that the noncoding regions provide important clues to the mechanisms of the generation of new alleles, and our results indicate that interlineage recombinations may be hidden and are perhaps more frequent than currently expected.     

Typing of artiodactyl MHC-DRB genes with the help of intronic simple repeated DNA sequences

An efficient oligonucleotide typing method for the highly polymorphic MHC-DRB genes is described for artiodactyls like cattle, sheep and goat. By means of the polymerase chain reaction, the second exon of MHC-DRB is amplified as well as part of the adjacent intron containing a mixed simple repeat sequence. Using this primer combination we were able to amplify the MHC-DRB exons 2 and adjacent introns from all of the investigated 10 species of the family of Bovidae and giraffes. Therefore, the DRB genes of novel artiodactyl species can also be readily studied. Oligonucleotide probes specific for the polymorphisms of ungulate DRB genes are used with which sequences differing in at least one single base can be distinguished. Exonic polymorphism was found to be correlated with the allele lengths and the patterns of the repeat structures. Hence oligonucleotide probes specific for different simple repeats and polymorphic positions serve also for typing across species barriers. The strict correlation of sequence length and exonic polymorphism permits a preselection of specific oligonucleotides for hybridization. Thus more than 20 alleles can already be differentiated from each of the three species.     

Mutations in the hepatocyte nuclear factor-4alpha gene in Japanese with non-insulin-dependent diabetes: a nucleotide substitution in the polypyrimidine tract of intron 1b.

Mutations of the hepatocyte nuclear factor 4 alpha (HNF-4alpha) gene have been demonstrated in maturity-onset diabetes of the young (MODY) 1 families. To investigate the possibility that the HNF-4alpha gene contributes to the onset of non-insulin-dependent diabetes mellitus (NIDDM) in Japanese patients, we screened all exons and flanking introns of this gene for mutations in 100 patients with NIDDM diagnosed after 25 years of age. We identified two missense mutations: M49V in exon 1c and T1301 in exon 4; and two nucleotide substitutions in introns: cytosine to thymidine at -5 nt in intron 1b and adenine to thymidine at -21 nt in intron 5. We screened an additional 220 diabetic subjects for the polymorphism in intron 1b. The c/t substitution in intron 1b was associated with NIDDM. This substitution in the polypyrimidine tract, an important cis-acting element directing intron removal, is likely to influence pre-mRNA splicing of this gene. T1301 in exon 4 was observed in only two diabetic subjects. This mutation could influence the conformation of this peptide, resulting in changes in ligand binding domain function. M49V in exon 1c was found in both diabetic and non-diabetic subjects; isoforms HNF-4alpha 4, 5, and 6 with this mutation may impair glucose metabolism in tissue. In contrast to the primary cause of nonsense and missense mutations of the HNF-4alpha gene in MODY1, the nucleotide substitution in intron 1b may partially contribute to development of NIDDM in combination with other genetic and environmental factors.