Molecular genotyping of the HLA-DQ α gene region

Restriction fragment analysis has been applied to genomic DNA extracted from human tumor cell lines. Polymorphic restriction fragments encompassing the HLA-DQ gene were observed upon digestion with Bgl II, Eco RI, and Hind III. Analysis of these polymorphic fragments (or allogenotopes) showed that for each restriction enzyme a series of three differently sized allogenotopes existed. Clusters of cosegregating allogenotopes belonging to the different allelic series defined three different allogenotypes. Each allogenotype exhibited a distinctive restriction map generated by digestion with five restriction enzymes. Comparison of these restriction maps showed that generation of the polymorphisms observed at the HLA-DQ region in these sets of cell lines is not caused by a single event. In some B- and T-lymphoma cell lines a fourth allogenotype was found. The restriction site map of genomic DNA from these cell lines suggested that the latter distribution of restriction enzyme sites was most probably generated by recombination between two of the previously observed allogenotypes at a crossover site(s) adjacent to the HLA-DQ gene.     

Exon-specific oligonucleotide probes localize HLA-DQ β allelic polymorphisms

The HLA genetic region consists of a large multigene complex which includes a number of highly homologous and genes encoding class II polypeptides, clustered in three major loci, DP, DQ, and DR. Analysis of genomic polymorphisms at each of these loci is of considerable interest due to the role of particular structural polymorphisms in immune function, but this analysis has been hampered by difficulty in distinguishing between such highly homologous loci. We have identified locus-specific and exon-specific class II gene sequences in order to produce synthetic oligonucleotide probes which hybridize specifically to DQ genes. Two such oligonucleotide probes are described which are specific for the ₁ and ₂exons of DQ (DC) which identify DQ genes in digests of cellular DNA and which can be used to characterize restriction sites flanking the two oligonucleotide-specific regions. By sequentially hybridizing these probes in modified Southern analyses, we have been able to generate a tentative restriction map of a newly identified DQ allele from digests of total genomic DNA. This oligonucleotide mapping technique discriminates between two HLA-DQw3 ⁺ alleles, DQ3.1 and DQ3.2, permitting the recognition of structural polymorphisms with DQ which are highly associated with type I diabetes mellitus.     

Taq I-generated HLA-DQ αpolymorphism in Japanese patients with narcolepsy

Taq I-generated HLA-DQrestriction fragment length polymorphism was examined in Japanese patients with narcolepsy. All patients were DR2 positive and shared a 6.0 kb fragment, although this fragment was found only in 54 % of the healthy DR2-positive Japanese. This finding added the DQ gene to the list of candidates for the possible narcolepsy-susceptibility gene. In contrast, there was no complete association between narcolepsy and DXrestriction fragment length polymorphism. These findings suggest that a narcolepsy-susceptibility gene is located closer to the DQ locus than to the DX locus.     

用聚合酶链反应和生物素寡核苷酸探针分析HLA-DQα的基因型

 来自不同人的染色体DNA经聚合酶链反应(PCR)将其HLA-DQα基因的多态区242bp人工地扩增30个循环。用1/10扩增产物点在尼龙膜上。将四种不同的等位基因特异的寡核苷酸(ASOs)经Bio-11-duTP进行3'末端标记得生物素探针。以此探针对不同扩增标本做狭缝印迹杂交。结果表明用此种非放射性的方法能探测出人白细胞表面抗原DQα基因的微多态性,从而得到不同个体的基因型。ASO分型是对血清HLA分型的重要补充和发展,它在骨髓或器官移植以及法医的个人识别中有一定实用价值。     

Sequence determination of a transcribed rabbit class II gene with homology to HLA-DQ α

The human HLA-DQ probe was used to screen genomic and cDNA libraries constructed from a rabbit T-cell line. Clones containing highly homologous sequences were obtained from both libraries and their sequences were determined. The organization of the RLA-DQ gene was determined by comparison of the nucleotide sequences of the genomic clone to that of the corresponding cDNA clone. This analysis allowed assignment of the complete structure of the RLA-DQ chain. Comparisons with human and mouse class II products revealed that RLA-DQ is more closely related to HLA-DQ/DX than to H-2 A. In contrast to the DQ/DX region of man, which contains at least two distinct alpha genes, the rabbit genome contains a single DQ gene which is equally distant from the HLA-DQ or -DX genes. The rabbit DQ gene, like human HLA-DQ, is transcribed in T cells.Abbreviations used in this paper RLA rabbit major histocompatibility complex - HLA human major histocompatibility complex - SSC 0.15 M sodium chloride, 0.015 M sodium citrate     

Analysis of HLA-DQ α sequences for prenatal diagnosis in single fetal cells from maternal blood

We have extended a previously developed method that allows prenatal DNA diagnosis of female fetuses through the isolation of single nucleated erythrocytes from maternal blood by developing a method that can distinguish between maternal and fetal nucleated erythrocytes. Nucleated erythrocytes were separated by a density-gradient method and then collected by micromanipulation. Sex was determined after primer extension preamplification (PEP) of the entire genome of a single cell, and human leukocyte antigen (HLA)-DQ α type was determined after further amplification of this gene. The HLA-DQ α genotype of fetal erythrocytes in maternal blood samples and their corresponding paternal and maternal lymphocytes were successfully determined in all cases. The accuracy of the method was determined by using single nucleated erythrocytes from umbilical cord blood from five normal deliveries. This is the first demonstration that the fetal HLA-DQ α gene sequences can be identified in a small aliquot of a single nucleated erythrocyte in maternal blood. We believe that this method ushers in a new era in which the reliability and accuracy of noninvasive prenatal DNA diagnosis from maternal blood is markedly improved. Received: 18 April 1997 / Accepted: 1 October 1997     

成纤维细胞生长因子8(FGF8)研究进展

成纤维细胞生长因子8(fibroblast growth factor 8,FGF8)是成纤维细胞生长因子(FGFs)家族的成员之一。其在人胚胎时期多种组织内进行表达,对各种器官的形成中起着重要的作用。在正常成人体内,FGF8的表达水平受到严格的限制,然而在某些癌细胞或炎症部位中大量表达,特别是在激素类癌症的发生和发展中起着重要的作用。因此应用FGF8抗体治疗激素类癌症,为临床提供了新的治疗途径。     

卷曲螺旋结构蛋白8(CCDC8)功能研究进展

卷曲螺旋结构蛋白8(coiled-coil domain containing 8,CCDC8)是一个由CCDC8基因编码目前功能未知的蛋白质。已有的证据表明,CCDC8是一个膜蛋白,与细胞骨架蛋白Obsl1(Obscurin-like protein1)、泛素E3连接酶Cul7(Cullin 7)在一个信号通路。CCDC8、Obsl1或者Cul7基因突变可以引起身材矮小的3-M综合征(3M syndrome)。CCDC8与锚蛋白ANKRA2(ankyrin-repeat family A protein 2)、p53通路相关的细胞凋亡有关;CCDC8与乳腺癌、肺癌等癌症有关;CCDC8还可与艾滋病毒(HIV-1)结构蛋白Gag相互作用,诱导Gag的内化、多泛素化和降解。     

山西省8种蝗虫8个种群的遗传学研究

采用水平淀粉凝胶电泳技术,对采自山西省蝗虫区系的优势种类;斑腿蝗科（Catantopidae）,斑翅蝗科（Oe-dipodidae）和网翅蝗科（Arcypteridae）3科7属8种蝗虫的11种酶进行了检测,共辨析出17个酶基因座位,并计算出等位基因频率和遗传距离,等位基因频率分析表明：Ao-I、Est-3,G3pd-1、Idh-2和Mdh-2基因座位的等位基因少,等位基因数目在种间变化较小,故推断其进化速率较慢,利用这些基因座位的保守特征,可作为分子标记研究较高级阶元的系统发育关系,而Gpi-1,Ldh-1和Me-1基因座位的等位基因多,等位基因数目在种内和种间差异较大,可以用作种,属间及种群间遗传结构的比较研究。对每个基因座位的各基因型进行χ^2检验,除Acp-1,Adk-1,Ao-1和Ao-2在部分蝗虫中符合Hardy-Weinberg平衡外,其余绝大多数基因座位的基因型频率显著偏离H-W平衡。在所研究的8种蝗虫中,多态位点百分率普遍较高（P=64.7%～94.1%）,但由于杂合子数目较少而使每个基因座位的平均杂合度降低（Ho=0.024～0.087）,对多态位点百分率分析发现：迁飞能力是影响蝗虫种间遗传变异的因素之一,具有迁飞能力的蝗虫（P=88.2%～94.1%）较非迁飞性蝗虫（P=64.7%～94.1%）表现出较高的遗传多态性,但也有例外,如中华稻蝗（Oxya chinensis）的P值高达94.1%,上述结果表明：由于迁飞行为可使个体暴露于各种不同环境,故而种群保持较高的遗传多态性能增强该物种在不同栖息地的生存和繁殖能力,因此,迁飞有利于维持迁飞性蝗虫遗传多态性的动态平衡。根据Nei的遗传一致度（I）和Roger的遗传距离（D）进行分析,结果与基于形态特征确定的分类阶元系统关系基本相符;即同属的小翅雏蝗（Chorthippus fallax）和白纹雏蝗（Chorthippus albonemus）具有最高的遗传一致度（I=0.813）和最小的遗传距离（D=0.336）,同位不同属间遗传一致度（I=0.798～0.559）和遗传距离（D=0.398～0.474）居中,科之间I值最小（I=0.523～0.479）,D值最大（D=0.505～0.523）,利用UPGMA对I值和D值进行聚类,所得两种聚类图在同属种间和同科属间的关系一致,但在科间关系有所差别,Roger的遗传距离（D）聚类树图表明：斑腿蝗科物种和斑翅蝗科物种间表现出较小的遗传距离（D=0.505）,而网翅蝗科与以上两科的遗传距离也极为接近（D=0.523）,综上所述,等位酶分析能较好地反映蝗虫同属种间和同科属间的亲缘关系,若能断更高阶元的系统发生,则需结合其他性状进行综合分析。     

拟南芥Atkin8a和Atkin8b基因表达特性

以拟南芥动蛋白(kinesin)kin-8家族的AtKin8a和AtKin8b这两个动蛋白基因作为研究对象,以组成型表达的肌动蛋白基因(Actin2)作为对照,利用半定量RT-PCR的方法,分析其在拟南芥各器官中的表达状况。结果表明:AtKin8a和AtKin8b基因主要在花器官中特异表达;随后克隆AtKin8a和AtKin8b基因启动子区域并与GUS基因融合,转基因植株花器官GUS染色表明:AtKin8a和AtKin8b基因的表达主要分别在胚珠、花药部位。由此推测它们可能分别在胚珠、花药发育过程中发挥作用。     

Procaspase-8/Caspase-8非凋亡作用的研究进展

Caspase-8作为细胞死亡信号诱导复合体中不可或缺的一员而一直备受关注,是细胞凋亡信号因子中研究的热门。然而,最近研究指出Caspase-8除了作为细胞凋亡信号中的一员外,还有很多非凋亡活性,比如促进细胞迁移、粘附、增殖分化等,甚至还有研究指出其与细胞炎症及免疫有关。本综述首先针对Caspase-8对细胞迁移信号通路中的影响做出了总结及分析,主要包括:Src对Caspase-8磷酸化的过程,Caspase-8对Rac1、Rab5及Calpain的影响。其次,本文还探讨了Caspase-8在其他信号通路中的非凋亡作用,为以Caspase-8为靶点抑制肿瘤扩散药物的研究提供新的思路。     

A telescopic method for photographing within 8×8 cm minirhizotrons

The volatile organic compounds produced during a sequence of soil incubations under controlled conditions, with either added NH₄ ⁺-N or NO₃ ^--N, were collected and identified. The nature and relative amounts of the volatile organic compounds produced by the microorganisms in the soils were remarkably reproducible and consistent.     

Both α and β chain polymorphisms determine the specificity of the disease-associated HLA-DQ2 molecules,with β chain residues being most influential

 We compared the peptide binding specificity of three HLA-DQ molecules; HLA-DQ(α1^*0501, β1^*0201), HLA-DQ(α1^*0201, β1^*0202), and HLA-DQ(α1^*0501, β1^*0301). The first of these molecules confers susceptibility to celiac disease and insulin-dependent diabetes mellitus, while the two latter molecules, which share either the α chain or the nearly identical β chain with HLA-DQ(α1^*0501, β1^*0201), do not predispose to these disorders. The binding of peptides was detected in biochemical binding assays as inhibition of binding of radiolabeled indicator peptides to affinity-purified HLA-DQ molecules. Binding experiments with several peptides demonstrated a clear difference in peptide binding specificity between the three HLA-DQ molecules. Further, single amino acid substitution analyses indicated that the HLA-DQ molecules have different peptide binding motifs. The experimental data were corroborated by computer modelling analysis. Our data suggest that the three HLA-DQ molecules prefer large hydrophobic residues in P1 of peptides with subtle differences in side-chain preferences. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202) both prefer large hydrophobic residues in P9, whereas HLA-DQ(α1^*0501, β1^*0301) prefers much smaller residues in this position. HLA-DQ(α1^*0501, β1^*0201) and HLA-DQ(α1^*0201, β1^*0202), in contrast to HLA-DQ(α1^*0501, β1^*0301), prefer negatively charged residues in P4 and P7. A less prominent P6 pocket also appears to differ between the three HLA-DQ molecules. Our results indicate that polymorphic residues of both the α and the β chain determine the peptide binding specificity of HLA-DQ(α1^*0501, β1^*0201), but that the β chain polymorphisms appears to play the most important role. The information on peptide residues which are advantageous and deleterious for binding to these HLA-DQ molecules may make possible the prediction of characteristic features of peptide that bind to HLA-DQ(α1^*0501, β1^*0201) and precipitate celiac disease. Received: 2 July 1996 / Revised: 7 August 1995     

HLA-DQ system and insulin-dependent diabetes mellitus in Japanese: does it contribute to the development of IDDM as it does in Caucasians?

Fifty-six unrelated Japanese patients with insulin-dependent diabetes mellitus (IDDM) were HLA-typed, and restriction fragment length polymorphism (RFLP) analysis was performed after enzyme digestion with Bam HI and Taq I by using both DR and DQ probes. As previously reported, increased frequencies of Bw54, Cw1, DR4, and DRw53, which are in strong linkage disequilibrium in the Japanese population and make the characteristic Japanese haplotype, were confirmed. DQw4, a new allele of the DQ system recognized by the monoclonal antibody HU-46 and in linkage disequilibrium with this haplotype, presented the highest IDDM association. The RFLP analysis also showed the strongest correlation to IDDM when the DQ probe was applied. These results indicate that HLA-DQ might play the most important role in the development of IDDM in Japanese as well as in Caucasians. The correlation of DQ amino acid sequences strongly associated with IDDM in Japanese are discussed in this study, and contrasting results were found when such sequences were compared with those of Caucasians.Abbreviations used in this paper IDDM insulin-dependent diabetes mellitus - RFLP restriction fragment length polymorphism - Asp aspartic acid - Asp-57 aspartic acid at the 57th residue of the DQ chain - non-Asp-57 nonaspartic acid at the 57th residue of the DQ chain - R.R. relative risk of Woolf and Haldane     

Correlation between an HLA-DQα length polymorphism of messenger RNA and serologically defined specificities (DQwl,DRw53, DR3 + 5)

mRNAs for the two chains of the HLA-DQ molecule were analyzed, in particular the DQ mRNA whose polymorphism had previously been suggested (Schenning et al. 1984). Northern blot transfers of the mRNA of 12 LCLs and of B lymphocytes from a healthy donor were carried out. We report that a length polymorphism of DQ mRNA exists, and we show that it can be correlated with serologically defined specificities (DQwl, DRw53, DR3 + 5). This correlation could be explained by a linkage disequilibrium, as these specificities are considered to be different from those carried by the DQ molecule (except for the DQw1 specificity).Abbreviations used in this paper DNA desoxyribonucleic acid - HLA human leukocyte antigen - LCL lymphoblastoid cell line - mRLP messenger RNA length polymorphism - mRNA messenger ribonucleic acid - RFLP restriction fragment length polymorphism - RNA ribonucleic acid