云南白族人群Y染色体DYF155S1基因座多态性研究

揭示云南白族136例无关男性个体Y染色体小卫星DYF155S1基因座多态性。用已建立的AmpFLP和MVRPCR方法分析DYF155S1基因座长度多态性、5′端多态性、3′端多态性。DYF155S1基因座具有高度多态性,基因多样性（h）达09996。研究表明将AmpFLP和荧光MVRPCR结合起来更能充分揭示DYF155S1基因座多态性,可为遗传学、人类学及法医学的研究提供有效的工具和基础资料。     

中国汉族群体5个STR分子遗传标记

为了解中国人5个STR基因座等位片段结构特征,获得汉族群体D2S2955、D3S4014、D20S604、D22S689和GATA198B05基因座的群体遗传学数据。采取成都地区无血缘关系汉族个体血样EDTA抗凝血。Chelex法提取DNA,PCR扩增,非变性聚丙烯酰胺凝胶不连续缓冲系统水平电泳分型,自动激光荧光测序仪测定DNA序列。序列分析显示,中国人D2S2955、D3S4014、D20S604基因座具有简单重复序列,而D22S689、GATA198B05基因座具有复杂重复序列。5个STR基因座在成都汉族群体中均具有遗传多态性。揭示了我国汉族人群5个STR基因座的等位基因片段结构特征,为人类群体遗传研究提供了数据,建立的不连续缓冲系统水平电泳分型方法为检测这5个STR基因座提供了简便技术。     

中国维吾尔族人群MSY1(DYF155S1)基因座多态性及其结构特点

应用荧光标记MVR-PCR、Amp-FLP与DNA序列分析技术等检测106例中国维吾尔族人群无关男性个体血纱样品,揭示了中国维吾尔族人群Y特异的小卫星MSY1 (DYF155S1)基因座5′和3′端多态性及其基因结构特点。DYF155S1基因座的多态性表现为3个方面：（1）长度多态性;（2）5′端多态性;（3）3′端多态性。106例无关个体共检出37个不同长度的片段,5′端检出68个类型,3′端检出23个类型。综合这3方面多态性,106例个体间没有相同,其基因多样性（h）超过0.9999。DNA序列分析发现该基因座5′端表现有7种模块结构,3′端有2种模块结构。DYF155S2片段缺失率约为4.7%。MVR-PCR、Amp-FLP与DNA序列分析技术结合起来可以更充分地揭示人群Y染色体特异的小卫星MSY1（DYF155S1）基因座多态性,并提出命名方式,从而为人类遗传学及法医学研究提供了有用的方法和基础资料。 Abstract:The study is to reveal the diversity and gene structure of 5′ and 3′ end of DYF155S1 locus in Y-chromosome minisatellite among Chinese Uygur population.Fluorescent MVR-PCR（minisatellite variant repeat by PCR）,Amp-FLP(Amplified fragment length polymorphism) and DNA sequencing methods were used repectively to detect 106 unrelated males among Chinese Uygur population.The polymorphisms of DYF155S1 locus could be revealed in three aspects:(1) polymorphic length:the sizes of amplified fragments ranged from 1405 to 2505bp.There are 37 types found among the 106 unrelated males.(2) polymorphism at 5′ end of DYF155S1 locus,68 types found among the 106 unrelated males.(3) polymorphism at 3′ end of DYF155S1 locus,23 types found among the 106 unrelated males.In combination of these three aspects of polymorphism,none of the 106 unrelated males tested had the same allele,and the gene diversity(h) was over 0.9999.Seven and two types of modular structure were founded in the 5′ and 3′ end of DYF155S1 locus,respectively,by DNA sequencing.The alleles at DYF155S2 locus showed yes/no dimorphism and the rate of deletion was 4.7%.The polymorphisms of DYF155S1 locus were fully revealed by using combination of MVR-PCR, Amp-FLP and DNA sequencing methods, and we suggested the nomenclature for alleles of MVR loci.These methods are useful tools and provide basic data for the study of human genetics and forensic medicine.     

用PCR-RFLP方法研究藏族HLA-DQA1和-DQB1基因多态性

应用目前HLA研究领域中成熟的,有效的PCR-RFLP基因分型技术,从DNA水平对藏族健康群体进行了HLA-DQA1(49人)和-DQB1(49人)基因分型,这在国内外属首次。所采用的PCR-RFLP基因分型技术是在HLA-DQA1和-DQB1各等位基因全部序列已知的情况下,对其第2个外显子碱基序列扩增进而进行RFLP分析的方法。这种方法得到的RFLP的所有片段都是已知序列,因而精确度很高,同时为发现新的等位基因提供了成熟而有效的分析方法。研究结果表明,在藏族DQA1的8个等位基因,DQA1*0301的基因频率最高(36.74％)。DQA1*0601(4.08％)、*0103(4.08％)和*0401(5.10％)最低。在DQB1的16个等位基因中,OQB1*0302(16.33％)、*0303(15.31％)和*0602(15.31％)为最常见,没有观察到*0504。统计分析表明,在DQA1各等位基因分布上,藏族与新疆汉族、北方汉族、上海汉族十分相近;与维吾尔族和哈萨克族也没有明显差异。在OQB1各等位基因的分布上,藏族与汉族、维族、哈族之间略有差异,而汉族、维族、哈族之间也存在一些差异。     

山东汉族人群中13个快速突变(RM)Y-STR基因座的多态性

本研究旨在调查13个快速突变Y染色体STR基因座(RMY-STR)在山东汉族人群中的等位基因频率以及遗传多态性。采集154个山东无关男性个体FTA卡血液样本。采用13个RMY-STRPCR复合扩增体系进行扩增以及AB3130XL遗传分析仪进行Y-STR分型,并对分型结果进行相关统计,检测13个RMY-STR位点遗传多态性分布。本研究在13个基因座上共检测出331个等位基因,基因型多态性(GD)分布在0.7643(DYS576)~0.9946(DYF399S1abc)之间。通过13个RMY-STR基因座在154名山东汉族男性无关个体中共检测出154种单倍型,无共享单倍型现象出现。总的单倍型多样性(HD值)为1,识别能力(DC值)为1。故13个RMY-STR基因座组成的分型系统在山东汉族人群中表现出很强的个体识别能力,具有重要的法医学应用价值。     

河南汉族群体6个STR基因座遗传多态性研究

对河南省河南籍汉族群体的6个短串联重复序列（Short tandem repeats,STR)基因座等位基因频率进行研究,得到河南汉族群体F13A1,F13B,D8S1179,CSF1PO,D5S818,TPOX基因座的群体遗传学依据。EDTA抗凝血样采自河南122名无血缘关系的汉族个体,采用Chelex法抽提DNA,PCR扩增,非变性聚丙烯酰胺垂直凝胶电泳,银染显色分析,得到6个基因座的等位基因频率,各基因座的杂合度分别为：0.62,0.46,0.83,0.59,0.78,0.65;人体识别率分别为0.78,0.66,0.95,0.79,0.92,0.82。6个STR基因座具有较高的杂合度,等位基因分布符合Hardy-Weinberg平衡,是较理想的遗传标记,可用于法医学个本识别和亲权鉴定。     

广州汉族人群D12S391和D11S554基因座序列变异

为研究高度变异的sTR基因座核心序列结构,对广州汉族人群突变率较高的D12S391和D11S554基因座等位基因进行了序列分析。结果显示D12S391基因座核心序列结构为(AGAT)8～17(AGAC)6～10(AGAT)0～1,其较小片段等位基因(15～18)仅表现为第1个重复单位(AGAT)数目的变异,而较大片段等位基因(19～27),可表现为第2或第3个重复单位数目的变异。发现有4种新的等位基因,分别命名为22″、23″、24′″和27。D11S554基因座核心序列结构更复杂,有5种核心序列,其中3种具有相同的基本结构(AAAGG)(AAAG)4(AAAGG)2～3,(AAAG)13～19。其大片段等位基因(219～249)核心序列结构中,既有四核苷酸重复,还有五核苷酸重复,以及单个硷基的变异、硷基插入或缺失。两基因座均存在序列异质性。结果表明D12S391和D11S554基因座属复杂重复类型,为其准确分型增加了难度,首先需建立相应群体的等位基因分型标准物。     

贵州地区汉族人群THO1、TPOX、CSF1PO基因座的遗传多态性

为了解贵州地区汉族群体中THO1、TPOX、CSF1PO基因座的遗传多态性,获得这3个基因座的群体遗传学数据和法医学相关数据。采自贵州地区汉族无关个体的110份EDTA抗凝血样用Chelex法提取DNA,应用PCR复合扩增技术扩增样本后,聚丙烯酰胺凝胶电泳分型。对3个STR基因座的等位基因频率进行了调查分析,并与其他汉族人群的等位基因频率进行了比较。在贵州汉族群体中,3个基因座的基因型分布符合Hardy-Weinberg平衡。3个STR基因座总个体识别率为0.9986,累积非父排除率为0.832。表明这3个基因座在法医学个体识别及亲子鉴定中是很有价值的遗传标记系统。 Abstract:To understand the genetic polymorphism at THO1,TPOX,CSF1PO STR loci for Han population in Guizhou Province,and construct a preliminary database,EDTA-blood specimens were collected from the 110 unrelated individuals in Han population from Guizhou.The DNA samples were extracted with Chelex method and amplified by multiplex polymerase chain reaction.The PAGE was used to type the PCR products.The allele frequencies were compared with other Han populations.The genotype distributions of THO1,TPOX and CSF1PO were in accordance with Hardy-Weinberg equilibrium.The combined PD and PE were 0.9986 and 0.832 respectively.All of the three loci in this study provide useful marker for forensic paternity test and individual identification.     

青海藏族HLA II类基因多态性的研究

用PCR-RFLP的技术进一步研究了青海藏族HLA-DPB1的多态性。在19个HLA-DPB1 的等位基因中,共检出18个等位基因。其中,*0501的频率最高（AF=38.0%）;其次为*0201（AF=20.0%）;未检出*1601。在HLA-DPB1各等位基因的分布上,藏族与中国南方汉族、中国北方汉族等无明显差异,而与高加索人及尼格罗人的差异则较为显著。综合隶属于三大人种11个群体中的HLA-DQA1、-DQB1和-DPB1基因座各等位基因的分布频率,用UPGMA方法构建了分子系统树, 实验结果进一步证实汉藏同源说。     

59个Y-STR基因座的法医学多态性应用效能评估

为了评估法医学DNA数据库建设中涉及的59个Y-STR基因座的遗传多态性和法医学应用效能,通过AGCU Y SUPP PLUS试剂盒和AGCU Y37试剂盒检测374个广东汉族无关男性个体,将检测出的59个Y-STR基因座按照突变率的高低进行分类组合和统计分析。结果显示,59个Y-STR基因座联合运用在374个无关男性个体中检出了374个单倍型,其中44个中低突变Y-STR组合、15个高快突变Y-STR组合分别检出373和372个单倍型。59个Y-STR的基因多态性数值分布在0.055 1 (DYS645)~0.958 0 (DYF387S1 a/b)之间。结果表明,这59个Y-STR在广东汉族群体中均具有良好的多态性,按中低突变Y-STR组合和高快突变Y-STR组合研发新的检测体系可更好地满足法医实践的不同需求。     

Stoichiometry studies reveal functional properties of KDC1 in plant shaker potassium channels

Functional heteromeric plant Shaker potassium channels can be formed by the assembly of subunits from different tissues, as well as from diverse plant species. KDC1 (K(+) Daucus carota 1) produces inward-rectifying currents in Xenopus oocytes when coexpressed with KAT1 and other subunits appertaining to different plant Shaker subfamilies. Owing to the presence of KDC1, resulting heteromeric channels display slower activation kinetics, a shift of the activation threshold toward more negative membrane potentials and current potentiation upon the addition of external zinc. Despite available information on heteromerization of plant Shaker channels, very little is known to date on the properties of the various stoichiometric configurations formed by different subunits. To investigate the functional properties of heteromeric nKDC1/mKAT1 configurations, we realized a series of dimeric constructs combining KDC1 and KAT1 alpha-subunits. We found that homomeric channels, formed by monomeric or dimeric alpha-subunit constructs, show identical biophysical characteristics. Coinjections of diverse tandem constructs, instead, displayed significantly different currents proving that KDC1 has high affinity for KAT1 and participates in the formation of functional channels with at most two KDC1 subunits, whereas three KDC1 subunits prevented the formation of functional channels. This article brings a contribution to the understanding of the molecular mechanisms regulating plant Shaker channel functionality by association of modulatory subunits.     

NPC1L1:固醇脂质吸收的关键蛋白质

NPC1L1是最近发现的一种与NPC1同源的蛋白质。在体内的分布有物种差异性,其亚细胞定位存在很大争议。近些年发现NPC1L1在固醇类脂质代谢途径中起着重要作用,是肠道吸收固醇类脂质尤其是胆固醇的关键蛋白质,这项新发现使得人们对固醇类脂质的吸收机制有了了解。高胆固醇血症是心血管系统疾病的一个高危因子,因此,对NPC1L1的研究具有重大的实际意义,正逐渐成为研究的热点。     

Variation in salinity tolerance and shoot sodium accumulation in Arabidopsis ecotypes linked to differences in the natural expression levels of transporters involved in sodium transport

Salinity tolerance can be attributed to three different mechanisms: Na⁺ exclusion from the shoot, Na⁺ tissue tolerance and osmotic tolerance. Although several key ion channels and transporters involved in these processes are known, the variation in expression profiles and the effects of these proteins on Na⁺ transport in different accessions of the same species are unknown. Here, expression profiles of the genes AtHKT1;1, AtSOS1, AtNHX1 and AtAVP1 are determined in four ecotypes of Arabidopsis thaliana. Not only are these genes differentially regulated between ecotypes, the expression levels of the genes can be linked to the concentration of Na⁺ in the plant. An inverse relationship was found between AtSOS1 expression in the root and total plant Na⁺ accumulation, supporting a role for AtSOS1 in Na⁺ efflux from the plant. Similarly, ecotypes with high expression levels of AtHKT1;1 in the root had lower shoot Na⁺ concentrations, due to the hypothesized role of AtHKT1;1 in retrieval of Na⁺ from the transpiration stream. The inverse relationship between shoot Na⁺ concentration and salinity tolerance typical of most cereal crop plants was not demonstrated, but a positive relationship was found between salt tolerance and levels of AtAVP1 expression, which may be related to tissue tolerance.     

人骨髓间充质干细胞向神经细胞分化过程中Notch通路信号分子表达的变化

本研究探讨Noah信号通路在人骨髓间充质干细胞（human mesenchymal stem cells,hMSCs）体外增殖及向神经细胞分化过程中的作用。采集健康自愿者骨髓,体外培养获得hMSCs,取第3代hMSCs,在诱导剂（β-ME,DMSO,BHA）作用下向神经细胞分化。诱导后用免疫细胞化学鉴定神经元特异性烯醇化酶（neuron-specific enolase,NSE）和尼氏体的表达以确定诱导效果：用流式细胞术检测细胞生长周期时相的变化。在诱导前后,用免疫荧光和RT-PCR方法检测Notch通路中Notch1受体蛋白、配体Jagged1（JAG1）、调节蛋白活化相关物早老素1（presenilin 1,PS1）、靶基因hairy and enhancer of split1（HES1）信号分子表达的变化。结果显示：诱导前,处于G0/G1期的hMSCs占58．5％,S＋G2/M期的细胞占41．5％;诱导后,G0/G1期细胞比例升高,而S＋G2/M期细胞比例下降,NSE阳性细胞率达（77±0．35）％,细胞质中可见深蓝色的块状或颗粒状尼氏体。免疫荧光显示,诱导前后hMSCs内Notch1和JAG1均呈阳性表达,但RT-PCR检测发现诱导后Notch1、JAG1、PSl和HES1 mRNA表达量较诱导前明显降低（均P〈0．05）。结果表明,诱导hMSCs向神经细胞分化能抑制Notch信号分子表达,低水平的Notch信号激活可能有利于神经细胞的分化。     

Carboxamide SIRT1 inhibitors block DBC1 binding via an acetylation-independent mechanism

SIRT1 is an NAD⁺-dependent deacetylase that counteracts multiple disease states associated with aging and may underlie some of the health benefits of calorie restriction. Understanding how SIRT1 is regulated in vivo could therefore lead to new strategies to treat age-related diseases. SIRT1 forms a stable complex with DBC1, an endogenous inhibitor. Little is known regarding the biochemical nature of SIRT1-DBC1 complex formation, how it is regulated and whether or not it is possible to block this interaction pharmacologically. In this study, we show that critical residues within the catalytic core of SIRT1 mediate binding to DBC1 via its N-terminal region, and that several carboxamide SIRT1 inhibitors, including EX-527, can completely block this interaction. We identify two acetylation sites on DBC1 that regulate its ability to bind SIRT1 and suppress its activity. Furthermore, we show that DBC1 itself is a substrate for SIRT1. Surprisingly, the effect of EX-527 on SIRT1-DBC1 binding is independent of DBC1 acetylation. Together, these data show that protein acetylation serves as an endogenous regulatory mechanism for SIRT1-DBC1 binding and illuminate a new path to developing small-molecule modulators of SIRT1.     

达菲药物对H1N1病毒作用研究

甲型H1N1病毒在全世界范围内爆发,引起人们广泛关注,而目前疫苗和新的药物正处于研发阶段,与此同时该病毒神经氨酸酶蛋白序列不断被报道。达菲作为治疗H1N1病毒的药物被患者广泛使用。通过同源性建模的方法比较神经氨酸酶的变异情况,从而预测达菲药物对变异前后的作用效果评价。通过AUTODOCK计算结合能,发现达菲药物与神经氨酸酶的结合能维持在2.4～4.2 kJ/mol范围内,动力学常数最高值达到18.2 mM。证明达菲药物对抑制病毒进入寄主细胞有明显效果。     

细胞色素P450 1A1基因多态性与我国某些肿瘤遗传易感性

近年来有关细胞色素P450基因多态性与肿瘤遗传易感性的研究正日益吸引越来越多的关注,本文对我国近年来有关细胞色素P450 1A1(CYP1Al)基因多态性与几种肿瘤遗传易感性的研究进行探讨,推测我国几种高发病率肿瘤的发生与我国CYP1A1基因多态分布状况有关,以此为进一步研究CYP1A1与肿瘤的关系作参考。     

一个中国Gilbert综合征家系的遗传学分析

对一个中国汉族Gilbert综合征遗传家系致病基因突变位点进行鉴定,以期了解该病的分子遗传学基础。首先提取先证者基因组DNA,PCR扩增尿苷二磷酸葡萄糖醛酸转移酶UGT1A1基因的5个外显子,以琼脂糖电泳鉴定PCR产物,纯化后直接测序鉴定。基因扫描显示,与血清胆红素水平密切相关的UGT1A1基因在第1和第5外显子存在纯合突变,而 UGT1A1基因启动子区域和内含子/外显子剪接边界位点序列未检测到突变。进一步对其他家系成员该基因的相应位点进行突变检测,结果显示他们在第1和第5外显子也存在杂合突变,其中还有两个成员在启动子区域检测到(TA)插入突变。对家系成员未抗凝新鲜血液进行生化检测证实了基因突变分析的结果。综合以上结果发现该家系三种突变并存,致病因素为第1和/或第5外显子突变,为显性遗传,两种突变位点纯合导致先证者出现严重胆红素代谢功能障碍。该家系因此成为Gilbert综合征突变位点及其致病机理研究的一个典型临床病例。     

eEF1A1基因克隆、原核分泌表达及融合蛋白纯化

eEF1A1作为蛋白合成中的重要翻译延伸因子,可与多种功能性蛋白如F-actin、BPOZ-2结合,并在细胞凋亡、蛋白降解方面起重要作用.以往原核基因工程蛋白表达系统大多为包涵体表达的变性分子,需要复性.为了获得eEF1A1原核分泌性可溶性蛋白分子,克隆了人eEF1A1蛋白编码序列(约1 300 bp),并成功构建pET22b-A原核分泌表达重组质粒,转化到大肠杆菌BL21(DE3)菌株,0.4 mmol/L终浓度IPTG诱导,经不同温度下包涵体与胞浆蛋白组分分析,快速明确蛋白表达情况,即诱导4 h后,37℃表达于包涵体组分,在30℃分泌表达至胞浆组分.通过His-Trap亲和层析纯化柱进行线性洗脱,Bradford法测定蛋白浓度高达620 mg/mL,SDS-PAGE分析纯度约为95%,蛋白大小符合50 kD,Western blotting显示目的蛋白能被eEF1A1抗体识别;质谱分析证实重组蛋白为人eEF1A1蛋白分子.为进一步研究其与重要功能性蛋白的相互作用及在细胞凋亡和蛋白降解中的作用奠定基础.