牛SRY蛋白表达纯化与体外功能鉴定

利用PCR技术从北京黑白花奶牛(Bostaurus)的基因组DNA中克隆了SRY(Sex-determiningregionontheYchromosome)基因编码区全长序列。序列分析表明牛SRY基因的HMG区(Highmobilitygroup)呈现高度的保守性,与人、小鼠、猪等的相似性达到70%。将SRY基因与pET-28a( )载体相连,构建表达载体pET-28a/SRY;把该表达载体转入大肠杆菌BL21(DE3),以IPTG诱导30℃诱导4h,SRY蛋白可高效表达,表达产物占总蛋白量的26%。对表达产物进行了Western-blotting检测,并采用亲和层析技术获得了高纯度的牛SRY蛋白。通过PCR技术分别获得牛、人、鼠的苗勒氏管抑制物(MullerianInhibitingsubstances,MIS)启动子,凝胶阻滞试验证明,牛SRY蛋白可与人及牛的MIS启动子结合,但与鼠的Mis启动子不发生相互作用。     

46,XY女性性反转患者SRY基因的两个新突变类型

Y染色体上的性别决定区域——SRY基因作为睾丸决定因子,可以调控男性性别发育过程。SRY基因是一种转录因子,属于带有高迁移率族蛋白家族,该家族成员包含能与DNA结合的HMG盒基序。已知SRY基因的缺失和点突变是造成XY女性性反转的病因之一。通过筛查10位中国46,XY女性性反转病人SRY基因的开放阅读框区域,探寻新的突变类型。用标准方法从外周血中抽提gDNA,通过聚合酶链式反应扩增SRY基因中部的609bp的DNA片段。扩增后的PCR片段被克隆到pUCm-T载体中,在ABI377-3自动测序仪上完成测序。运用限制性内切酶酶切分析的方法验证DNA测序的结果。结果表明,在两个患者的SRY基因中分别发现了新的核苷酸点突变,并都导致氨基酸替代。一个突变发生在SRY基因的5’端HMG盒外的核苷酸第113位腺嘌呤(A)被鸟嘌呤(G)取代,并导致谷氨酸被甘氨酸替换;另一个突变是第387位核苷酸发生T被A替换,该突变引起第129位的酪氨酸变成终止密码,她父亲的SRY序列被证明是正常的野生型。通过查询文献和人类基因突变数据库(HGMD),这两个突变都是以前未见报道过的新型SRY基因突变,并使因核苷酸替换引起SRY基因突变总数增加到45。     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

中国人SRY基因的分离、克隆和核苷酸序列分析

睾丸决定因子基因(Testis-determining factor,TDF)位于Y染色体短臂上,它的表达产物诱导睾丸组织的发生。SRY基因(Sex-determining Region of the Y)位于Y染色体的性别决定区内,许多特征显示SRY就是TDF。我们选用与SRY基因相应的引物,用PCR技术对正常人男女各10例的基因组DNA进行扩增。将特异扩增的男性SRY基因片段重组到质粒PUC12中,得到含有中国人SRY基因片段的克隆,命名为PSY-1、PSY-2。用[~(32)p]标记重组质粒中的SRY基因片段作探针,与PCR结果进行Southern杂交,男性样品均显示特异杂交带,女性样品为阴性。用末端终止法测定克隆的SRY基因片段的全部核苷酸序列为299bp,含有SRY基因中高度保守及功能特异性区域的240bp,我们对此进行了讨论。     

牛SRY同源基因的PCR扩增和克隆

本文采用人SRY基因的一对引物,通过PCR扩增获得了雄性牛(Bos taurus)SRY同源基因片段。进一步证实牛存在与人SRY基因同源的相应基因。将PCR产物与载体pUC—Eco—T连接后,用以转化JM109菌,经过与人SRY基因探针菌落杂交,筛选获得了牛SRY同源基因克隆pBosY O.6后者的插入片段为相应于人SRY基因保守区在内的一段约609bp DNA。此外还比较分析了人和牛SRY同源基因片段限制酶图谱。     

赤麂SRY基因的测序及其与部分偶蹄目动物SRY基因序列的比较

采用人SRY基因的一段保守序列的引物,通过PCR在雄性赤麂中扩增出了赤麂SRY基因的特异片段,通过DNA斑点杂交证实其扩增产物与人SRY基因探针进行菌落杂交筛选出赤麂SRY基因的阳性克隆,并对其进行了,将其序列与基因库中录入的所有偶蹄目动物的SRY基因序列进行同源性比较,用UPGMA法构建了其系统进化树,从分类和进化上对赤麂SRY基因进行分析。     

SRY基因及其性别决定

对近十年来关于脊椎动物尤其是哺乳动物的SRY基因及其性别决定机制的研究进展作了简要的综述。扼要阐述了哺乳动物的SRY基因的结构、转录、表达及其在性别决定中的作用模式及相关的基因家族。     

六例性反转综合征患者的分子遗传学分析

对六例性反转综合征患者(3例XX男性)(3例XY女性)用Y-特异性DNA探针进行了Southern印迹杂交分析,并用PCR技术扩增了SRY基因部分序列。结果表明,1例XX男性缺乏源于Y染色体的杂交信号,也无SRY基因;其余2例XX男性和3例XY女性都检测到Yp-DNA序列和SRY基因。这对进一步阐明性反转综合征的病因和SRY基因的作用机制具有重要意义。     

雄牛特异的SRY同源序列的扩增和分析

利用人、兔、鼠SRY序列设计引物,应用PCR扩增牛的SRY序列,获得200bp的雄牛特异的扩增片段。克隆该扩增片段,获得重组质粒pCH21,进行序列分析,并与人、兔和鼠SRY的对应区域比较,具有高度同源性。用pCH21 DNA作探针与牛的基因组DNA酶切图谱杂交,显示了雄牛特异的I．7kb的杂交带。分析200bp的PCR扩增片段是牛的SRY基因片段。用同一对引物扩增人和山羊的DNA样品,也获得雄性特异的200bp的扩增片段。     

小麂Sry基因的克隆和测序

应用人的性别决定基因SRY(Sex-determining Region Y gene,SRY)中HMG框内的一对引物,对小麂细胞株的基因组DNA进行PCR扩增,得到雄性小麂细胞的220bp扩增产物,而在雌性小麂细胞中未发现扩增产物。将雄性小麂细胞的220bp扩增产物通过T-A互补法克隆到质粒pGEM-T 载体中,筛选阳性克隆进行DNA测序。测序结果表明小麂Sry基因保守序列与人的SRY基因保守区相同碱基的比值为152/184,达到82.6%。提示小麂Sry基因与人的SRY基因存在着较高的同源性,说明SRY基因在进化过程中高度保守。 Abstract:Using the primers from SRY gene——HMG Box for PCR amplification in genomic DNA of Muntiacus reevesi cell strains,a 220bp fragment was obtained in the male but not in the female.The 220bp fragment was cloned into the pGEM-T vector using T/A clone method.The identified positive clone was sequenced.The result shows that 82.6% nucleotides(152bp/184bp) are homologous between Muntiacus Sry and human SRY gene.It suggests that SRY is highly conserved during evolution.     

水稻AT-hook基因家族生物信息学分析

AT-hook是一种小型的DNA结合蛋白基序, 在哺乳动物非组蛋白染色体的高移动组蛋白(HMG-I/Y)中首次发现。对其它生物的研究表明, AT-hook蛋白在染色质结构组装、靶细胞特异性结合、转录调控和生长发育的调控中发挥重要作用。利用生物信息学方法, 从水稻(Oryza sativa)基因组中鉴定出了45个编码AT-hook蛋白的基因, 并对这些基因的系统进化、染色体定位及其编码蛋白的结构和功能等进行了系统的生物信息学分析。结果表明, 水稻AT-hook蛋白的结构和特性分化不显著; 45个水稻AT-hook基因可划分成5个亚族; 染色体复制是基因家族成员扩增的进化途径之一。基因数字表达分析结果显示, AT-hook基因主要在水稻幼穗中表达, 并通过qRT-PCR验证了部分基因的数字表达结果。     

苏云金芽胞杆菌以色列亚种20kDa蛋白质对CytA蛋白溶细胞作用的影响

为了证明苏云金芽胞杆菌以色列亚种２０ｋＤｅ蛋白质对ＣｙｔＡ蛋白溶细胞作用的影响, 根据２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因的核苷酸序列,用ＡＭＰＬＩＦＹ程序设计了一套带有酶切位 点的引物,经ＰＣＲ扩增分别获得了２０ｋＤｅ蛋白质和ｃｙｔＡ蛋白基因。将其基因与表达载体 ｐＵＨＥ２４连接并转化到大肠杆菌ＸＬＩ和ＤＨＳ 分别获得含２０ｋＤａ蛋白质基因的克隆子 ＬＺ２９;含ｃｙｔＡ基因的克隆子ＬＺｃｙｔＡ和含有二者基因的重组子ＬＺ２０Ａ．在ＩＰＴＧ诱导下,测定 了不同克隆株基因表达产物对大肠杆菌细胞生长的影响。结果表明：ＬＺ２０的细胞生长不受影 响;ＬＺｃｙｔＡ的细胞被杀死;ＬＺ２０Ａ的细胞生长也不受影响。这表明２０ｋＤａ蛋白质基因与ｃｙｔＡ 蛋白基因重组后,２０ｋＤａ蛋白质基因表达产物可保护ＣｙｔＡ蛋白对大肠杆菌的溶细胞作用,而 巳这种作用并不因不同大肠杆菌受体而改变。     

Determination of the cleavage site of the phage T4 prohead protease in gene product 68. Influence of protein secondary structure on cleavage specificity

The cleavage site of the T4 prohead protease in gene product 68 of bacteriophage T4 has been determined by direct protein sequencing. It is located close to the carboxy-terminal end of a predicted alpha-helix in the sequence Asn-Val-Glu-Ala between the Glu and Ala residues. Secondary structure seems to be more important in determining cleavage than the presence of an aliphatic amino acid three residues before the cleavage site that was proposed earlier. In this case, that position is occupied by Asn, a hydrophilic residue. A second potentially cleavable Glu-Ala is found five residues after the cleaved sequence and this is preceded by an Ile at the -3 position. Despite this, the sequences of the amino and carboxyl termini of the uncleaved protein are identical to those previously proposed from an analysis of the DNA sequence of the gene.     

重组SARS病毒N蛋白可与SARS患者血清发生特异反应

N蛋白是SARS CoV病毒基因组编码病毒核衣壳蛋白 ,它不同于现已知的任何蛋白质 .对它的深入研究对揭示SARS -CoV的致病机理和疫苗及诊断试剂的研制有重要意义 .灭活的病毒经逆转录后 ,用根据已知的病毒的基因组序列所设计的引物PCR扩增N蛋白基因 .扩增出的基因经序列分析表明和已知的序列完全一致 ,共编码 4 2 2个氨基酸残基 .将N蛋白基因克隆入原核表达载体pET2 8a构建成表达质粒pET2 8a N .表达质粒转化大肠杆菌BL2 1 (DE3) ,并用IPTG诱导后 ,获得了高表达N蛋白的重组菌株 .目的蛋白经一步金属离子螯合层析纯化后获得了纯度超过 90 %的样品 .Western印迹及ELISA分析表明 ,SARS患者体内有特异性的针对N蛋白的抗体 ,并具有较高的特异性 .这为临床上诊断SARS患者提供了新方法 ,并为SARS疫苗的研制提供了研究思路     

基因是什么? 分子遗传学教学中的体会和理解

随着分子遗传学的飞速发展,基因概念也在不断更新。笔者从事分子遗传学、分子生物学、遗传学教学以及基因与基因表达调控方面研究多年,对基因的本质和概念有较深的理解和认识。回顾了经典基因概念的形成和发展过程,并讨论了真核生物中的RNA遗传和朊病毒复制现象,提出了新的基因概念。认为基因是携带遗传信息的、可遗传的核酸片段或者多肽分子,它们可以编码具功能的RNA分子或多肽分子。     

Importance of the B2 domain of the Arabidopsis ABI3 protein for Em and 2S albumin gene regulation

Genetic and molecular studies have shown that the Arabidopsis ABSCISIC ACID-INSENSITIVE3 (ABI3) protein plays a prominent role in the control of seed maturation. The ABI3 protein and its orthologues from various other plant species share four domains of high sequence identity, including three basic domains designated as B1, B2 and B3. The leaky abi3-1 mutation is a single amino acid substitution within the B3 domain. A new abi3 allele, abi3-7, was generated by mutagenizing abi3-1 seeds. The abi3-7 line contains, in addition to the abi3-1 mutation, a point mutation that converts residue Ala-458 into Thr within the B2 domain of the ABI3 protein. This Ala residue is absolutely conserved in all known ABI3 orthologues. Abi3-7 seeds display reductions in dormancy and in sensitivity to abscisic acid which are intermediate between those of the leaky abi3-1 and of the severe abi3-4 and abi3-5 mutants. Accumulation and distribution of At2S1 and At2S2 albumin mRNA as well as of AtEm1 and AtEm6 late embryogenesis-abundant proteins and mRNA have been analyzed. Both At2S1 and At2S2 mRNA are reduced in abi3-7, but distribution of At2S2 is spatially restricted. Accumulation of AtEm6 protein is more sensitive to abi3-7 mutation than AtEm1. However both mRNAs are considerably reduced in this mutant. Their distribution is also differentially affected. These results provide genetic evidence for the importance of the conserved B2 domain for ABI3 function in vivo.     

Structure of phage P22 coat protein aggregates formed in the absence of the scaffolding protein.

Previous studies have shown that the assembly of the precursor shell (prohead) of bacteriophage P22 requires the copolymerization of the gene 5 coat protein with the gene 8 scaffolding protein. Removal of the scaffolding protein by mutation prevents efficient coat protein assembly, but some aberrant particles do form. We have now isolated these structures and characterized them with respect to morphology, protein composition, and small-angle X-ray scattering properties.The aberrant particles fall into three morphological classes, i.e. complex spirals and closed shells of two sizes. Small-angle X-ray scattering studies confirm that the larger particles are hollow shells with the radius of proheads ($\text{r = 260}\text{A}\text{?}$), and not of the mature virus ($\text{r = 285}\text{A}\text{?}$). These structures lack the inner shell of scaffolding protein found in proheads. The small particles have a radius of 195 Å, smaller than proheads, and appear to contain material, not scaffolding protein, within the outer shell.The aberrant particles contain two minor protein species, the gene 9 tail-spike protein, and an unidentified 67,000 molecular weight polypeptide, probably from the host. Neither is found in normal proheads. Removal of gene.9 product by mutation did not affect the formation of the aggregates. Fractionation of the morphological classes of particles revealed that the 67,000 molecular weight band was associated with the closed shells. It may be serving as a pseudo-initiator.Earlier studies had shown that treatment of proheads with sodium dodecyl sulfate in vitro resulted in loss of the scaffolding protein, and expansion of the shell to the mature radius of 285 Å. When the 8^? prohead-sized shells were treated similarly, they also expanded to the mature-sized shell. These results support the idea that there are at least two stable states of the coat protein, one of which, the prohead form, is an obligatory precursor of the mature form.