种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA文库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33.其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶(PTP)家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区.Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达.结果提示,人源PTP-TD14是一个新的蛋白酪氨酸磷酸酶。 Abstract:A human cDNA of 5248bp encoding a novel protein tyrosine phosphatase PTP-TD14(1636aa) has been isolated from fetal liver.The gene is located at chromosome 3p21.3,an area frequently deleted in many types of cancer,and composed of at least 26 exons and 25 introns.The phosphatase has unique features in its domain structure:a tyrosine phosphatase domain,a C-terminal PEST motif,two SH3-binding motifs,two proline-rich region and an N-terminal domain similar to yeast BRO1 (a yeast protein that is involved in the mitogen-activated protein kinase signaling pathway).Northern blot and dot blot hybridizations indicate that it is expressed ubiquitously in human 50 tissues and 7 cancer cell lines.Thus,it is a novel protein tyrosine phosphatase gene located on 3p21.3.     

一种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33。其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶（PTP）家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区。Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达。结果提示,人源PTP－TD14是一个新的蛋白酪氨酸磷酸酶。     

应用RH技术定位人胎肝的7个新基因

辐射杂交细胞系（RH）技术是一种体细胞遗传学技术,是继荧光原位杂交技术（FISH）后新建立的染色体定位方法。本文应用该技术将发现于22周孕龄人胎肝的7个新基因进行了染色体定位：肝细胞生成素（hepatopoietin,HPO）定位于Chr.16q22.1～22.3,HQ0508定位于Chr.22q13.31～13.32,HQ0750定位于Chr.11q24.3,HQ0915定位于Chr.16p13.3,HQ1880定位于Chr.11q13.2～13.4,HQ2180定位于Chr.19q13.2～13.3,HQ3091定位于Chr.12q21.33。 Abstract：Seven novel genes, which were isolated from human fetal liver (22weeks) cDNA library, were successfully mapped to their appropriate chromosomal positions by using radiation hybrid (RH) panel. The novel gene HPO was mapped to Chr.16q22.1～22.3, HQ0508 to Chr.22q13.31～13.32, HQ0750 to Chr.11q24.3, HQ0915 to Chr.16p13.3, HQ1880 to Chr.11q13.2～13.4, HQ2180 to Chr.19q13.2～13.3, and HQ3091 to Chr.12q21.33, respectively.     

Islet1基因与心脏发育

心脏发育及心脏疾病干细胞的治疗要求对心脏发育过程中的控制细胞增殖及分化的相关基因的作用机制进行深入了解.Islet1基因(Isl1基因)含有6个外显子和5个内含子,定位于人类5号染色体5q11.2.该基因在基因组内约占12kb,目前所知其最长可读框(ORF)至少由5个外显子组成,编码一个由384个氨基酸组成的转录因子蛋白.最近研究发现,不同的心脏细胞可能源于同一种多能心脏祖细胞—Isl1+细胞,心脏的这一发育模式与血液细胞的形成模式非常相像.另外有研究结果显示,Isl1是与心脏发育密切相关的转录因子之一,其表达随着心脏发育成熟而逐渐下调.虽然针对Isl1基因做了较多的研究工作,但是它表达调控的具体模式及发挥功能的详细作用机制目前仍未完全清楚,本文对最近几年Isl1基因的研究进展作一综述.     

小鼠睾丸凋亡基因MSRG-11的人类同源基因SPATA17的克隆

利用生物信息学方法结合实验手段克隆了一个在睾丸组织中特异高表达的小鼠生精细胞凋亡相关基因Spata17的人类同源基因SPATA17（GenBank登录号为AY963797）。应用生物信息学分析显示该基因定位在染色体1q41,包含11个外显子,内含子和外显子交界区均符合gt—ag规则;该基因开放阅读框为1083bp编码一个由361个氨基酸组成的、分子量为43．49kD、等电点为9．71、含有三个保守IQ功能域的蛋白;对SPATA17编码蛋白质进行生物信息学分析,无跨膜区,无信号肽序列,推测为一非分泌性蛋白。多组织和Northern blot结果显示该基因只在睾丸组织中特异高表达,转录本大小为2．0kb。总之,研究表明SPATA17在睾丸组织生精细胞凋亡起重要作用。     

人类生殖相关新基因的定位和组织表达

基因定位对研究基因之间以及基因与疾病之间的相互关系具有重要意义。应用辐射杂种细胞系技术（RH）对我们克隆的人类新基因HBRP（Human BSP-Related Protein）进行了染色体定位,结果将该基因定位于19q13.2～13.3,同时应用生物信息学方法在人类基因组重叠片段数据库进行该基因的定位,结果相吻合。研究证明,RH技术具有快速、精确、简便等优点,是基因定位研究中一强有力的技术。同时通过RT-PCR方法研究了HBRP基因在人体各组织中的表达分布,结果显示该基因在睾丸、肠、肾、肝、脾、胃、胰腺组织有较高的表达,而在检测的脑、肺、骨骼肌、心肌组织中表达较弱。 Abstract:Gene localization is significant in elucidating the interaction between genes,gene and diseases.Using radiation hybrid (RH) technique,we cloned and localized a novel gene,designated human BSP-related protein (HBRP) on 19q13.2～13.3,in line with its localization in data bank of overlapping fragment of human genome through bioinformatics method.It is suggested RH is rapid,precise,simple and powerful in gene localization.In addition,we detected the expression and distribution of HBRP in human tissues by RT-PCR.The results showed HBRP was highly expressed in intestine,kidney,liver,spleen,stomach and pancreas,whereas lowly in brain,lung,muscle and heart.     

棉花咖啡酰辅酶A-O-甲基转移酶基因的克隆及表达

根据棉花纤维特异表达cDNA文库分析得到的咖啡酰辅酶A-O-甲基转移酶(CCoAOMT)基因EST序列设计引物,采用RT-PCR技术首次从棉花中克隆了一个CCoAOMT基因,命名为GhCCoAOMT1(GenBank登录号为FJ848871).研究结果表明:GhCCoAOMT1基因cDNA全长960 bp,具有一个753 bp的开放阅读框,5'非编码区为9 bp,3'非编码区为198 bp,编码250个氨基酸,预测分子量约为28.306 kDa,等电点为5.39.利用PCR方法克隆了GhCCoAOMT1基因的基因组序列,长度为1 311 bp,包含5个外显子和4个内含子.氨基酸同源性分析发现,GhCCoAOMT1与来自毛白杨、烟草和苎麻的CCoAOMT同源性较高.半定量RT-PCR检测表明,GhCCoAOMT1基因在棉花各个组织中都有表达,其中茎部的表达量最高,其次表达量依次为根>花瓣>子叶>10 d纤维>雄蕊>胚珠>叶.     

黑麂Y染色体的鉴别和Sry基因的克隆及定位

以流式细胞仪分离小麂(Muntiacus reevesi)Y染色体和黑麂(Muntiacus crinifrons)Y1,Y2,X+4和1号染色体,利用DOP-PCR技术富集了分离的各单条染色体。然后,将小麂的Y染色体的DOP-PCR产物经Cy3标记后直接作为涂染探针,应用染色体涂染技术与雌雄黑麂的核型标本进行杂交,确认了黑麂真正的Y染色体为Y2染色体。再以黑麂的Y1,Y2,X+4和1号染色体的DOP-PCR产物为模板,用人的特异性的SRY（sex determining region of the Y chromosome）基因引物对其进行扩增,结果表明黑麂只有Y2染色体出现了SRY扩增片段。然后扩增产物克隆和测序,比较它与人的同源性,初步把黑麂的Sry基因定位在Y2染色体上。最后提取雄性黑麂的基因组DNA,并用同一对引物对其进行扩增,亦得到Sry基因的片段,对此扩增片段进行克隆,测序,结果表明其与Y2染色体得到的Sry基因片段完全一样,与人SRY基因的同源性均为83%。 Abstract：The single Y chromosome of Muntiacus reevesi and Y1,Y2 ,X+4,1 chromosome of Muntiacus crinifrons were obtained by flow-sorting ,then they were amplified through DOP-PCR . After that, the metaphase karyotype of Muntiacus crinifrons were painted by using the product of the DOP-PCR of the Y chromosome of Muntiacus reevesi as a special probe and the result showed that Y2 chromosome was the real Y chromosome of Muntiacus crinifrons. Secondly the product of the DOP-PCR of Y1,Y2,X+4,1 chromosome of Muntiacus crinifrons were used as the templates of the next amplification using the special primer devised according to the human SRY gene .One band was obtained only from Y2 chromosome, then it was cloned to the T-vector and sequenced. The Sry gene sequence of Muntiacus crinifrons was acquired and the conclution was that there are 83% homology between the human and Muntiacus crinifrons. It was testified that in all mammal Sry gene is consertive. On the other side the Sry gene was located to the Y2 chromosome of the Muntiacus crinifrons.     

丹参ACC氧化酶基因(SmACO1)的克隆与序列分析

依据丹参转录组数据库序列信息,采用RT-PCR和染色体步移技术从丹参中首次克隆得到ACC氧化酶基因,命名为SmACO1(GenBank注册号为JQ026111)。该基因gDNA序列长1 347 bp,由3个外显子和2个内含子组成;cDNA全长1 117 bp,包含945 bp的开放阅读框,编码314个氨基酸残基。生物信息学分析显示SmACO1为无信号肽与跨膜结构域,且定位于细胞质的稳定亲水蛋白,含有Fe²⁺依赖的加氧酶结构域。实时荧光定量PCR结果表明,SmACO1基因在丹参不同组织器官中差异表达,花中表达量最高;其表达受到病原菌和茉莉酸甲酯的诱导,表明SmACO1基因可能在植物防御反应中发挥作用。     

Mapping of the Menkes locus to Xq13.3 distal to the X-inactivation center by an intrachromosomal insertion of the segment Xq13.3-q21.2

Summary During a systematic chromosomal survey of 167 unrelated boys with the X-linked recessive Menkes disease (MIM 309400), a unique rearrangement of the X chromosome was detected, involving an insertion of the long arm segment Xq13.3-q21.2 into the short arm at band Xp11.4, giving the karyotype 46,XY,ins(X) (p11.4q13.3q21.2). The same rearranged X chromosome was present de novo in the subject's phenotypically normal mother, where it was preferentially inactivated. The restriction fragment length polymorphism and methylation patterns at DXS255 indicated that the rearrangement originated from the maternal grandfather. Together with a previously described X;autosomal translocation in a female Menkes patient, the present finding supports the localization of the Menkes locus (MNK) to Xq13, with a suggested fine mapping to sub-band Xq13.3. This localization is compatible with linkage data in both man and mouse. The chromosomal bend associated with the X-inactivation center (XIC) was present on the proximal long arm of the rearranged X chromosome, in line with a location of XIC proximal to MNK. Combined data suggest the following order: Xcen-XIST(XIC), DXS128-DXS171, DXS56-MNK-PGK1-Xqter.     

Precise chromosomal locations of the genes for dentatorubral-pallidoluysian atrophy (DRPLA), von Willebrand factor (F8vWF) and parathyroid hormone-like hormone (PTHLH) in human chromosome 12p by deletion mapping

The precise chromosomal localization of the gene for dentatorubral-pallidoluysian atrophy (DRPLA) was detected by deletion mapping. Segregation patterns of genotypes of polymerase chain reaction products of DRPLA, von Willebrand factor (F8vWF), antigen CD4(p55) (CD4) and parathyroid hormone-like hormone (PTHLH) loci were studied in patients with del(12)(p13.3p13.3), del(12)(p12.3-p11.2), del(12)(p12.1-p11.2), del(12) (p11.2p11.2) and their parents. The gene for DRPLA was assigned to p13.1-p12.3 of chromosome 12. In addition, genes for F8vWF and PTHLH were mapped to p13.2 and p11.2 of chromosome 12, respectively.     

Genomic organization and mapping of the gene encoding the PP2A B56gamma regulatory subunit

Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase that regulates a wide variety of cellular processes. The enzymatic activity and intracellular localization of PP2A are determined by three distinct families of cellular regulatory subunits (B, B', and B'). The B' subunit, also known as B56, is the most diverse, consisting of five isoforms (alpha, beta, gamma, delta, and epsilon). The gene encoding B56gamma has been designated as PPP2R5C and encodes three differentially spliced variants: B56gamma1, -gamma2, and -gamma3. However, conflicting chromosomal loci have been reported in human genomic databases. The original cytogenetic mapping placed the gene on chromosome 3p21.3, whereas subsequent studies using radiation hybrid analysis localized PPP2R5C to chromosome 14q. In this study, by radiation hybrid mapping, FISH analysis, BAC clone sequencing, and RT-PCR analysis, we show that the functional gene PPP2R5C exists at 14q32.2 and gives rise to three splicing variants, B56gamma1, -gamma2, and -gamma3, whereas a nonfunctional B56gamma1 pseudogene, PPP2R5CP, is present at 3p21.3. We also report the genomic organization of both the functional gene and the pseudogene.     

Characterization and chromosomal mapping of a human steroid 5 alpha-reductase gene and pseudogene and mapping of the mouse homologue.

The enzyme steroid 5 alpha-reductase catalyzes the conversion of testosterone into the more powerful androgen, dihydrotestosterone. We previously described the cloning of rat and human cDNAs that encode steroid 5 alpha-reductase and their expression in oocytes and cultured cells. Here, we report the isolation, characterization, and chromosomal mapping of two human steroid 5 alpha-reductase genes. One gene (symbol SRD5A1) is functional, contains five exons separated by four introns, and maps to the distal short arm of chromosome 5. Two informative restriction fragment length polymorphisms are present in exons 1 and 2 of this gene. A second gene (symbol SRD5AP1) has all of the hallmarks of a processed pseudogene and was mapped to the q24-qter region of the X chromosome. In the mouse, a single steroid 5 alpha-reductase gene (Srd5 alpha-1) is linked to Xmv-13 on chromosome 13.