虎纹蛙Sox基因的PCR扩增及SSCP分析

本文采用PCR技术,以特异扩增人SRY基因HMG－box保守区的一对引物,扩增了虎纹 蛙Sox基因（SRY－box gene）。并对扩增产物进行了SSCP分析。结果雌雄虎纹蛙个体均扩增 出一条带,大小为221 bp,表明虎纹蛙Sox基因在雌雄个性间无性别特异性。SSCP分析结果 显示虎纹蛙Sox基因雌雄个体片段的单链迁移率无差异,而与人的有较大差异。本文为探讨 虎纹蛙的性别决定机制及Sox基因的进行提供了分子资料。     

东北虎Sox3和Sox4基因的保守区序列分析

应用特异于HMG－box区域的兼并引物，以基因组DNA为模板扩增了东北虎的SOX基因，在扩增产物中发现一条220bp的扩增带。经过克隆、序列测定和同源性检索，发现一个基因片段与人、小鼠、鸡和爪蟾Sox3的同源性分别为95％、95％、92％和88％；与人和小鼠SRY基因的同源性分别为75％和73％。因此该片段应当为东北虎的Sox3基因片段，被命名为PtSox3。另一片段与人、小鼠、爪蟾和大鼠Sox4的同源性分别为98％、97％、95％和94％；与人和小鼠SRY基因的同源性分别为56％和54％被命名为PtSox4。     

黑斑蛙Sox基因的PCR扩增

以特异扩增人SRY基因HMG -box保守区的一对引物 ,扩增了黑斑蛙的Sox基因 (SRY -boxgene) .结果表明 ,雌雄黑斑蛙个体均扩增出了两条带 ,大小分别为 4 50bp和 90 0bp ,显示了Sox基因在黑斑蛙雌雄个体间无性别特异性 ,且可能含有内含子 .本文为探讨黑斑蛙的性别决定机制及Sox基因的进化提供了分子资料 .     

黄鳝性别决定与SRY基因不相关

以人和鼠的SRY片断为探针与黄鳝基因组DNA进行杂交,发现雌雄黄鳝中均有相同的杂交带.用一对SRY基因保守区HMGbox的引物,在雌雄黄鳝的基因组DNA中均扩增出约200bp片段.将此片断克隆测序后发现雌雄个体中此片断仅相差1个bp,且与人的SRY基因HMG盒基因极相似.以该片段与雌雄个体的RNA进行Northern杂交未能检测到杂交带,RT-PCR反应扩增出一条微弱的200bp片段.以上结果表明:在雌雄黄鳝的基因组DNA中均存在SRY同源片断;黄鳝中的SRY同源片断可能与其性别无直接关系,而是具备其他功能,由此推测低等脊椎动物的性别决定可能由其他基因控制.     

两对引物PCR扩增黑斑蛙Sox基因

以人的SRY基因的HMG-box保守区设计的两对引物,对黑斑蛙Sox基因的扩增.结果如下:1)雌雄黑斑蛙均扩增出一条带,大小约为220 bp,说明Sox基因的长度在黑斑蛙雌雄个体间无性别差异;2)两对引物的扩增结果相同,表明Sox基因有高度保守性.     

利用SRY基因鉴定绵羊成纤维细胞性别的研究

SRY是哺乳动物性别决定的重要基因，利用绵羊SRY基因序列设计PCR引物，对绵羊胚胎来源的成纤维细胞提取总DNA进行PCR扩增，鉴定6个不同胚胎来源的成纤维细胞的性别．结果表明，3个有扩增带为雄性，3个无扩增带为雌性，为核移植选取雄性或雌性转基因成纤维细胞提供一项可行的方法．同时提取牛、山羊、小鼠的总DNA，利用绵羊SRY基因引物进行扩增，对SRY基因在哺乳动物中的保守性进行研究．结果表明，绵羊SRY基因保守区外的一对引物对雄性山羊、绵羊、牛及小鼠均能扩增出特异的DNA片段，说明SRY基因序列有很强的保守性．     

一个在中华绒螯蟹性腺中特异表达的Sox基因HMG盒区的克隆与鉴定

根据人的睾丸决定因子（SRY）的高速泳动组蛋白区设计简并引物,以中华绒螯蟹雌雄个体的基因组DNA为模板进行PCR扩增,从雌雄体均扩出220bp左右的条带,说明中华绒螯蟹中存在SRY基因的同源体,且无性别差异．经克隆、测序后得到两条SRY盒区相关基因（Sox基因）片段,ES1和ES2,其序列与人相应Sox基因保守区的最高同源性分别为66％和93％．ES1和ES2在成体8种组织中的检测结果表明,ES1在精巢、肌肉、心脏、肝脏、鳃、胸神经团、不同时期的卵巢及排出的成熟卵等组织中均表达,而ES2只在精巢、成熟的卵巢以及排出的成熟卵中有表达,说明ES2基因可能参与性腺的发育和早期胚胎发育过程．     

王锦蛇(Elaphe carinata)Sox基因保守区的克隆及测序

参照人SRY基因HMG-box保守区序列设计一对兼并引物,PCR扩增了王锦蛇的Sox基因,采用SSCP技术筛选阳性克隆,并对其进行了测序.结果在雌雄个体中共筛选出4个Sox基因,其中一个为雌性独有,显示出性别差异性;4个Sox基因DNA序列及编码的氨基酸序列与人相应SOX基因的相似性分别为91%、91%、92%、91%和96%、98%、96%、96%,显示出高度的保守性.实验结果为王锦蛇的性别决定机制研究提供了分子资料.     

鲤鱼中5个Sox基因保守区的克隆和比较

SRY/Sry基因已被公认为是哺乳动物的睾丸决定因子(TestisDeterminingFactor,TDF)基因,它的正确的时空表达是雄性生殖腺形成的关键,即导致哺乳动物胚胎性别决定的开关基因.作为一个大基因家族的首位成员,它的发现诱发了Sox基因家族的研究热潮.Sox基因家族是在动物中发现的一类新的编码转录因子的基因家族,其产物具有一个HMG基序保守区,参与诸如性别决定、骨组织的发育、血细胞生成过程、神经系统的发育、晶状体的发育等多种早期胚胎发育过程.鱼类是脊椎动物中进化地位较低的一类生物,除了个别种类出现了与性别相关的染色体外,绝大多数都无异形性染色体,说明了鱼类正处于性别染色体进化的重要时刻.研究鱼类中的Sox基因对于研究SRY的发生、性别染色体的进化以及性别的决定机制有着重要的意义.本实验利用兼并引物PCR的方法,参照Sox基因的HMG-box区氨基酸序列设计简并引物,对鲤鱼(Cyrinuscarpio)的基因组进行扩增,获得5个新的基因片段.经过在Genbank中进行同源性比较和分析,证明它们是鲤鱼的Sox基因并分别命名为CcSox3、CcSox4、CcSox11、CcSox14、CcSox21.与鲤鱼中的这些Sox基因具有最高同源性的基因分别是OlSox3,同源性为94.03%;CvSox4基因,同源性为88.06%;DrSox11基因,同源性为97.01%;MmSox14和HsSox14基?     

扬子鳄DMRT1基因DM保守区的克隆及序列分析

DMRT1基因是DMRT基因家族的一个重要成员,在动物性别决定过程中发挥重要作用.本文参照人DMRT1基因DM保守区及有关文献,设计了一对简并引物,扩增了扬子鳄的DMRT1基因,并对扩增产物进行了亚克隆与测序.结果在雌雄扬子鳄个体中各获得一个预期的基因片段,长度为140bp,序列无性别差异性,命名为扬子鳄AsDMRT1基因.序列同源性分析表明,扬子鳄DMRT1基因DM盒区核苷酸序列与人和鼠相应基因的同源性分别为87%、86%;编码的氨基酸序列与人和鼠相应基因编码序列的同源性分别为95%、97%.充分显示了DMRT1基因在进化上的高度保守性.     

Sex determination involves synergistic action of SRY and SF1 on a specific Sox9 enhancer

The mammalian Y chromosome acts as a dominant male determinant as a result of the action of a single gene, Sry, whose role in sex determination is to initiate testis rather than ovary development from early bipotential gonads. It does so by triggering the differentiation of Sertoli cells from supporting cell precursors, which would otherwise give follicle cells. The related autosomal gene Sox9 is also known from loss-of-function mutations in mice and humans to be essential for Sertoli cell differentiation; moreover, its abnormal expression in an XX gonad can lead to male development in the absence of Sry. These genetic data, together with the finding that Sox9 is upregulated in Sertoli cell precursors just after SRY expression begins, has led to the proposal that Sox9 could be directly regulated by SRY. However, the mechanism by which SRY action might affect Sox9 expression was not understood. Here we show that SRY binds to multiple elements within a Sox9 gonad-specific enhancer in mice, and that it does so along with steroidogenic factor 1 (SF1, encoded by the gene Nr5a1 (Sf1)), an orphan nuclear receptor. Mutation, co-transfection and sex-reversal studies all point to a feedforward, self-reinforcing pathway in which SF1 and SRY cooperatively upregulate Sox9 and then, together with SF1, SOX9 also binds to the enhancer to help maintain its own expression after that of SRY has ceased. Our results open up the field, permitting further characterization of the molecular mechanisms regulating sex determination and how they have evolved, as well as how they fail in cases of sex reversal.     

Genetic evidence equating SRY and the testis-determining factor

The testis-determining factor gene (TDF) lies on the Y chromosome and is responsible for initiating male sex determination. SRY is a gene located in the sex-determining region of the human and mouse Y chromosomes and has many of the properties expected for TDF. Sex reversal in XY females results from the failure of the testis determination or differentiation pathways. Some XY females, with gonadal dysgenesis, have lost the sex-determining region from the Y chromosome by terminal exchange between the sex chromosomes or by other deletions. If SRY is TDF, it would be predicted that some sex-reversed XY females, without Y chromosome deletions, will have suffered mutations in SRY. We have tested human XY females and normal XY males for alterations in SRY using the single-strand conformation polymorphism assay and subsequent DNA sequencing. A de novo mutation was found in the SRY gene of one XY female: this mutation was not present in the patient's normal father and brother. A second variant was found in the SRY gene of another XY female, but in this case the normal father shared the same alteration. The variant in the second case may be fortuitously associated with, or predisposing towards sex reversal; the de novo mutation associated with sex reversal provides compelling evidence that SRY is required for male sex determination.     

A human XY female with a frame shift mutation in the candidate testis-determining gene SRY

The primary decision about male or female sexual development of the human embryo depends on the presence of the Y chromosome, more specifically on a gene on the Y chromosome encoding a testis-determining factor, TDF. The human sex-determining region has been delimited to a 35-kilobase interval near the Y pseudoautosomal boundary. In this region there is a candidate gene for TDF, termed SRY, which is conserved and specific to the Y chromosome in all mammals tested. The corresponding gene from the mouse Y chromosome is deleted in a line of XY female mutant mice, and is expressed at the expected stage during male gonadal development. We have now identified a mutation in SRY in one out of 12 sex-inversed XY females with gonadal dysgenesis who do not lack large segments of the short arm of the Y chromosome. The four-nucleotide deletion occurs in a sequence of SRY encoding a conserved DNA-binding motif and results in a frame shift presumably leading to a non-functional protein. The mutation occurred de novo, because the father of the sporadic XY female that bears it has the normal sequence at the corresponding position. These results provide strong evidence for SRY being TDF.     

PCR amplification of Sox genes inMisgurnus anguillicaudatus andParamisgurnus dabryanus (Cypriniforms; Cobitidae)

Using the primers which specially amplify the conservative motif of Human SRY gene, we studied the PCR amplification of Sox genes in genomic DNA of two species of mud loach:Misgurnus anguillicaudatus andParamisgurnus dabryanus. Four bands with the length of 200,550,940 and 1000 bp respectively, were presented in the PCR products ofMisgurnus anguillicaudatus. Three bands with the length of 200,550 and 900 bp were presented in that ofParamisgurnus dabryanus. Southern blotting results indicated that the 200 and 550 bp bands are specially positive. There is no difference between male and female individuals as well as between these two species. Chang zhongjie: Born in Nov. 1965, Ph. D. graduate student     

The sex reversal of two mosaics with 45, XO/46, XX and 46, XY/47, XXY

Sex-reversed males with 45, XO and 46, XX are associated with patients carrying a small male-determining region-SRY gene of the Y chromosome, while some 46, XY females can be the result of SRY gene mutations on Y chromosome. Duplication of DSS gene on Xp21 can also cause 46, XY individual with normal Y chromosome and an intact SRY gene develops as phenotypically sex reversed female. We report two patients of the sex reversal, one develops as a male carrying the karyotype 45, XO/46, XX, the other is a phenotypically female with karyotype 46, XY/47, XXY. The mosaic with 46, XY/47, XXY female might be reported for the first time. Xu Yaoxian: born in Dec., 1956, Lecturer