牦牛SRY和TRO基因部分序列克隆与测序分析

对牦牛SRY和TRO的部分基因克隆和序列分析,以期为牦牛X精子和Y精子鉴定、性染色体的基因定位以及分子标记辅助选择研究提供理论依据.从牦牛和西门塔尔牛冷冻精液中提取DNA,用特定引物对SRY、TRO基因部分序列进行扩增并进行TA克隆和测序.结果表明,这两个基因区域在牛种中有极高的保守性,牦牛与普通牛SRY和TRO基因这两个区域的核酸同源性分别高达99.08%和99.39%.     

人类Y染色体上与精子发生相关的基因的研究进展

Y染色体是男性特有的染色体，其上存大精子发生相关基因，在Y染色体长臂上的AZF区的缺失与男性不育密切相关，AZF分为a,b,c,d四个亚区，其中的精子发生相关的基因有DFFRY、DBY，RBMY1和DAZ，在AZF区外，Y染色体长臂的SMCY基因也与精子发生相关，Y染色体短臂上精子发生原基因主要有SRY、ZFY和TSPY基因，以上基因的缺失或表达异常都会造成精子发生障碍或性发育障碍，这些基因的研究在临床上有重要的意义。     

赤麂Sry基因的定位及Y染色体的鉴别

应用流式细胞仪分离赤麂的1,Y1,Y2染色体,通过简并寡核苷酸引物聚合酶链式反应(DOP-PCR)增加模板数量.用人的性别决定基因HMG框内设计1对引物进行PCR扩增.在雄性赤麂Y2染色体DOP-PCR产物中扩增出与人SRY基因同源的Sry基因片段,经克隆测序后,初步证明赤麂Y2染色体是真正的Y染色体,同时对赤麂Sry基因进行了初步定位.     

Boule基因在精子发生过程中的作用

精子发生是一个复杂的细胞分化过程,生精细胞经过有丝分裂和减数分裂产生单倍型的精细胞,精细胞再经过复杂的形态变化而形成成熟的精子。这一过程需要众多基因表达的精确调控。目前已发现了一些与男性不育相关的候选基因,这些候选基因中的DAZ(Deleted in azoospermia)基因家族在精子发生过程中发挥关键性的调控作用。Boule基因是2001年发现的DAZ家族的新成员,是人类精子发生过程中重要的调控因子。Boule表达的改变或BOULE蛋白的缺乏可引起减数分裂阻滞和精子生成障碍,从而导致无精子症并产生不育。     

动物性别鉴定与控制研究进展

哺乳动物性别决定是以位于雄性Y染色体短肴上被称为SRY基因为调控中心、多基因参与的级联调控过程。但染色体理论并非性别决定机制的全部，外部环境中的某些因素也是性别决定机制的重要条件。本中对家畜性别控制技术的一些方法的原理、理论基础基本方法和性反转等进行了综述，介绍了家畜的精子和早期胚胎性别鉴定与控制的一些方法。     

应用FQ-PCR检测孕妇外周血中胎儿细胞含量

合成一对染色体单拷贝基因GAPDH特异的引物和一对Y染色体单拷贝基因SRY特异的引物,并合成两条特异性TaqMan探针,加等量的DNA到含有上述两个引物探针的PCR反应体系中,行荧光定量PCR,获得两种基因的定量值,通过这两种基因含量的比值,计算孕妇外周血中胎儿细胞含量.在30 μL FQ-PCR反应体系中,加入最少约0.05 ng 正常男性基因组DNA能够稳定地获得扩增曲线;在60例临床孕妇外周血标本DNA 的DCFQPCR扩增中有35例出现SRY扩增阳性,阳性率为58.3%;在这些标本中,GAPDH 基因Ct值范围在20~22之间;SRY 基因Ct值范围在33~40;经计算,胎儿细胞含量范围在1/10.4~1/10.6.     

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。     

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.     

利用妊娠奶牛血液快速鉴定不同发育阶段的胚胎性别

采集妊娠奶牛静脉血液，从血浆中提取胎儿DNA，利用牛Y染色体上性别决定基因（sex-determining region Ylinked gene, SRY gene）的特异性序列设计引物，应用巢式PCR扩增SRY基因特异性片段，同时以牛肌动蛋白（β-Actin）特异性片段的扩增产物作为内参照，对32头妊娠奶牛早期胚胎性别进行鉴定。结果表明：从不同妊娠期奶牛血浆中提取胎儿游离DNA进行胚胎性别鉴定的总准确率为80％，其中1～2月龄、2～3月龄、4～8月龄胚胎性别鉴定的准确率分别为60％、85．7％、92．3％。     

奶牛性别鉴定

叙述了利用性别决定基因（SRY基因）PCR扩增进行奶牛性别鉴定的发展历史,在此基础上阐述了PCR技术和方法在奶牛胎儿性别鉴定中的应用及其存在的问题和应用前景.     

SRY基因在部分动物类群系统进化中保守性研究

用特异于人 HMG- box区域的一对引物 ,对脊推动物 5个纲 10个物种及 2种无脊推动物的基因组 DNA进行 PCR扩增 ,并以 dig标记的人 SRY基因为探针 ,与扩增产物进行 Southern杂交 ,结果表明 :在这 12个物种中都存在 SRY基因的同源序列 ,无脊推动物克氏螯虾及背角无齿蚌杂交中显色较慢 ,表明 SRY基因在系统进化中具有高度的保守性且同源程度与物种在进化上的地位有关     

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.     

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.     

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.     

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     

两重巢式PCR检测母体外周血中胎儿游离DNA

使用两重巢式PCR技术检测母体外周血中胎儿游离DNA,为非损伤性产前鉴定胎儿性别和诊断单基因疾病奠定基础.采用改良的chelex100方法提取60例孕龄为16~36周孕妇的外周血浆中游离胎儿DNA,依据NCBIX染色体长臂ATL1位点,Y染色体上DYS14位点序列,设计并合成引物,用巢式PCR同时扩增2个基因片段,以鉴定胎儿性别.结果表明:有ATL1位点和DYS14位点的扩增产物261bp和198bp鉴定为男性胎儿,而仅有261bp扩增产物鉴定为女性胎儿.均以真实出生性别进行确认后发现,此方法能有效提高检测特异性.两重巢式PCR能够简便并准确地鉴定胎儿性别,对单基因疾病特别是X-连锁遗传病的诊断有积极意义.     

扬子鳄Sox基因的PCR-SSCP分析

参照人SRY基因HMG－box保守区的序列,设计一对引物,扩增了扬子鳄的Sox基因,并进行了SSCP分析,结果显示扬子鳄Sox基因的扩增片段与人SRY基因扩增片断大小相同,为220bp左右;且雌雄个体间该基因片段的单链迁移率无差异,而与人的有较大差异,本研究为扬子鳄的性别决定机制的探讨及Sox基因的进化保守性分析提供分子资料。     

A candidate spermatogenesis gene on the mouse Y chromosome is homologous to ubiquitin-activating enzyme E1.

The human X-linked gene A1S9 complements a temperature-sensitive cell-cycle mutation in mouse L cells, and encodes the ubiquitin-activating enzyme E1. The gene has been reported to escape X-chromosome inactivation, but there is some conflicting evidence. We have isolated part of the mouse A1s9 gene, mapped it to the proximal portion of the X chromosome and shown that it undergoes normal X-inactivation. We also detected two copies of the gene on the short arm of the mouse Y chromosome (A1s9Y-1 and A1s9Y-2). The functional A1s9Y gene (A1s9Y-1) is expressed in testis and is lost in the deletion mutant Sxrb. Therefore A1s9Y-1 is a candidate for the spermatogenesis gene, Spy, which maps to this region. A1s9X is similar to the Zfx gene in undergoing X-inactivation, yet having homologous sequences on the short arm of the Y chromosome, which are expressed in the testis. These Y-linked genes may form part of a coregulated group of genes which function during spermatogenesis.