小鼠睾丸生精细胞凋亡相关基因mTSARG3的克隆

从在小鼠隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 37)出发 ,利用GeneScan软件分析该片段所在染色体基因组序列 ,获得一个包含该EST的新基因序列。设计该基因特异性引物从小鼠睾丸cDNA文库中进行PCR扩增 ,分离出小鼠睾丸生精细胞凋亡相关基因mTSARG3(GenBank登录号为AF4 192 92 )。该基因定位于小鼠 7号染色体 7E1 E2区带 ,全长为 11kb ,cDNA全长为 132 8bp ,包含 8个外显子 ,编码由 316个氨基酸组成的、分子量为 36kD的蛋白质。该蛋白质含有DnaJ区和DnaJ- c区 ,与热激蛋白 4 0家族多种蛋白质有较高相似性 ,其中与小鼠DJB4 - MOUSE在 336aa的范围内有 4 6 %的相似性 ,属热激蛋白 4 0家族新成员。多组织RT PCR和Northern印迹结果显示 ,该基因在小鼠睾丸组织高表达 ,转录本大小约为 1.35kb ;Southern杂交结果显示 ,该基因在小鼠正常睾丸和隐睾组织无缺失和重排。实验结果证明成功克隆到了一个小鼠睾丸生精细胞凋亡相关基因mT SARG3。     

利用巢式PCR技术和人类基因组草图搜索法快速获得人类睾丸生精细胞凋亡相关基因TSARG2

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 44 5 42 )入手 ,设计了基因特异性引物和载体特异性引物进行巢式PCR扩增 ,结合人类基因组草图搜索法 ,从睾丸cDNA文库中快速分离出人类睾丸凋亡相关基因TSARG2的 5′末端而获得全长cDNA ,GenBank登录号为AY0 40 2 0 4(保密期为 1年 ) ,同时应用生物信息学的方法克隆了该基因在小鼠中的同源基因 ,GenBank登录号为AF395 0 83。TSARG2基因的cDNA全长为 12 33bp ,包含 6个外显子 ,基因组跨越 115kb ,编码由 30 5个氨基酸组成的、分子量为 34 75 1的蛋白质 ,与已知蛋白质无明显同源性。查询最新的人类基因组工作草图 ,该基因定位在染色体 4q33～ 34 .1。     

一个新的睾丸特异表达基因的克隆及功能的初步分析

运用“数据库消减杂交”(digital differential display)方法来筛选人类睾丸特异表达新基因,获得了有差异显示的代表新基因的克隆重叠群。挑选其中一个克隆重叠群HS．326528进行多组织RT—PCR,初步获得该重叠群在人睾丸中有高表达。从该重叠群的IMAGE出发,采用生物信息学的方法快速克隆了一个人类新基因的全长cDNA序列,其全长1044bp,开放阅读框为214～529bp,定位于15q26．2,编码由105个氨基酸组成、分子量为11．7kD、等电点为10．09的一个碱性蛋白,该蛋白与已知蛋白无明显的同源性,克隆实验证明该基因的阅读框完全正确,RT—PCR和Northern blot显示该基因在人类睾丸中特异表达,实时PCR结果表明：该基因在成人睾丸中高表达,在精子中有中度表达,在胚胎睾丸中低表达,推测该基因与精子的生成有关,命名为SRG8(homo sapiens spermatogenesis—related gene 8)(GenBank登录号：AY489187),该基因编码的蛋白定位于细胞核。流式结果分析表明,SRG8基因能够促使HeLa细胞由S期向G2期的转变,从而加速细胞的分裂。这些结果表明SRG8基因可能在睾丸的发育及精子的形成过程中起重要的作用。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段 (BE6 4 4 5 4 2 )入手 ,利用网上生物信息学克隆了该基因全长 ,GenBank登录号为AF395 0 83。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA ,SRG2基因的cDNA全长为 10 5 8bp ,编码由 2 95个氨基酸组成、分子量为 335 79、等电点为 9.6 4的蛋白质 ,与人类同源基因TSARG2相似性为 78% ,与已知蛋白质无明显同源性。RT PCR结果表明该基因只在睾丸中有高表达。     

人类生精相关基因TSARG4的cDNA克隆

为了探索精子生成的分子机制 ,从人精子外部致密纤维蛋白相关基因SPAG4(spermantigen 4)和小鼠精母细胞中表达的AK0 0 62 2 5基因出发 ,找到两个人类EST ,BG72 0 5 64和AI70 0 45 4,其中BG72 0 5 64在人睾丸中表达。运用“间隙填充法”填平这两个EST之间的间隙 ,从人睾丸文库中快速克隆了同源于SPAG4和AK0 0 62 2 5基因的人类TSARG4基因 (testisandspermatogenesisrelatedgene 4) (GenBank登录号为AF40 13 5 0 ) ,并用RT PCR对该基因阅读框进行验证。TSARG4基因全长 12 5 2bp ,开放阅读框为 94～ 12 3 3bp ,定位于 2 0q11.2 ,推定编码 3 79个氨基酸 ,预计分子量为 43 0 81.45 ,等电点为 8.61,该基因与小鼠精母细胞基因AK0 0 62 2 5编码的氨基酸序列同源性 74% ,与人类SPAG4基因编码的氨基酸序列同源性 45 %。RT PCR表明人类TSARG4基因在多个组织中均有表达 ,而同源的小鼠AK0 0 62 2 5基因仅在睾丸中表达     

睾丸生精细胞凋亡相关基因TSARG1与Mtsarg1的分子克隆及Mtsarg1基因的表达谱分析

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段（GenBank登录号：BE644538）出发,利用生物信息学和实验技术,克隆了小鼠睾丸生精细胞凋亡相关新基因Mtsarg1及相应的人类新基因TSARG1,Gen-Bank登录号分别为AF399971和AY032925。小鼠Mtsargl与人类TSARGl基因在氨基酸水平有55％的一致性和61％相似性,与其他已知蛋白质无明显同源性。小鼠10种组织的RT-PCR分析结果表明,Mtsargl基因在睾丸中高表达,在附睾中呈微弱表达,在其他组织不表达,提示Mtsargl和TSARGl基因在生精细胞凋亡或精子发生中具有潜在的重要作用。     

小鼠睾丸生精细胞凋亡相关基因SRG2的分子克隆及其在隐睾中的表达特征

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段(BE644542)入手,利用网上生物信息学克隆了SRG2基因全长,GenBank登录号为AF395083。从小鼠睾丸cDNA文库中分离出该基因完整阅读框cDNA,SRG2基因的cDNA全长为1088bp,为编码295个氨基酸、分子量为33579kD、等电点为9．64的蛋白质,与人类同源基因TSARG2相似性为78％,而与其他已知蛋白质无明显同源性。RT-PCR结果表明：该基因只在睾丸中有高表达。应用新型的分子信标检测该基因在不同时期隐睾中的mRNA表达水平,发现该基因呈明显上调,证明该基因在隐睾的发生发展中具重要作用。     

运用数字差异展示方法克隆一个人类新的锌指蛋白基因ZNF474

运用数字差异展示方法,克隆一个与生精相关的睾丸高表达基因。借助公共ESTs数据库,利用DDD软件比较分析各种睾丸文库与其他组织或细胞系文库有差异表达的ESTs,成功克隆到一个在人类睾丸中高表达的新基因。结合实验获得新基因cDNA全长,该基因被国际人类基因命名委员会命名为ZNF474(GeneBank登陆号AY461732)。ZNF474的cDNA全长为1 972 bp,定位在5 q23.2。通过RT-PCR及测序验证,其开放阅读框的位置在377 bp~1 471 bp处,编码364个氨基酸,在氨基酸水平与小鼠同源基因有66%的一致性,而与其他已知蛋白质无明显同源性。Northern杂交分析显示ZNF474在成体睾丸组织特异高表达,卵巢组织弱表达,在多种其他组织中不表达,为单一转录本。原位杂交显示ZNF474基因在正常成人睾丸组织各级生精细胞、隐睾组织以及精原细胞癌组织中均有较高表达。综上考虑,推测ZNF474作为生殖细胞中特异的转录因子,对人类的精子发生和卵母细胞的发育可能起重要作用。     

蒙古绵羊 tnp1基因cDNA全编码区的克隆及序列分析

过渡蛋白1基因(tnp1)是圆形精子细胞特异表达的基因.绵羊tnp1基因的DNA序列至今尚未报道.为了开展绵羊圆形精子细胞标记基因的研究,根据其他物种tnp1基因cDNA的保守序列设计引物,从成年蒙古绵羊睾丸中提取总RNA,采用RT-PCR和分子克隆方法,克隆了蒙古绵羊tnp1基因cDNA全编码区.该基因cDNA 长246 bp,包含一个168 bp的ORF,编码含有54个氨基酸的多肽链.DNA序列测定结果与牛的核苷酸序列比对,同源性为94.0%.绵羊tnp1基因的cDNA克隆和序列测定为进一步研究绵羊精子发生过程奠定了基础.     

利用电子差异展示方法克隆人类睾丸高表达新基因SPATA11

利用NCBI中的电子差异展示(digital differential display,DDD)软件,比较来自睾丸(包括睾丸癌)与来自其它组织的EST文库,从筛查人类睾丸中高表达而在其他组织中不表达或低表达的差异ESTs入手,成功克隆了一个在人类睾丸中高表达的新基因SPATA11．RT-PCR实验证实其在成人睾丸高表达．序列分析表明该基因含4个外显子,基因组跨越2．6kb,定位于19pl3．3．cDNA编码一个含221个氨基酸,相对分子质量为24．5kD的新蛋白．Northern杂交结果显示：该基因含有1．1kb大小的唯一转录本,主要在睾丸中强表达．肝脏、肺、卵巢和肾脏中有微弱表达．而其他组织中该基因无表达．     

Molecular Cloning and Preliminary Function Study of a Novel Human Gene, TSARG7, Related to Spermatogenesis

A novel human gene TSARG7 (GenBank accession No. AY513610) was identified from a human testis cDNA library by using the m TSARG7 gene (GenBank accession No. AY489184) as an electronic probe. The gene whose full cDNA length is 2 463 bp containing 12 exons and 11 introns is located in the human chromosome 8p11.21. The predicted protein encoded by this gene contains 456 amino acids with a theoretical molecular weight of 56 295 dalton and isoelectric point of 9.13. It is a new member of the acyltransferase family since its sequence possesses the highly conserved PlsC domain existing in all acyltransferase-like proteins. Two groups, the TSARG7 and mTSARG7, the TSARG7 and Au041707, share 97% identity in the 456 amino acids. Expression of the TSARG7 gene is restricted to the testis. Subcellular localization studies show that the EGFP-tagged TSARG7 protein was localized in the cytoplasm of GC-1 cells. The TSARG7 mRNA expression was initiated in the testis of a 13-year-old boy, and its level increased steadily along with spermatogenesis and sexual maturation of the human. The results of heat stress experiment demonstrate that TSARG7 expression has a relation with temperature. In conclusion, our study suggests that we have cloned a novel human gene and this gene may play an important role in human spermatogenesis and sexual maturation.     

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and.ll introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC- 1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.     

Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family.

Beginning with a mouse gene mTSARG3, which was related to apoptosis of spermatogenic cells, bioinformatics was applied and a predicted novel rat gene full-length cDNA sequence was attained. Gene-specific primers were designed for PCR in rat testis cDNA library. A new gene Tsarg1 (GenBank Accession No. AY380804) was cloned, which is related to apoptosis in rat spermatogenic cells. The gene whose full cDNA length is 1176 bp containing 8 exons and 7 introns is located in rat chromosome 1q32-1q33, which encoded a protein containing 316 amino acid residues and being a new member of HSP40 protein family since the sequence contains the highly conserved J domain, which is present in all DnaJ-like proteins and is supported to have a critical role in DnaJ-DnaK protein-protein interactions. The results of RT-PCR and Northern blot analysis showed that Tsarg1 was specifically expressed in rat testis, which probably inhibits rat testis spermatogenic cell apoptosis.     

Cloning, characterization and primary function study of a novel gene, Cymg1, related to family 2 cystatins

Cystatins are cysteine proteinase inhibitors. We found two expression sequence tags (ESTs), CA463109 and AV042522, from a mouse testis library using Digital differential display (DDD). By electrical hybridization, a novel gene, Cymg1 (GenBank accession No. AY600990), which has a full length of 0.78kb, and contains four exons and three introns, was cloned from a mouse testis cDNA library. The gene is located in the 2G3 area of chromosome 2. The full cDNA encompasses the entire open reading frame, encoding 141 amino acid residues. The protein has a cysteine protease inhibitor domain that is related to the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the CRES subfamily, which are related to the family 2 cystatins and are expressed specifically in the male reproductive tract. CYMG1 has a 44% (48/108) identity with mouse CRES and 30% (42/140) identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 is specifically expressed in adult mouse testes. Cell location studies showed that the GFP-tagged CYMG1 protein was localized in the cytoplasm of HeLa cells. Immunohistochemistry revealed that the CYMG1 protein was expressed in mouse testes spermatogonium, spermatocytes, round spermatids, elongating spermatids and spermatozoa. RT-PCR results also showed that Cymg1 was expressed in mouse testes and spermatogonium. The Cymg1 expression level varied in different developmental stages: it was low 1 week postpartum, steadily increased 2 to 5 weeks postpartum, and was highest 7 weeks postpartum. The expression level at 5 weeks postpartum was maintained during 13 to 57 weeks postpartum. The Cymg1 expression level in the testes over different developmental stages correlates with the mouse spermatogenesis and sexual maturation process. All these indicate that Cymg1 might play an important role in mouse spermatogenesis and sexual maturation.     

Cloning of a novel gene,<Emphasis Type="Italic">Cymg1</Emphasis>, related to family 2 cystatins and expressed at specific stages of mouse testis development

We have cloned a novel gene,Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library.Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that theCymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed thatCymg1 was expressed in testis and spermatogonial cells.Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate thatCymg1 may play important roles in mouse spermatogenesis and sex maturation