目的克隆一个新的人睾丸特异性基因。方法运用“数据库消减杂交”(Digital Differential Display,DDD)方法筛选人类睾丸特异表达新基囚,获得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替群Hs．180197进行多组织RT-PCR验证...目的克隆一个新的人睾丸特异性基因。方法运用“数据库消减杂交”(Digital Differential Display,DDD)方法筛选人类睾丸特异表达新基囚,获得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替群Hs．180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重叠群的IMAGE克隆出发,采用生物信息学的方法克隆一个人类新基因的全长cDNA序列。结果新基因全长1197bp,开放阅读框为504～806bp,定位于6p21．1-p21．2,编码由100个氨基酸组成,分子量为10KD,等电点为6．81的一个蛋白,该蛋白与已知蛋白无同源性。克隆实验验证该基因阅读框完全正确,推测其可能与精子生成相关,暂命名为TDRGI(Testis Development Related Gene 1),GenBank登录号为DQ168992。结论“数据库消减杂交”与实验验证相结合用于发现更多人类功能新基因是行之有效的。展开更多
目的克隆一个新的人睾丸特异性基因。方法运用电子差异展示方法筛选人类睾丸特异表达新基因,获得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.180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重...目的克隆一个新的人睾丸特异性基因。方法运用电子差异展示方法筛选人类睾丸特异表达新基因,获得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.180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重叠群的IMAGE克隆出发,采用生物信息学方法克隆一个人类新基因的全长cDNA序列。结果新基因全长1197bp,开放阅读框为504～806bp,定位于6p21.1-p21.2,编码由100个氨基酸组成,相对分子质量为10000,等电点为6.81的一个蛋白,该蛋白与已知蛋白无同源性。克隆实验验证该基因阅读框完全正确,推测其可能与精子生成相关,暂命名为TDRG1(testis development related gene1),GenBank登录号为DQ168992。结论电子差异展示方法与实验验证相结合用于发现人类功能新基因是行之有效的。展开更多
Background Evidence for the importance of genetic factors in male infertility is accumulating This study was designed to identify a novel testis specific gene related to spermatogenesis by a new strategy of digital di...Background Evidence for the importance of genetic factors in male infertility is accumulating This study was designed to identify a novel testis specific gene related to spermatogenesis by a new strategy of digital differential display (DDD) Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene Multi tissue RT PCR was performed to analyse its tissue specific expression The full length cDNA of the new gene was obtained using the BLAST program Sequencing was performed and the result was analysed Semi quantitative RT PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene The sequence of the opening reading frame was integrated into the pQE 30 vector expressed in Escherichia coil strain M15(pREP4) With IPTG induction, the target protein was detected Results A full length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified SPATA12 was 2430 bp in length, located in chromosome 3p21 1 3p21 2 The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT PCR and sequencing The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20 417 8 and isoelectric point of 5 23 The sequence has no significant homology with any known protein in databases Semi quantitative RT PCR and Northern blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis The expression recombinant of SPATA12 was constructed and a high level of the histidine tagged fusion protein was obtained Conclusions DDD can be confirmed by SPATA12 as a novel computational biology based approach for identification of the testis specific expression genes SPATA12 may function as a testicular germ cell associated gene that 展开更多
文摘目的克隆一个新的人睾丸特异性基因。方法运用“数据库消减杂交”(Digital Differential Display,DDD)方法筛选人类睾丸特异表达新基囚,获得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替群Hs．180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重叠群的IMAGE克隆出发,采用生物信息学的方法克隆一个人类新基因的全长cDNA序列。结果新基因全长1197bp,开放阅读框为504～806bp,定位于6p21．1-p21．2,编码由100个氨基酸组成,分子量为10KD,等电点为6．81的一个蛋白,该蛋白与已知蛋白无同源性。克隆实验验证该基因阅读框完全正确,推测其可能与精子生成相关,暂命名为TDRGI(Testis Development Related Gene 1),GenBank登录号为DQ168992。结论“数据库消减杂交”与实验验证相结合用于发现更多人类功能新基因是行之有效的。
文摘目的克隆一个新的人睾丸特异性基因。方法运用电子差异展示方法筛选人类睾丸特异表达新基因,获得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.180197进行多组织RT-PCR验证该重叠群在人睾丸中的表达。然后从包含该重叠群的IMAGE克隆出发,采用生物信息学方法克隆一个人类新基因的全长cDNA序列。结果新基因全长1197bp,开放阅读框为504～806bp,定位于6p21.1-p21.2,编码由100个氨基酸组成,相对分子质量为10000,等电点为6.81的一个蛋白,该蛋白与已知蛋白无同源性。克隆实验验证该基因阅读框完全正确,推测其可能与精子生成相关,暂命名为TDRG1(testis development related gene1),GenBank登录号为DQ168992。结论电子差异展示方法与实验验证相结合用于发现人类功能新基因是行之有效的。
文摘Background Evidence for the importance of genetic factors in male infertility is accumulating This study was designed to identify a novel testis specific gene related to spermatogenesis by a new strategy of digital differential display (DDD) Methods Based on the generation of expressed sequenced tags (ESTs), comparing the testis libraries with other tissue or cell line libraries by the DDD program, we identified a new contig of the ESTs which were derived from testis libraries and represented a novel gene Multi tissue RT PCR was performed to analyse its tissue specific expression The full length cDNA of the new gene was obtained using the BLAST program Sequencing was performed and the result was analysed Semi quantitative RT PCR and Northern blot analyseis of mRNA from differential normal tissues were performed to clarify the expression pattern of the new gene The sequence of the opening reading frame was integrated into the pQE 30 vector expressed in Escherichia coil strain M15(pREP4) With IPTG induction, the target protein was detected Results A full length cDNA sequence of the new gene named SPATA12 (GeneBank accession number AY221117) in human testis was identified SPATA12 was 2430 bp in length, located in chromosome 3p21 1 3p21 2 The sequence of the opening reading frame was 676-1248 bp, as was confirmed by RT PCR and sequencing The cDNA encodes a novel protein of 190 amino acids with a theoretical molecular weight of 20 417 8 and isoelectric point of 5 23 The sequence has no significant homology with any known protein in databases Semi quantitative RT PCR and Northern blot analyses of multiple tissues showed that SPATA12 was expressed significantly in normal human testis The expression recombinant of SPATA12 was constructed and a high level of the histidine tagged fusion protein was obtained Conclusions DDD can be confirmed by SPATA12 as a novel computational biology based approach for identification of the testis specific expression genes SPATA12 may function as a testicular germ cell associated gene that
