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。     

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 electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe 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 Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation. 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 electricalhybridization,a novel gene,Cymgl(GenBank accession No.AY600990),which has a full length of 0.78 kb,and contains four exons and three introns,was cloned from a mouse testis eDNA library.The gene is locatedin the 2G3 area of chromosome 2.The full eDNA encompasses the entire open reading frame,encoding 141amino acid residues.The protein has a cysteine protease inhibitor domain that is related to the family 2cystatins but lacks critical consensus sites important for cysteine protease inhibition.These characteristicsare seen in the CRES subfamily,which are related to the family 2 cystatins and are expressed specifically inthe 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 Cymgl is specifically expressed inadult mouse testes.Cell location studies showed that the GFP-tagged CYMG 1 protein was localized in thecytoplasm of HeLa cells,lmmunohistochemistry revealed that the CYMG1 protein was expressed in mousetestes spermatogonium,spermatocytes,round spermatids,elongating spermatids and spermatozoa.RT-PCRresults also showed that Cymgl was expressed in mouse testes and spermatogonium.The Cymgl expressionlevel varied in different developmental stages:it was low 1 week postpartum,steadily increased 2 to 5 weekspostpartum,and was highest 7 weeks postpartum.The expression level at 5 weeks postpartum was main-tained during 13 to 57 weeks postpartum.The Cymgl expression level in the testes over different develop-mental stages correlates with the mouse spermatogenesis and sexual maturation process.All these indicatethat Cymgl might play an important role in mouse spermatogenesis and sexual maturation.     

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.     

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.     

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。     

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．     

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.     

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.     

NYD-SP16, a novel gene associated with spermatogenesis of human testis

By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, a novel human testis gene NYD-SP16 was identified. NYD-SP16 expression was 6.44-fold higher in adult testis than in fetal testis. NYD-SP16 contains 1595 base pairs (bp) and a 762-bp open reading frame encoding a 254-amino acid protein with 73% amino acid sequence identity with the mouse testis homologous protein. The NYD-SP16 gene was localized to human chromosome 5q14. The deduced structure of the NYD-SP16 protein contains one transmembrane domain, which was confirmed by GFP/NYD-SP16 fusion protein expression in the cytomembrane of the transfected human choriocarcinoma JAR cells, suggesting that it is a transmembrane protein. Multiple tissue distribution indicated that NYD-SP16 mRNA is highly expressed in the testes and pancreas, with little or no expression elsewhere. Further analysis of abnormal expression in infertile male patients revealed complete absence of NYD-SP16 in the testes of patients with Sertoli-cell-only syndrome and variable expression in patients with spermatogenic arrest. Homologous gene expression in mouse testis was confirmed in spermatogenic cells by in situ hybridization. The results of cDNA microarray, in situ hybridization, and semiquantitative polymerase chain reaction in mouse testis of different stages indicated that NYD-SP16 expression is developmentally regulated. These results suggest that the putative NYD-SP16 protein may play an important role in testicular development/spermatogenesis and may be an important factor in male infertility.     

Overexpression a novel zebra fish spermatogenesis-associated gene 17 (<Emphasis Type="Italic">SPATA17</Emphasis>) induces apoptosis in GC-1 cells

The spermatogenesis-associated 17 gene (SPATA17, previously named MSRG-11) was reported to be a candidate spermatocyte apoptosis-related gene which may play a critical role in human spermatogenesis, especially in meiosis. Analysis of SPATA17 expression and regulation in zebra fish may provide insight into the understanding of the complicated process of gonadogenesis and its potential function in spermatocyte cell apoptosis. In this study, we cloned and characterized the SPATA17 gene from zebra fish which consists of nine exons separated by eight introns. The consensus open reading frame (1258 bp) encodes a polypeptide of 357 amino acids which shares 44% identity with the human SPATA17 gene. Bioinformatic analysis reveals that SPATA17 protein contains three short calmodulin-binding motifs (IQ motif) and is considered to play a critical role in interactions with CaM proteins. Multi-tissue RT-PCR and Northern blot results demonstrated that the zebra fish SPATA17 gene was expressed strongly in testis and a slight amount of expression in ovary. Flow cytometry analysis and genomic DNA ladders result showed that the expression of SPATA17 protein in the GC-1 cell line could accelerate cell apoptosis. Analysis of the SPATA17 sequence and its spatial expression pattern indicate that this gene is highly conserved and may play an important role in the process of zebra fish gonadogenesis.