一个人类精子发生相关新基因TSARG7的克隆和初步功能研究

精子发生是一个多基因参与的复杂的生理过程,虽然人们克隆了一些与精子发生相关的基因,但迄今为止,人们对精子发生的分子机制了解有限,因而克隆相关的新基因,并研究其在理论上和实践上的功能仍然十分重要。该文从人类睾丸cDNA文库出发,以小鼠精子发生相关基因mTSARG7基因为电子探针,得到1个与人类的精子发生相关的新基因TSARG7（GenBank登录号为AY513610）,该基因的cDNA全长为2463bp,含有12个外显子和11个内含子,定位在人类8号染色体8p11.21上。TSARG7编码的蛋白质含有456个氨基酸,分子量为56．295kDa,等电点为9．13,为胞浆中非分泌性蛋白,具有磷酸酰基转移酶（Hsc）的结构域,属于酰基转移酶家族的新成员,该家族成员具有脂质合成的功能。TSARG7和mTSARG7,TSARG7和Au041707分别具有97％的同源性。该基因在睾丸中特异表达,亚细胞定位在胞浆中表达。TSARG7 mRNA在13岁的睾丸中开始表达,并且随着精子的发生和性成熟而稳定增加,热应急实验显示该基因的表达与温度相关。综上所述,该文克隆了人的1个新基因,该基因与精子的发生和性成熟相关。     

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.     

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。     

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     

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。     

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.     

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 and bioinformatics analysis of a novel spliced variant of survivin from human breast cancer cells.

Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Delta Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.     

恒河猴Fas基因的克隆

目的研究猴艾滋病发病机制中细胞凋亡的作用,克隆凋亡相关基因Ｆａｓ的ｃＤＮＡ序列。方法从恒河猴腹股沟淋巴结中提取总ＲＮＡ,根据人的Ｆａｓ ｃＤＮＡ序列设计上、下游引物,通过ＲＴ－ＰＣＲ扩增出目的ｃＤＮＡ片段,将这一片段克隆到ｐＧＦＭ－Ｔ Ｅａｓｙ载体中,筛选阳性克隆并进行序列测定。结果首次克隆了恒河猴Ｆａｓ基因,编码序列为１００５ ｂｐ,将其序列提交ＧｅｎＢａｎｋ,收录号为ＡＹ００７５７２。结论克隆的新序列与ＧｅｎＢａｎｋ已收录的人和食蟹猴的Ｆａｓ基因在编码序列的长度上稍有区别,人的编码序列为１００８ｂｐ,食蟹猴的为９９６ ｂｐ。     

小鼠植入前胚胎紧密化相关基因Crg1的克隆

从已获得的运用抑制消减杂交技术(Suppression Subtractive Hybridization,SSH)分离、克隆和筛选代表8-细胞早期胚胎和紧密化8-细胞胚胎差别表达基因的ESTs片段(GenBank登录号：BQ740263、BQ740251)入手,经比较二者的同源性发现这两个EST末端反向互补,拼接成一个cDNA片段,经分析此序列包含一个完整的阅读框,提交给GenBank,登录号为AY134859。根据此序列设计引物从小鼠8-细胞紧密化胚胎cDNA中经PCR扩增出目的片段,克隆入pUCm—T载体后测序而获得全长cDNA,为小鼠植入前胚胎紧密化相关基因Crg1,分析比较证明Crg1基因与AY134859基本吻合。Crg1基因的cDNA全长为810bp,只有一个外显子,编码由150个氨基酸组成,分子量理论值为17．67kD的蛋白质。与最新的小鼠基因组工作草图进行电子杂交,该基因被定位在小鼠的14号染色体上。RT—PCR实验证明在小鼠植入前各个时期的胚胎、小鼠胚胎干细胞中均有表达,在小鼠胚胎成纤维细胞中没有表达。半定量RT—PCR实验证明Crg1基因在紧密化胚胎中表达较8—细胞胚胎高。采用Northern—blot手段分析Crg1基因在成年小鼠的8种组织中的表达情况,结果表明该基因只在小鼠卵巢中有微弱的表达,转录本大小为1．2kh,而在成年小鼠的脑、心脏、肾、睾丸、肝脏、肺、脾等中没有表达。研究表明,Crg1基因可能与小鼠胚胎紧密化及保持细胞的全能性相关。     

Cloning, structural organization and chromosomal mapping of rat costimulatory molecule 4-1BBL

4-1BBL （TNFSF9） is a member of the tumor necrosis factor （TNF） ligand superfamily, which is expressed on some activated antigen presenting cells and B cells. We isolated a rat cDNA clone encoding the rat homologue of the human 4-1BBL （GenBank accession No. AY259541）. The deduced rat 4-1BBL protein, consisting of 308 amino acids with a molecular weight of 33,469 Da, was a typical type II transmembrane glycoprotein, the same as human and murine 4-1BBL. “SDAA” in the cytoplasmic domain of rat 4-1BBL was deduced to act as the phosphorylation site for casein kinase I （“SXXS” motif）, which is present in the cytoplasmic domains of human and murine 4-1BBL, and all other TNF ligand family members known to utilize reverse signaling. The two introns of 4-1BBL were also cloned （GenBank accession No. AY332409）. Rat 4-1BBL is much more homologous with murine 4-1BBL than with human 4-1BBL, in both nucleotide and amino acid sequences. Rat 4-1BBL was expressed in all tested tissues： brain, lung, colon, liver, thymus, testicle, kidney, adrenal, stomach, spleen and heart. The chromosomal location of rat 4-1BBL was first identified by bioinformatics, then by fluorescence in situ hybridization at 9q11 （GenBank accession UniGene No. Rn.46783）. Rat, murine and human 4-1BBL genes are evolved from a common gene. The identification and characterization of the rat counterpart of human 4-1BBL will facilitate studies of the biological function of this molecule.     

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。     

小鼠睾丸特异表达基因TSEG-1的克隆及序列分析

从表达序列标签(expressed sequence tags, ESTs)数据库ZooDDD中获得小鼠正常睾丸表达的EST, 通过dbEST数据库检索出与其高度同源的EST序列, 构建EST叠加群(contigs), Biolign软件拼接, GeneScan软件预测contigs对应的基因组序列中的外显子、内含子; 针对开放阅读框设计引物序列, 采用RT-PCR从小鼠睾丸组织中克隆新基因的cDNA, 分析该基因在小鼠各脏器中的mRNA表达, 并对测序结果进行生物信息学分析。结果表明: 在小鼠X染色体的1 668~2 011 kb间克隆出一新基因TSEG-1, 全长为510 bp, 开放阅读框为336 bp, 编码111氨基酸, 分子量12.84258 kDa, 等电点11.4000。RT-PCR证实该基因开放阅读框正确, 在小鼠睾丸组织中特异性表达, 且与小鼠其他cDNA 无同源性, 获得GenBank 登录号EU079024。功能区分析发现TSEG-1蛋白可能为一种跨膜蛋白, 跨膜区位于第41~61氨基酸残基。TSEG-1基因与人类睾丸特异性组蛋白2a变异体基因有较高同源性, 在TSEG-1基因5′-端非编码侧翼预测发现存在1个启动子区域, 范围为680 bp。 TSEG-1蛋白可能有4个抗原性位点, 2个特异性蛋白激酶的磷酸化位点, 其亚细胞定位可能位于线粒体。小鼠睾丸特异性基因TSEG-1的克隆为进一步研究其生物学功能和表达调控奠定了基础。     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

Leucine-rich repeat C4 protein is involved in nervous tissue development and neurite outgrowth, and induction of glioma cell differentiation

LRRC4, leucine-rich repeat C4 protein, has been identified in human （GenBank accession No. AF196976）, mouse （GenBank accession No. DQ177325）, rat （GenBank accession No. DQ119102） and bovine （GenBank accession No. DQ 164537） with identical domains. In terms of their similarity, the genes encoding LRRC4 in these four mammalian species are orthogs and therefore correspond to the same gene entity. Based on previous research, and using in situ hybridization, we found that LRRC4 had the strongest expression in hippocampal CA1 and CA2, the granule cells of the dentate gyrus region, the mediodoral thalamic nucleus, and cerebella Purkinje cell layers. Using a P19 cell model, we also found that LRRC4 participates in the differentiation of neuron and glia cells. In addition, extracellular proteins containing both an LRR cassette and immunoglobulin domains have been shown to participate in axon guidance. Our data from neurite outgrowth assays indicated that LRRC4 promoted neurite extension of hippocampal neurons, and induced differentiation of glioblastoma U251 cells into astrocyte-like cells, confirmed by morphology observation and glial fibrillary acidic protein expression.     

毛竹抗逆锌指蛋白基因cDNA克隆与序列分析

根据水稻抗逆相关锌指蛋白基因的保守区序列设计引物,以毛竹基因组DNA和cDNA为模板,采用PCR方法,成功扩增出1个含有完整阅读框架的cDNA序列,长度为495bp,序列无内含子,共编码164个氨基酸,将其命名为PeZFP基因(GenBank登录号:FJ472953)。氨基酸序列(GenBank登录号:ACL01101)的分析结果表明,PeZFP与其他锌指结构蛋白有较高的同源性,同水稻锌指结构抗逆蛋白OSIAP1序列相似性高达87.7%,且其序列C端具有典型的AN1类型的锌指结构Cx2-4Cx9-12Cx2Cx4Cx2Hx5HxC,在N端具有典型的A20类型锌指蛋白结构,推测此PeZFP为植物抗逆OsIAP1类似基因,在功能上与毛竹抗逆性相关。