cDNA代表性差异分析法分离鼻咽癌上皮细胞株HNE1表达差异cDNA序列的初步研究

目的寻找鼻咽癌中差异性表达基因，包括与鼻咽癌发病相关的候选抑瘤基因。方法应用ｃＤＮＡ代表性差异分析法（ＲＤＡ），分离原代培养的正常人鼻咽上皮细胞与鼻咽癌细胞株ＨＮＥ１中差异表达的ｃＤＮＡ序列，Ｓｏｕｔｈｅｒｎ杂交和Ｎｏｒｔｈｅｒｎ杂交被用来分析差异性表达产物的来源，最后，将这些序列克隆到ｐＧＥＭ－Ｔｅａｓｙ载体中，并用链终止法测序。结果在第４轮杂交及扩增反应后，获得４条差异性条带。Ｓｏｕｔｈｅｒｎ杂交及Ｎｏｒｔｈｅｒｎ杂交证明，这些差异性片段来自作为“检测”扩增子的正常人鼻咽上皮，在鼻咽癌细胞株ＨＮＥ１中不表达或表达降低。序列分析这些差异性片段的克隆，发现一些序列是与已知基因高度同源的基因，包括一些看家基因；另有一些基因则为新基因序列。结论鼻咽癌的发生是一个多基因参与的过程，所获得的差异性片段中，与之同源的一些已知基因具有抑瘤功能。     

从EST克隆中筛选肝癌内高表达的基因片段

应用Ｎｏｒｔｈｅｒｎ杂交技术，从表达序列标记（Ｅｘｐｒｅｓｓｅｄｓｅｑｕｅｎｃｅｔａｇ，ＥＳＴ）克隆中筛选出一个在肝癌组织内高表达，而在相应癌旁肝及正常肝组织内低表达或不表达的基因片段。ＤＮＡ序列测定表明，该基因片段为一未知新基因的部分序列。此基因片段可作为探针，进一步筛选ｃＤＮＡ文库，以得到新的癌基因的候选基因。     

人类睾丸生精细胞凋亡相关基因TSARG3的克隆

目的：克隆人类睾丸生精细胞凋亡相关基因TSARG3。方法：从已获得的小鼠稳睾和正常睾丸对照中表达量有明显差异的表达序列标签片段（BE644537）入手，构建人同源表达序列标签重叠群，应用基因特异性引物和载体特异性引物，在睾丸cDNA文库的DN或进行巢式PCR扩增、测序，对测序结果进行生物信息学分析。结果：从睾丸cDNA文库中分离出人类睾丸凋亡相关基因的5’末端而获得全长cDNA,命名为TSARG3，GenBank登录号为AF419291（保密期为1年），同时应用相同方法克隆了该基因在小鼠中的同源基因，GenBank登录号为AF419292。结论：获得人类睾丸生精细胞凋亡相关基因TSARG3，该基因可能与人类睾丸生精细胞凋亡有关。     

大鼠Fas配体基因的克隆

目的 研究Ｆａｓ配体（Ｆａｓ ｌｉｇａｎｄ,ＦａｓＬ）在移植免疫方面的作用,克隆其编码的全部ｃＤＮＡ序列,并添加Ｋｏｚａｋ序列,以便在直核细胞高效表达大鼠ＦａｓＬ。方法 从大鼠的睾丸组织中提取总ＲＮＡ。下游引物以ＲＴ－ＰＣＲ的方法扩增出－９３１ｂｐ的ＤＮＡ征段,将这一片段重组于ｐＢＲ－ＲＳＶ载体中,酶切鉴定插人片段正确后进行全列分析。结果 证实该研究所克隆的ｃＤＮＡ是编码正确的大鼠ＦａｓＬ基因,与     

mRNA差异显示法筛选小鼠精子发生相关基因

目的筛选小鼠精子发生相关基因。方法应用改良的mRNA差异显示技术对1、2、3、4周龄小鼠睾丸进行了基因表达的差异显示分析,并对差异表达的cDNA片段进行了克隆和测序。结果获得了27个在不同周龄小鼠睾丸组织存在明显表达差异的基因片段,其中3个片段分别与Genbank中已知基因LTBP-3、rjs、P38 MAPK基因高度同源,其余基因片段为无同源序列的新基因片段。结论上述结果表明精子发生是一个多基因参与的过程,筛选出的这些基因可能在精子发生的不同阶段调控着精子发生的全过程。     

热休克蛋白90α的cDNA克隆

本文从构建ｃＤＮＡ有限文库中分离出含有全部编码区＋４／＋２８３０ｂｐ的Ｈｓｐ９０αｃＤＮＡ，经ＤＮａｓｅＩ有限酶解片段的定向亚克隆和序列测定，获得了含有人Ｈｓｐ９０α的ｃＤＮＡ全序列克隆。     

胃癌基因表达的免疫组化研究

应用免疫组化方法对４２例胃癌和相位癌旁组织以及１１例良性胃病组织的ｐ５３、ｃ－ｍｙｃ和ｒａｓ基因编码产物进行检测。结果发现；３种基因在胃癌中的阳性率分别是３３．３％、２３．８％和４２．９％；在相应癌旁组织的阳性率依次分别是１６．７％、１１．９％和１６．７％；ｐ５３和ｃ－ｍｙｃ在在良性胃病组织中无表达，ｒａｓ基因在良性胃病组织中阳性率２．４％。     

从rds小鼠视网膜克隆视网膜色素变性相关差异片段

目的 从遗传性视网膜色素变性动物模型rds小鼠中克隆视网膜色素变性发病过程中特异表达的基因。方法 应用差异显示技术,分析rds小鼠发病过程中视网膜的mRNA。对特异性表达的mRNA片段进行克隆测序。结果 在视网膜色素变性发病过程中,rds小鼠的视网膜存在着相当明显的基因表达差异。在所测序分析的5个差异显示片段中其中一个与GenBank刚登录的功能未知的、从成年男性睾丸组织中克隆出来的cDNA序列较高同源（同一率为86％）另外4个为低同源序列。25天rds小鼠高表达的一个差异片段,与37天正常鼠表达而rds小鼠不表达的另一个片段,长度相同,177个碱基只相差两个。结论 视网膜色素变性这类慢性病变,存在着多个基因的表达及调控。     

重组人截短型胰岛素样生长因子1在大肠杆菌中的高效表达

目的：建立人截短型胰岛素生长因子１的原核高效表达系统。方法：利用基因重组技术将人人截短胰岛素样生长因子１「Ｄｅｓ（１－３）ＩＧＦ１」的ｃＤＮＡ片段克隆到融合蛋白表达载体ｐＭＴＹ４中,用离子交换层析法纯化蛋白并经ＳＤＳ聚丙烯酰胺凝胶电泳,放射免疫检测,Ｎ－末端前１６位氨基酸序列测定及生物活性检测等方法对所获蛋白进行了鉴定。结果;获得含人Ｄｅｓ（１－３）ＩＧＦ１基因的重组质粒,在大肠杆菌中高效表达出含     

胃癌高表达cDNA片段W41的克隆及组织表达谱分析

目的：从人胃癌组织中克隆新的相关易感基因。方法：利用cDNA末端快速扩增PCR技术得到了扩增片段，将其克隆、核苷酸序列分析，并将序列在GenBank进行同源性比较。利用Northern杂交、多组织Northern及基因表达系列分析了所得基因片段的组织表达谱。结果：获得1条533bp带有poly(A)尾的cDNA片段，可见加尾信号AATAAA，与GenBank基因数据库同源比较，该序列未见与任何已知基因同源，登录GenBank(登录号为AF325202）。该序列在胃癌组织的表达强度高于对应正常组织，多组织Northern及基因表达系列分析表明该序列在多种肿瘤组织中高表达，在正常组织中表达减弱。结论：得到了1条与胃癌可能相关的新的cDNA序列。     

mRNA差异显示法克隆小鼠精子发生相关基因p38MAPK基因

目的：克隆1、2、3、4周龄小鼠睾丸之间差异表达的基因。方法：应用改良的mRNA差异显示技术,对1、2、3、4周龄小鼠睾丸进行基因表达的差异显示分析,并对其中1个从2周龄小鼠中高表达的cDNA片段进行了克隆和测序。结果：所克隆的cDNA片段与小鼠大脑、造血干细胞p38MAPK（p38beta mitogen-activated protein knase,p38MAPK-β）基因的同源性分别为91％和85％.Northern dot blot结果显示该基因在小鼠睾丸和脑组织 中表达最高。结论：小鼠p38MAPK基因基因可能与小鼠精子发生相关。     

The human autosomal gene DAZLA: testis specificity and a candidate for male infertility

The DAZ (Deleted in AZoospermia) and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. We report the isolation of the human DAZLA gene by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologues. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility.      

Susceptibility locus for non-obstructive azoospermia is localized within the HLA-DR/DQ subregion: primary role of DQB1*0604

Non-obstructive azoospermia is a male infertility characterized by no or little sperm in semen as a result of a congenital dysfunction in spermatogenesis. Previous studies have reported a higher prevalence of particular human leukocyte antigen (HLA) antigens in non-obstructive azoospermia. As the expression of the RING3 gene located in the HLA class II region was predominant in the testis, mainly around spermatids and pachytene spermatocytes, it is tempting to speculate that RING3 is one of the strong candidate genes responsible for the pathogenesis of the disease. In this study, the genetic polymorphism in the RING3 gene was investigated by the direct sequencing technique. As a result, a total of 14 single nucleotide polymorphisms were identified. Among them, six were localized in the coding region but none of them was accompanied by an amino-acid substitution. No significant difference in the allelic distribution at these 14 polymorphic sites was observed between the patients and healthy controls, suggesting that the susceptible gene for non-obstructive azoospermia is not the RING3 gene. Then, in order to map the susceptibility locus for non-obstructive azoospermia precisely within the HLA region, 11 polymorphic microsatellite markers distributed from the SACM2L gene just outside the HLA class II region (187 kb telomeric of the DPB1 gene) to the OTF3 gene in the HLA class I region were subjected to association analysis in the patients. Statistical analysis of distribution in the allelic frequency at each microsatellite locus demonstrated that the pathogenic gene for non-obstructive azoospermia is located within the HLA-DR/DQ subregion. In fact, DRB1*1302 and DQB1*0604 were found to be strongly associated with non-obstructive azoospermia by polymerase chain reaction-based DNA typing. Further, haplotype analysis suggested that the DQB1*0604 allele may play a decisive role in the pathogenesis of non-obstructive azoospermia.     

Spermatogenesis-specific association of SMCY and MSH5

The status of chromatin during spermatogenesis is dynamically regulated by specific histone codes or stage-specific histone changes. The functional links between such epigenetic regulation and proteins regulating meiosis are largely unknown. In mammals, genes encoded on the Y chromosome are thought to possess male-specific biological functions. While genes located within the azoospermia factor region (AZF) are known to be involved in spermatogenesis, the physiological function of individual genes is not known. SMCY is a gene mapped to the AZF, and in this report, we analyzed the function of SMCY protein during spermatogenesis. Biochemical identification of the proteins with which it interacted showed that SMCY formed a distinct complex with MSH5, a critical meiosis-regulatory protein in the human testicular germ cell line, NEC8. As anticipated, histone H3K4 demethylase activity was detected. Immunohistochemical analysis revealed the co-localization of SMCY with MSH5 at a specific stage of meiotic prophase progression during murine spermatogenesis. Our results suggest that SMCY may have a male-specific function as a histone H3K4 demethylase by recruiting a meiosis-regulatory protein to condensed DNA.     

Characterization of the genomic organization, localization and expression of four PRY genes (PRY1, PRY2, PRY3 and PRY4).

PRY (PTP-BL related on the Y chromosome) has been proposed as a candidate spermatogenesis gene. We report the characterization of the genomic structure, the number of copies on the Y chromosome and the expression of the gene. By comparison of the cDNA sequence with the genomic sequence, five exons were identified. Analysis of GenBank-derived clones on the Y chromosome revealed the presence of two full-length copies in azoospermia factor region b (AZFb) (PRY1 and PRY2) and two shorter versions of the PRY gene containing exons 3, 4 and 5 in AZFc (PRY3 and PRY4). A clone containing sequences homologous to exons 3, 4 and 5 is located in area 5L (between AZFa and AZFb), a clone containing a sequence homologous to exon 5 is located in area 5M (in AZFb) and a clone containing a fragment homologous to exon 3 is located in 6F. A repeat structure of exons 1 and 2 is present on the short arm of the Y chromosome as well as on the long arm. PRY1 and PRY2, two gene copies that are located in AZFb, a region often deleted in patients with severe male infertility, were shown to be expressed in the testis. PRY may therefore play an important role in spermatogenesis.     

Presence of the AZF region in a female with an idic(Y)(q11)

In a child with some features of Turner's syndrome, gonosomal mosaicism with an isodicentric nonfluorescent (idic)Y chromosome was detected (mos 45,X/47,X,idic(Y)(q11),idic(Y)(11)/46,X,idic(Y)(q11)). Histopathological examination showed streak gonads with some evidence of ovarian stroma and no sign of gonadoblastoma. Polymerase chain reaction (PCR) analysis in blood lymphocytes and gonadal tissues using primers of seven loci along the Y chromosome, including the sex determined region (SRY), azoospermia factor region (AZF) and the deleted in azoospermia ( DAZ ) gene was positive for all loci tested, confirming the isodicentric character of the Y chromosome and indicating the presence of the AZF region. It is remarkable that the existence of spermatogenesis controlling genes does not play an important role in gonadal development and differentiation in a phenotypic female with some Turner stigmata. The data presented here are briefly discussed with previously-described patients.     

Deletion of RBM and DAZ in azoospermia: evaluation by PRINS.

Molecular and cytogenetic studies from infertile men have shown that one or more genes controlling spermatogenesis are located in proximal Yq11.2 in interval 6 of the Y chromosome. Microdeletions within the azoospermia factor region (AZF) are often associated with azoospermia and severe oligospermia in men with idiopathic infertility. We evaluated cells from a normal-appearing 27-year-old man with infertility and initial karyotype of 45,der(X)t(X;Y)(p22.3;p11.2)[8]/46,t(X;Y)(p22.3;p11.2)[12]. By fluorescence in situ hybridization with dual-color whole chromosome paint probes for X and Y chromosomes, we confirmed the Xp-Yp interchange. By primed in situ labeling, we identified translocation of the SRY gene from its original location on Yp to the patient's X chromosome at band Xp22. We also obtained evidence that the apparent marker was a der(Y) (possibly a ring) containing X and Y domains, and observed that the patient's genome was deleted for RBM and DAZ, two candidate genes for AZF.     

Deletion of RBM and DAZ in azoospermia: Evaluation by PRINS

Molecular and cytogenetic studies from infertile men have shown that one or more genes controlling spermatogenesis are located in proximal Yq11.2 in interval 6 of the Y chromosome. Microdeletions within the azoospermia factor region (AZF) are often associated with azoospermia and severe oligospermia in men with idiopathic infertility. We evaluated cells from a normal‐appearing 27‐year‐old man with infertility and initial karyotype of 45,der(X)t(X;Y)(p22.3;p11.2)[8]/46,t(X;Y)(p22.3;p11.2)[12]. By fluorescence in situ hybridization with dual‐color whole chromosome paint probes for X and Y chromosomes, we confirmed the Xp‐Yp interchange. By primed in situ labeling, we identified translocation of the SRY gene from its original location on Yp to the patient's X chromosome at band Xp22. We also obtained evidence that the apparent marker was a der(Y) (possibly a ring) containing X and Y domains, and observed that the patient's genome was deleted for RBM and DAZ, two candidate genes for AZF. © 2001 Wiley‐Liss, Inc.