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1.
体外(in vitro)生化研究已证明哺乳动物Tra2蛋白是前体mRNA剪接的重要调控因子,但是,对该蛋白在in vivo条件下的剪接功能,尤其是在神经特异性基因剪接中的功能及其细胞特异性,目前所知甚少。本文采用in vivo分析模型,在COS-l和PFSK两种不同类型的细胞中,研究了两个神经特异性基因(GluR-B,SMN2)剪接的细胞特异性,同时分析了Tra2β1在这两个基因剪接中的功能及其细胞特异性。结果表明,在研究的两种细胞中,GluR-B和SMN2“小基因”的剪接均具有明显的细胞特异性;而Tra2β1蛋白的过量表达在这两种不同的细胞中对“小基因”的剪接有显著的相同倾向的调节作用,提示Tra2β1蛋白对该两个基因剪接的调节作用可能没有细胞特异性。  相似文献   

2.
体外(in vitro)生化研究已证明哺乳动物Tra2蛋白是前体mRNA剪接的重要调控因子,但是,对该蛋白在in vivo条件下的剪接功能,尤其是在神经特异性基因剪接中的功能及其细胞特异性,目前所知甚少。本文采用in vivo分析模型,在COS-1和PFSK两种不同类型的细胞中,研究了两个神经特异性基因(GluR-B,SMN2)剪接的细胞特异性,同时分析了Tra2β1在这两个基因剪接中的功能及其细胞特异性。结果表明,在研究的两种细胞中,GluR-B和SMN2“小基因”的剪接均具有明显的细胞特异性;而Tra2β1蛋白的过量表达在这两种不同的细胞中对“小基因”的剪接有显著的相同倾向的调节作用,提示Tra2β1蛋白对该两个基因剪接的调节作用可能没有细胞特异性。  相似文献   

3.
鉴定9个新的RHD基因mRNA可变剪接体   总被引:1,自引:0,他引:1  
许先国  吴俊杰  洪小珍  朱发明  严力行 《遗传》2006,28(10):1213-1218
为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。  相似文献   

4.
NPCEDRG基因是采用基因定位候选克隆策略获得的1个鼻咽癌候选抑瘤基因. NPCEDRG在鼻咽癌细胞和组织中表达下调,重新恢复NPCEDRG基因在CNE2细胞系的表达,可部分逆转CNE2 的恶性表型. 本研究对CNE2细胞所表达的NPCEDRG基因mRNA剪接变异体克隆、鉴定,发现NPCEDRG基因至少有7个转录起始位点,其中NM_032316的TSS位于ATG上游-85 nt处,AF538150和AK094248的TSS位于-25 nt处;AF538150不存在第2外显子中6核苷酸序列(5′-TTGCAG-3′)的缺失,其CDs为516 bp,编码1种由171个氨基酸组成的蛋白质(而非GenBank中公布的CDs为510 bp,1种由169个氨基酸组成的蛋白质). 本研究成功克隆得到1种新的NPCEDRG基因的mRNA剪接变异体V2,其TSS位于-23 nt处,其CDs为297 bp,编码1种由98个氨基酸组成的蛋白质.  相似文献   

5.
目的:传统Ras家族由Kras,Hras和Nras基因组成,这类基因的点突变经常在人类肿瘤中发现,突变热点位于12,13,61位密码子。ERas基因是2003年在鼠胚胎干(ES)细胞中发现的,其cDNA编码的蛋白与Kras,Hras和Nras分别有46%,43%和47%的相似性,故属于新的Ras家族成员,近几年发现ERas基因的表达与胃癌密切相关,而传统Ras基因在胃癌细胞中的表达及突变情况系统报道较少,本文旨在研究传统Ras基因Kras,Hras,Nras及其家族新成员ERas基因在胃癌细胞中的表达和突变情况。方法:选用7株不同来源不同分化程度的胃癌细胞系,利用RT—PCR及real-timePCR检测Ras基因在这些胃癌细胞系中的表达,并通过测序对传统Ras基因突变热点12,13,61位密码子及ERas基因全长进行突变分析。结果:QRas基因在这些胃癌细胞系中均有不同程度的表达,其中Hras和Nms基因在各株细胞中表达水平均一,而Kras和ERas基因则呈差异性表达;②在这些胃癌细胞中传统Ras基因突变热点12,13,61位密码子不存在突变,ERas基因全长亦未检测到突变.③发现Kras基因一新的剪接型,特点为第一、三外显子直接拼接,缺失第二外显子,命名为Kras△E2。结论:与在其他肿瘤中不同,传统Ras基因在胃癌细胞中不存在突变热点,家族新成员ERas基因全长亦无突变,在国际上首次报道新剪接型Kras△E2,从而得出创新性结论:Ras基因家族在胃癌细胞中并不是通过热点突变导致持续活化而致癌,而可能是通过ERas基因表达量的调节或形成新的剪接型KrasAE2而致癌。另外,Kras基因是一被受国际关注的肿瘤基因,新剪接型的发现可能会对Kras基因致癌机制产生新的认识,意义重大。  相似文献   

6.
目的:传统Ras家族由Kras,Hras 和Nras 基因组成,这类基因的点突变经常在人类肿瘤中发现,突变热点位于12,13,61 位 密码子。ERas 基因是2003 年在鼠胚胎干(ES)细胞中发现的,其cDNA编码的蛋白与Kras,Hras 和Nras分别有46%,43%和47% 的相似性,故属于新的Ras 家族成员,近几年发现ERas 基因的表达与胃癌密切相关,而传统Ras 基因在胃癌细胞中的表达及突 变情况系统报道较少,本文旨在研究传统Ras基因Kras,Hras,Nras及其家族新成员ERas 基因在胃癌细胞中的表达和突变情况。 方法:选用7 株不同来源不同分化程度的胃癌细胞系,利用RT-PCR 及real-timePCR 检测Ras基因在这些胃癌细胞系中的表达, 并通过测序对传统Ras基因突变热点12,13,61 位密码子及ERas 基因全长进行突变分析。结果:①Ras基因在这些胃癌细胞系中 均有不同程度的表达,其中Hras 和Nras 基因在各株细胞中表达水平均一,而Kras 和ERas 基因则呈差异性表达;②在这些胃癌 细胞中传统Ras基因突变热点12,13,61位密码子不存在突变,ERas基因全长亦未检测到突变.③发现Kras 基因一新的剪接型, 特点为第一、三外显子直接拼接,缺失第二外显子,命名为Kras△E2。结论:与在其他肿瘤中不同,传统Ras 基因在胃癌细胞中不 存在突变热点,家族新成员ERas 基因全长亦无突变,在国际上首次报道新剪接型Kras△E2,从而得出创新性结论:Ras基因家族 在胃癌细胞中并不是通过热点突变导致持续活化而致癌,而可能是通过ERas 基因表达量的调节或形成新的剪接型Kras△E2 而 致癌。另外,Kras 基因是一被受国际关注的肿瘤基因,新剪接型的发现可能会对Kras 基因致癌机制产生新的认识,意义重大。  相似文献   

7.
为了寻找新的Down’s综合征相关基因,利用生物信息学分析与实验技术相结合的方法,从定位于Down’s综合征关键区内(21q22.3)的EST(GenBank登录号H77399)出发,从人类睾丸组织cDNA文库内克隆到含同源盒结构域转录因子PKNOX1的一种新剪接型全长cDNA,命名为PKNOX1B,GenBank登陆号AYl42115。PKNOX1B基因跨越58.4kb,全长cDNA约2.8kb,有11个外显子和10个内含子,编码405个氨基酸残基的酸性蛋白质,分子量为44.628kDa,等电点6.28。PKNOX1B与PKNOX1的前9个外显子及9个内含子完全相同,由于PKNOX1B在第10与11外显子之间发生了差异剪接,以致其在3’端cDNA序列被截短约2kb,所编码的蛋白质在C端较PKNOX1短30个氨基酸残基。但PKNOX1B保留了与PKNOX1完全相同的同源盒结构域,因而它可能与其他含同源盒结构域基因家族成员一样参与了发育的遗传调控。RT-PCR结果显示PKNOX1B除骨髓组织外在人体组织广泛表达。在睾丸组织中PKNOX1可见5kb,2.9kb,2kb 3种转录本,而在其他组织中仅发现2个较大的转录本,2kb的转录本在睾丸组织呈现特异性的表达,它有可能参与了精子的发生过程。  相似文献   

8.
鸡Z染色体上DMRT1基因的多重跨染色体剪接   总被引:1,自引:0,他引:1  
性别决定与分化发育是同时涉及生命现象中两种细胞分裂(有丝分裂和减数分裂)形式的惟一的分化发育过程。对该过程中关键基因DMRT1的转录分析,发现位于鸡Z染色体上的DMRT1基因分别同时与4号染色体上的CENPC1基因、5号染色体上CD5R基因和2号染色体上37LRP/p40基因发生跨染色体剪接,由此构成了新的跨染色体剪接本DMRT1-CENPC1、DMRT1-CD5R和DMRT1-37LRP/p40。对其剪接位点的分析,发现两段染色体序列存在的重叠区可能在这种剪接中起着重要作用。DMRT1基因在转录过程中同时与多个染色体上基因发生多次跨染色体剪接的发现,无疑有助于对在转录水平上的多样性基因调控以及性别决定与分化发育等的认识开辟另一新途径。  相似文献   

9.
水稻NBS-LRR基因选择性剪接的全基因组检测及分析   总被引:1,自引:0,他引:1  
顾连峰  郭荣发 《遗传学报》2007,34(3):247-257
选择性剪接是促进基因组复杂性和蛋白质组多样性的一种主要机制,但是对水稻NBS-LRR序列选择性剪接的全基因组分析却未见报道。通过隐马尔柯夫模型搜索,从TIGR数据库里得到了855条编码NBS-LRR基序的序列。利用这些序列在KOME、TIGR基因索引及UniProt三个数据库中进行同源搜索,获得同源的完整cDNA序列、假设一致性序列和蛋白质序列。再利用Spidey和SIM4程序把完整cDNA序列和假设一致性序列联配到相应的BAC序列上来预测选择性剪接。蛋白质序列和基因组序列之间的联配使用tBLASTn。在这875个NBS-LRR基因中,119个基因具有选择性剪接现象,其中包括71内含子保留,20个外显子跳跃,25个选择性起始,16个选择性终止,12个5′端的选择性剪接和16个3′端选择性剪接。大多数选择性剪接都为两个和多个转录本所支持。可以通过访问http://www.bioinfor.org查询这些数据。进而通过生物信息学分析剪接边界发现外显子跳跃和内含子保留的‘GT…AG’的规则不如组成型的保守。这暗示了它们是通过不同的调控机制来指导剪接变构体的形成。通过分析内含子保留对蛋白质的影响,发现选择性剪接的蛋白更倾向于改变其C端氨基酸序列。最后对选择性剪接的组织分布和蛋白质定位进行分析,结果表明选择性剪接的最大类的组织分布是根和愈伤组织。超过1/3剪接变构体的蛋白质定位是质膜和细胞质。这些选择性剪接蛋白可能在抗病信号转导中起到重要作用。  相似文献   

10.
河豚毒-抵抗性(TTX-R)Nav1.5 Na 通道是心肌的特异性Na 通道,虽然研究发现神经元中也存在河豚毒-抵抗性Na 电流及Nav1.5/SCN5A mRNA的表达,但其确切的cDNA序列尚不清楚.采用RT-PCR法对人脑组织Nav1.5/SCN5A基因cDNA进行克隆发现:人脑组织Nav1.5/SCN5A基因cDNA有2种变构体,hB1和hB2(accession number EF629346,EF629347),其中hB1全长6201个碱基,其开放读码框架(ORF)参与编码2016个氨基酸,和人心肌Nav1.5 Na 通道氨基酸序列相同率高达98%,共有28个不同的氨基酸,其中7个集中位于第6A外显子与第6外显子编码区.与人心肌Nav1.5/SCN5A基因cDNA不同的是,在对人脑组织Nav1.5/SCN5A基因cDNA的克隆中未发现该基因第18外显子的选择性剪接,但却发现其第24外显子的选择性剪接,2种选择性剪接体(hB1和hB2)在脑组织中基本同时表达,表达比率接近1∶1,但在心脏中二者的表达比率却与年龄有关.人Nav1.5/SCN5A基因的第24外显子定位于染色体3P21区,共有54个碱基,参与编码18个氨基酸.RT-PCR法证实第24外显子的选择性剪接也可发生在大鼠心脑之外的其他组织中,竞争性PCR法证明,不同组织中2种选择性剪接体的表达比率不同,且随着周龄的增加,2种选择性剪接体在各组织中表达的变化趋势不同.此外,RT-PCR法还发现Wistar大鼠全身16种组织中均可检测到Nav1.5/SCN5A mRNA的表达.上述实验结果说明,Nav1.5 Na 通道在全身组织中分布广泛,但编码人脑组织Nav1.5 Na 通道与心肌组织该离子通道的cDNA序列不同,是Nav1.5/SCN5A基因的2种变构体,这为深入研究不同组织中Nav1.5 Na 通道的功能提供了基础.  相似文献   

11.
Loss of hPot1 function leads to telomere instability and a cut-like phenotype   总被引:15,自引:0,他引:15  
The human telomere binding protein hPot1 binds to the most distal single-stranded extension of telomeric DNA in vitro, and probably in vivo, as well as associating with the double-stranded telomeric DNA binding proteins TRF1 and TRF2 through the bridging proteins PTOP (also known as PIP1 or TINT1) and TIN2. Disrupting either the DNA binding activity of hPot1 or its association with PTOP results in elongated telomeres, suggesting a role for hPot1 in telomere length regulation. However, mutations to POT1 and Cdc13p, the fission and budding yeast genes encoding the structural orthologs of this protein, leads to telomere instability and cell death. Thus, it is possible that the hPot1 protein may also serve to cap and protect telomeres in humans. Indeed, we now find that knocking down the expression of hPot1 in human cells causes apoptosis or senescence, as well as an increase in telomere associations and anaphase bridges, telltale signs of telomere instability. In addition, knockdown cells also displayed chromatin bridges between interphase cells, reminiscent of the cut phenotype that was first described in fission yeast and in which cytokinesis progresses despite a failure of chromatid separation. However, unlike the yeast cut phenotypes, we suggest that the cut-like phenotype observed in hPot1 knockdown cells is a consequence of the fusion of chromosome ends and that this fusion impedes proper chromosomal segregation. We conclude that hPot1 protects chromosome ends from illegitimate recombination, catastrophic chromosome instability, and abnormal chromosome segregation.  相似文献   

12.
The protein hPot1 shares homology with telomere-binding proteins in lower eukaryotes and associates with single-stranded telomeric DNA in vitro as well as colocalizing with telomere-binding proteins in vivo. We now show that hPot1 is coimmunoprecipitated with telomeric DNA and that stable expression of this protein in telomerase-positive cells results in telomere elongation, supporting the idea that hPot1 is a bona fide mammalian telomere-binding protein. We previously found that mutations in the N-terminal DAT domain of the hTERT catalytic subunit of telomerase rendered the enzyme catalytically active but unable to elongate telomeres in vivo. This phenotype could be partially rescued by fusion with the double-stranded telomeric protein hTRF2. Given that hPot1 binds to single-stranded DNA in vitro (at the same site that hTERT binds to in vivo), we addressed whether fusion of hPot1 can rescue the DAT mutations more efficiently than that of hTRF2. We now report that a DAT mutant of hTERT is indeed efficiently rescued upon fusion to hPot1. However, this rescue depended on the ability of hPot1 to localize to telomeres rather than binding to DNA per se. These data support a model whereby the DAT domain of hTERT is implicated in telomere-telomerase associations.  相似文献   

13.
14.
15.
Feng Q  Li P  Leung PC  Auersperg N 《Genomics》2004,84(3):587-591
Five alternatively spliced mRNA isoforms of human caspase-1 have been identified previously and we report here the cloning of a new isoform, named CASP1 zeta (zeta), from human ovarian surface epithelial cell cDNA. The new isoform zeta is identical to the alpha isoform but missing 79 nucleotides in the coding region of the prodomain of procaspase-1. Analysis of the cDNA sequence of the zeta isoform revealed an ORF of a shorter protein missing the 39 amino acids at the amino terminal of procaspase-1alpha, which comprises the important caspase activating recruitment domain (CARD), which is required for interactions between caspases and other proteins. Secondary structure analysis of procaspase-1 CARD predicted the truncation of the alpha1, the alpha2, and part of the alpha3 helix in the zeta isoform in comparison to the full-length alpha isoform. The new zeta isoform was expressed in many, but not all, adult human tissues by RT-PCR. In HEK293 cells, transient overexpression of wild-type caspase-1zeta induced apoptosis to levels similar to those of caspase-1alpha. However, mutational change at the caspase-1 active center of the Cys 246 of caspase-1zeta, as well as Cys 285 of caspase-1alpha, completely abolished their apoptotic activity. Our findings suggest that caspase-1zeta is a widespread, new proapoptotic isoform of caspase-1. They also demonstrate that the first 39 amino acids of the N-terminal of the CARD in procaspase-1 are not required for its apoptotic activity.  相似文献   

16.
Adriamycin, an anticancer agent acting on topoisomerase II, promotes the arrest of cell division and neurite extension in a "neurite-minus" murine neuroblastoma cell line, N1A-103. This morphological differentiation is accompanied by a blockade in the S phase of the cell cycle, modification of the amount of peripherin, and appearance of the beta 7-tubulin isoform. Yet, adriamycin-induced N1A-103 cells fail to express other neuronal markers, such as long-lasting Ca2+ channels, synaptophysin, and the shift in the proportion of the beta'1 tubulin isoform to the beta'2 isoform, whose appearance parallels the terminal differentiation of the wild type neuroblastoma cell line N1E-115. Hence, a comparison of the behavior of these two cell lines leads to the proposal that there are two programs of neuroblastoma differentiation: one where expression is triggered by the arrest of cell division and which is observed in adriamycin-induced N1A-103 variant cells, and the other, presumably occurring further downstream, which would involve further changes in morphogenesis and acquisition of new electrophysiological properties.  相似文献   

17.
We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na+/H+ exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na+/H+ exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 cell line). We found that Aquaporin-2 modifies Na+/H+ exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na+/H+ exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na+/H+ exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na+/H+ exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na+/H+ exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na+/H+ exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na+/H+ exchanger isoform 1 function is proliferative. We propose that Na+/H+ exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation.  相似文献   

18.
We previously characterized RNA polymerase II-associated protein 3 (RPAP3) as a cell death enhancer. Here we report the identification and characterization of splicing isoform of RPAP3, isoform 1 and 2. We investigated the interaction between RPAP3 and PIH1 domain containing protein 1 (PIH1D1), and found that RPAP3 isoform 1, but not isoform 2, interacted with PIH1D1. Furthermore, knockdown of RPAP3 isoform 1 by small interfering RNA down-regulated PIH1D1 protein level without affecting PIH1D1 mRNA. RPAP3 isoform 2 potentiated doxorubicin-induced cell death in human breast cancer T-47 cells although isoform 1 showed no effect. These results suggest that R2TP complex is composed of RPAP3 isoform 1 for its stabilization, and that RPAP3 isoform 2 may have a dominant negative effect on the survival potency of R2TP complex.  相似文献   

19.
FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. The human isoform 2 of FADS (hFADS2), which is the product of FLAD1 gene, was over-expressed in Escherichia coli as a T7-tagged protein and identified by MALDI-TOF MS analysis. Its molecular mass, calculated by SDS-PAGE, was approx. 55 kDa. The expressed protein accounted for more than 40% of the total protein extracted from the cell culture; 10% of it was recovered in a soluble and nearly pure form by Triton X-100 treatment of the insoluble cell fraction. hFADS2 possesses FADS activity and has a strict requirement for MgCl2, as demonstrated in a spectrophotometric assay. The purified recombinant isoform 2 showed a kcat of 3.6 x 10(-3)s(-1) and exhibited a KM value for FMN of about 0.4 microM. The expression of the hFADS2 isoform opens new perspectives in the structural studies of this enzyme and in the design of antibiotics based on the functional differences between the bacterial and the human enzymes.  相似文献   

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