K562细胞DHRS4 L1基因新选择性剪接亚型的克隆及二级结构预测

目的： K562细胞DHRS4L1[ dehydrogenase／reductase （ SDR family） member 4 like 1]的表达及其二级结构分析。方法以K562细胞cDNA为模板, PCR扩增DHRS4[ dehydrogenase／reductase （ SDR fami-ly） member 4]基因簇Ea2转录本。将PCR产物A-T克隆至pGEMT-Easy质粒,对质粒进行DNA Sanger测序。将测序所得序列用NCBI ORF finder分析是否存在编码区,用Clustal Omega分析RNA序列系统树,用RNAfold web server分析RNA最小自由能二级结构。结果用RT-PCR和Sanger测序方法发现, K562表达DHRS4L1 Ea2转录本,未检测到其表达DHRS4L2[dehydrogenase／reductase （SDR family） member 4 like 2] Ea1转录本。 K562表达的DHRS4L1 Ea2至少有8种选择性剪接亚型（ KU058702、 KU058703、 KU058704、KU058705、 KU058706、 KU058707、 KU058708、 KU058709）,其中6种为首次发现。 KU058702、 KU058703、KU058704、 KU058705和KU058707第二外显子受到多种形式的选择性剪接,恰好仅影响第2臂的二级结构,不影响其他臂的二级结构,提示这些不同亚型的二级结构有一定相似性,第二外显子所对应的二级结构臂可能在区分不同 DHRS4L1亚型功能中发挥关键作用。结论研究发现 K562细胞至少表达8种DHRS4L1选择性剪接亚型,为长链非编码RNA。其二级结构分析为后续研究DHRS4L1在白血病细胞中的潜在功能奠定基础。     

DHRS4L1基因选择性剪接亚型的克隆及分析

目的 检测分析DHRS4基因簇新命名基因DHRS4L1的RNA选择性剪接亚型和转录特点,并预测其功能.方法 cDNA末端快速扩增克隆DHRS4L1转录本剪接亚型的全长序列,Jellyfish软件比对RNA亚型的外显子组成,RNAStructure和在线开放读码框预测其二级结构和蛋白编码功能,定量PCR检测DHRs4L1...     

BS69的亚细胞定位及其核输出信号序列的分析鉴定

目的: 分析腺病毒E1A相关蛋白BS69不同亚型的DNA序列,寻找新的核输出信号序列,并在Cos7细胞中表达确定其亚细胞定位。方法: 分析数据库中不同BS69亚型DNA序列,与传统和输出信号序列比对,寻找可能的新的核输出信号序列。采用DNA重组技术把BS69不同亚型片段的cDNA插入到真核表达载体pcDNA3.1上,转染Cos7细胞,用免疫荧光染色方法确定其亚细胞定位,用Western blotting方法验证不同亚型BS69在细胞中的功能。结果: 在BS69亚型2上面发现1段富含亮氨酸的基因序列,与核输出信号序列极为相似。免疫荧光染色方法显示BS69亚型2定位于细胞浆中,而BS69亚型1和2个亚型共同序列编码的蛋白质则定位于细胞核中。BS69亚型2参与了EB病毒潜伏膜蛋白1(Epstein-Barr virus latent membrane protein 1,LMP1)介导的信号转导途径。结论: BS69不同亚型具有不同的亚细胞定位,因此具有不同的生物学功能,其中核蛋白是转录调控因子,细胞浆蛋白则可能参与鼻咽癌发生发展的调控。     

HBV Pre S1蛋白在酵母细胞中的转录激活功能

目的 探索应用双杂交体系统克隆与乙型肝炎病毒（HBV）Pre S1蛋白结合的肝细胞受体的可行性。方法 构建编码HBV Pre S1全序列或部分序列与酵母蛋白GAL 4 DNA结合区域融合蛋白的酵母表达质粒，应用这些质粒转化酵母报道菌株SFY526，检测β－半乳糖苷酶活性作为酵母中转录激活的指标。构建编码Pre S1全序列与GAL4 DNA结合区域融合蛋白的哺乳动物细胞表达质粒，与CAT报道质粒共转染肝癌细胞系Huh-7，使用薄层层析法检测细胞提取物的氯霉素乙酰转移酶活性。结果 全序列Pre S1蛋白与GAL4 DNA结合区域的融合蛋白在酵母细胞中呈现转录激活功能，其转录激活序列被定位于Pre S1第21－47氨基酸之间。全序列Pre S1蛋白与GAL 4 DNA结合区域的融合蛋白在哺乳动物细胞中未呈现转录激活功能。结论 HBV Pre S1蛋白在酵母细胞中的转录激活功能，限制了研究人员应用酵母双杂交体系统克隆与Pre S1结合的HBV受体。然而，Pre S1蛋白在哺乳动物细胞未呈现转录激活功能。哺乳动物细胞双杂交体系统可能是克隆与Pre S1蛋白作用的肝细胞受体的可行途径。     

DHRS4基因簇缺失型神经母细胞瘤细胞株的克隆和鉴定

目的 单克隆培养人神经母细胞瘤SH-SY5Y-细胞株,并鉴定其在基因水平缺失DHRS4基因簇,包括DHRS4、DHRS4L2和DHRS4L1的基因序列.方法 有限稀释法培养实验室偶得的低表达DHRS4基因的人神经母细胞瘤SH-SY5Y细胞,相差显微镜观察细胞的生长和增殖,PCR、RT-PCR检测DHRS4、DHRS4L...     

DHRS4反义RNA结合蛋白的高通量检测与分析

目的 DHRS4是细胞内NADP(H)依赖性视黄醇脱氢/还原酶家族成员4的编码基因。与人类DHRS4基因以头对头形式存在的反义转录本AS1DHRS4,可以调控DHRS4基因的转录。本研究高通量检测分析DHRS4反义RNA结合的蛋白以探讨DHRS4反义转录本调控正义基因表达的作用机制。方法采用甲醛交联细胞内的核酸-蛋白复合物,然后通过生物素标记的奇数组和偶数组寡核苷酸作为探针pull-down AS1DHRS4结合的蛋白,LCMS/MS鉴定后数据库比对分析DHRS4反义RNA结合的蛋白。结果奇数组和偶数组探针pull-down后分别鉴定得到650种和508种蛋白,两组探针检测的共同蛋白434种。大部分蛋白为结合蛋白并与RNA的加工和修饰相关。结论 DHRS4反义RNA结合的蛋白的检测和分析为今后进一步研究反义RNA功能和机制提供了线索和基础。     

人IgE重链恒定区2～4区蛋白的真核表达、纯化及其相互作用分子捕获

目的真核表达、纯化人IgE重链恒定区2～4区(IgECε2-4)蛋白,利用表面等离子体共振技术(SPR)捕获其相互作用蛋白。方法构建3个含不同信号肽序列的IgECε2-4重组真核表达载体,分别瞬时转染HEK293FT悬浮细胞,优化选用表达量最高的重组质粒进行大量瞬转表达,镍柱亲和层析纯化。免疫荧光技术鉴定重组蛋白与KU812细胞表面IgE受体FcεRⅠ的结合,采用SPR技术捕捉人血清中与IgECε2-4相互作用的蛋白。结果含信号肽3(SP3)的重组质粒表达量最高,表达量约为6.2 mg/L;亲和纯化获得高纯度的人IgECε2-4蛋白;免疫荧光技术鉴定显示重组蛋白IgECε2-4可与KU812细胞表面受体结合。通过SPR技术捕获到人血清中39种可能与IgECε2-4结合的蛋白。结论获得纯化的重组蛋白IgECε2-4,IgECε2-4能与KU812细胞表面FcεR1α受体结合,靶标垂钓结果显示IgECε2-4能与人血清中的39种蛋白存在相互作用或有间接结合作用。     

CD45分子选择性剪接亚型的功能和调节

白细胞共同抗原CD45分子由一类结构相似、相对分子质量较大的跨膜蛋白组成,其胞浆区段具有蛋白质酪氨酸磷酸酶的作用,因而在细胞的信号传导及功能调节中发挥重要作用。CD45mRNA外显子的不同剪切拼接方式,能编码产生CD45RA、CD45RB、CD45RC、CD45RO等多种蛋白亚型。CD45亚型分子的表达与T淋巴细胞功能相关,且随着细胞的分化、发育和激活,分子亚型可发生转换。近年研究显示,CD45基因DNA的表观遗传学修饰、DNA结合的蛋白以及RNA剪接位点的突变等多种因素可以调控CD45不同外显子的剪接,从而调节淋巴细胞表面CD45分子不同亚型的表达。调控CD45外显子在成熟转录本的保留或者缺失的因素,也可能最终调控淋巴细胞CD45分子亚型的表达以及淋巴细胞的功能,并与临床自身免疫性疾病、血液病、肿瘤等疾病的发病相关。     

CPT-Ⅱ基因4号外显子质粒构建与变异分析

背景：肉毒碱棕榈酰转移酶Ⅱ(Carnitine palmitoyltransferase-Ⅱ, CPT-Ⅱ)位于细胞线粒体内膜上,是脂肪酸氧化过程中起关键作用的酶之一。目前关于流感病毒感染与CPT-Ⅱ基因变异尚未见报道。 目的：构建CPT-Ⅱ第4号外显子(E4)表达质粒及其核苷酸变异分析。 方法：从流感病毒感染2例患者外周血中提取DNA,以PCR法扩增CPT-Ⅱ基因的第4号外显子,将其片段插入pGEM-T载体,以T4 DNA连接酶,构建T-CPT-Ⅱ E4,转化大肠杆菌DH5α细胞,增菌,制备质粒,EcoR I酶切、测序和变异分析。 结果与结论：成功构建重组表达质粒pGEM-T-CPT-ⅡE4,经DNA测序,证实质粒中插入CPT-Ⅱ第4号外显子完整序列,全长1 305个核苷酸,编码435个氨基酸;与Genebank源序列对照比较,发现含有两个变异位点：1 618(GTC→ATC)和1 858 (TTT→TCT),对应氨基酸为V368I和F448L。     

肿瘤转移抑制相关基因TMSG-1核仁定位信号序列的鉴定

目的 鉴定肿瘤转移抑制相关基因TMSG-1蛋白中潜在的特异性定位信号序列并探索其亚细胞定位机制.方法 聚合酶链反应(PCR)扩增TMSG-1开放读码框全长及不同长度的截断片段,定向克隆于绿色荧光蛋白(GFP)表达质粒pEGFP-N1;各融合蛋白表达质粒转染人胚肾细胞系HEK293细胞;转染48 h后提取细胞总蛋白进行GFP的Western blot检测或用冷丙酮固定细胞后激光共聚焦显微镜观察融合蛋白的亚细胞定位.结果 GFP分别融合TMSG-1全长蛋白(aa1-380)及其截断片段T1(aa1-70)、T2(aa1-128)、T3(aa129-380)、T4(aa71-128)、T5(aa71-179)和T6(aa71-380),Western blot检测结果显示成功表达了各融合蛋白.激光共聚焦显微镜观察亚细胞定位显示融合蛋白T4(aa71-128)主要定位于细胞核内的核仁部位,融合蛋白T6(aa71-380)以细胞核内弥散分布为主,而TMSG-1全长融合蛋白及融合蛋白T1、T2、T3、T5则定位于胞质.进一步的序列缺失去除T4( aa71-128)羧基末端10个氨基酸得到截断片段T4Δ119-128,T4Δ119-128融合的GFP仍位于细胞核,但核仁内的绿色荧光信号明显减弱.结论 肿瘤转移抑制相关基因TMSG-1存在潜在的核仁定位信号,位于aa119-128(RRRRNQDRPS),这一发现为深入研究TMSG-1的亚细胞定位及相关功能奠定了基础.     

A Golgi localization signal identified in the Menkes recombinant protein

Menkes disease arises from a genetic impairment in copper transport. The gene responsible for the phenotype has been identified as a copper transporting ATPase ( ATP7A ). Recently, the protein encoded by the ATP7A gene has been localized to the Golgi complex. In order to investigate the role of the Menkes disease protein in copper transport, recombinant constructs containing both the full-length open reading frame and an alternatively spliced form have been successfully expressed and localized in mammalian cells. Other studies of a patient with occipital horn syndrome, an allelic variant of Menkes disease, have demonstrated that only this alternatively spliced isoform and not the full-length form is expressed in this patient. The milder form of this patient's phenotype suggests that the alternatively spliced isoform has some functional role in copper transport. In the present study the full-length recombinant Menkes protein was shown by immunofluorescence to localize to the Golgi apparatus and the alternatively spliced form, lacking sequences for transmembrane domains 3 and 4 encoded by exon 10, was shown to localize to the endoplasmic reticulum. Using sequences from exon 10 fused to a non-Golgi reporter molecule, a 38 amino acid sequence containing transmembrane domain 3 of the Menkes protein was found to be sufficient for localization to the Golgi complex. Therefore, the protein sequence encoded by exon 10 may be responsible for this differential localization and both isoforms may be required for comprehensive transport of copper within the cell.      

An alternatively-spliced mRNA in the carboxy terminus of the neurofibromatosis type 1 (NF1) gene is expressed in muscle

The gene for neurofibromatosis type 1 (NF1) was identified bypositional cloning and found to contain two alternatively splicedexons. The first described alternatively spliced exon (exon23a) is located within the GAP-related domain of the gene andinserts an additional 63 nucleotides into the NF1 mRNA. Thesecond alternatively spliced exon (exon 48a) is located nearthe extreme carboxy terminus of the gene and inserts an additional54 nucleotides into the mRNA. This second isoform, termed 3'ALT,was originally detected while screening a fetal brain cDNA library.Examination of its expression by reverse-transcribed RNA PCRdemonstrates high level of expression in cardiac muscle, skeletalmuscle and smooth muscle. Trace levels of expression are detectedin brain and nerve. The 3'ALT isoform is expressed in fetalcardiac muscle, adult left ventricle and cardiac Purkinje cells.Further confirmation of the existence of this isoform was obtainedby blotting the PCR products with a radiolabeled oligonucleotideentirely derived from sequences contained within exon 48a andby direct sequencing of the PCR products. Additionally, thisisoform is expressed in muscle tissues from other vertebratespecies. The expression of this isoform in muscle suggests thatthe NF1 gene may play additional tissue-specific roles in muscledevelopment and signal transduction.     

Cancer-associated missplicing of exon 4 influences the subnuclear distribution of the DNA replication factor CIZ1

Cip1-interacting zinc finger protein 1 (CIZ1, also known as CDKN1A-interacting zinc finger protein 1) stimulates initiation of mammalian DNA replication and is normally tethered to the nuclear matrix within DNA replication foci. Here, we show that an alternatively spliced human CIZ1 variant, lacking exon 4 (Delta E4), is misexpressed as a consequence of intronic mutation in Ewing tumor (ET) cell lines. In all ET lines tested, exon 4 is skipped and an upstream mononucleotide repeat element is expanded to contain up to 28 thymidines, compared to 16 in controls. In exon-trap experiments, a 24T variant produced three-fold more exon skipping than a 16T variant, demonstrating a direct effect on splicing. In functional assays, Delta E4 protein retains replication activity, but fails to form subnuclear foci. Furthermore, coexpression of mouse Delta E4 with Ciz1 prevents Ciz1 from localizing appropriately, having a dominant negative effect on foci formation. The data show that conditional exclusion of exon 4 influences the spatial distribution of the Ciz1 protein within the nucleus, and raise the possibility that CIZ1 alternative splicing could influence organized patterns of DNA replication.     

SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation

CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO.     

Investigation of the alternatively spliced insert region of the D2L dopamine receptor by epitope substitution

Alternatively spliced variants of the D2 dopamine receptor have distinct neuronal function and localization. The long isoform (D2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop-a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Galphai/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D2L mutant is indistinguishable from the wild type D2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D2 receptor function.