6q25区域内一个新基因MTLC的克隆及特性分析

目的：克隆6q25区域内喉癌相关新基因。方法：以表达序列标签为电子探针，在人类基因组数据库中进行电子杂交，钓出相就基因组DNA克隆后进行基因预测。根据获得的基因序列设计引物，逆转录－聚合酶链反应扩增新基因cDNA。结果：克隆了1个6q25区域内的新基因。该基因全长约21kb，含两个外显子，其cDNA序列长1006bp，编码蛋白包括235个氨基酸。其5’侧翼序列存在癌蛋白c-Myc的结合位点，将其命名为MTLC（c-Myc target from laryngeal cancer cells）基因。同源性分析表明该基因编码蛋白与小鼠MT－MC1蛋白同源性达78％。MTLC蛋白一级结构含有一个核定位信号结构域，亚细胞定位实验证实该基因主要在细胞核表达，Northern印迹分析表明MTLC基因在心肌、肝、肾、脑等多个组织有表达。结论：成功克隆了1个新基因MTLC，该基因可能作为c-Myc的靶基因以转录因子形式参与维持细胞正常生理功能。     

一个新的锌指蛋白基因ZNF333 cDNA的分离及表达谱分析

本研究通过生物信息学和分子生物学相结合的方法成功克隆了一个新的锌指蛋白基因ZNF333 cDNA。ZNF333基因包含14个外显子，编码665个氨基酸。蛋白质N端包含2个保守的KRAB－A盒，2个多态的KRAB－B盒，而C端包含10个重复的C2H2型锌指基序。ZNF333的表达在心脏组织中最高，在其他组织中也可检测到表达。同时还发现该基因存在选择性剪切形式。通过与人类基因组框架图的比较将ZNF333定位在19p13.1。     

一个新的小鼠腭裂候选基因cDNA序列的克隆

目的 克隆并筛选与小鼠腭裂发生相关候选新基因。方法 利用聚合酶链反应改良扣除杂交技术，克隆正常组与腭裂组小鼠腭突组织中差异表达的基因，经反向点杂交鉴定后挑选阳性克隆并测序及同源性分析，Northern印迹杂交检测所获的新基因mRNA的全长。结果 经大规模测序后得4个新的表达序列标签，其中1个片段全长为809bp，Northern印迹杂交结果显示为该基因的eDNA全长。结论 获得一个新的与小鼠腭裂发生相关的候选基因的cDNA全长。     

一种含多个CCP结构域的新型基因的克隆及生物学特性初探

目的克隆并分析新型基因CCP22，研究其结构及初探生物学特性。方法常规分子克隆、生物信息学分析、Western blot、RT—PCR。结果利用酵母双杂交技术从人乳腺文库中分离到一种含有22个补体调控蛋白（complement control protein，CCP）序列元件的新基因，命名为CCP22。生物信息学分析发现，该基因定位于人染色体9q31．2-32，共15个外显子，基因编码序列全长4494bp，编码1497个氨基酸。将CCP22基因全长编码序列克隆于真核载体中，该表达载体转染人胚肾细胞293T后获得的表达产物经Western blot证实CCP22属于分泌蛋白。将CCP22与绿色荧光蛋白（EGFP）标签融合后证实CCP22主要分布在细胞核外。Northern blot证实，内源性CCP22 mRNA转录本全长约6kb。RT-PCR检测表明，CCP22在正常乳腺细胞株中高表达，而在多种乳腺肿瘤细胞株中不表达或低表达。结论CCP22在乳腺肿瘤发生发展中可能发挥重要作用。     

人类survivin基因的克隆及在胃粘膜中表达的初步分析

目的：克隆人类Survivin(SVV)基因并分析其在胃粘膜中的表达情况。方法：采用逆转录聚合酶链反应从人胃癌组织中克隆SVV基因，地高辛标记cRNA探针，原位杂交检测其在胃粘膜中的表达。结果：得到两个SVV基因cDNA克隆、即SVV－S4A与SVV－S1B，前者与已知SVV基因cDNA序列相同，后者发生了SVV第3外显子丢失。原位杂交显示SVV－S4A主要表达于胃癌细胞的胞质，SVV－S1B主要表达于正常胃组织。结论：SVV－S4A和SVV－S1B在胃癌组织与正常胃粘膜组织中的表达存在不同特征。     

SEA基因与GPI信号肽序列融合基因的真核表达载体的构建和序列分析

目的：将超抗原金黄色葡萄球菌肠毒素A（staphylococcus enterotoxinA,SEA)基因的人胎盘碱性磷酸酶（hPLAP-1)C末端信号肽序列，即糖基化磷脂酰肌醇（glycosyl-phosphatidyl inositol,GPI)附着信号序列，拼接成融合基因，并构建该融合基因的真核表达载体。方法：利用基因SOEing法，分别扩增hPLAP-1 C末端信号肽序列和SEA基因，然后将二段基因拼接起来，拼接后与pGEM-T克隆载体连接，再亚克隆到真核表达载体pcDNA3.1( )中，测序鉴定。结果：分别成功扩增出hPLAP-1 C末端信号肽序列131bp和SEA基因789bp，并将二段基因序列成功拼接（901bp），且成功构建了真核表达载体pcDNA3.1( )/SEA-GPI，经测序是正确的。结论；真核表达载体pcDNA3.1( )/SEA-GPI的构建，为研究SEA－GPI抗肿瘤作用奠定了基础。     

人类胚胎干细胞特异表达新基因HPESCRG1的克隆和特性分析

目的克隆一个人类胚胎干细胞特异表达的新基因并对其特性进行分析。方法从一个在人类胚胎干细胞(embryonicstem,ES)中特异表达的表达序列标签CF948547出发,应用生物信息学方法和分子生物学技术克隆一个新基因,采用逆转录-聚合酶链反应分析新基因的表达谱,并应用增强型绿色荧光蛋白真核表达系统分析新基因的亚细胞定位。结果成功克隆了一个新基因HPESCRG1,GenBank登录号为AY283672。该基因cDNA长1395bp,包含9个外显子和8个内含子,开放阅读框为250～1146bp,定位在3q13.13,预测编码297个氨基酸,预计分子量为33784,等电点为9.35。其编码蛋白有一个SAP功能域,该蛋白定位在细胞核内。该基因仅在人类ES细胞中表达,而在其分化细胞不表达,在人胚胎成纤维细胞、人间充质干细胞、成人和流产胚胎的多种正常组织中不表达。结论HPESCRG1基因是一个人类ES细胞中表达特异性新基因,很可能与人类ES细胞的自我更新和维持其未分化状态密切相关。     

激活素受体相互作用蛋白3的基因克隆及其免疫学特性研究

目的：研究新克隆的激活素受体相互作用蛋白3（ActRIP3）的免疫学特性和其介导Ser／Thr激酶型受体胞内信号传导的作用。方法：以酵母双杂交法发现的ActRIP基因片段作为探针，从小鼠脑cDNA文库中克隆ActRIP3基因。EIA法分析其与激活素ⅡA型受体（ActRⅡA）结合能力，Westernblot杂交检测成熟蛋白在组织中的表达，免疫组化染色分析其在脑组织中的分布，采用pcDNA-AetRIP3与CAGA-1ux报告基因质粒共转染HEK293细胞分析信号传导作用。结果：克隆的ActRIP3基因全长1197bp，编码101个氨基酸残基，EIA分析显示ActRIP3与ActRⅡA具有特异性结合作用，这种作用与ActRIP3的N末端氨基酸序列有关。Westernblot杂交显示天然ActRIP3相对分子质量约为14000，在多种组织表达，免疫组化染色显示其在脑组织中的分布以海马及下丘脑为主。通过表达ActRIP3可促进激活索诱导的特异性基因转录活性。结论：ActRIP3属于ActRIP家族新成员，具有特异结合ActRⅡA的能力，并具有促进激活素信号传导的作用。     

乳腺增生病p53基因第5外显子突变及其蛋白表达

目的：探讨p53基因在乳腺癌发生早期的作用。方法：用免疫组化方法检测36例乳腺单纯性增生、31例不典型增生、14例原位癌和16例浸润癌中p53蛋白的表达，用PCR－SSCP检测了上述组织中p53基因第5外显子突变。结果：p53蛋白在单纯性增生、不典型增生、导管内癌、浸润癌中的表达率分别为0、22.6%(7/31)、42.8%(6/14)、50％（8／16），PCR－SSCP在各组中均未检测到该基因第5外显子突变。结论：乳腺癌发生早期阶段有p53基因的参与，但与第5外显子突变无明显关系。     

X连锁淋巴组织增生征基因的克隆

X连锁淋巴细胞增生征（XLP）基因已分别被两个研究小组定位克隆，分别命名为SH2D1A和SAP。证实该基因有四个外显子，编码一种128个氨基酸的蛋白质，其中含有一个SH2区，它能特异地结合于信号蛋白磷酸化的酪氨酸处，该基因的突变是发生XLP纷原因。     

SH2A 基因对细胞信号转导的影响及其亚细胞定位

目的 研究Src同源域2(src homology 2，SH2)A基因在细胞信号转导中的作用并进行亚细胞定位。方法 通过RT—PCR方法扩增SH2A cDNA编码序列，构建真核重组表达载体pcDNA3．1-SH2A，利用脂质体转染肝癌Bel7402细胞、COS7细胞，检测蛋白酶C(protein kinaseC，PKC)、酪氨酸蛋白激酶(tyrosine protein kinase，TPK)、丝裂原激活蛋白激酶(mitogen activated protein kinase，MAPK)活性的改变；另构建pEGEP—SH2A，转染同前，荧光显微镜观察荧光定位。结果 测序结果显示真核重组表达载体pcDNA3．1-SH2A及pEGFP—SH2A中均含有SH2AcDNA编码序列；肝癌Bel7402细胞、COS7细胞转染pcDNA3．1-SH2A后，胞浆PKC的活性下降了40％左右，MAPK和TPK活性未见明显改变。荧光显微镜观察发现SH2A基因在细胞质中表达。结论 SH2A基因编码蛋白在PKC信号转导通路中起抑制作用；SH2A基因编码蛋白定位于细胞质。     

Clinical,In Silico,and Experimental Evidence for Pathogenicity of Two Novel Splice Site Mutations in the SH3TC2 Gene

Abstract: Charcot-Marie-Tooth (CMT) neuropathy is the most common inherited neuromuscular disorder. CMT is genetically very heterogeneous. Mutations in the SH3TC2 gene cause Charcot-Marie-Tooth neuropathy type 4C (CMT4C), a demyelinating form with autosomal recessive inheritance. In this study, two novel splice site mutations in the SH3TC2 gene have been studied (c.279G → A, c.3676–8G → A). Mutation c.279G → A was detected on one allele in two unrelated families with CMT4C in combination with a known pathogenic mutation (c.2860 C →T in one family, c.505T → C in the other) on the second allele of SH3TC2 gene. Variant c.3676–8G → A was detected in two patients from unrelated families on one allele of the SH3TC2 gene in combination with c.2860C →T mutation on the other allele. Several in silico tests were performed and exon trap experiments were undertaken in order to prove the effect of both mutations on proper splicing of SH3TC2. Fragments of SH3TC2 were subcloned into pET01 exon trap vector (Mobitec) and transfected into COS-7 cells. Aberrant splicing was predicted in silico for both mutations, which was confirmed by exon trap analysis. For c.279G → A mutation, 19 bases from intron 3 are retained in cDNA. The mutation c.3676–8G→ A produces a novel splice acceptor site for exon 17 and complex changes in splicing were observed. We present evidence that mutations c.279G → A and c.3676–8G →A in the SH3TC2 gene cause aberrant splicing and are therefore pathogenic and causal for CMT4C.     

Identification of a novel mutation of SH3BP2 in cherubism and demonstration that SH3BP2 mutations lead to increased NFAT activation

We describe a novel missense mutation (Aspartic acid to Asparagine, p.D419N (g.1371G>A, c.1255G>A) within exon 9 of SH3BP2 in a patient with cherubism, an autosomal dominant syndrome characterized by excessive osteoclastic bone resorption of the jaw. Two siblings and the father were carriers but lacked phenotypic features. Transient expression of p.D419N (c.1255G>A), as well as three previously described exon 9 mutations from cherubism patients (p.R415Q (c.1244G>A), p.D420E (c.1259G>A), and p.P418R (c.1253C>G)) increased activity of NFAT (nuclear factor of activated T-cells), an osteoclastogenic mediator, indicating that cherubism results from gain of function mutations in SH3BP2.     

Characterization of a new disease-causing mutation of SH2D1A in a family with X-linked lymphoproliferative disease

Males with an expressed mutation in the SH2D1A gene that encodes an SH2 domain protein named SH2D1A or SAP (NP_002342; signaling lymphocyte activating molecule [SLAM]-associated protein), have an X-linked syndrome characterized by an increased vulnerability to infection with Epstein-Barr virus (EBV). We evaluated two related male patients with fatal infectious mononucleosis (FIM) and mutation in the SH2D1A gene. Sequence analysis revealed a hemizygous c.47G>A mutation in one of the patients, and heterozygosity for this mutation in the genomic DNA from his mother and maternal grandmother. This mutation resulted in p.G16D amino acid change in the sequence of the SAP protein. To analyze the effect of this missense mutation on protein function cDNA was generated by site-directed mutagenesis and expressed in COS cells. We found that half-life of the p.G16D protein was comparable to that of wild type SAP. However, the mutant protein was defective in binding to its physiological ligands SLAM and 2B4. These results suggest that a defect in ligand binding contributes to the loss of function of the SAP protein in patients carrying p.G16D mutation.     

Identification and characterization of LASP2 gene in silico

LASP1 (also known as MLN50) gene, located centromeric to the PPP1R1B-ERBB2-GRB7 locus on human chromosome 17q12, is amplified and over-expressed in breast cancer. Here, we identified and characterized a novel LASP1-related gene, LASP2, by using bioinformatics. Nucleotide sequence of human LASP2 cDNA was determined in silico by assembling EST BF699808 and 5'-truncated FLJ39221 cDNA. Nucleotide sequence of mouse Lasp2 cDNA was derived from 1200007O21Rik cDNA. Human LASP2 (270 aa) showed 97.4% and 63.7% total-amino-acid identity with mouse Lasp2 and human LASP1, respectively. LASP2 and LASP1 were the LASP family proteins consisting of LIM domain, Nebulin repeat, and SH3 domain. LASP2 and NEBL mRNAs were transcribed from the LASP2/NEBL gene on human chromosome 10p12 due to alternative splicing. LASP2 mRNA consists of exons 1a-4a, 24, 27, and 28 of the LASP2/NEBL gene, while NEBL mRNA consists of exons 1-28. Exon 1a-4a of the LASP2/NEBL gene were more homologous to exon 1-4 of the LASP1 gene on human chromosome 17q12, while exon 1-28 of the LASP2/NEBL gene were more homologous to exons of NEB gene on human chromosome 2q23. Some part of the LASP2/ NEBL-TEM7L-ARL8-CACNB2 locus on 10p12 was paralogous to the LASP1-TEM7-CACNB1 locus on 17q12, while the other part of the LASP2/NEBL-TEM7L-ARL8-CACNB2 locus was paralogous to the NEB-ARL5-CACNB4 locus on 2q23. These facts indicate that the LASP2/NEBL-TEM7L-ARL8-CACNB2 is a chimeric locus, which might be generated through the homologous recombination between the ancestral lasp2-tem7l-cacnb2 locus and the ancestral nebl-arl8 locus. Therefore, gene fusion during evolution is one of the mechanisms to generate alternative splicing.     

High frequency of SH3TC2 mutations in Czech HMSN I patients

La??uthová P, Mazanec R, Vondrá?ek P, ?i?ková D, Haberlová J, Sabová J, Seeman P. High frequency of SH3TC2 mutations in Czech HMSN I patients. Charcot–Marie–Tooth (CMT) neuropathy type 4C (CMT4C) is an autosomal recessive (AR), demyelinating neuropathy with early spine deformities caused by mutations in the SH3TC2 gene. To determine the spectrum of SH3TC2 mutations in the Czech population, the entire coding region of SH3TC2 was sequenced in 60 unrelated Czech patients. The prevalent mutation was shown to be the p.Arg954Stop. Therefore, 412 additional patients referred for CMT testing were tested for the presence of p.Arg954Stop only. Of 60 patients in whom the SH3TC2 gene was sequenced, at least one mutation was detected in 13 (21.7%) patients and biallelic pathogenic mutations were detected in 7 (11.6%) patients. Of the 412 patients tested for p.Arg954Stop, the mutation was found in 8 patients (1.94%), 6 were homozygous and 2 were heterozygous. The second causative mutation was detected by sequencing in one of the patients but not in the other. Nine novel sequence variants were detected. Their pathogenicity was further tested in silico and in control samples. Mutations in the SH3TC2 gene are a frequent cause of demyelinating hereditary neuropathy among Czech patients. In total, at least one mutation was found in 21 unrelated patients. CMT4C seems to be the most frequent type of AR CMT and one of the most frequent of all CMT types. Mutation p.Arg954Stop is highly prevalent in the Czech population. Patients with demyelinating neuropathy along with non‐dominant mode of inheritance and negative for CMT1A/hereditary neuropathy with liability to pressure palsy should be tested for the presence of the p.Arg954Stop mutation or other mutations in the SH3TC2 gene.     

X-连锁迟发性脊椎骨骺发育不良SEDL基因剪接受体突变导致潜在剪接位点激活

目的 深入研究X-连锁迟发性脊椎骨骺发育不良（X-linked spondyloepiphyseal dysplasia tarda,SEDL）的发病机理，为最终防治本病提供依据。方法 应用逆转录-PCR及克隆测序方法对1例涉及SEDL基因第5内含子剪接受体缺失的SEDL患者进行mRNA表达研究。结果 该患者存在2个不同片段长度的mRNA表达产物，与GenBank正常序列进行BLAST比较后发现，393bp的表达产物是第6外显子内一个新的潜在剪接位点激活后形成的产物；433bp的表达产物与8号染色体的部分基因组序列完全一致。结论 SEDL基因第5内含子剪接受体位点及其后的第6外显子共13个碱基的缺失突变导致第6外显子内一个新的潜在剪接受体位点激活，使转录后的mRNA丢失了第6外显子内47bp的编码序列，并使紧接其后的2个密码子产生移码，导致翻译的提前终止（D109-S123del；S124fsX126）。另外，该突变可能激活了8号染色体上假基因SEDLP2的转录，从而部分地补偿了SEDL蛋白的功能。     

A G-->A transition creates a branch point sequence and activation of a cryptic exon, resulting in the hereditary disorder neurofibromatosis 2

We describe a G-->A transition within intron 5 of the NF2 gene. This mutation creates a consensus splice branch point sequence. To our knowledge this is the first report of a mutation that creates a functional branch point sequence in a human hereditary disorder. The new branch point sequence is located 18 bp upstream of a consensus splice acceptor site. A consensus splice donor site is found 106 bp 3' of the acceptor site. Asa consequence the G-->A transition results in an alternatively spliced mRNA containing an additional exon 5a of 106 bp derived from intron sequences. We cloned the mutant cDNA and show that due to an in-frame stop codon the cDNA codes for a truncated NF2 protein. The mutation was observed in three affected members of an NF2 family. In a tumour of one of the family members both alternatively spliced and wild-type mRNA were found, although the wild-type allele of the gene is absent due to an interstitial deletion on chromosome 22. We also show that immunoprecipitations reveal the presence of full-length wild-type NF2 protein in the tumour lysate. These data support the hypothesis that some degree of normal splicing of the mutant precursor RNA is taking place. It is therefore likely that this residual activity of the mutant allele explains the relatively mild phenotype in the family. These data also indicate that complete inactivation of the gene is not required for tumour formation.