HP8：增强子结合蛋白家族的一个新基因的cDNA克隆

以大鼠增强子结合蛋白（Ｃ／ＥＢＰ）ｃＤＮＡ为探针，筛选人胎肝ｃＤＮＡ库，得到一个含有２４８０碱基命名为ＨＰ８的ｃＤＮＡ克隆，其中８４－１１０９位碱基属编码区。经基因数据库（ＥＭＢＬ，９５．６版本）查对未发现同源基因。该基因编码的蛋白Ｃ－末端含有亮氨酸拉链结构，和Ｃ／ＥＢＰ功能区（Ｃ／ＥＢＰ基因家族新成员。基因组Ｓｏｕｔｈｅｒｎ杂交证实该基因为单拷贝基因。在１４种胎儿组织中显示小肠、皮肤高表达，肾     

增强子结合蛋白家庭的一个新基因HP8定位于人19号染色体长臂1…

探讨增强子结合蛋白家族一个新基因ＨＰ８的人染色体定位。按常规方法制备分裂期 淋巴细胞切片，用生物素标记ＨＰ８基因３‘－端２．１ｋｂｃＤＮＡ作探针、进行荧光原位杂交对ＦＩＳＨ信号及染色体上ＤＡＰＩ结合条带同时拍照，二者重迭信号进行计数。     

增强子结合蛋白家庭的一个新基因HP8定位于人19号染色体长臂13.1-13.2区带

目的:探讨增强子结合蛋白(C/EBP)家族一个新基因HP8的人染色体定位.方法:按常规方法制备分裂期人淋巴细胞切片,用生物素标记HP8基因 3’-端 2.1kb cDNA作探针,进行荧光原位杂交(FISH).对FISH信号及染色体上DAPI结合条带同时拍照,二者重迭信号进行计数,结果:100个有丝分裂图中,有72个显示阳性信号位于同一对染色体,并且无其他非特异性杂交带,杂交效率约为70%.参照DAPI结合条带,阳性信号位于人19号染色体长臂13.1-13.2区带.结论:HP8基因定位于人19号染色体长臂 13.1-13.2区带.     

肝癌内高表达新基因的部分cDNA克隆

目的寻找人肝癌相关的基因。方法应用Ｎｏｒｔｈｅｒｎ杂交技术，对表达序列标记（ＥｘｐｒｅｓｓｅｄＳｅ－ｑｕｅｎｃｅｔａｇｓ，ＥＳＴ）基因克隆进行筛选，以得到的基因片段为探针，筛选人胎肝ｃＤＮＡ文库。结果得到一个在肝癌内高表达的ＥＳＴ基因片段。进一步筛选人胎肝ｃＤＮＡ文库，得到一命名为ＦＬ６的ｃＤ－ＮＡ克隆，核苷酸序列测定表明，ＦＬ６长度为１４６４个碱基，检索最新版本ＧｅｎｅＤａｔａｂａｓｅｓ（ＥＭＢＬ９６．６），未发现同源基因。实验结果还显示该基因在胎儿各组织内有不同的表达特性。结论该基因可能与细胞的增殖、癌变过程相关。     

新基因TMBP-1的全长cDNA克隆

目的：克隆胸腺细胞发育相关新基因。方法：本研究建立了抑制性差减杂交技术（SSH）、构建了胸腺基质细胞系cDNA差减杂交文库。应用cDNA末端快速扩增方法（RACE）进一步克隆新基因的cDNA全长序列。结果：筛选两个不同胸腺基质细胞系的差异表达基因,获得了新基因cDNA片段C91,应用RACE成功克隆新基因TMBP－1cDNA全长序列。它拥有一个969bp的开放读码框架,编码323个氨基酸。同源序列 比较发现,它编码一个未知功能的膜结合蛋白。Northem杂交分析显示,mRNA转录本长度为1．5kb;在两种胸腺基质细胞系中均有表达。新基因的cDNA全长序列。已被GenBank所接受。结论：抑制性差减杂交技术是克隆新基因片的有效方法;成功克隆新基因TMBP－1的cDNA全长序列。     

人类Quaking基因的cDNA克隆

研究发现 ,在Ｑｕａｋｉｎｇ(ＱＫ)突变小鼠 (战栗者 )中 ,由于位于 17号染色体ＱＫ基因 (ＱＫ ,ＱＫ 1～ 7,ＧｅｎＢａｎｋ登记号Ｕ44 940 )的缺失突变或点突变 ,导致其神经系统 (包括中枢及周围神经系统 )的髓鞘形成障碍 ,临床表现为以后半身为主的震颤等 ,严重者可于胚胎早期死亡[1,2 ] 。人类ＱＫ基因尚未克隆 ,为此。我们通过同源序列分析方法以小鼠的ＱＫ 1～ 7基因的编码区序列作为信息探针 ,成功地克隆了人类ＱＫ的 6种剪接型。一、材料和方法1 信息分析方法 ;信息分析方法在ＳＵＮ工作站采用ＧＣＧ软件包进行 ,根据大规模测序提供…     

人类新基因eola1全长cDNA序列的克隆

人类基因组计划完成后 ,功能基因和蛋白质组的研究成为当今生命科学研究的热点[1] 。克隆差异表达基因对于深入理解基因功能及阐明重大疾病的发病机制都有极其重要的意义。本文报道了我们最近应用抑制消减杂交 (ＳＳＨ) [2 ] 和ｃＤＮＡ末端扩增技术 (ＲＡＣＥ) [3 ] 从内毒素 (ＬＰＳ)刺激后人脐静脉内皮细胞中克隆一个人类新基因ｅｏｌａ1全长ｃＤＮＡ序列。1　材料和方法1.1　细胞培养和ＬＰＳ处理　　培养人脐静脉内皮细胞系ＥＣＶ3 0 4,分成ＬＰＳ处理组和对照组。ＬＰＳ处理系在培养基中加入ＬＰＳ(0 111∶Ｂ4,10 0ｎｇ/ｍｌ)刺激6ｈ…     

胰岛素基因增强子结合蛋白1基因与糖尿病及肥胖的研究进展

转录因子对于胰岛B细胞的分化和胰岛素的分泌具有重要的调控作用.迄今为止,国际上已经确定的6种青少年成人发病型糖尿病(MODY)基因中,5种为转录因子,分别为肝细胞核因子(HNF)-4α/MODY1、HNF-1α/MODY3、胰岛素启动子因子(IPF)-1/MODY4、HNF-1β/MODY5和神经分化因子1(NeuroD-1)/β细胞E盒转录液活因子2(BETA-2)/MODY6[1],因此确立了转录因子在单基因突变型、呈常染色体显性遗传的MODY中引发致病的重要性.     

家族性急性髓系白血病相关新基因ELF2C cDNA全长的克隆

目的克隆家族性急性髓系白血病相关新基因全长,在分子水平上探讨急性白血病发生发展的机制。方法以构建的家族性急性髓系白血病抑制性？肖减性文库中1个有差异表达的新基因EST序列（zywb4）为基础,综合应用电子克隆和cDNA末端快速扩增法（RACE）技术克隆家族性急性髓系白血病相关新基因的全长cDNA。结果获得家族性急性髓系白血病差异表达新基因ELF2C的全长cDNA和432bp的开放阅读框（ORF）,Blast检索为一功能未知基因,被GenBank收录,注册号为DQ359746。结论成功获得一个家族性急性髓系白血病相关新基因ELF2CcDNA全长,为进一步研究其功能提供了依据。     

HP8: The cDNA clone of a novel member of C/EBP gene family

ＨＰ８：ＴｈｅｃＤＮＡｃｌｏｎｅｏｆａｎｏｖｅｌｍｅｍｂｅｒｏｆＣ／ＥＢＰｇｅｎｅｆａｍｉｌｙ￥（徐砺新）（万大方）（刘彦仿）（李宏年）（张萍萍）（隋延仿）（顾健人）ＸｕＬｉｘｉｎ，ＷａｎＤａｆａｎｇ，ＬｉｕＹａｎｆａｎｇ，ＬｉＨｏｎｇｎｉａｎ，Ｚｈ...     

cDNA microarray in isolation of novel differentially expressed genes related to human glioma and clone of a novel full-length gene

Background This investigation was undertaken to obtain differentially expressed genes related to human glioma using cDNA microarray and the characterization of one novel full-length gene. Methods Total RNA was extracted from human glioma tissues and normal brain tissues, and mRNA was used to make probes. After hybridization and washing, the results were scanned using a computer system. The gene named 681F05 clone was an expressed gene to human glioma through four-time hybridization and scanning. Subsequently northern blot analysis was performed by northern blot, 5‘RACE and bioinformatics. Results Fifteen differentially expressed genes to human glioma were obtained through four-time hybridization and scanning. Northern blot analysis confirmed that 681F05 clone was low-expressed in human brain tissues and over-expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that 681F05 clone is two cDNA clones encoding two novel proteins that are highly identified to the cyclophilin isoform 10 of C. Elgans, respectively. Sequence analysis revealed the two cDNA clones are two different splicing variants of a novel cycophilin-like gene (PPIL3a and PPIL3b). Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human PPIL3 may be correlated with the formation of human glioma.     

常染色体隐性遗传Alport综合征基因突变分析方法研究

目的 通过一例常染色体隐性遗传型Alport综合征患者家系的基因突变分析,探讨常染色体隐性遗传Alport综合征的基因诊断方法.方法 提取该患者外周血细胞的基因组DNA,应用基因组DNA PCR测序法对COL4A3和COL4A4基因各外显子进行突变筛查.对于基因组DNA序列异常者,从外周血细胞和EB病毒转染的细胞中提取总RNA,应用cDNA RT-PCR测序方法分析突变.同时在家系和正常人群中进行验证.结果 使用基因组DNA样本经PCR直接测序法检测到COL4A3基因上两个致病性新突变(IVS 39+1 G＞A剪接突变和c.1729-1737 de19bp缺失突变);经RT-PCR测序方法验证其与基因组DNA样本中检测到的突变一致,并证实剪接突变产生异常的转录产物.结论 基因组DNA PCR测序法和cDNA RT-PCR-测序法均检测到Alport综合征患者COL4A3的致病突变,两种方法结果一致;cDNA RT-PCR测序法因为其相对快捷,并且可以了解突变转录情况而优于经典的基因组筛查突变方法.     

中国北方一马凡氏综合征家系致病基因突变研究

目的研究确定我国北方一马凡氏综合征(MFS)家系致病基因突变位点。方法对马凡氏综合征一家系进行临床研究和系谱分析。采集家系中3例患者和3例健康成员的静脉血,提取基因组DNA。应用聚合酶链反应(PCR)扩增马凡氏综合征致病基因原纤维蛋白1(FBN1)基因外显子及外显子-内含子接头处,直接测序确定致病的基因突变。结果马凡氏综合征患者的FBN1基因外显子6发生了错义突变,cDNA 640位的鸟嘌呤被腺嘌呤取代(G640A);对应的甘氨酸改变为丝氨酸。突变后EagⅠ内切酶位点消失。该突变在家系中表现为与疾病共分离。结论 FBN1基因突变G640A是该马凡氏综合征家系的致病基因突变。     

Schwannomatosis: a new member of neurofibromatosis family

Background Schwannomatosis is a recently recognized peripheral nerve polyneoplasm with clinical characteristics and a genetic background that differ from those of neurofibromatosis 2 (NF2).The diagnost...     

Schwannomatosis: a new member of neurofibromatosis family

Background Schwannomatosis is a recently recognized peripheral nerve polyneoplasm with clinical characteristics and a genetic background that differ from those of neurofibromatosis 2 (NF2). The diagnostic and treatment criteria of this rare disorder are herein discussed.

Methods The data of 180 patients who underwent operations for benign schwannomas from 2003 to 2012 in our center were reviewed. Eight of them were classified as schwannomatosis according to the diagnostic criteria suggested by MacCollin. The demographic characteristics were documented and compared between the two groups of patients. The patients’ clinical presentations, imaging characteristics, histological features, and treatment results were retrospectively investigated and summarized.

Results Of the 180 cases of benign schwannomas we reviewed this time, eight patients presented with schwannomatosis (4.44%). The mean age of the two groups was not significantly different (40.0 vs. 44.7 years, t=0.88, P=0.378). However, schwannnomatosis seems to more generally occur in females (75% vs. 48% were females, P=0.162), although the difference was not statistically significant. The initial main symptom was pain. The neurological examination was otherwise normal. Magnetic resonance imaging (MRI) revealed multiple discrete, well-defined round, or oval lesions distributed along the course of the peripheral nerves in the extremities with low-to-intermediate signal intensity on T1-weighted images and high-signal intensity on T2-weighted images. Vestibular schwannomas were excluded in four patients by cranial MRI. The lesions in all patients were resected and were pathologically proven to be schwannomas. The average follow-up period was 26 months. Six individuals obtained a good result without symptoms or function loss.

Conclusions Schwannomatosis is characterized by the development of multiple schwannomas without evidence of the vestibular tumors that are diagnostic for NF2. It commonly occurs in middle-aged females. It has similar demographic features to solitary benign schwannoma. Surgical resection always results in a good outcome.

     

F-Box蛋白家族新成员FBXO30基因的融合表达

采用非定向克隆技术将FBXO3 0 (F boxonlyprotein 3 0 )基因编码区cDNA序列克隆到哺乳类融合表达载体pEGFP C2中 ,通过脂质体转染 ,将pEGFP C2 /FBX0 3 0导入NIH 3T3细胞株 ,并在荧光显微镜下观察融合蛋白的表达。采用定向克隆技术将FBXO3 0基因编码区cDNA序列克隆到表达载体pGEX 4T 2中 ,转化大肠杆菌。结果显示FBXO3 0蛋白主要存在于胞浆中。十二烷基硫酸钠 聚丙烯酰胺凝胶电泳和免疫印迹均检测到一条约 6 5 5kD左右的新增蛋白泳带。此外 ,还用谷胱甘肽Sepharose 4B亲和层析柱获得了纯化的该融合表达产物。FBXO3 0蛋白为胞浆蛋白 ,以可溶性的形式存在 ,它能在原核表达体系中稳定表达 ,这对制备其特异性抗体和从蛋白水平研究其功能均具有十分重要的意义