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Identification of the Alternative Promoters of the KChIP4 Subfamily

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Acta Biochimica et Biophysica Sinica 2005年第4期37卷 241-247页

作者： xiao-Yun deNG Fang CAI kun xia qian pan zhi-gao long ling-qian wu de-sheng liang he-ping dai zhuo-Hua ZHANG jia-hui xia National Laboratory of Medical Genetics,Central South University,Changsha 410078,China National Laboratory of Medical Genetics Central South University Changsha 410078 China

The subfamily of voltage-dependent potassium (Kv) channel interacting protein 4 (KChIP4)is made up of the auxiliary interacting protein of voltage-dependent potassium *** this study,thestructure of four splicing variants of the human KChIP4 gene was *** of the four isoforms ofthe KChIP4 gene,KChIP4.1,KChIP4.2 and KChIP4.4,were amplified from mouse and human fetal braintissues by reverse transcription-polymerase chain reaction and then *** on the bioinformaticsanalysis of the genomic sequences of the gene,we cloned and characterized two promoter fragments fromthe KChIP4 *** was a 325 bp fragment upstream of the 5’ end of the KChIP4.1 mRNA sequence andthe other was an 818 bp fragment located immediately at the 5’ end of the KChIP4.4 *** of themcan initiate the transcription of the reporter gene in HT1080 cells and Sprague-Dawley (SD) rat fetal brainneurons,and they contain C+G islands,except typical TATA boxes and CAAT *** shows that theKChIP4 gene expression is regulated by an alternative promoter.

关键词： splicing variant alternative promoter transcriptional regulation

Cx31 is assembled and trafficked to cell surface by ER-Golgi pathway and degraded by proteasomal or lysosomal pathways

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Cell Research 2005年第6期15卷 455-464页

作者： Li qiang he*, Fang CAI, Yu LIU, Mu Jun LIU, zhi ping TAN, qian pan, Fai yan FANG, de sheng liang,ling qian wu, zhi gao long, he ping dai, kun xia**, jia hui xia, zhuo Hua ZHANGNational Laboratory of Medical Genetics, Central South University, Changsha 410078, China. National Laboratory of Medical Genetics Central South University Changsha China Institute of Medical Genetics Medical School of Shanghai Jiao Tong University Shanghai China

Gap junctions, consisting of connexins, allow the exchange of small molecules (<1 kD) between adjacent cells, thusproviding a mechanism for synchronizing the responses of groups of cells to environmental stimuli. Conn... 详细信息

Gap junctions, consisting of connexins, allow the exchange of small molecules (he responses of groups of cells to environmental stimuli. Connexin 31 is amember of the connexin family. Mutations on connexin 31 are associated with erythrokeratodermia variabilis, hearingimpairment and peripheral neuropathy. However, the pathological mechanism for connexin 31 mutants in these diseasesare still unknown. In this study, we analyzed the assembly, trafficking and metabolism of connexin 31 in heLa cellsstably expressing connexin 31. Calcein transfer assay showed that calcein transfer was inhibited when cells weretreated with Brefeldin A or cytochalasin D, but not when treated with nocodazole or α-glycyrrhetinic acid, suggestingthat Golgi apparatus and actin filaments, but not microtubules, are crucial to the trafficking and assembly of connexin31, as well as the formation of gap junction intercellular communication by connexin 31. Additionally, α-glycyrrhetinicacid did not effectively inhibit gap junctional intercellular communication formed by connexin 31. Pulse-chase assayrevealed that connexin 31 had a half-life of about 6 h. Moreover, Western blotting and fluorescent staining demonstratedthat in heLa cells stably expressing connexin 31, the amount of connexin 31 was significantly increased after these cellswere treated with proteasomal or lysosomal inhibitors. These findings indicate that connexin 31 was rapidly renewed,and possibly degraded by both proteasomal and lysosomal pathways.

关键词： connexin Cx31 assembly trafficking metabolism.

Rapid genetic diagnosis and prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography

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Chinese Medical Journal 2006年第14期119卷 1222-1225页

作者： zhu hai-yan wu ling-qian pan qian TANG Bei-sha liang de-sheng long zhi-gao dai he-ping xia kun xia jia-hui National Laboratory of Medical Genetics Xiangya Hospital Central South University Changsha 410078 China department of Neurology Xiangya Hospital Central South University Changsha 410078 China

Spinal muscular atrophy （SMA） is a common Pautosomal recessive neuromuscular disorder （1in 6000 to 10 000 births） caused by mutations in the SMN1 gene at 5q13. More than 90%-98% of SMA patients show homozygous deletion of SMN1, which has proved to be useful in the diagnosis of SMA. But it is hampered because of the existence of a highly homologous gene, SMN2. Based on nucleotide mismatches between SMN1 and SMN2, the following two DNA tests are usually performed： single-strand conformational polymorphism （SSCP） and polymerase chain reaction （PCR） followed by a restriction enzyme *** this study we developed a new method for rapid genetic diagnosis of SMA by denaturing high-performance liquid chromatography （DHPLC）, which is based on different retention of homoduplexes and heteroduplexes in detecting the homozygous deletion of SMN1. Both genetic and prenatal diagnoses were performed successfully for a SMA family by DHPLC, which was confirmed as a rapid and effective technique for detecting the deletion of SMN1.

关键词： spinal muscular atrophy SMA SMN1 SMN2 DHPLC, gene diagnosis prenatal diagnosis

Intracellular Distribution,Assembly and Effect of Disease-associated Connexin 31 Mutants in heLa Cells

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Acta Biochimica et Biophysica Sinica 2005年第8期37卷 547-554页

作者： Li-qiang he~& Yu LIU Fang CAI zhi-ping TAN qian pan de-sheng liang zhi-gao long ling-qian wu liang-Qun HUANG he-ping dai kun xia~* jia-hui xia zhuo-Hua ZHANG National Laboratory of Medical Genetics,Central South University,Changsha 410078,China 24#,Lane 1400,West Beijing Road,Medical Genetics Institute of Shanghai jiaotong University.Shanghai,China National Laboratory of Medical Genetics Central South UniversityChangsha 410078 China

Mutations in connexin 31(Cx31)are associated with erythrokeratodermia variabilis(EKV),hearing impairment and peripheral neuropathy;however,the pathological mechanism of Cx31 mutantsremains *** study analyzed 11 disease-associated Cx31 variants and one non-disease-associatedCx31 variant and compared their intracellular distribution and assembly in heLa cells and their effect onthese *** fluorescent localization assay showed no gap junction plaque formation in the cells expressingthe recessive EKV-associated mutant(L34P)and four hearing impairment-associated mutants(66de1D,141dell,R180X and E183K),significantly reduced plaque formation in the cells with five EKV-associateddominant mutants(G12R,G12D,R42P,C86S and F137L)and no obvious change in the cells with twoother mutants(Ⅰ 141V and 652dei 12).Immunoblotting analysis showed that 12 mutated Cx31 s,like WT-Cx31,are able to form the Triton X-100 insoluble complex;however,the quantity of Triton X-100 insolublecomplex in the transfected heLa cells varied among different Cx31 ***,the expression offive EKV-associated dominant mutants(G12R,G12D,R42P,C86S and F137L)caused cell death in ***,the five hearing impairment-associated mutants did not induce cell *** above resultssuggest that disease-associated mutants gain deleterious functions *** summary,disease-associated Cx31 mutants impair the formation of normal gap junctions at different levels,and the diseasesassociated with Cx31 mutations may result from the abnormal assembly,trafficking and metabolism of theCx31 mutants.

关键词： connexin 31 erythrokeratodermia variabilis hearing impairment peripheral neuropathy gap junctional intercellular communication(GJIC)

1例7p21.2→pter部分三体综合征患者及其表型定位研究

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Acta Genetica Sinica 2005年第2期32卷 124-129页

作者：梁德生邬玲仟蔡芳夏昆龙志高潘乾戴和平夏家辉中南大学医学遗传学国家重点实验室长沙410078

采用人类染色体G显带技术、高分辨显带技术、荧光原位杂交技术 (FISH) ,对一例 7p2 1 .2→pter部分三体综合征患者的染色体进行研究 ,并综合文献报道的 1 4例 7p部分三体综合征的临床资料 ,就 7p部分三体综合征患者的核型与表型的相关... 详细信息

采用人类染色体G显带技术、高分辨显带技术、荧光原位杂交技术 (FISH) ,对一例 7p2 1 .2→pter部分三体综合征患者的染色体进行研究 ,并综合文献报道的 1 4例 7p部分三体综合征的临床资料 ,就 7p部分三体综合征患者的核型与表型的相关关系、核型与疾病基因的相关关系等问题进行探讨。通过 1例染色体 7p2 1 .2→pter部分三体患者的染色体分析、表型定位研究和相关文献复习比较 ,探索染色体区带与表型的关系。结果表明 ,7p2 1 .2→p2 2是 7p部分三体综合征的关键片段 ,生殖器畸形、髋关节脱位与 7p1 5重复有关 ,心脏畸形与 7p多个基因作用有关 ,颅缝早闭基因可能位于 7p2 1 .2→p2 1 .3。

关键词： 7p21.2→pter部分三体高分辨显带荧光原位杂交(FISH) 表型定位

基于人类遗传资源收集、保藏及生物信息学技术的遗传病致病基因定位与克隆(英文)

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北京大学学报（医学版） 2006年第1期38卷 21-24页

作者：夏家辉夏昆潘乾龙志高戴和平邬玲仟梁德生蔡芳中南大学医学遗传学国家重点实验室长沙410078

Mapping and identification of disease associated genes will demonstrate the genetic basis for the human geneticdisorders, and provide the fundamental data for elucidation of pathogenesis mechanismof the disorders. Genetic re-sources, including pedigree information, blood sample, and tissues, etc., are essential materials for finding of thelinked locus and gene for certain genetic disease. Genome wide scanning, positional cloning and candidate ap-proach are most widely used methods or strategy, by which, thousands of diseases responsible genes have been i-dentified. National laboratory of medical genetics of China (NLMG) has initiated the study on genetic resourcescollection, mapping and identification of disease associated gene since 1970s, here we summarize the major find-ings in this area achieved by NLMG.

关键词：核型分析染色体图克隆分子基因组人类遗传遗传病致病基因

人源基因载体pHrn介导p53基因在Bel-7402中的抗癌研究(英文)

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生物化学与生物物理进展 2007年第5期34卷 465-470页

作者：薛志刚李剑尹彪张雅坤刘雄昊潘乾龙志高戴和平夏昆邬玲仟梁德生夏家辉中南大学中国医学遗传学国家重点实验室长沙410078

利用人源基因载体结合人巨细胞病毒(CMV)增强子,人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子及肿瘤抑制基因p53构建肿瘤抑制载体,研究它们在肝癌细胞株Bel-7402中的功能.将CMV增强子(CMVe)与肿瘤特异性启动... 详细信息

利用人源基因载体结合人巨细胞病毒(CMV)增强子,人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子及肿瘤抑制基因p53构建肿瘤抑制载体,研究它们在肝癌细胞株Bel-7402中的功能.将CMV增强子(CMVe)与肿瘤特异性启动子hTERTp组合,分别结合GFP及精氨酸型和脯氨酸型肿瘤抑制基因p53,构建GFP表达载体pcheGFP以及p53表达载体pchp53Arg和pchp53Pro;脂质体2000转染肝癌细胞株Bel-7402;24h后利用荧光显微镜以及流式细胞仪检测GFP的表达;RT-PCR、蛋白质印迹检测p53基因的表达;利用Hoechst33258、Annexin V/PI染色,检测p53促使肿瘤细胞的促凋亡情况.结果表明:GFP荧光表达载体在Bel-7402细胞中能有效表达;RT-PCR及蛋白质印迹检测到精氨酸型和脯氨酸型p53基因在Bel-7402细胞内表达;Hoechst33258染色及Annexin V/PI染色检测细胞凋亡实验没有得到明显的细胞凋亡结果.结果表明:实验所构建的肿瘤抑制载体在肝癌细胞Bel-7402能有表达,并能形成外源性p53蛋白,但没有检测到明显的p53促使肝癌细胞调亡的结果.这可能与p53对hTERT的负调控作用有关.

关键词：人源基因载体人端粒酶逆转录酶(hTERT) p53

人源载体介导的minidystrophin-EGFP融合基因在Cos-7细胞中的表达(英文)

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中华医学遗传学杂志 2005年第5期22卷 493-496页

作者：梁羽梁德生薛志刚龙志高邬玲仟潘乾胡艺俏戴和平夏昆夏家辉中南大学中国医学遗传学国家重点实验室长沙410078

目的构建微小肌营养不良蛋白(minidystrophin)和增强绿色荧光蛋白(enhanced greenfluoresce protein, EGFP)融合基因的人源载体,观察该载体在Cos-7细胞中的表达。方法以正常人肌营养不良基因cDNA(GenBank NM 004006)为模板,通过PCR克隆... 详细信息

目的构建微小肌营养不良蛋白(minidystrophin)和增强绿色荧光蛋白(enhanced greenfluoresce protein, EGFP)融合基因的人源载体,观察该载体在Cos-7细胞中的表达。方法以正常人肌营养不良基因cDNA(GenBank NM 004006)为模板,通过PCR克隆的方法构建minidystrophin基因,融合EGFP基因后连接到人源载体pHrneo,大量提取重组质粒,转染Cos-7细胞,通过逆转录聚合酶链反应、荧光显微镜观察等方法检测该载体在细胞内的表达。结果成功构建pHrnDysG载体,转染Cos-7后,逆转录聚合酶链反应可扩增出735 bp特异条带,荧光显微镜观察可见表达蛋白分布于细胞膜上。结论pHrn载体介导的minidystrophin基因可以在真核细胞表达,并被有效地转运至细胞膜,可望用于杜氏肌营养不良基因治疗的研究。

关键词：人源载体融合基因 Cos-7细胞基因表达微小肌营养不良蛋白增强绿色荧光蛋白杜氏肌营养不良

一常染色体显性遗传寻常型鱼鳞病家系致病基因的定位

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中国医学科学院学报 2007年第3期29卷 302-306,I0001页

作者：胡正茂谢志国邬玲仟梁德生朱海燕潘乾龙志高戴和平夏家辉夏昆中南大学湘雅医院医学遗传学国家重点实验室长沙410078 南京市鼓楼医院妇产科南京210008

目的研究一常染色体显性遗传寻常型鱼鳞病家系的致病基因。方法采用基因组扫描方法,利用1号染色体上的微卫星标记对该家系进行连锁分析,然后对候选基因FLG的部分编码区及外显子与内含子交界处进行突变检测。结果在D1S2696得到最大两点连... 详细信息

目的研究一常染色体显性遗传寻常型鱼鳞病家系的致病基因。方法采用基因组扫描方法,利用1号染色体上的微卫星标记对该家系进行连锁分析,然后对候选基因FLG的部分编码区及外显子与内含子交界处进行突变检测。结果在D1S2696得到最大两点连锁LOD值3.46(θ=0),单体型分析将疾病基因定位在D1S2726-D1S305之间约15cM范围内;在FLG基因的外显子非重复序列及部分重复序列未发现与疾病相关的突变。结论该寻常型鱼鳞病家系的致病基因位于D1S2696附近,其致病基因可能是除FLG以外的其他基因。

关键词：寻常型鱼鳞病微卫星标记连锁分析突变分析