人凝血Ⅸ因子cDNA在转基因小鼠内的表达调控

为了研究人凝血IX因子cDNA在转基因小鼠内的表达,特别是顺式调节顺序的存在位置,本文将3种具有不同结构的人凝血IX因子cDNA的重组基因导入离体培养细胞,发现外源的人IX因子cDNA都能表达人IX因子蛋白,进而将它们分别作成转基因小鼠,结果发现在转基因小鼠内都失去了表达特性。这些结果证实了在人凝血IX因子cDNA中存在着决定其在转基因小鼠内表达的顺式调节顺序,同时也排除了这种顺式调节顺序存在于3′端非编码顺序中的可能性。作者再结合一些相关研究的结果,认为其顺式调节顺序可能存在于5′端的非编码顺序中。     

IDENTIFICATION OF PARTIAL MUTATIONAL SITES IN HUMAN MITOCHONDRIAL DNA IN THE CHINESE AND ITS SIGNIFICANCE

A simple method for the identification of mutational sites in human mitochondrial DNA (mtDNA) was described. It was based on the human Cambridge sequence as a relative standard sequence and a single base pair substitution in mtDNA as a unique mutational form. The partial mutational sites can be determined using this method which was characterized by combining the restriction mapping with the analysis for the table of human mtDNA potential mutational sites with rapidity and simplicity. In the meanwhile, six mtDNA mutational sites found in Chinese population were identified by means of this method.     

带有人Ⅸ因子cDNA的反转录病毒载体的构建及其在血友病B患者皮肤成纤维细胞中的高效转移和表达

本文报道对血友病B实施基因治疗的可能性,首先将由SV40早期启动子,小鼠MT-1启动子和反转录病毒LTR启动子控制的Ⅸ因子cDNA构建到反转录病毒载体,然后用电穿孔法将构建的4个反转录病毒载体分别转入一株Amphotropic辅助细胞,PA317细胞,再用一株人纤维肉瘤细胞,HT1080细胞,测定这些辅助细胞的产病毒滴度,可得到2×10~4CFU/ml到5×10~5CFU/ml左右的病毒感染颗粒,用ELISA分别测定转有不同病毒载体的PA317细胞的Ⅸ因子蛋白产量,发现LTR启动子的表达效率最高,Ⅸ因子蛋白的分泌速率可达584ng/10~6细胞/24h,而SV40早期启动子和MT-1启动子的表达效率分别只有它的1/10和1/20,将表达效率最高的反转录病毒载体pXL—Ⅸ 1转入一株取自血友病B患者原代培养的皮肤成纤维细胞后同样能产生较高浓度的Ⅸ因子蛋白,其分泌速率可达549ng/10~6细胞/24h,其中75％以上的Ⅸ因子具有凝血活性,从而达到了首先在体外培养细胞纠正Ⅸ因子基因缺陷的目的,实现了血友病B基因治疗的第一步。     

THE DISTRIBUTION OF P ELEMENTS IN D. melanogaster ALONG THE COAST OF CHINA STUDIED BY DNA HYBRIDIZATION METHOD

The distribution of the P-M system in four local Drosophila melanogaster populations obtained insouthern China has been examined by mating tests. There was no P strain. A HindⅢfragnment ofthe P element has been cloned, named as pPH 0.86, and used as probe in this study. The distribu-tion of the P element itself in thirteen local D. melanogaster populations along the eastern coast ofChina has been studied by the method of DNA hybridization. It is found that all the populations ob-tained from the north of Shanghai carry P elements but few populations from the south of Shanghai carry Pelements. In comparison with the mating test, the method of DNA hybridization is more direct andreliable.The origin and trends of distribution of P elements in China are discussed in this paper.     

HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN CLOTTING FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of f     

中国汉族、维吾尔族、哈萨克族和回族群体线粒体DNA多态性的研究

本文应用线粒体(mtDNA)作为研究工具,对我国汉族、维吾尔族、哈萨克族和回族群体的遗传结构进行了系统分析。通过对四个民族群体mtDNA限制性类型的调查,发现了一批新的类型,其中包括一些只存在于某一种族和民族中的突变类型,表明mtDNA限制酶切片段多态性类型的分布在不同民族和种族中有显著差别。在与灵长类动物比较后发现,现代世界上只有少数东方人种中保留着mtDNA的祖先类型,这为亚洲可能是人类的起源地之一提供了间接证据。我们根据限制性位点差计算出各民族间的遗传距离,绘制成系统树,表明维吾尔族和哈萨克族群体最先汇聚成一支,而汉族与回族汇聚成一支,最后两支再汇聚在一起。同时发现,维吾尔族和哈萨克族群体内部交异比汉族和回族大,从分子水平揭示了这四个民族群体问的相互关系。     

中国人群线粒体DNA部分突变位点的测定和意义

本文报道了一种简易的人体线粒体DNA(mtDNA)突变位点测定方法。这一方法以剑桥顺序为标准顺序。在确定不同限制酶的识别位置及其片段大小后,用不同限制酶对不同个体的酶切图谱作比较。在发现长度突变型后,利用已知限制酶识别顺序,确定人mtDNA剑桥顺序上所具有的潜在性突变位点。根据标准的剑桥顺序和被检酶切片段大小以及潜在性突变情况,估计出被检mtDNA发生突变的确切位置或大概位置。应用这一方法,我们对在中国汉族、维吾尔族、哈萨克族和回族人群中发现的六种 mtDNA变异型的突变位点进行了分析。     

应用DNA分子杂交研究黑腹果蝇P因子在中国沿海的分布

本文通过黑腹果蝇(Drosophila melanogaster)的交配试验研究了我国南部四个地区黑腹果蝇群体中P-M系统的分布,没有发现P品系。同时建立了P因子Hind Ⅲ片段克隆——pPH0.86。以此为探针,用DNA分子杂交法研究了我国沿海13个地区黑腹果蝇群体中P因子的分布,发现上海以北均带有P因子,上海以南则极少有P因子。比较了交配试验和DNA分子杂交两种方法,表明后一方法更为直观可靠。就P因子的传播途径,中国大陆P因子的来源及其传播趋向等作了讨论。     

MITOCHONDRIAL DNA POLYMORPHISM IN CHINESE

Human mitochondrial DNA (mtDNA) restriction endonuclease fragment patterns were analyzed using placenta DNA isolated from 273 individuals representing four different nationalities, the Han, the Uygur, the Kazakh and the Hui populations. Thirty-eight fragment patterns (morphs) were observed with the enzyme ApaI, BamHI, EcoRl, HindIII, HinfI, HhaI, HapII, KpnI, MboI, PstI, PvuII, SacI, ScaL and XhoI. Fourteen new morphs, including some only existing in individual racial and national populations were observed, which indicates that there is a significant difference in the distribution of mtDNA morphs among various national and racial populations. By comparison with the mtDNA sequences in primate species, some mtDNA ancestral morphs were found to be retained in Oriental population today. This result provided indirect evidence that Asia may be one of the human original sources. Genetic distances among four national populations computed and employed in construction of an average linkage tree suggested that the