Distribution of factor VIII mRNA and antigen in human liver and other tissues

The cellular site of synthesis of factor VIII (FVIII:C; anti-haemophilic factor) has long been sought. Previous studies suggested the liver as a major site of synthesis, but extrahepatic sources such as spleen and lung have been implicated. Using an immunoradiometric assay (IRMA), we recently localized factor VIII antigen (FVIII:Ag, formerly FVIII:CAg), to whole perfused guinea pig liver and spleen, and to isolated hepatocytes, with lesser or trace amounts in other tissues. Using an immunohistological technique, Stel et al. detected FVIII:Ag in normal human liver sinusoidal endothelial cells, while Exner et al. detected FVIII:Ag by IRMA in extracts of human lymph nodes, lung, liver and spleen. The localization of antigen in tissues does not, however, distinguish sites of factor VIII synthesis from those of storage, and such experiments are subject to misinterpretation due to entrapment of plasma factor VIII in tissues. The recent cloning of the human factor VIII gene provides hybridization probes for the detection of factor VIII messenger RNA in cells, thus directly determining sites of synthesis. During complementary DNA cloning, we detected factor VIII mRNA in liver, and it has been localized by others in liver and placenta and in liver and kidney. In the present study, we detected factor VIII mRNA in isolated human hepatocytes, in spleen and in numerous tissues including lymph nodes and kidney, but not in white blood cells or cultured endothelial cells. We also found that the factor VIII, factor VII, factor IX and protein C antigens in liver are predominantly localized in hepatocytes, while very little von Willebrand factor antigen (vWF:Ag, formerly FVIIIR Ag) is detectable in this organ.     

50例中国人IX因子基因的限制性片段长度多态性

用IX因子基因内探针F9(VⅢ)对TaqI,BamHI和EcoRI酶切的50例中国人基因组DNA进行杂交分析。结果表明,所有个体经TaqI酶切的杂交片段为4.5kb和1.8kb,BamHI和EcoRI酶切的杂交片段分别为23kb和5.0 kb。基因组DNA样本中未发现限制性片段长度多态性(RFLP),这与欧美国家的民族群体中存在着IX因子基因内TaqI和BamHI的RFLP的结论不同。造成不同种族间DNA水平差异的原因,很可能与长期在不同地理环境中的进化适应有关。     

Genetic mapping and diagnosis of haemophilia A achieved through a BclI polymorphism in the factor VIII gene

Haemophilia A is the most common inherited bleeding disorder in man, affecting approximately 1 male in 10,000. The disease is caused by a deficiency in the gene for factor VIII, a component of the intrinsic coagulation pathway. Due to the broad range of clotting activity in normal and heterozygous females, it is often difficult to confirm the status of women at risk for carrying the disease. A genetic marker in the form of a restriction fragment length polymorphism (RFLP) within or tightly linked to the factor VIII gene would serve as a tag for the haemophilia gene, thus allowing both accurate carrier detection and improved, earlier prenatal diagnosis by chorionic villi sampling. The recent isolation of the factor VIII gene has allowed a search for RFLPs within the gene, and we report here the identification of a common polymorphism within the factor VIII gene, revealed by the restriction enzyme BclI, which can be used diagnostically in about 42% of all families. Although the disease haemophilia A has been mapped to the distal portion of Xq, the BclI RFLP makes possible higher-resolution genetic linkage mapping with respect to other polymorphic markers on this portion of the X chromosome. We have established close linkage of the factor VIII gene to several useful RFLP markers, including the highly informative marker St14. These markers should also be useful for prenatal diagnosis of haemophilia A and for detection of its carriers.     

Cloning, sequencing and expression of a swine IgG H chain gene inE. coli

A DNA fragment about 1.5 kb has been isolated from spleen of adult Chinese swine by RT-PCR. The DNA fragment encodes immunoglobulin IgG H chain gene. Sequencing analysis showed that the DNA fragment is 1 425 bp long, complete CDS. The C region of the gene has been classified as Subclass Ig γ3, and is the same as reported by Sun et al., but V region of the present gene is 42 bp less by comparison. The gene has been ligated into expression vector pET-3b (NSEB)( - ). A protein about 52 ku has been expressed in E. coli with an expression level of about 21 % .     

短柄五加(Acanthopanax brachypus)rbcL基因的结构分析

克隆了含完整短柄五加rbcL基因的3.2kb EcoRI片段,测定了该基因的核苷酸序列.所测核苷酸序列总长度为1924bp,其中编码区1428bp,编码475个氨基酸的蛋白质.测定的基因5’上游区共278bp,包含原核性质-35区(TTGCGC),-10区(TACAAT)及类似真核的TATA box元件(TATATA).5’前导区长194bp,其中SD序列为GGAGG,紧邻起始密码子上游.测定的3’下游区共218bp,含2个相邻的转录后可形成茎环结构的反向重复序列.短柄五加rbcL基因编码区推导的氨基酸序列与烟草、菠菜、豌豆、苜蓿、玉米、水稻、松树、地钱、衣藻和Anacystis的同源性分别为93.5％、94.11％、94.53％、94.74％、89.68％、92.21％、92.21％、92.63％、87.58％和80.84％.本文还对不同植物rbcL基因的启动区及部分5’和3’非编码区进行了比较分析.     

Detection and sequence of mutations in the factor VIII gene of haemophiliacs

The most common inherited bleeding disorder in man, haemophilia A, is caused by defect in factor VIII, a component in the blood coagulation pathway. The X-chromosome-linked disease almost certainly stems from a heterogeneous collection of genetic lesions. Because, without proper treatment, haemophilia can be a fatal disease, new mutations are necessary to account for its constant frequency in the population. In addition, haemophilia A displays a wide range of severity, and some 15% of haemophiliacs generate high levels of antibodies against factor VIII ('inhibitor patients'). The present work elucidates the molecular genetic basis of haemophilia in some individuals. Using the recently cloned factor VIII gene as a probe, we have identified two different nonsense point mutations in the factor VIII gene of haemophiliacs, as well as two different partial deletions of the gene. Our survey of 92 haemophiliacs indicates no firm correlation between antibody (inhibitor) production and gross gene defects.     

Expression of the intact C<Subscript>4</Subscript> type<Emphasis Type="Italic">pepc</Emphasis> gene cloned from maize in transgenic winter wheat

Maize intact C4-pepc gene was amplified through LA-PCR and successfully sub-cloned into modified vector pGreen0029 to form a stable expression construct named as pBAC214 (12 kb), which contains CaMV 35S promoter driven bar gene as selection marker. Comparing the cloned DNA sequences (6.7 kb) with published maize C4-pepc gene (GenBank accession E17154) sequences, the identity of DNA sequence alignment is 98.96%. There are only 49 differences between these two intact DNA sequences, of which 13 occur in the region of promoter, 18 in introns, and 18 in exons. The homology of mRNA sequence alignment is 99.38%, and the putative amino acids sequence identity is 99.38%. There are only 15 differences between these two mRNA, and these differences bring 4 sites mutant on the putative amino acids of PEPC protein. Through biolistic bombardment of PDS1000/He system, expression vector pBAC214 has been transformed into winter wheat. Southern blotting results show that the intact C4-pepc gene has been integrated into genome of winter wheat. SDS-PAGE analysis of leaf soluble protein in transgenic wheat showed that the intact C4opepc gene was well transcribed, spliced and translated as in maize. The enzyme activity of leaf PEPC in transgenic wheat has been detected. The activities of leaf PEPC increased over 3-5 times in some transgenic plants. The data of photosynthesis rate and transpiration rate of transgenic wheat flag leaves showed that the C4-pepc gene can increase the photosynthesis rate and transpiration rate of transgenic wheat.     

BMP7基因真核表达载体的构建及荷瘤小鼠制备

目的克隆BMP7基因及构建BMP7基因真核表达载体,稳定转染于H22细胞系,制备荷瘤小鼠。方法应用RT-PCR技术从人胚肾细胞系293T细胞总RNA中成功扩增出1 293 bp包含BMP7基因编码区的cDNA序列,在上下游加上BamHI及HindⅢ双酶切位点后形成带酶切位点的BMP7基因,然后连入含BaraH I及HindⅢ双酶切位点的pEGFP-C1表达质粒上,构建BMP7基因的重组真核表达质粒pEGFP-C1-BMP7。脂质体介导方法转染细胞,RT-PCR、western-blot方法检测其mRNA及蛋白表达,皮下注射制备荷瘤小鼠。结果扩增的cDNA经测序比较与基因文库完全一致;BamHI及HindⅢ双酶切后,质粒形成约4.7 kb和约1.3 kb两条带,与理论计算值完全一致。注射转染BMP7基因后H22细胞小鼠瘤体明显增大。结论成功克隆BMP7基因及构建BMP7基因的真核表达载体。BMP7基因高表达可能是细胞癌变的原因之一。     

Cloning of a novel gene encoding human thioredoxin-like protein

mRNA differential display (DDRT-PCR) has been used to analyze different human fetal brain tissues of different developmental stages (13- and 33-week). According to the sequence of one EST obtained in this assay, a pair of primers have been designed to screen the arrayed human fetal brain cDNA library. A1 .2-kb cDNA clone has been found. This cDNA consists of an 867 bp open reading frame, a 132 bp 5' untranslated sequence and a 209 bp 3' untranslated sequence with a typical polyadenylation signal. The coding region predicts a protein of 289 amino acids. Its N-terminal of 105 residues is highly homologous to human thioredoxin, while no homology has been found in the databases with its C-terminal of 184 residues. Its N-terminal region also contains the conserved active site sequence CGPC (Cys-Gly-Pro-Cys) of thioredoxin. It was named human Thioredoxin-like gene (hTRXL).     

Expression of active human factor VIII from recombinant DNA clones

DNA clones encoding the complete 2,351 amino acid sequence for human factor VIII have been isolated and used to produce biologically active factor VIII in cultured mammalian cells. The recombinant protein corrects the clotting time of plasma from haemophiliacs and has many of the biochemical and immunological characteristics of serum-derived factor VIII.     

Ultrastructural localization of factor VIII procoagulant antigen in human liver hepatocytes

Factor VIII is generally believed to circulate in blood as a multimeric complex of two glycoproteins which are physiologically and immunologically distinct. One component of the factor VIII complex is factor VIII procoagulant activity (FVIII:C) which is associated with factor VIII/procoagulant antigen (FVIII:Ag, formerly FVIII/CAg). The second, larger unit of the complex is factor VIII/von Willebrand factor (vWF:Ag, formerly factor VIII-related antigen or FVIIIRAg). FVIII:C has anti-haemophilic activity and is defective or deficient in patients with classical haemophilia, and vWF:Ag is absent in patients with von Willebrand disease. FVIII:Ag was demonstrated recently in endothelial cells lining hepatic sinusoids, by using immunoperoxidase staining and light microscopy, whereas biochemical data had indicated its presence predominantly in the hepatocyte fractions and in lesser amounts in endothelial cells. Moreover, recent hybridization experiments detected FVIII:C messenger RNA in liver and kidney tissues. Despite several efforts, the cells responsible for FVIII:C synthesis have not been unequivocally identified. Here we use protein A-gold complex labelling to demonstrate the ultrastructural localization of FVIII:C in human liver cells; the results indicate that hepatocytes may synthesize FVIII:Ag.     

黑曲霉β-葡萄糖苷酶基因的分离

以黑曲霉基因组DNA、mRNA为模板,利用种属相似性设计了一对引物,采用PCR与RT-PCR技术得到β-葡萄糖苷酶基因的DNA和cDNA全序列.测序表明:克隆得到的DNA序列全长2 924 bp,cDNA序列全长2 583 bp,编码860个氨基酸.编码序列推导的蛋白质序列相似性分析显示:完整蛋白质序列同源性高达97%～99%.该研究为β-葡萄糖苷酶基因的遗传转化及应用 奠定了基础.     

黄瓜全雌性状相关的RAPD分子标记筛选

用CTAB法提取黄瓜总基因组DNA，采用RAPD技术探寻与黄瓜全雌性相关的分子标记.共筛选39条随机引物，其中有12条引物显示多态性.进一步筛选表明：S10引物能在全雌系黄瓜中扩增出一条约2000 bp的稳定、清晰的特异条带，而在雌雄同株的单株中不存在.经回收、克隆并测序，获得全长2003 bp的序列.在NCBI上进行Blast分析，表明为新发现序列，含有编码62个氨基酸的开放阅读框.其编码短肽的功能尚不确定.     

Structure of the C2 domain of human factor VIII at 1.5 A resolution

Human factor VIII is a plasma glycoprotein that has a critical role in blood coagulation. Factor VIII circulates as a complex with von Willebrand factor. After cleavage by thrombin, factor VIIIa associates with factor IXa at the surface of activated platelets or endothelial cells. This complex activates factor X (refs 6, 7), which in turn converts prothrombin to thrombin in the presence of factor Va (refs 8, 9). The carboxyl-terminal C2 domain of factor VIII contains sites that are essential for its binding to von Willebrand factor and to negatively charged phospholipid surfaces. Here we report the structure of human factor VIII C2 domain at 1.5 A resolution. The structure reveals a beta-sandwich core, from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues. Behind the hydrophobic surface lies a ring of positively charged residues. This motif suggests a mechanism for membrane binding involving both hydrophobic and electrostatic interactions. The structure explains, in part, mutations in the C2 region of factor VIII that lead to bleeding disorders in haemophilia A.     

Linkage of human apolipoproteins A-I and C-III genes

It has recently been suggested that polymorphisms in the human apolipoprotein A-I (apo A-I) gene locus may be related to the development of premature atherosclerosis and hypertriglyceridaemia. To understand if and how these polymorphisms affect apo A-I gene expression, we studied the genomic sequences flanking the apo A-I gene. Here we show the presence of another apolipoprotein gene, apolipoprotein C-III (apo C-III), approximately 2.6 kilobases (kb) downstream of the 3' end of the apo A-I gene. We also show that the apo A-I and apo C-III genes are convergently transcribed and that a polymorphism previously reported to be associated with hypertriglyceridaemia may be due to a single base pair substitution in the 3'-noncoding region of apo C-III mRNA.     

一种优化的mRNA差异显示技术分析白刺盐碱胁迫表达相关基因研究

mRNA差别显示PCR技术是分离、检测差异基因表达的有力工具 我们研究了存在的可能影响DDRT-PCR研究结果的因素 通过改变DDRT-PCR方法中的引物、PCR反应中的退火温度等条件,得到了一种优化的DDRT-PCR技术,运用该项技术研究了白刺盐碱胁迫相关基因的差异表达 结果发现,运用这种方法得到的差异表达基因片段假阳性率较低,且差别条带较长,多为800bp以上,克服了DDRT-PCR技术中的主要问题,为更广泛地运用该项技术奠定了基础