Characterization of the human factor VIII gene

The complete 186,000 base-pair (bp) human factor VIII gene has been isolated and consists of 26 exons ranging in size from 69 to 3,106 bp and introns as large as 32.4 kilobases (kb). Nine kb of mRNA and protein-coding DNA has been sequenced and the mRNA termini have been mapped. The relationship between internal duplications in factor VIII and evolution of the gene is discussed.     

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.     

Nucleotide sequence of the gene encoding human atrial natriuretic factor precursor

The mammalian atrium is an endocrine organ that may be involved in the control of blood pressure and extracellular fluid volume. A series of peptides, which seem to be associated with atrium-specific secretory granules, have potent natriuretic, diuretic and smooth muscle relaxant activities. Sequence determination of several of these peptides, which range from 21 to 126 amino acids long, shows that they form a family, derived from a common precursor. Rat and human complementary DNAs that encode the precursor to the various peptides, collectively called atrial natriuretic factors (ANFs), have been cloned. Nucleotide sequencing showed that the ANFs are located at the C-terminus of a polypeptide of relative molecular mass 13,000. We describe here the isolation and characterization of the corresponding human gene. Two introns interrupt the gene; one is located in the region coding for the N-terminus of the precursor and the other separates the codon for the C-terminal tyrosine from the rest of the peptide.     

Genetic engineering of factor VIII

Genetic manipulation of mammalian cells has provided a means of producing unlimited quantities of a high purity, virus-free preparation of factor VIII--the most complex protein manufactured through rDNA technology to date.     

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.     

The effect of a secretion-enhanced heavy chain on improving intein-based dual-vector co-delivery of a full-length factor VIII gene

Treatment of hemophilia A by gene therapy is adversely affected by inefficient FVIII secretion and the large FVIII gene, which is difficult to package in the promising adeno-associated virus (AAV) vectors. Inhibited secretion of FVIII is caused mainly by inefficient secretion of its heavy chain. Previously, we have employed a protein splicing-based dual-vector to co-transfer a B-domain-deleted FVIII (BDD-FVIII) gene, suggesting that the light chain, covalently ligated to a co-expressed heavy chain can improve the secretion of spliced BDD-FVIII. However, its level of secretion was affected by inefficient secretion the heavy chain. Here, we studied the effect of a mutant heavy chain with L303E/F309S substitutions, which enhance FVIII secretion on the heavy chain itself and spliced FVIII when using a protein splicing-based split-delivery of a full-length FVIII gene. Eukaryotic vectors expressing Ssp DnaB intein-fused mutant heavy and light chains were transiently co-transfected into cultured COS-7 cells. A spliced FVIII protein was seen in co-transfected cells by Western blot analysis. The heavy chain was secreted by cells transfected with the mutant heavy chain gene alone at (39±11) ng/mL and this secretion increased to (123±13) ng/mL when cells were co-transfected with the light chain gene, which was greater than the secretion of wild-type heavy chain. The amount of spliced FVIII in the culture supernatant of co-transfected cells was (86±14) ng/mL, with an activity of (0.61±0.08) IU/mL, which was greater than that of wild-type FVIII co-transfected cells. Spliced FVIII and bioactivity were also detected in the combined culture supernatant of cells individually transfected with mutant heavy and light chain gene at a higher level than that of combined wild-type heavy and light chain transfections. This suggested that the heavy chain with improved secretion markedly increased the efficacy of protein splicing-based split delivery of the full-length FVIII gene using a dual-vector. These results encourage the transfer of this technology to an animal model using a dual-AAV vector.     

Human epidermal growth factor receptor cDNA sequence and aberrant expression of the amplified gene in A431 epidermoid carcinoma cells

The complete 1,210-amino acid sequence of the human epidermal growth factor (EGF) receptor precursor, deduced from cDNA clones derived from placental and A431 carcinoma cells, reveals close similarity between the entire predicted v-erb-B mRNA oncogene product and the receptor transmembrane and cytoplasmic domains. A single transmembrane region of 23 amino acids separates the extracellular EGF binding and cytoplasmic domains. The receptor gene is amplified and apparently rearranged in A431 cells, generating a truncated 2.8-kilobase mRNA which encodes only the extracellular EGF binding domain.     

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.     

Parental origin of mutations of the retinoblastoma gene

Retinoblastoma and osteosarcoma arise from cells that have lost both functional copies of the retinoblastoma gene. Using the cloned retinoblastoma gene and other linked polymorphic loci, it is possible to reconstruct the sequential loss of the two homologous gene copies that precedes the development of these tumours. In non-hereditary tumours, the loss of each of the two homologues occurs somatically; in hereditary cases, the initial mutation is in the germline. Recently, Toguchida et al. reported that the paternally derived copy is preferentially the first one to become mutant during the genesis of non-hereditary osteosarcomas. We report here a similar analysis of patients with retinoblastoma in which we find no such predilection for initial somatic mutations. In contrast, when an initial mutation was a new germline mutation, it was derived from the father, a result which is consistent with new germline mutations arising primarily during spermatogenesis.     

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.     

Alteration of voltage-dependence of Shaker potassium channel by mutations in the S4 sequence.

Voltage-dependent potassium, sodium and calcium ion channels may share a common mechanism of activation, in which the conserved S4 sequence acts as the primary voltage sensor. Site-directed mutagenesis of the S4 sequence of the Shaker potassium channel and electrophysiological analysis suggest that voltage-dependent activation involves the S4 sequence but is not solely due to electrostatic interactions.     

人转铁蛋白基因的克隆及序列分析

目的:克隆人转铁蛋白基因并对其编码序列进行分析.方法:以人胎肝cDNA为模板,利用PCR方法克隆人转铁蛋白基因;通过与基因组序列对比分析基因组结构;通过TargetP 1.1和SignalP 3.0预测信号肽;通过Clustal X(1.81)进行蛋白序列联配.结果:PCR扩增了一个长2 160 bp的基因片断,序列分析表明其覆盖了完整编码框,编码由698个氨基酸组成的人转铁蛋白.进一步分析发现人转铁蛋白基因有19个外显子和18个内含子,编码人转铁蛋白N端具有19个氨基酸组成的信号肽序列.人转铁蛋白与猩猩、猴子、兔子和老鼠的转铁蛋白氨基酸相似率分别为94%、91%、78%、73%.生物信息分析表明,人转铁蛋白含有高度保守的参与蛋白二硫键形成的半胱氨酸以及铁离子结合位点,有两个序列较同源的结构域.结论:成功克隆人转铁蛋白基因,人转铁蛋白与其它物种转铁蛋白同源.     

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.     

Prevalence of ras gene mutations in human colorectal cancers

A combination of DNA hybridization analyses and tissue sectioning techniques demonstrate that ras gene mutations occur in over a third of human colorectal cancers, that most of the mutations are at codon 12 of the c-Ki-ras gene and that the mutations usually precede the development of malignancy.     

Nucleotide sequence of the rat skeletal muscle actin gene

The actins constitute a family of highly conserved proteins found in all eukaryotic cells. Their conservation through a very wide range of taxonomic groups and the existence of tissue-specific isoforms make the actin genes very interesting for the study of the evolution of genes and their controlling elements. On the basis of amino acid sequence data, at least six different mammalian actins have been identified (skeletal muscle, cardiac muscle, two smooth muscle actins and the cytoplasmic beta- and gamma-actins). Rat spleen DNA digested by the EcoRI restriction enzyme contains at least 12 different fragments with actin-like sequences but only one which hybridized, in very stringent conditions, with the skeletal muscle cloned cDNA probe. Here we describe the sequence of the actin gene in that fragment. The nucleotide sequence codes for two amino acids, Met-Cys, preceding the known N-terminal Asp of the mature protein. There are five small introns in the coding region and a large intron in the 5'-untranslated region. Comparison of the structure of the rat skeletal muscle actin gene with available data on actin genes from other organisms shows that while the sequenced actin genes from Drosophila and yeast have introns at different locations, introns located at codons specifying amino acids 41, 121, 204 and 267 have been preserved at least from the echinoderm to the vertebrates. A similar analysis has been done by Davidson. An intron at codon 150 is common to a plant actin gene and the skeletal muscle acting gene.     

Organization and sequence studies of the 17-piece chicken conalbumin gene

The conalbumin gene has been cloned and shown to consist of at least 17 exons approximately 60-200 base pairs long. The DNA sequence upstream from the region coding for the 5' end of the mRNA shows similarities with sequences present in homologous positions in other genes. High and low frequency repetitive sequences are found both upstream from the conalbumin gene and within one intron.     

BmNPV半胱氨酸蛋白酶基因的核苷酸序列研究

对家蚕核型多角体病毒苏州株（BmNPVsu）半胺氨酸蛋白酶基因（CP）的序列分析表明，该基因读码框为972个核苷酸，编码323个氨。同源性分析表明，BmNPVsu CP的氨基酸序列与不同来源的木瓜蛋白酶超家庭的CP具有较高的同源性，特别与Trpanosomabrucei的CP具有较高的一致性，达32％。在组织蛋白酶B、H、L、S以及木瓜蛋白酶的36个保守氨基酸残基中有31种出现在BmNPUsu的CP中，BmNPVsuCP同其他杆状病毒CP一样，可看作木瓜蛋白酶超家庭成员。