Molecular cloning of a cDNA encoding human antihaemophilic factor

Jj, Toole; Jl, Knopf; Jm, Wozney; La, Sultzman; Jl, Buecker; Dd, Pittman; Rj, Kaufman; Brown, Eugene L.; Shoemaker, Charles B.; Ec, Orr

doi:10.1038/312342a0

Cited by 917 publications

(530 citation statements)

References 30 publications

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“…The F8c probe used to mark the long arm (Xq) telomere is a 1.4-kb EcoRI fragment containing exon 26 of the blood-clotting factor VIII locus (21), which hybridizes exclusively to the locus in Xq28, near the telomere.…”

Section: Methodsmentioning

confidence: 99%

The Barr body is a looped X chromosome formed by telomere association.

Walker

Cargile

Floy

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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We examined Barr bodies formed by isodicentric human X chromosomes in cultured human cells and in mouse-human hybrids using confocal microscopy and DNA probes for centromere and subtelomere regions. At interphase, the two ends of these chromosomes are only a micron apart, indicating that these inactive X chromosomes are in a nonlinear configuration. Additional studies of normal X chromosomes reveal the same telomere association for the inactive X but not for the active X chromosome. This nonlinear configuration is maintained during mitosis and in a murine environment. (4) or in situ hybridization (2) places the Barr body adjacent to the nuclear envelope in 75-80% of interphase cells. Comings (5) suggested that inactive X chromosomes attach randomly to the nuclear membrane, and the multiple Barr bodies in aneuploid cells are widely distributed (6, 7). Nuclear matrix attachment sites are similar for the active and inactive X chromosomes (8). Yet, the configuration of the Barr body has been relatively unexplored. DNA hybridization, in situ (9), has provided a powerful method to examine chromosomes during interphase, revealing an orderly arrangement of chromosomes in the interphase nucleus (10-13) and tissue-specific variation (14, 15). Using such methods to explore the human inactive X chromosome, we find that the Barr body consists of a condensed X chromosome in a nonlinear configuration, with telomeres in close proximity.We examined the Barr body in interphase and mitotic cells using fluorescent probes for centromere and telomere regions of human X chromosomes. In addition to normal X chromosomes, we studied isodicentric X chromosomes (16), which form bipartite Barr bodies (16). Always inactive, they are mirror image duplications with two centromeres (one nonfunctional) and with two identical telomeres (see Fig. 1). The duplicate centromeres as well as common telomeres and their longer length facilitate structural analysis. To compare distance between hybridization signals with relative physical length we examined three isodicentrics, two joined by their long arms (3935 and 7213) and the third attached at the short arms (411). We isolated these dicentric chromosomes from their normal homologue in hybrid cells so that all signals would come from the dicentric X chromosome and to examine the human Barr body in a mouse cell environ. Finally, we simultaneously hybridized centromere and subtelomere probes using differential labels and confocal microscopy. MATERIALS AND METHODSCell Lines. These are characterized in Table 1. The hybrids derived from A9 mouse fibroblasts were selected in hypoxanthine/aminopterin/thymidine medium, back selected in 6-thioguanine to eliminate the active X; to retain the inactive X, the silent HPRT locus was reactivated by 5-azacytidine. Inactive X hybrids derived from tsA1S9T mouse cells were selected directly at 390C for activity of the AJS9T locus at Xpll (17).Preparation of Slides. Interphase cells. Confluent cells in LabTek slide chambers were fixed in methanol/acetic acid (3:1...

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Section: Methodsmentioning

confidence: 99%

The Barr body is a looped X chromosome formed by telomere association.

Walker

Cargile

Floy

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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“…Mutagenesis was performed within the mammalian expression vector pMT 2 (27) containing the FVIII cDNA (pMT 2 VIII). Mutant plasmids were generated through oligonucleotide site-directed mutagenesis by using the PCR as described (28).…”

Section: Methodsmentioning

confidence: 99%

“…FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-aa single-chain glycoprotein of 280 kDa from which a signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (1)(2)(3). Whereas the A and C domains exhibit 35-40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein.…”

mentioning

confidence: 99%

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Pipe

Kaufman

1997

Proc. Natl. Acad. Sci. U.S.A.

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125

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Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690-2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII), necessitating exogenous replacement by either plasma-or recombinant-derived FVIII preparations. FVIII has a domain organization of A1-A2-B-A3-C1-C2 and is synthesized as a 2,351-aa single-chain glycoprotein of 280 kDa from which a signal peptide is cleaved upon translocation into the lumen of the endoplasmic reticulum (1-3). Whereas the A and C domains exhibit 35-40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein. Intracellular proteolytic processing within the B domain after residue Arg-1648 generates an 80-kDa light chain (domains A3-C1-C2) and a heterogeneous-sized heavy chain of 90-200 kDa (domains A1-A2-B). The heavy and light chains are associated as a heterodimer through a divalent metal-ion-dependent linkage between the A1 and A3 domains.In plasma, FVIII circulates in an inacti...

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“…This is probably due to the functional and physiological differences of various regions of the fallopian tube. No MspI sites exist within this region of the F8 gene Toole et al, 1984;Wood et al, 1984); therefore, MspI/HpaII Southern blot methods were not informative for examining methylation patterns at this locus (data not shown).…”

Section: Cell Type-specific Rfmps In Human and Mouse Genesmentioning

confidence: 99%

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

Laprise

Gray

2007

Gene

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Several approaches are used to survey genomic DNA methylation patterns, including Southern blot, PCR, and microarray strategies. All of these methods are based on the use of methylation-sensitive isoschizomer restriction enzyme pairs and/or sodium bisulfite treatment of genomic DNA. They have many limitations, including PCR bias, lack of comprehensive assessment of methylated sites, laborintensive protocols, and/or the need for expensive equipment. Since the presence of 5-methylcytosine alters the melting properties of DNA molecules, denaturing gradient gel blots (DGG blots), a gene scanning technique which detects differences in DNA fragments based on differential melting behavior, were used to examine genomic modification patterns in normal tissues. Variations in melting behavior, observed as restriction fragment melting polymorphisms (RFMPs), were detected in various tissues from single individuals in all human and mouse genes tested, suggesting the presence of widespread differential cell type-specific DNA modification. Additional DGG blot experiments comparing genomic DNA to unmethylated cloned DNA suggested that the melting variants were most likely caused by DNA methylation differences. The results suggest that the use of DGG blots can provide a comprehensive and rapid method for comparing complex in vivo DNA modification patterns in normal adult somatic cells.

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Molecular cloning of a cDNA encoding human antihaemophilic factor

Cited by 917 publications

References 30 publications

The Barr body is a looped X chromosome formed by telomere association.

The Barr body is a looped X chromosome formed by telomere association.

Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

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