Construction of cDNA coding for human von Willebrand factor using antibody probes for colony-screening and mapping of the chromosomal gene

Abstract

Von Willebrand Factor (vWF) mRNA was identified in fractionated polyA+ RNA preparations isolated from cultured human endothelial cells. Micro-injection of specific polyA+ RNA fractions in Xenopus laevis oocytes provoked the synthesis of a vWF-like product which could be detected with an immunoradiometric assay relying on Sepharose-linked monoclonal anti-vWF IgG and different radiolabeled monoclonal anti-vWF IgGs. A vWF-mRNA-containing polyA+ RNA preparation served as substrate for a size-selected cDNA-expression library of 60 000 colonies which was screened for the synthesis of antigens related to vWF, using polyclonal anti-vWF IgG and a second antibody conjugated with peroxidase. Eight positive colonies were detected of which two reacted strongly in the enzyme-linked assay. Immunoblotting of bacterial extracts of "expression clones" with a monoclonal anti-vWF IgG revealed polypeptides which size fits within the length of the cDNA insertions. Northern blotting of human endothelial RNA, employing fragments of vWF cDNA as probes, showed specific hybridization with a mRNA of about 9000 nucleotides. DNA-sequence analysis of a vWF-cDNA insertion revealed an open reading frame followed by a translation stopcodon. It is argued that the cDNA insertions encode the carboxy-terminal part of the vWF protein. vWF-cDNA probes were employed to map the von Willebrand factor gene on chromosome 12 using a panel of 35 human-rodent somatic cell hybrids.