Molecular cloning of cDNA for human von Willebrand factor: authentication by a new method.
We have identified a 2.4 kb partial cDNA clone (pDL34) for human von Willebrand factor (vWf) mRNA. pDL34 was selected by screening an endothelial cell cDNA library with a radiolabeled reverse transcript of mRNA obtained by specific immunoisolation of vWf polysomes from endothelial cells. pDL34 selectively hybridized to an endothelial cell-associated 9.5 kb mRNA. To confirm its identity, SP6 RNA polymerase was used to generate in vitro transcripts of the cDNA. This synthetic RNA, truncated at its 5' end, directed the synthesis of several unique polypeptides in rabbit reticulocyte lysates. These polypeptides were immunoprecipitated by polyclonal and monoclonal anti-vWf antibodies. These results indicate that pDL34 contains an authentic partial copy of vWf mRNA. In vitro transcription of partial cDNA clones and translation of the resulting RNAs may be a useful general method of verifying the identity of various cDNA clones in circumstances where antibodies are available and protein sequence is not.
D000911:Antibodies, Monoclonal; D001779:Blood Coagulation Factors / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA; D004727:Endothelium / Q000032:analysis / Q000166:cytology; D006801:Humans; D010957:Plasmids; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic; D014841:von Willebrand Factor / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D001779:Blood Coagulation Factors; D012333:RNA, Messenger; D014841:von Willebrand Factor; D004247:DNA