Cloning of Human Polyubiquitin cDNAs and a Ubiquitin-Binding Assay Involving Its In Vitro Translation Product

During large-scale in vitro translation analysis of a human full-length cDNA bank, we found a clone producing a remarkably smaller translation product than that expected from the open reading frame. The cDNA encodes a polyubiquitin, UbC, composed of nine tandem repeats of the ubiquitin unit. The bank contained twelve UbC cDNAs including four full-length ones. Sequencing analysis of these clones showed that UbC cDNAs can be classified into two types, UbC1 and UbC2, in each of which there are six polymorphic nucleotide variations. The present UbC cDNA was in vitro translated in a rabbit reticulocyte or wheat germ extract to produce a free ubiquitin labeled with [36S]methionine. The labeled ubiquitin could be used as a substrate for thiol ester formation with ubiquitinactivating enzyme E1 or ubiquitin-conjugating enzyme E2.