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-lengt h cDNA bank, we found a clone producing a remarkably smaller translati on product than that expected from the open reading frame. The cDNA en codes a polyubiquitin, UbC, composed of nine tandem repeats of the ubi quitin unit. The bank contained twelve UbC cDNAs including four full-l ength ones. Sequencing analysis of these clones showed that UbC cDNAs can be classified into two types, UbC1 and UbC2, in each of which ther e 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 [S-35]methionine. The labeled u biquitin could be used as a substrate for thiol ester formation with u biquitin-activating enzyme El or ubiquitin-conjugating enzyme E2.