CHARACTERIZATION OF A MONOCLONAL-ANTIBODY AND A CDNA FOR POLYUBIQUITIN OF AMEBA-PROTEUS

A monoclonal antibody was obtained that reacts with many different pro teins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence m icroscopy we found the antigens to be dispersed throughout the cytopla sm but were more concentrated in the nucleus. The antibody cross-react ed with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages . Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquit in showed the most variations among the nineteen polyubiquitins of oth er organisms compared. The well-conserved (20)Ser and (55)Thr residues were replaced with Gly and Ser, respectively. The (28)Ala residue fou nd in most organisms was replaced with Gin or Glu in the amoeba. Amoeb ae contained two ubiquitin-mRNAs that could be detected by Northern bl ot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiqu itinated in response to cellular stresses.