Premium
Characterization of a Monoclonal Antibody and a cDNA for Polyubiquitin of Amoeba proteus
Author(s) -
Lee So Young,
Kim Hyun Joon,
Yoo So Yeun,
Ahn Tae In
Publication year - 1998
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.1998.tb05095.x
Subject(s) - amoeba proteus , biology , complementary dna , microbiology and biotechnology , monoclonal antibody , cytoplasm , ubiquitin , antibody , immunofluorescence , amoeba (genus) , tetrahymena pyriformis , biochemistry , tetrahymena , gene , genetics
A monoclonal antibody was obtained that reacts with many different proteins (14‐200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross‐reacted 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 polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well‐conserved 20 Ser and 55 Thr residues were replaced with Gly and Ser. respectively. The 28 Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin‐mRNAs that could be detected by Northern blot 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 ubiquitinated in response to cellular stresses.