Characterization of a monoclonal antibody and a cDNA for polyubiquitin of Amoeba proteus.
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 20Ser and 55Thr residues were replaced with Gly and Ser, respectively. The 28Ala 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.
Lee
S Y
SY
Department of Biology Education, Seoul National University, Korea.
Kim
H J
HJ
Yoo
S Y
SY
Ahn
T I
TI
D000595:Amino Acid Sequence; D000656:Amoeba / Q000235:genetics* / Q000276:immunology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000913:Antibodies, Protozoan / Q000276:immunology*; D000918:Antibody Specificity; D000953:Antigens, Protozoan / Q000235:genetics* / Q000276:immunology; D001704:Biopolymers / Q000235:genetics* / Q000276:immunology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D016054:DNA, Protozoan; D005260:Female; D018869:Heat-Shock Response; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D025821:Polyubiquitin; D014452:Ubiquitins / Q000235:genetics* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D001704:Biopolymers; D018076:DNA, Complementary; D016054:DNA, Protozoan; D014452:Ubiquitins; D025821:Polyubiquitin