A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase.
A hnRNP-free nuclear matrix prepared from chicken MSB-1 cells was used to raise monoclonal antibodies. The monoclonal antibodies 2H3 and 3B7 showed identical non-homogeneous immunofluorescence staining patterns of nuclei in MSB-1 cells and chicken embryonic fibroblasts. In a synchronized culture of MSB-1 cells, the immunoreactivity of nuclei with 2H3, but not with 3B7, antibody decreased markedly during the progression of S phase, but returned to the normal level at the next G1 phase. When cells were treated with Triton X-100 prior to fixation with paraformaldehyde or cells were fixed in methanol, nuclei were reactive with 2H3 antibody throughout the S phase. Both 2H3 and 3B7 antibodies recognized a high molecular mass nuclear antigen (HMNA) of approximately 550 kDa, which was associated with the nuclear matrix. HMNA was resistant to extraction with 0.5 M NaCl from the nuclei at the G1/S boundary but became extractable by the end of S phase. A cDNA clone, pBHB36, containing a partial sequence for HMNA was isolated by immunoscreening as a double positive clone with 2H3 and 3B7 antibodies. The deduced 1,150 residue-long sequence of pBHB36 shows no homology with any molecules in the nucleotide and protein sequence databases, and contains different epitope regions for 2H3 and 3B7 antibodies. A possibility of hydrophobic association of HMNA with nuclear protein(s) during the progression of S phase is discussed.
Shimada
K
K
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981 Japan.
Harata
M
M
Mizuno
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D034961:Antigens, Nuclear; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D002645:Chickens; D018076:DNA, Complementary; D000939:Epitopes / Q000276:immunology; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000276:immunology*; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D012333:RNA, Messenger / Q000235:genetics; D016196:S Phase / Q000276:immunology
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D018076:DNA, Complementary; D000939:Epitopes; D009687:Nuclear Proteins; D012333:RNA, Messenger