A nuclear matrix-associated high molecular mass nuclear antigen, HMNA, of chicken and marked decrease of its immunoreactivity during the progression of S phase
Shimada, K; Harata, M; Mizuno, S
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 nonhomogeneous 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 G(1) 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 G(1)/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.