Proteins recognized by antibodies against isolated cytological heterochromatin from rat liver cells change their localization between cell species and between stages of mitosis (interphase vs metaphase).
Heterochromatin in the cell nucleus seems to concentrate various proteins, such as Drosophila heterochromatin protein 1, which maintain the repressed state of gene expression. However, it still remains obscure how protein composition related to chromatin structure is different between heterochromatin and euchromatin in interphase nuclei. We isolated cytological heterochromatin from sonicated interphase nuclei obtained from rat liver cells and prepared antisera against it. The dense heterochromatic bodies seen in the preparation of intact nuclei were duplicated in a relatively pure form during the preparation of heterochromatin. In the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, differences between the fractions of heterochromatin and euchromatin were noted by their protein composition. Isolated heterochromatin was then digested by DNase after partial digestion with trypsin and its dense structure changed to become highly sensitive to DNase. The prepared antibodies reacted with the heterochromatin region of rat liver cell nuclei and isolated cytological heterochromatin; however, they did not react with euchromatin. Using immunohistochemistry, the antibodies bound to each cell nucleus in all tissues observed; some cell types were distinguished by their differential stainability (e.g. staining in the cytoplasm). Staining of the mitotic cells showed that the proteins recognized by the antibodies were localized in the cytoplasm and, in part, on the chromosomes. Based on the results of molecular cloning from rat liver cDNA library using the antibodies as a probe, it seemed that the antibodies mainly recognized two proteins similar to arginase and general vesicular transport factor p115, respectively. The results obtained from these experiments reveal that some proteins located in the heterochromatin of interphase liver cell nuclei seem to play important roles in condensing a portion of the chromatin structure during interphase and suggest that proteins composing heterochromatin might be changed according to cell types or the stage of the cell cycle.
Hara
M
M
Department of Anatomy, St Marianna University School of Medicine, Kawasaki, Japan. m5hara@marianna-u.ac.jp
Igarashi
J
J
Yamashita
K
K
Iigo
M
M
Yokosuka
M
M
Ohtani-Kaneko
R
R
Hirata
K
K
Herbert
D C
DC
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D022041:Euchromatin; D006570:Heterochromatin / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D007150:Immunohistochemistry; D007399:Interphase; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008297:Male; D008677:Metaphase; D051381:Rats; D017208:Rats, Wistar; D012261:Ribonucleoproteins / Q000032:analysis*; D013045:Species Specificity; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D002843:Chromatin; D022041:Euchromatin; D006570:Heterochromatin; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins; D003851:Deoxyribonucleases; D014357:Trypsin