The human autoantigen MCP1 is required during early stages of DNA replication.
Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed chromosomes throughout mitosis. During interphase, this antigen shows a speckle distribution in the nucleus, excluding the nucleolus. Additionally, MCP1 binds tightly to the scaffold/matrix component of nuclei and isolated chromosomes. In order to determine the in-vivo localization of the antigen, we have expressed MCP1 fused to EGFP in tissue culture cells. The results demonstrate that MCP1 is located in the nucleus during interphase and during mitosis associates tightly to condensed chromosomes. Furthermore, microinjection of specific antibody confirms these results. We have used a specific monoclonal antibody (mAb 402) against MCP1 to assess the function of this antigen during cell cycle progression. HeLa and Ptk-2 cells that were microinjected into the nucleus and/or cytoplasm at G1/S and very early S phase were not able to progress and complete DNA replication. However, injection of mAb 402 at mid or late S phase does not prevent completion of DNA replication and subsequent progression into mitosis. Microinjection of mAb 402 in Ptk-2 cells synchronized in mitosis did not interfere with progression of mitosis and cells divided. Our results suggest that MCP1 is required at the G1/S transition and during early S phase.
Bronze-da-Rocha
E
E
Laboratório de Genética Molecular da Mitose, Instituto de Biologia Molecular e Celular da Universidade do Porto, Portugal. erocha@ibmc.up.pt
Nóvoa
A
A
Cunha
C
C
do Carmo-Fonseca
M
M
Staines
N A
NA
Sunkel
C E
CE
D000818:Animals; D000911:Antibodies, Monoclonal / Q000008:administration & dosage; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism*; D002453:Cell Cycle / Q000187:drug effects; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D006801:Humans; D007399:Interphase / Q000187:drug effects / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D007614:Macropodidae; D008845:Microinjections; D008938:Mitosis / Q000187:drug effects / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016196:S Phase / Q000187:drug effects
D000911:Antibodies, Monoclonal; D001324:Autoantigens; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C487774:metaphase chromosome protein 1, human; D049452:Green Fluorescent Proteins