RESUMO
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.
Assuntos
Autoantígenos/genética , Autoantígenos/metabolismo , Replicação do DNA/genética , Animais , Anticorpos Monoclonais/administração & dosagem , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Citoplasma/efeitos dos fármacos , Citoplasma/metabolismo , Replicação do DNA/efeitos dos fármacos , Expressão Gênica , Proteínas de Fluorescência Verde , Humanos , Interfase/efeitos dos fármacos , Interfase/genética , Proteínas Luminescentes/genética , Macropodidae , Microinjeções , Mitose/efeitos dos fármacos , Mitose/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fase S/efeitos dos fármacosRESUMO
Aims-To directly visualise immunoglobulin (Ig) heavy (H) and light chain genes (kappa and lambda) in metaphase chromosomes and interphase nuclei of normal and malignant lymphocytes using small genomic probes targeted to intragenic sequences.Methods-Cytogenetic preparations from phytohaemagglutinin stimulated lymphocytes, B-chronic lymphocytic leukaemia (B-CLL) cells, and a B-prolymphocytic leukaemia (B-PLL) cell line, containing a t(11;14), were hybridised in situ using biotin or digoxigenin labelled plasmid probes. The kappa genes were visualised with a combination of probes for the Ckappa, Jkappa, Vkappa1, and Vkappa2 segments, the lambda genes with a probe containing the Jlambda2-Clambda2, Jlambda3-Clambda3 segments and the H genes with a probe for Clambda2. Hybridisation sites were visualised using appropriate fluorochrome conjugates and images were analysed by digital microscopy.Results-In both normal and malignant lymphoid cells, the kappa and lambda genes were visualised as a single dot signal, whereas the H lambda genes were resolved as either two or three separate signals per chromatid in metaphase chromosomes or per allele in interphase nuclei. In the malignant PLL cells, double hybridisation experiments with a painting library specific for the chromosome 11 showed that the lambda region was retained in the translocated chromosome, with an in situ resolution pattern similar to that of the normal allele.Conclusions-This study shows that a high resolution in situ analysis of the three Ig loci can be efficiently performed with small size genomic probes on both normal and malignant lymphoid cells. Such an approach offers a flexible tool for the molecular characterisations of these loci on chromosomes and individual neoplastic cells.
