Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed ... more 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.
Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human c... more Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31--33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.
The allelic muscle disorders known as limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi my... more The allelic muscle disorders known as limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy and distal anterior compartment myopathy result from defects in dysferlin-a sarcolemma-associated protein involved in membrane repair. Mutation screening in the dysferlin gene (DYSF) enabled the identification of seven Portuguese patients presenting the variant c.5492G>A, which was observed to promote skipping of exon 49 (p.Gly1802ValfsX17). Several residually expressed products of alternative splicing also involving exons 50 and 51 were detected in the leukocytes and muscle of both patients and normal controls. Quantitative transcript analysis confirmed these results and revealed that Delta49/Delta50 transcripts were predominant in blood. Although the patients were apparently unrelated, the c.5492G>A mutation was found in linkage disequilibrium with a particularly rare haplotype in the population, corroborating the hypothesis of a common origin. Despite the presence of the same mutation on the same haplotype background, onset of the disease was heterogeneous, with either proximal or distal muscle involvement.
Metaphase chromosome protein 1 (MCP1) is a nuclear autoantigen that is associated with condensed ... more 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.
Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human c... more Systemic lupus erythematosus autoantibodies were used to identify and to characterize new human chromosome-associated proteins. Previous immunolocalization studies in human and murine tissue culture cells showed that some of these monoclonal antibodies recognize nuclear antigens that associate with condensed chromosomes during mitosis. One antibody was selected for screening a human HeLa S3 cDNA expression library, and cDNAs that code for an antigen of 31--33 kDa were isolated. Immunological, biochemical and cell fractionation data indicate that the 31- to 33-kDa antigen corresponds to the chromosome-associated protein recognized by the original monoclonal antibody. Sequence analysis shows that we isolated a novel human gene. Immunolocalization to human tissue culture cells shows that during interphase the antigen is dispersed in the nucleus and that during mitosis it associates exclusively with condensed chromosomes. A similar pattern of localization was also observed in mouse fibroblasts, suggesting that the antigen is conserved among different species. Finally, we show that part of the antigen remains bound to the scaffold/matrix component, even after high salt extraction.
The allelic muscle disorders known as limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi my... more The allelic muscle disorders known as limb-girdle muscular dystrophy type 2B (LGMD2B), Miyoshi myopathy and distal anterior compartment myopathy result from defects in dysferlin-a sarcolemma-associated protein involved in membrane repair. Mutation screening in the dysferlin gene (DYSF) enabled the identification of seven Portuguese patients presenting the variant c.5492G>A, which was observed to promote skipping of exon 49 (p.Gly1802ValfsX17). Several residually expressed products of alternative splicing also involving exons 50 and 51 were detected in the leukocytes and muscle of both patients and normal controls. Quantitative transcript analysis confirmed these results and revealed that Delta49/Delta50 transcripts were predominant in blood. Although the patients were apparently unrelated, the c.5492G>A mutation was found in linkage disequilibrium with a particularly rare haplotype in the population, corroborating the hypothesis of a common origin. Despite the presence of the same mutation on the same haplotype background, onset of the disease was heterogeneous, with either proximal or distal muscle involvement.
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Papers by Elsa Rocha