PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES THAT RECOGNIZE NUCLEAR COMPONENTS
XIAO YAN
XIAO YAN
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Abstract
By adopting a monoclonal antibody approach, cell nuclei were isolated from rat liver and treated with DNase I and RNase A. Different nuclear subfractions were prepared by salt extraction and used as immunogens for the generation of hybridomas secreting monoclonal antibodies that bind specifically to different domains of the cell nucleus. Three of the antibodies, designated 408, 202, and 3H7, were found to label the nuclei with respective patterns. In interphase cells, these antibodies give a speckle nuclear staining. During metaphase, however, the 4G8 and 2G2 antigens were diffusely distributed in the nucleus, and reimported into the daughter nuclei postmitotically, but the 3H7 antigen was associated with condensed chromosomes with strong intensity, dissociated from chromosomes in the late anaphase and telophase, and finally reimported into the daughter nuclei. Indirect imunofluoresence showed that mAb 408 and 3H7 labeling is conserved in many cell lines and tissues from different species, but the mAb 202 only labels the rat cell lines. Double labeling experiments suggest that the speckles labeled by mAb 408 and 202 colocalized with splicing factor SC-35 and Sm. On the basis of these observations, mAb 4G8 and 2G2 might recognize splicing factors or accessory proteins. Western blotting with these antibodies revealed that 4G8, 2G2 and 3H7 antigens have a size of 62, 19, and 34 kDa, respectively. The 4G8 antigen was phosphorylated as demonstrated by immunoprecipitation of 32p labeled cell extracts. The 4G8 antigen was identified to be the spliceosome associated protein SAP 62 by 2-Dimensional immunoblotting and immunoprecipitation or the in vitro translated SAP 62. The 408 antibody specifically immunoprecipitated not only the expected spliceosome complex B, but also the complex E, indicating the involvment of U2snRNP in the E complex formation. In addition, SAP 62 is spatially localized in the spliceosome and tightly associated with nuclear speckle domain. The 3H7 antigen was tightly bound to the subnuclear structure shown by using the biochemical fractionation procedure which removed bulk of nuclear components. Together with the association of this antigen with condensed chromosomes, 3H7 antigen may be involved in the process of chromatin condensation during mitosis.
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1996
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