PubMed · 10891806
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N Le Forestier, P Bouche. 2000. [Motor conduction block].. https://pubmed.ncbi.nlm.nih.gov/10891806/
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The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.
Cells infected by herpes simplex virus type 1 in the G2 phase of the cell cycle become stalled at an unusual stage of mitosis defined as pseudoprometaphase. This block correlates with the viral immediate-early protein ICP0-induced degradation of the centromere protein CENP-C. However, the observed pseudoprometaphase phenotype of infected mitotic cells suggests that the stability of other centromere proteins may also be affected. Here, we demonstrate that ICP0 also induces the proteasome-dependent degradation of the centromere protein CENP-A. By a series of Western blot and immunofluorescence experiments we show that the endogenous 17-kDa CENP-A and an exogenous tagged version of CENP-A are lost from centromeres and degraded in infected and transfected cells as a result of ICP0 expression. CENP-A is a histone H3-like protein associated with nucleosome structures in the inner plate of the kinetochore. Unlike fully transcribed lytic viral DNA, the transcriptionally repressed latent herpes simplex virus type 1 genome has been reported to have a nucleosomal structure similar to that of cellular chromatin. Because ICP0 plays an essential part in controlling the balance between the lytic and latent outcomes of infection, the ICP0-induced degradation of CENP-A is an intriguing feature connecting different aspects of viral and/or cellular genome regulation.
The target antigens of the oligoclonal bands in the cerebrospinal fluid (CSF) of patients with multiple sclerosis (MS) are unknown but may reflect important autoantigens in MS. One approach to identify candidate antigens is to allow CSF to select peptide motifs from a random phage library. To determine whether selected peptide motifs are related to the pathogenesis of MS, it is important to know if other MS patients and appropriate control patients have antibodies reactive with these sequences either in CSF or sera. Unfortunately, serologic screening of such sequences directly in phage clones gave non-specific reactions. Western blotting was found to obviate the non-specificity problem and together with isoelectric focusing, could also be used to demonstrate the co-migration of antigen specific oligoclonal bands with individual total IgG bands. Using 2D gel electrophoresis, absorption of CSF antibodies by specific peptide sequences selected from the phage library could be demonstrated. These techniques should facilitate the systematic study of the targets of the oligoclonal bands in CSF of patients with MS.