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Origin recognition complex (ORC), CDC6, and MCM proteins assemble sequentially to form prereplication chromatin. However, their organization remains largely unclear in mammalian cells. Here we show that ORC1 proteins are associated with non-chromatin nuclear structures and assemble in nuclear foci in mammalian cells using an in vivo chemical cross-linking method. CDC6 proteins were also found to assemble in nuclear foci on non-chromatin nuclear structures, although their physical association with ORC1 has been undetectable. In contrast to the situation in yeast cells, CDC6 was found to remain associated with non-chromatin nuclear structures even after cells entered into S phase. Instead, ORC1 proteins were found to be degraded by a proteasome-dependent pathway during S phase. We also found that some ORC2 proteins are associated with non-chromatin nuclear structures like ORC1, although the remainder binds to nuclease-sensitive chromatin. Further analyses indicate that ORC2 physically interacts with ORC1 on non-chromatin nuclear structures. On the other hand, our results suggest that although a small proportion of MCM complexes are loaded onto chromatin regions near ORC foci, most of them are more widely distributed. Possible relations between such organization of prereplication chromatin and complicated origin specification in higher eukaryotic cells are discussed.It has been suggested that nucleic acid metabolism is carried out on spatially organized domain structures in the cell nucleus (reviewed in Ref. 1). Non-chromatin nuclear structures such as the nuclear matrix, the scaffold, and the nucleoskeleton have been suggested as key players in organizing high order chromatin/nuclear structures (reviewed in Refs. 2 and 3). In the case of DNA replication, for example, fluorescence microscopic analyses have revealed discrete granular sites of replication, i.e. replication sites or replication foci (4 -7). Replication foci may be constructed based on non-chromatin nuclear structures, since nascent DNA and many proteins involved in DNA synthesis have been found to attach to these (Refs. 8 and 9; also reviewed in Refs. 2 and 3). In addition, non-chromatin nuclear elements have also been implicated in the initiation step of DNA replication (reviewed in Ref. 10); replication origins are often situated close to matrix/scaffold attachment regions (11)(12)(13)(14). In this regard, it is of interest to ascertain how replication initiation proteins such as the origin recognition complex (ORC), 1 CDC6, and MCM interact with non-chromatin nuclear structures.The ORC, consisting of six subunits ORC1 to -6, was originally identified as binding specifically to definitive origin sequences in budding yeast (15). Subsequent studies with yeast and Xenopus egg extracts suggested stepwise assembly of ORC, CDC6, and MCM, a six-member protein complex, onto chromatin to form prereplication complexes (reviewed in Refs. 16 and 17). An additional protein(s) is also required for the prereplication complex formation (18,19). However, the mode of...

No abstract

Intracellular localization of 73,000 and 72,000 dalton heat shock proteins (HSP73/72) in HeLa cells that were heat shocked or treated with chemical stressors was investigated using indirect immunofluorescent staining. The antiserum used specifically recognized the HSP73/72 in HeLa cells, and HSPs were increased by heating cells at 42 degrees C for 2 or 4 h and by prior treatment with chemical stressors (sodium arsenite, cadmium chloride, 8-hydroxyquinoline and ethanol). There was diffuse cytoplasmic staining at 37 degrees C, whereas nucleoli were stained brightly when cells were heated at 42 degrees C for 2 h. This rapid accumulation of HSP73/72 in the nucleoli was not inhibited by cycloheximide (50 micrograms/ml). Translocation of HSPs to the nucleoli was specific for heat because no translocation was induced by treatment with chemical stressors. When the cells were returned to 37 degrees C after heating, the HSPs in their nucleoli disappeared rapidly and diffuse cytoplasmic staining was present after 6-9 h. Our results suggest that the transient accumulation of HSP73/72 in HeLa cell nucleoli that is induced by heat shock is not correlated with the development of thermotolerance obtained in other cell systems.

ABSTRACT.A monoclonal antibody was raised against the highest molecular weight protein associated with microtubules (MAP-1). Its specific binding to MAP-1 was determined by immunoblotting of the gel electrophoretogram of microtubule proteins prepared from porcine brain. The antibody reacted only with MAP-1, not with MAP-2, tau or tubulin. Indirect Immunofluorescent staining by this antibody showed bright intranuclear spots, the centrosome and the faint meshwork of the cytoplasm in several types of cultured mammalian cells; HeLa, PtK2, human skin fibroblasts, mouse melanoma cells, Chinese hamster ovary cells. The nulcear spots in the interphase cells, were replaced by diffuse enhanced fluorescence throughout the cell except for chromosomes during mitosis. They reappeared in late telophase, first in the cytoplasm, late in the nucleus. The punctate pattern of nuclear immunofluorescence was not affected by microtubule-depolymerizing agents. The result that it persisted on residual cell structures after extraction with a high salt concentration buffer containing Triton X-100 followed by digestion with DNase I and RNase A suggests that the antigen is associated with the nuclear skeleton.