西谷 秀男

In most eukaryotes, replication origins are composed of long chromosome regions, and the exact sequences required for origin recognition complex (ORC) and minichromosome maintenance (MCM) complex association remain elusive. Here, we show that two stretches of adenine/thymine residues are collectively essential for a fission yeast chromosomal origin. Chromatin immunoprecipitation assays revealed that the ORC subunits are located within a 1 kb region of ori2004. Analyses of deletion derivatives of ori2004 showed that adenine stretches are required for ORC binding in vivo. Synergistic interaction between ORC and adenine stretches was observed. On the other hand, MCM subunits were localized preferentially to a region near the initiation site, which is distant from adenine stretches. This association was dependent on adenine stretches and stimulated by a non-adenine element. Our results suggest that association of multiple ORC molecules with a replication origin is required for efficient MCM loading and origin firing in fission yeast.

A truncated human RanBPM has been isolated as a protein binding to Ran, Ras-like nuclear small GTPase. Full-sized human RanBPM cDNA which was recently isolated, was found to encode a protein of 90 kDa which comprises a large protein complex. Consistent with this finding, several proteins were found to be co-precipitated with RanBPM by immunoprecipitation analysis. Accordingly, in the present study, we screened the human cDNA library by the two-hybrid method using RanBPM cDNA as bait. One novel protein designated as Twa1 (Two hybrid associated protein No. I with RanBPM), and two known proteins, a human homologue (hMuskelin) of mouse Muskelin and HSMpp8 were isolated repeatedly. Twa1 was well conserved through evolution and was localized within the nucleus. Interestingly, in addition to Muskelin and RanBPM, Twa1 was found to possess the LisH-CTLH motif which is detected in proteins involved in microtubule dynamics, cell migration, nucleokinesis and chromosome segregation. These functions overlap with functions suggested for the RanGTPase cycle. Immunoprecipitation and gel-filtration analyses indicated that both Twa1 and hMuskelin did indeed comprise a protein complex with RanBPM. Taken together with the fact that RanBPM interacts with Ran, our present findings suggested that there is an as yet uncovered function of the RanGTPasc cycle. (C) 2002 Elsevier Science B.V. All rights reserved.

The androgen receptor (AR) is a ligand-dependent transcription factor that has an essential role in the normal growth, development, and maintenance of the prostate gland. The AR is part of a large family of steroid receptors that also includes the glucocorticoid, progesterone, and mineralocorticoid receptors. Steroid receptor family members share significant homology at their DNA and ligand-binding domains. However, these receptors exhibit a high degree of sequence variability at their NH2-terminal domain, which suggests the possibility of receptor-specific interactions with co-regulator proteins. Transcriptional co-regulators that interact with the AR may have a role in defining AR activity and may be involved in directing AR-specific responses. Here we have identified Ran-binding protein in the microtubule-organizing center (RanBPM) to be a novel AR-interacting protein by yeast two-hybrid assay and have confirmed this interaction by glutathione S-transferase- and His-tagged pull-down assays. In addition, transient overexpression of RanBPM in prostate cancer cell lines resulted in enhanced AR activity in a ligand-dependent fashion. Glucocorticoid receptor activity was also enhanced when RanBPM was overexpressed, whereas estrogen receptor activity remained unchanged. These data demonstrate that RanBPM interacts with steroid receptors to selectively modify their activity.

To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Its control has been uncovered by the discovery of the CDKs (cyclin dependent kinases) as master regulators of the cell cycle and the initiator proteins of DNA replication, such as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At the end of mitosis, the MCM complex is loaded on to chromatin with the aid of ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs, together with the Cdc7 kinase, trigger the initiation of replication, recruiting the DNA replicating enzymes on sites of replication. The activated MCM complex appears to play a key role in the DNA unwinding step, acting as a replicating helicase and moves along with the replication fork, at the same time bringing the origins to the unlicensed state. The cycling of CDK activity in the cell cycle separates the two states of replication origins, the licensed state in G1-phase and the unlicensed state for the rest of the cell cycle. Only when CDK drops at the completion of mitosis, is the restriction on licensing relieved and a new round of replication is allowed. Such a CDK-regulated licensing control is conserved from yeast to higher eukaryotes, and ensures that DNA replication takes place only once in a cycle. Xenopus laevis and mammalian cells have an additional system to control licensing. Geminin, whose degradation at the end of mitosis is essential for a new round of licensing, has been shown to bind Cdt1 and negatively regulate it, providing a new insight into the regulation of DNA replication in higher eukaryotes.

S-phase onset is controlled, so that it occurs only once every cell cycle. DNA is licensed for replication after mitosis in Gl, and passage through S-phase removes the license to replicate. In fission yeast, Cdc6/18 and Cdt1, two factors required for licensing, are central to ensuring that replication occurs once per cell cycle. We show that the human Cdt1 homologue (hCdt1), a nuclear protein, is present only during G(1). After S-phase onset, hCdt1 levels decrease, and it is hardly detected in cells in early S-phase or G(2). hCdt1 can associate with the DNA replication inhibitor Geminin, however these two proteins are mostly expressed at different cell cycle stages. hCdt1 mRNA, in contrast to hCdt1 protein, is expressed in S-phase-arrested cells, and its levels do not change dramatically during a cell cycle, suggesting that proteolytic rather than transcriptional controls ensure the timely accumulation of hCdt1. Consistent with this view, proteasome inhibitors stabilize hCdt1 in S-phase. In contrast, hCdc6/18 levels are constant through most of the cell cycle and are only low for a brief period at the end of mitosis. These results suggest that the presence of active hCdt1 may be crucial for determining when licensing is legitimate in human cells.

Previously isolated RanBPM, a Ran-binding protein in the microtubule-organizing center, which had been thought to play a role in Ran-stimulated microtubule assembly, turned out to be a truncated protein. To clarify the function of RanBPM, we cloned the full-sized RanBPM cDNA that encodes a 90 kDa protein. compared to the previously isolated cDNA that encoded a 55 kDa protein. The newly cloned 5 ' coding region contains a great number of cytidine and guanidine nucleotides, like the CpG island. Thus, full-sized RanBPM cDNA encodes a long stretch of proline and glutamine residues in the N-terminal region. It comprises a protein complex of more than 670 kDa. Ran was detected in this complex when RanBPM and Ran were both ectopically expressed. New antibodies to RanBPM were prepared against three different regions of RanBPM. All of them detected a 90 kDa protein that is predominantly localized both in the nucleus and in the cytoplasmic region surrounding the centrosome, but none of them stained the centrosome, In this context, our previous notion that RanBPM is a centrosomal protein should be discarded. RanBPM is well conserved in the animal kingdom. It may play an important role in uncovering Ran-dependent nuclear events. (C) 2001 Elsevier Science B.V. All rights reserved.

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle(1). This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin(2-10). The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis(12). Other factors acting in this control have been postulated(13) and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

The nucleotide exchange activity of RCC1,the only known nucleotide exchange factor for Ran, a Ras-like small guanosine triphosphatase, was required for microtubule aster formation with or without demembranated sperm in Xenopus egg extracts arrested in meiosis II. Consistently, in the RCC1-depleted egg extracts, Ran guanosine triphosphate (RanGTP), but not Ran guanosine diphosphate (RanGDP), induced self-organization of microtubule asters, and the process required the activity of dynein. Thus, Ran was shown to regulate formation of the microtubule network.

The DNA replication checkpoint is required to maintain the integrity of the genome, inhibiting mitosis until S phase has been successfully completed. The checkpoint preventing premature mitosis in Schizosaccharomyces pombe relies on phosphorylation of the tyrosine-15 residue on cdc2p to prevent its activation and hence mitosis, The cdc18 gene is essential for both generating the DNA replication checkpoint and the initiation of S phase, thus providing a key role for the overall control and coordination of the cell cycle. We show that the C terminus of the protein is capable of both initiating DNA replication and the checkpoint function of cdc18p. The C terminus of cdc18p acts upstream of the DNA replication checkpoint genes rad1, rad3, rad9, rad17, hus1 and cut5 and requires the wee1p/mik1p tyrosine kinases to block mitosis, The N terminus of cdc18p can also block mitosis but does so in the absence of the DNA replication checkpoint genes and the wee1p/mik1p kinases therefore acting downstream of these genes. Because the N terminus of cdc18p associates with cdc2p in vivo, we suggest that by binding the cdc2p/cdc13p mitotic kinase directly, it exerts an effect independently of the normal checkpoint control, probably in an unphysiological manner.

In fission yeast, cdc18p plays a critical role in bringing about the onset of S phase. We show that cdc18p expression is subject to a complex sequence of cell cycle controls which ensure that cdc18p levels rise dramatically as cells exit mitosis, before the appearance of CDK activity in G(1). We find that transcription of cdc18, together with the transcription of other cdc10p/res1p targets, is first initiated as cells enter mitosis and continues even in cells arrested in mitosis with highly condensed chromatin, However, cdc18p cannot accumulate during mitosis because it is targeted for proteolysis by mitotic cdc2p-protein kinase-mediated phosphorylation. On exit from mitosis, the cdc2p mitotic kinase activity falls, stabilizing cdc18p, which then rapidly accumulates. This combination of mitotic transcription and CDK-mediated proteolysis ensures that progression through mitosis simultaneously prepares cells for DNA replication. During S phase, cdc18 transcription is then switched off, preventing the reinitiation of DNA synthesis until the completion of the next round of mitosis.

By incubating at 30 degrees C in the presence of an energy source, p34(cdc2)/cyclin B was activated in the extract prepared from a temperature-sensitive mutant, tsBN2, which prematurely enters mitosis at 40 degrees C, the nonpermissive temperature (Nishimoto, T., E. Eilen, and C. Basilico. 1978. Cell. 15:475-483), and wild-type cells of the hamster BHK21 cell line arrested in S phase, without protein synthesis. Such an in vitro activation of p34(cdc2)/cyclin B, however, did not occur in the extract prepared from cells pretreated with protein synthesis inhibitor cycloheximide, although this extract still retained the ability to inhibit p34(cdc2)/cyclin B activation. When tsBN2 cells arrested in S phase were incubated at 40 degrees C in the presence of cycloheximide, Cdc25B, but not Cdc25A and C, among a family of dual-specificity phosphatases, Cdc25, was lost coincidentally with the lack of the activation of p34(cdc2)/cyclin B. Consistently, the immunodepletion of Cdc25B from the extract inhibited the activation of p34(cdc2)/cyclin B. Cdc25B was found to be unstable (half-life < 30 min). Cdc25B, but not Cdc25C, immunoprecipitated from the extract directly activated the p34(cdc2)/cyclin B of cycloheximide-treated cells as well as that of nontreated cells, although Cdc25C immunoprecipitated from the extract of mitotic cells activated the p34(cdc2)/cyclin B within the extract of cycloheximide-treated cells. Our data suggest that Cdc25B made an initial activation of p34(cdc2)/cyclin B, which initiates mitosis through the activation of Cdc25C.

Background: We have previously isolated a series of temperature-sensitive mutants for cell-proliferation from the BHK21 cell line, derived from the golden hamster. These mutants proliferate at 33.5 degrees C, the permissive temperature, but not at 39.5 degrees C the restrictive temperature. Using DNA-mediated gene transfer, human genes complementing these ts mutants were cloned. Results: At 39.5 degrees C the tsBN250 cell line, a temperature-sensitive mutant of the BHK21 cell line, had a defect in the G1 phase, but not in the S phase. The human gene complementing tsBN250 cells was found to encode histidyl-tRNA synthetase. Indeed, the tsBN250 cell line had a single base change-guanine to adenine at the second position of the 362nd codon of hamster histidyl-tRNA-synthetase, converting arginine to histidine. Following release from serum starvation, cyclin E, but not cyclin D1, was accumulated, while, at 39.5 degrees C, the mRNA of cyclin D1 was normally expressed in tsBN250 cells. A similar inhibition of cyclin D1 accumulation was observed in another ts mutant, tsBN269, which has a single point mutation in lysyl-tRNA synthetase. Overexpression of cyclin D1 enabled tsBN250 cells to enter the S phase. Conclusion: tsBN250 cells have a single point mutation in histidyl tRNA synthetase that causes a loss of histidyl-tRNA synthetase activity which in turn reduces the content of cyclin D1, but not of cyclin E, thereby resulting in G1 arrest.

A key problem in the cell cycle is understanding what brings about the initiation of DNA replication and how this is linked with global cell cycle controls. The fission yeast gene cdc18 is required for DNA replication and is transcriptionally activated by the cdc10/res1/res2 control acting at START in late G1. We show here that overexpressing cdc18 is able to bring about repeated rounds of DNA synthesis in the absence of mitosis and of continuing protein synthesis. The level of the cdc18-encoded protein p65(cdc18) is periodic in the cell cycle, peaking at the G1 to S phase transition, and p65(cdc18) is located in the nucleus when cdc18 is overexpressed. We propose that p65(cdc18) acts af the initiation of DNA replication and plays a major role in controlling the onset of S phase.

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage (''peeling off''). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of . OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha(5) beta(1), vinculin, stress fibers, and phosphotyrosine (p-Tyr)-containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content, Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+](i)) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+](i), both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+](i) is also necessary for peeling off, Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert-butyl)-1,4-benzohydroquino (BHQ) plus EGTA prevented both NH2Cl-induced increases in [Ca2+](i) and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl-induced [Ca2+](i) increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+](i) by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+](i) in NH2Cl-induced peeling off.

Homologous recombination on the chromosome is in many cases uniform, but in both procaryotes and eucaryotes there are specific regions Or sites, named ''hotspots'', where homologous recombination occurs at a higher rate. DNA replication origin in procaryotes (phage) is one example (1). Another example is the ''HOT1'' site in yeast, which has activity to stimulate recombination, homologously, in adjacent regions (2). The molecular mechanisms involved in enhancing homologous recombinations are not well understood. Microscopically, there is a site, the homologous recombination of the surrounding region of which is stimulated. The ''Chi'' site is such a recombinational hotspot and was first identified in lambda phage (3-5). The Chi site enhances recombination not just in its vicinity but even as far away as 10 kb (6, 7). The Chi consists of an 8 bp specific sequence, 5'-GCTGGTGG-3', distributed in Escherichia coli chromosomal DNA (one per 5 to 15 kb on the average) (8, 9). RecBCD, which is a Chi responsive enzyme, enters into duplex DNA, probably through a double-stranded (ds)-break (''cos'' site in the case of lambda phage) and moves in it with concomitant DNA degradation. The exonuclease activity of the enzyme seems to be modulated by Chi only when the enzyme approaches Chi from the correct side, the result being an enhancement of homologous recombination at the surrounding region (6, 10-18). For various analyses, the lambda phage system has been most extensively used, because in the E. coli system there are numerous Chi sites and the entrance for the enzyme on the circular chromosome has yet to be identified (6, 19). While a recombinational hotspot site on the E. coli chromosome has been identified, mechanisms related to enhancement remain unknown (20). Recently, we identified a new type of recombination hotspot, termed Hot, and a detailed analysis was made (21, 22). We obtained evidence that the homologous recombination is closely linked with DNA replication fork blocking events.

In Escherichia coli, eight kinds of chromosome-derived DNA fragments (named Hot DNA) were found to exhibit homologous recombinational hotspot activity, with the following properties. (i) The Hot activities of all Hot DNAs were enhanced extensively under RNase H-defective (mh) conditions. (ii) Seven Hot DNAs were clustered at the DNA replication terminus region on the E. coli chromosome and had Chi activities (H. Nishitani, M. Hidaka, and T. Horiuchi, Mel. Gen. Genet. 240:307-314, 1993). Hot activities of HotA, -B, and -C, the locations of which were close to three DNA replication terminus sites, the TerB, -A, and -C sites, respectively, disappeared when terminus-binding (Tau or Tus) protein was defective, thereby suggesting that their Hot activities are termination event dependent. Other Hot groups showed termination-independent Hot activities. In addition, at least HotA activity proved to be dependent on a Chi sequence, because mutational destruction of the Chi sequence on the HotA DNA fragment resulted in disappearance of the HotA activity. The HotA activity,which had disappeared was reactivated by insertion of a new, property oriented Chi sequence at the position between the HotA DNA and the TerB site. On the basis of these observations and positional and orientational relationships between the Chi and the Ter sequences, we propose a model in which the DNA replication fork blocked at the Ter site provides an entrance for the RecBCD enzyme into duplex DNA.

To clone new replication origin(s) activated under RNase H-defective (rnh-) conditions in Escherichia coli cells, whole chromosomal DNA digested with EcoRI was to with a Km(r) DNA fragment and transformed into an rnh- derivative host. From the Km(r) transformants, we obtained eight kinds of plasmid-like DNA, each of which contained a specific DNA fragment, termed ''Hot'', derived from the E. coli genome. Seven of the Hot DNAs (HotA-G) mapped to various sites within a narrow DNA replication termination region (about 280 kb), without any particular selection. Because Hot DNA could not be transformed into a mutant strain in which the corresponding Hot region had been deleted from the chromosome, the Hot DNA, though obtained as covalently closed circular (ccc) DNA, must have arisen by excision from the host chromosome into which it had initially integrated, rather than by autonomous replication of the transformed species. While Hot DNA does not have a weak replication origin it does have a strong recombinational hotspot active in the absence of RNase H. This notion is supported by the finding that Chi activity was present on all Hot DNAs tested and no Hot-positive clone without Chi activity was obtained, with the exception of a DNA clone carrying the dif site.

We cloned the hamster cdc25C cDNA by using the human cdc25C cDNA as a probe and prepared an antibody to Escherichia coli-produced hamster cdc25C protein that is specific to the human cdc25C protein. The microinjected antibody inhibited a chromosome condensation induced by tsBN2 mutation, indicating that the cdc25C protein is required for an activation of p34cdc2 kinase caused by loss of RCC1 function. The hamster cdc25C protein located in the cytoplasm, prominently in a periphery of the nuclei of cells arrested with hydroxyurea, and seemed to move into the nuclei by loss of RCC1 function. Also, we found a molecular shift of the cdc25C protein in cells showing premature chromosome condensation (PCC), in addition to normal mitotic cells. This molecular-shift appeared depending on an activation of p34cdc2 kinase.

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.

Bovine heart 67-kd protein (p67) was coisolated with calpactin I complex by cycles of Ca2+-dependent precipitation followed by solubilization with EGTA-containing buffer. Using affinity-purified anti-p67 antibody and anti-p36 (36-kd subunit of calpactin I) antibody, we examined the localization of the two proteins in secretory atrial myocytes and other endocrine tissues of adult rats. Immunofluorescence microscopy showed that p67 was expressed both in the atrial myocytes in situ and in cultured atrial myocytes in which we failed to detect p36 and that p67 appeared to be closely associated with the cell surface. We also found that p67 was colocalized with p36 in the thyroid follicle epithelium and zona reticularis of the adrenal gland. On the other hand, neither p67 nor p36 was detectable in pancreas islet cells. Immunoelectron microscopy revealed that p67 was localized at the sarcolemma in the atrial myocytes in situ. The p67, which was shown to be a globular molecule with a diameter of 18-25 nm by a low-angle rotary shadowing method, bound radioactive Ca2+ on a nitrocellulose membrane. The results suggest that Ca2+-binding proteins expressed in endocrine cells seem to vary from tissue to tissue and that p67 may function in Ca2+-mediated events at the plasma membrane of secretory atrial myocytes and some types of endocrine cells expressing this protein.

The temperature-sensitive mutant cell line tsBN2, was derived from the BHK21 cell line and has a point mutation in the RCC1 gene. In tsBN2 cells, the RCC1 protein disappeared after a shift to the non-permissive temperature at any time in the cell cycle. From S phase onwards, once RCC1 function was lost at the non-permissive temperature, p34cdc2 was dephosphorylated and M-phase specific histone H1 kinase was activated. However, in G1 phase, shifting to the non-permissive temperature did not activate p34cdc2 histone H1 kinase. The activation of p34cdc2 histone H1 kinase required protein synthesis in addition to the presence of a complex between p34cdc2 and cyclin B. Upon the loss of RCC1 in S phase of tsBN2 cells and the consequent p34cdc2 histone H1 kinase activation, a normal mitotic cycle is induced, including the formation of a mitotic spindle and subsequent reformation of the interphase-microtubule network. Exit from mitosis was accompanied by the disappearance of cyclin B, and a decrease in p34cdc2 histone H1 kinase activity. The kinetics of p34cdc2 histone H1 kinase activation correlated well with the appearance of premature mitotic cells and was not affected by the presence of a DNA synthesis inhibitor. Thus the normal inhibition of p34cdc2 activation by incompletely replicated DNA is abrogated by the loss of RCC1.

The RCC1 gene has been isolated from several vertebrates, including human, hamster and Xenopus. Genes similar to RCC1, namely BJ1 and SRM1/PRP20, have been isolated from the insect Drosophila and from the budding yeast Saccharomyces cerevisiae. A mutation of the RCC1 gene in the hamster BHK21 cell line, tsBN2, confers pleiotropic phenotypes, including G1 arrest and premature induction of mitosis in cells synchronized at the G1/S boundary. Similarly, mutations of the SRM1/PRP20 gene are pleiotropic: the srm1 mutant shows G1 arrest and suppression of the mating defect of mutants lacking pheromone receptors, and the prp20 mutant shows an alteration in mRNA metabolism. Here we show that both BJ1 and SRM1/PRP20 complement the temperature sensitive phenotype of the tsBN2 cells. Like RCC1 proteins of vertebrates, the protein products of the Drosophila and yeast RCC1 homologues were located in the nuclei of the mammalian cells. These results suggest that the BJ1 and SRM1/PRP20 genes are functionally equivalent to the vertebrate RCC1 genes, and that the RCC1 gene plays an important role in the regulation of gene expression in the eukaryotic cell cycle.

When BHK21 cells synchronized in early S phase were exposed to okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, mitosis specific events such as premature chromosome condensation, the productions of MPM-2 antigens, dispersion of nuclear lamins and the appearance of mitotic asters were induced, and then disappeared upon further incubation. These mitosis specific events occurred even in the presence of cycloheximide. Within 1 h of exposure to OA, cdc2/histone H1 kinase activity rose 10-fold compared with untreated controls, but returned to the control level upon further incubation. Using antibodies against either p34cdc2 or cyclin B it was found that p34cdc2 complexed with cyclin B was dephosphorylated after OA treatment concomitant with the activation of cdc2 kinase, and that cyclin B was subsequently degraded concomitant with a decrease in cdc2 kinase activity, as in normal mitosis. In contrast, when cells in G1 phase were treated with OA no increase in cdc2 kinase activity was observed. Moreover when cells in pseudo-metaphase induced by nocodazole were treated with OA, cdc2 kinase was inactivated. These results suggest that OA sensitive protein phosphatases control both the activation and inactivation of the p34cdc2 kinase.