The synthesis of protein(S) for chromosome condensation may be regulated by a post‐transcriptional mechanism

Nishimoto, Tetsuya; Ishida, Ryoji; Ajiro, Kozo; Yamamoto, Susumu; Takahashi, Taijo

doi:10.1002/jcp.1041090213

Cited by 63 publications

(58 citation statements)

References 25 publications

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“…The finding that protein-synthesis inhibitors prevent both PCC-induction and an activation of p34cdc kinase caused by tsBN2-mutation (Nishimoto et al, 1981;Ajiro and Nishimoto, 1985;Nishitani et al, 1991) indicates that the protein(s) synthesized by loss of RCC1 function is essential for an activation of p34cdc2 kinase. So far, two proteins, cyclin B and cdc25 have been known to be required for an activation of p34cdc2 kinase (Nurse, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…to the nonpermissive one (Nishimoto et al, , 1981Nishitani et al, 1991). In this mutant, therefore, a negative feedback control ensuring a coupling between S and M phases is defective at the nonpermissive temperature.…”

Section: Introductionmentioning

confidence: 93%

“…Such an activation of p34cdc2 kinase and PCCinduction are prevented by emetine and cycloheximide, inhibitors of protein synthesis. (Nishimoto et al, 1981;Ajiro and Nishimoto, 1985;Nishitani et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

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Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Seki

Yamashita

Nishitani

et al. 1992

MBoC

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 93%

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Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Seki

Yamashita

Nishitani

et al. 1992

MBoC

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“…For the induction of chromosome condensation, new protein but not DNA synthesis is required, thereby suggesting that a chromosome-condensing protein(s) is produced in tsBN2 cells as a result of the temperature shift. With premature condensation of the chromosome, tsBN2 cells cease to proliferate and die (9,10). Thus, using tsBN2 cells as a recipient of DNA-mediated gene transfer, a eucaryotic gene required for the regulatory process of chromosome condensation could be cloned.…”

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confidence: 99%

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

Ren¹,

Ohtsubo²,

Sekiguchi³

et al. 1986

Mol. Cell. Biol.

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The tsBN2 cell line, a temperature-sensitive (ts) mutant of baby hamster kidney cell line BHK21/13, seems to possess a mutation in the gene that controls initiation of chromosome condensation. At the nonpermissive temperature (39.5°C), the chromatin of tsBN2 cells is prematurely condensed, and the cells die. Using tsBN2 cells as a recipient of DNA-mediated gene transfer, we investigated a human gene that is responsible for regulation of chromosome condensation and cell proliferation. We found that the human gene complementing the tsBN2 mutation resides in the area of the 40-to 50-kilobase HindHI fragment, derived from HeLa cells. Based on this finding, we initiated cloning of a human gene complementing the tsBN2 mutation. From A and cosmid libraries carrying partial digests of DNA from the secondary transformants, the 41.8-kilobase Hindm fragment containing the human DNA was isolated. The cloned human DNA was conserved in ts+ transformants through primary and secondary transfections. Two cosmid clones convert the ts-phenotype of tsBN2 cells to ts+ with more than 100 times a higher efficiency, compared with cases of transfection with total human DNA. Thus, the cloned DNA fragments contain an active human gene that complements the tsBN2 mutation.In metaphase chromosomes, the DNA is packed between 5,000 and 10,000 times (3); this packaging of DNA is performed reversibly during the cell cycle. The interphase chromatin becomes condensed at mitosis, and the chromatids become increasingly shorter from prophase to metaphase and become decondensed at telophase. Since both replication and transcription of DNA are severely inhibited by chromosome condensation, the cells must possess a mechanism regulating this process of the cell cycle (11). Among the temperature-sensitive (ts) mutants of the BHK21 cell line for cell proliferation (8), we found a ts mutant, the tsBN2 cell line, which seems to have a ts defect with regard to the regulatory mechanism for chromosome condensation. At 39.5°C, a nonpermissive temperature, the chromatin of tsBN2 cells is condensed, even in the interphase. For the induction of chromosome condensation, new protein but not DNA synthesis is required, thereby suggesting that a chromosome-condensing protein(s) is produced in tsBN2 cells as a result of the temperature shift. With premature condensation of the chromosome, tsBN2 cells cease to proliferate and die (9, 10). Thus, using tsBN2 cells as a recipient of DNA-mediated gene transfer, a eucaryotic gene required for the regulatory process of chromosome condensation could be cloned. We reported that DNAs derived from hamster and mouse cells can convert the ts phenotype of tsBN2 cells to ts+ (6). In the present work, we found that human DNA also possesses such an activity. Using the human-specific alu sequences (5) as a probe, we cloned a human DNA fragment which complements the tsBN2 mutation. MATERIALS AND METHODSCell lines and media. The cell line tsBN2 is a ts mutant of the BHK21 cell line (8). The cell line tsBN2-N9 is a thymidine kinase-negative ...

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“…Our results are too preliminary to attribute the stimulation to complementation, although they are consistent with this possibility. It has been suggested that the mutation in ts BN-2 affects DNA initiation (1,8) and chromosome condensation (4,8,9). The system described herein provides the potential for identification of the factors and perhaps of the genes involved in these important biological processes.…”

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confidence: 99%

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

Närkhammar¹,

Hand²

1985

Mol. Cell. Biol.

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ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three-to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.The hamster cell line ts BN-2, a mammalian temperaturesensitive mutant defective in DNA replication (7,8), has a defect in the initiation of DNA synthesis on individual replicons (1; M. Narkhammar and R. Hand, submitted for publication). The mutant cells also display premature chromosome condensation under restrictive conditions (8, 9).We have found that the ts BN-2 phenotype is expressed in vitro (submitted for publication). The in vitro replication system used is well characterized and active for a number of rodent cell lines (2,3,(10)(11)(12). It continues semiconservative DNA synthesis at an initial rate of up to 50% of that in intact cells (10) and probably supports some initiation as well (2, 4). Other temperature-sensitive DNA mutants express their defect in this system (12). Such a system could be useful for isolation of mutant gene products and their wild-type (WT) counterparts, as has been the case in procaryotic systems (5). We report here that the defect in ts BN-2 DNA synthesis is restored in vitro and perhaps complemented by nuclear extracts from nonmutant cells.In preliminary experiments, we found that when extracts were prepared from WT or revertant cells (8; submitted for publication), their ability to stimulate dTTP incorporation was dose dependent up to an amount of protein (6) We next determined the stimulatory effect of whole-cell extracts from WT revertant and temperature-sensitive mutant cells on dTTP incorporation in lysed mutant cells (Table 1).Addition of extracts prepared as described previously (10) from WT BHK, revertant RBN-2, or CHO cells had no significant effect on dTTP incorporation in WT BHK or revertant RBN-2 cells that had been incubated at 39.5°C for 3 h. In contrast, each of the three extracts increased incorporation by more than 200% in ts BN-2 cells subjected to the same incubation time at 39.5°C. Considering the depression of DNA synthesis in ts BN-2 cells by heat inactivation, the addition of extracts restored dTTP incorporation to more than 85% of that in nonmutant cells. The increases to more than 200% were reproducible and significant (P < 0.05).

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The synthesis of protein(S) for chromosome condensation may be regulated by a post‐transcriptional mechanism

Cited by 63 publications

References 25 publications

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Chromosome condensation caused by loss of RCC1 function requires the cdc25C protein that is located in the cytoplasm.

Molecular cloning of a human gene that regulates chromosome condensation and is essential for cell proliferation.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells.

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