Human cyclin A is required for mitosis until mid prophase.

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21(Cip1/Waf1/Sdi1) (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

Isolation of a cDNA encoding the X enopus homologue of mammalian Cdc25A that can induce meiotic maturation of oocytes.

From a cDNA library of Xenopus laevis (Xl) oocytes, we isolated a cDNA encoding a putative protein phosphatase homologous to mammalian Cdc25A. Sequence analysis predicts that the Xl cdc25A gene product (Xl Cdc25A) consists of 521 amino acid residues and shares overall 55% identity with human Cdc25A. When its mRNA is injected into Xl oocytes, Xl Cdc25A can act as a potent M phase inducer.

Intracranial self-stimulation induces Fos expression in GABAergic neurons in the rat mesopontine tegmentum.

The cholinergic neurons which originate in the mesopontine tegmentum and innervate the midbrain ventral tegmental area have been proposed to play a key role in intracranial self-stimulation reward. This mesopontine area also contains GABA neurons. Detailed information is still lacking, however, about the relationship of cholinergic and GABAergic neurons in this region to self-stimulation reward. Therefore, using double immunostaining for Fos as a marker of neuronal activity and choline acetyltransferase as a marker of cholinergic neurons, or for Fos and GABA, we investigated whether self-stimulation of the medial forebrain bundle induces Fos expression within cholinergic and GABAergic neurons in two regions of the mesopontine tegmentum, i.e., pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus. Self-stimulation of the medial forebrain bundle for 1 h induced a large increase in the number of cells expressing Fos in both the pedunculopontine tegmental nucleus and laterodorsal tegmental nucleus, when compared to control brains. However, the self-stimulation-induced expression of Fos was restricted mostly to GABA-, but not choline acetyltransferase-, immunostained cells. We also examined, using microdialysis, whether self-stimulation increases acetylcholine efflux in the ventral tegmental area, a terminal region of the mesopontine tegmentum cholinergic pathway. One hour of self-stimulation significantly increased acetylcholine efflux from this terminal area. These results indicate that intracranial self-stimulation of the medial forebrain bundle may increase acetylcholine release without affecting expression of Fos in cholinergic neurons, while the same stimulation may induce Fos expression in GABAergic neurons of the mesopontine tegmentum. GABAergic as well as cholinergic neurons in this area appear to be activated by self-stimulation reward in the medial forebrain bundle.

Intracranial self-stimulation increases differentially in vivo hydroxylation of tyrosine but similarly in vivo hydroxylation of tryptophan in rat medial prefrontal cortex, nucleus accumbens and striatum.

We have examined using microdialysis the effect of intracranial self-stimulation (ICSS) on the in vivo hydroxylation rate of tyrosine and tryptophan in the medial prefrontal cortex (mPFC), nucleus accumbens (NAC) and striatum (STR). A decarboxylase inhibitor NSD-1015 was included in the perfusate, which enabled the simultaneous measurement of 3,4-dihydroxyphenylalanine (DOPA) and 5-hydroxytryptophan (5-HTP) as an index of the in vivo hydroxylation level of tyrosine and tryptophan. When rats were exposed to 1 h of ICSS at the medial forebrain bundle (MFB), their extracellular levels of DOPA significantly increased in the mPFC, NAC and STR, but with a different magnitude and time course. The same stimulation produced a delayed increase in extracellular 5-HTP, compared to DOPA, in these brain regions. The profile of 5-HTP response demonstrated no apparent difference among the regions. These findings indicate that ICSS of the MFB can increase differentially the in vivo hydroxylation of tyrosine but similarly the in vivo hydroxylation of tryptophan in the mPFC, NAC and STR.

Replacement of maternal 40S rRNA precursor by newly synthesized precursor in early embryos of Xenopus laevis.

The amount of intact 40S rRNA precursor was followed by Northern hybridization in the course of the early embryogenesis of wild-type Xenopus laevis and its anucleolate mutant. The total amount of 40S rRNA precursor did not alter appreciably until the midblastula stage, decreased at the late blastula stage, and then increased. In the anucleolate mutant, in which no rRNA synthesis occurs, the 40S rRNA precursor decreased at the late blastula stage and disappeared after the gastrula stage. In the nuclear fraction of the wild type, the 40S rRNA precursor was detectable after the midblastula stage. Therefore, the 40S rRNA precursor in the pre-blastula embryos is maternal and decreases at the late blastula stage. New synthesis of 40S rRNA precursor apparently occurs after the midblastula stage.

Diurnal changes of blood pressure, heart rate and body temperature during sleep in the rat.

We have studied diurnal changes in mean arterial pressure (MAP), heart rate (HR) and body temperature (Tb) during wake (W), non-rapid eye movement sleep (NREMS) and REM sleep (REMS) in the rat. Although HR and Tb show a similar sinusoidal diurnal variation during all vigilance states, the diurnal profile for the MAP is vigilance-state dependent. During W, MAP values are higher during the dark phase, during NREMS, no significant diurnal change is seen, and during REMS, the MAP exhibits a reversed diurnal change, being higher during the light phase. The low frequency component (0.25 - 0.725 Hz) in the power spectral density of the blood pressure, and index of sympathetic activity, is also higher during the light phase than the dark phase in REMS. The present findings suggest that diurnal changes in MAP in the rat result from the wake rhythm, and that the mechanism for the diurnal control of MAP may be different from that for HR or Tb.

Overexpression of the cytoplasmic retention signal region of cyclin B2, but not of cyclin B1, inhibits bipolar spindle formation in Xenopus oocytes.

Cyclin B, a regulatory subunit of maturation/M-phase promoting factor (MPF), has several subtypes in many vertebrate species. However, it is not known whether the different B-type cyclins have any different functions in vertebrate cells, although their subcellular localizations seem to differ largely from each other. To examine the roles of two major B-type cyclins, B1 and B2, in spindle formation in M phase, we overexpressed their N-termini in Xenopus oocytes; the N-termini of cyclins B1 and B2 contained a cytoplasmic retention signal (CRS), and hence their overexpressions were expected to competitively inhibit the subcellular localizations of the endogenous cyclins B1 and B2, respectively. Upon entry into meiosis I, oocytes overexpressing the cyclin B1 N-terminus formed an apparently normal bipolar spindle, but those oocytes overexpressing the cyclin B2 N-terminus formed a monopolar (or monoastral) spindle. This defect in bipolar spindle formation was observed only when the cyclin B2 N-terminus contained its own CRS sequence, and was able to be rescued by overexpression of full-length cyclin B2. These results suggest, for the first time, that the correct subcellular localization of cyclin B2, but not of cyclin B1, is essential for (the initiation of) bipolar spindle formation in Xenopus oocytes.

Correlation between physiological and transforming activities of the c-mos proto-oncogene product and identification of an essential Mos domain for these activities.

Using Xenopus eggs and NIH3T3 cells as assay systems, we have compared the physiological (i.e., maturation-inducing and cleavage-arresting) and in vitro transforming activities of the c-mos genes from various species as well as their mutant genes. These analyses show that the three biological activities all depend upon the intrinsic protein kinase activity of Mos and correlate well with each other. Furthermore, our results demonstrate that a well conserved N-terminal 14-amino acid sequence of Mos, termed the Mos-box, is essential for all three activities. These results indicate that the in vitro transforming activity of Mos can be ascribed to the same kinase activity of Mos that exerts the physiological activities.

Degradation of Mos by the N-terminal proline (Pro2)-dependent ubiquitin pathway on fertilization of Xenopus eggs: possible significance of natural selection for Pro2 in Mos.

The c-mos proto-oncogene product (Mos), an essential component of the cytostatic factor responsible for meiotic arrest in vertebrate eggs, undergoes specific proteolysis soon after fertilization or activation of Xenopus eggs. To determine the degradation pathway of Mos on egg activation, various Mos mutants were expressed in Xenopus eggs and their degradation on egg activation was examined. Mos degradation absolutely required its penultimate proline (Pro2) residue and dephosphorylation of the adjacent serine (Ser3) residue. These degradation signals were essentially the same as those of Mos in meiosis I of Xenopus oocyte maturation, where Mos has been shown to be degraded by the 'second-codon rule'-based ubiquitin pathway. To test whether Mos degradation on egg activation is also mediated by the ubiquitin pathway, we attempted to identify and abrogate a specific ubiquitination site(s) in Mos. We show that the major ubiquitination site in Mos is a Lys34 residue and that replacement of this residue with a non-ubiquitinatable Arg residue markedly enhances the stability of Mos on egg activation. These results indicate that the degradation of Mos on egg activation or fertilization is mediated primarily by the N-terminal Pro2-dependent ubiquitin pathway, as in meiosis I of oocyte maturation. The N-terminal Pro2 residue of Mos appears to be naturally selected primarily for its degradation on fertilization, rather than that in meiosis I.

Differential occurrence of CSF-like activity and transforming activity of Mos during the cell cycle in fibroblasts.

The Xenopus c-mos proto-oncogene product, Mosxe, possesses cytostatic factor (CSF) activity to arrest maturing oocytes in metaphase II and has weak transforming activity in mouse NIH3T3 cells. We show that Mosxe mutants bearing 'stabilizing' penultimate N-terminal amino acids are strongly transforming and can retard progression through the G2-M phases in Mosxe-transformed cells, probably via their CSF activity. On the other hand, a cyclin-Mosxe fusion protein, which undergoes abrupt degradation at the end of mitosis and is restored to its normal levels only after the G1 phase, transforms cells much less efficiently than a mutated cyclin-Mosxe fusion protein that is stable during M-G1 transition. Moreover, in low-serum medium, cells transformed by the unstable cyclin-Mosxe require a long period to enter the S phase, in contrast with the rapid entry into the S phase of cells transformed by the stable cyclin-Mosxe. These results provide strong evidence that unlike the physiological CSF activity, the transforming activity of Mos is exerted in the G1 phase of the cell cycle.

The 'second-codon rule' and autophosphorylation govern the stability and activity of Mos during the meiotic cell cycle in Xenopus oocytes.

The c-mos proto-oncogene product, Mos, functions in both early (germinal vesicle breakdown) and late (metaphase II arrest) steps during meiotic maturation in Xenopus oocytes. In the early step, Mos is only partially phosphorylated and metabolically unstable, while in the late step it is fully phosphorylated and highly stable. Using a number of Mos mutants expressed in oocytes, we show here that the instability of Mos in the early step is determined primarily by its penultimate N-terminal residue, or by a rule referred to here as the 'second-codon rule'. We demonstrate that unstable Mos is degraded by the ubiquitin-dependent pathway. In the late step, on the other hand, Mos is stabilized by autophosphorylation at Ser3, which probably acts to prevent the N-terminus of Mos from being recognized by a ubiquitin-protein ligase. Moreover, we show that Ser3 phosphorylation is essential for Mos to exert its full cytostatic factor (CSF) activity in fully mature oocytes. Thus, a few N-terminal amino acids are primary determinants of both the metabolic stability and physiological activity of Mos during the meiotic cell cycle.

Meiotic cell cycle in Xenopus oocytes is independent of cdk2 kinase.

In vertebrates, M phase-promoting factor (MPF), a universal G2/M regulator in eukaryotic cells, drives meiotic maturation of oocytes, while cytostatic factor (CSF) arrests mature oocytes at metaphase II until fertilization. Cdk2 kinase, a G1/S regulator in higher eukaryotic cells, is activated during meiotic maturation of Xenopus oocytes and, like Mos (an essential component of CSF), is proposed to be involved in metaphase II arrest in mature oocytes. In addition, cdk2 kinase has been shown recently to be essential for MPF activation in Xenopus embryonic mitosis. Here we report injection of Xenopus oocytes with the cdk2 kinase inhibitor p21Cip in order to (re)evaluate the role of cdk2 kinase in oocyte meiosis. Immature oocytes injected with p21Cip can enter both meiosis I and meiosis II normally, as evidenced by the typical fluctuations in MPF activity. Moreover, mature oocytes injected with p21Cip are retained normally in metaphase II for a prolonged period, whereas those injected with neutralizing anti-Mos antibody are released readily from metaphase II arrest. These results argue strongly against a role for cdk2 kinase in MPF activation and its proposed role in metaphase II arrest, in Xenopus oocyte meiosis. We discuss the possibility that cdk2 kinase stored in oocytes may function, as a maternal protein, solely for early embryonic cell cycles.

Complete nucleotide sequence of the human RCC1 gene involved in coupling between DNA replication and mitosis.

Total genomic DNA of the human RCC1 gene was isolated from HeLa DNA and its complete nucleotide sequence (34,641 bp) was determined by the shotgun sequencing method. The exon-intron junctions were precisely assigned to this sequence by comparing the nucleotide sequence of RCC1 genomic DNA with that of its cDNA. The RCC1 gene was found to have 14 exons, 8 of which (starting from the seventh one) coded the seven repeated sequences of RCC1 protein. A single exon corresponded roughly to each repeat of the RCC1 protein except for the middle one, indicating that the RCC1 gene was generated through amplification of a primordial exon. Primer extension analysis revealed the presence of an internal promoter.

Molecular cloning of the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identity of CCG2 with the human X chromosomal SCAR/RPS4X gene.

A temperature-sensitive mutant tsBN63 cell line was isolated by the fluorodeoxyuridine method from the BHK21/13 cell line after mutagenesis with nitrosoguanidine. When cultures of tsBN63 cells growing asynchronously at 33.5 degrees C were shifted to 39.5 degrees C, a nonpermissive temperature, the ability for protein synthesis was rapidly reduced and cell proliferation stopped mainly at G1 phase, and partly at G2 phase. Synchronized cultures of tsBN63 cells did not commence DNA synthesis when shifted up in G1 phase. The human gene complementing the tsBN63 mutation was cloned by DNA-mediated gene transfer and its cDNA of 1.1 kb conferring ts+ phenotype on tsBN63 cells was isolated from the cDNA library of Raj (mer+) cells with a frequency of 10(-3). On the basis of the determined nucleotide sequence, the isolated human gene turned out to be the X chromosomal RPS4X encoding the ribosomal protein S4. The size of the CCG2 gene was estimated to be about 12 kb by complementation analysis of the tsBN63 mutation with cloned genomic DNA.

Suppression of DNA replication via Mos function during meiotic divisions in Xenopus oocytes.

Meiosis is characterized by the absence of DNA replication between the two successive divisions. In Xenopus eggs, the ability to replicate DNA develops during meiotic maturation, but is normally suppressed until fertilization. Here we show that development of the DNA-replicating ability depends on new protein synthesis during meiosis I, and that mere ablation of the endogenous c-mos product Mos allows maturing oocytes to enter interphase and replicate DNA just after meiosis I. Moreover, we demonstrate that during normal maturation cdc2 kinase undergoes precocious inactivation in meiosis I and then premature reactivation before meiosis II; importantly, this premature cdc2 reactivation absolutely requires Mos function and its direct inhibition by a dominant-negative cdc2 mutant also results in nuclear reformation and DNA replication immediately after meiosis I. These findings indicate that suppression of DNA replication during meiotic divisions in Xenopus oocytes is accomplished by the Mos-mediated premature reactivation of cdc2 kinase. We suggest that these mechanisms for suppressing DNA replication may be specific for meiosis in animal oocytes, and that the ultimate biological function, including the well known cytostatic factor activity, of Mos during meiotic maturation may be to prevent undesirable DNA replication or parthenogenetic activation before fertilization.

Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation.

The human RCC1 gene was cloned after DNA-mediated gene transfer into the tsBN2 cell line, which shows premature chromosome condensation at nonpermissive temperatures (39.5-40 degrees C). This gene codes for a 2.5-kb poly(A)+ RNA that is well conserved in hamsters and humans. We isolated 15 cDNA clones from the Okayama-Berg human cDNA library, and found two that can complement the tsBN2 mutation with an efficiency comparable to that of the genomic DNA clone. The base sequences of these two active cDNA clones differ at the 5' proximal end, yet both have a common open reading frame, encoding a protein of 421 amino acids with a calculated molecular weight of 44,847 and with seven homologous repeated domains of about 60 amino acids. This human RCC1 gene was located to human chromosome 1 using sorted chromosomal fractions.