scholarly journals The replication origin decision point is a mitogen-independent, 2-aminopurine-sensitive, G1-phase event that precedes restriction point control.

1997 ◽  
Vol 17 (8) ◽  
pp. 4312-4321 ◽  
Author(s):  
J R Wu ◽  
D M Gilbert
Keyword(s):  
Protein Kinase ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Decision Point ◽  
Cell Nuclei ◽  
Phase Selection ◽  
Restriction Point ◽  
Xenopus Egg ◽  
Point Control

At a distinct point during G1 phase (the origin decision point [ODP]), Chinese hamster ovary (CHO) cell nuclei experience a transition (origin choice) that is required for specific recognition of the dihydrofolate reductase (DHFR) origin locus by Xenopus egg extracts. We have investigated the relationship between the ODP and progression of CHO cells through G1 phase. Selection of the DHFR origin at the ODP was rapidly inhibited by treatment of early G1-phase cells with the protein kinase inhibitor 2-aminopurine (2-AP). Inhibition of the ODP required administration of 2-AP at least 3 h prior to phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and the restriction point (R point). Cells deprived of either serum or isoleucine from metaphase throughout early G1 phase acquired the capacity to replicate in Xenopus egg extract (replication licensing) and subsequently passed through the ODP on the same schedule as cells cultured in complete growth medium. After growth arrest at the R point with hypophosphorylated Rb protein, serum- or isoleucine-deprived cells experienced a gradual loss of replication licensing. However, recognition of the DHFR origin by Xenopus egg cytosol remained stable in growth-arrested cells until the point at which all nuclei had lost the capacity to initiate replication. These results provide evidence that the ODP requires a mitogen-independent protein kinase that is activated after replication licensing and prior to R-point control.

Regulation of mammalian replication origin usage in Xenopus egg extract

1998 ◽  
Vol 111 (19) ◽  
pp. 2989-2998 ◽  
Author(s):  
D.S. Dimitrova ◽  
D.M. Gilbert
Keyword(s):  
Replication Origin ◽  
Chinese Hamster ◽  
G1 Phase ◽  
Cell Nuclei ◽  
Entire Genome ◽  
Xenopus Egg Extract ◽  
Xenopus Embryos ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg

Xenopus embryos initiate replication at random closely spaced sites until a certain concentration of nuclei is achieved within the embryo, after which fewer, more specific chromosomal sites are utilized as origins. We have examined the relationship between nucleo-cytosolic ratio and origin specification when Chinese hamster ovary (CHO) cell nuclei are introduced into Xenopus egg extracts. At concentrations of intact late-G1-phase nuclei that approximate early Xenopus embryos, the entire genome was duplicated nearly 4 times faster than in culture, accompanied by a de-localization of initiation sites at the dihydrofolate reductase (DHFR) locus. As the concentration of nuclei was increased, the number of initiation sites per nucleus decreased and initiation at the DHFR locus became localized to the physiologically utilized DHFR origin. Origin specification was optimal at nuclear concentrations that approximate the Xenopus mid-blastula transition (MBT). Higher concentrations resulted in an overall inhibition of DNA synthesis. By contrast, with intact early G1-phase nuclei, replication initiated at apparently random sites at all concentrations, despite an identical relationship between nucleo-cytosolic ratio and replicon size. Furthermore, permeabilization of late-G1-phase nuclei, using newly defined conditions that preserve the overall rate of replication, eliminated site-specificity, even at nuclear concentrations optimal for DHFR origin recognition. These data show that both nucleo-cytosolic ratio and nuclear structure play important but independent roles in the regulation of replication origin usage. Nucleo-cytosolic ratio clearly influences the number of replication origins selected. However, titration of cytosolic factors is not sufficient to focus initiation to specific sites. An independent mechanism, effecting changes within G1-phase nuclei, dictates which of many potential initiation sites will function as an origin.

scholarly journals Negative regulation of α2-adrenergic receptor-mediated Gi signalling by a novel pathway

1999 ◽  
Vol 343 (1) ◽  
pp. 77-85 ◽  
Author(s):  
Aya TAKESONO ◽  
Joe ZAHNER ◽  
Kendall J. BLUMER ◽  
Taku NAGAO ◽  
Hitoshi KUROSE
Keyword(s):  
Protein Kinase ◽  
Cho Cells ◽  
Adrenergic Receptor ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
Negative Regulation ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Synthesis Inhibitor ◽  
Cytosolic Factor

Chinese hamster ovary (CHO) cells stably expressing α2 adrenergic receptor (α2AR) were pretreated with cholera toxin (CTX) and then treated with or without PMA. The α2AAR-mediated inhibition of forskolin-stimulated cAMP accumulation was completely ablated by CTX pretreatment only after additional treatment with PMA. Although the addition of cycloheximide (protein synthesis inhibitor) and H-89 (cAMP dependent protein kinase inhibitor) did not completely counteract the negative regulation, the elevation of cAMP was a primary factor for negative regulation by treatment with CTX and PMA. In contrast with the cAMP response, the inhibition of membrane adenylate cyclase activity and the agonist competition curve were not influenced by treatment with CTX or PMA, suggesting that a cytosolic factor was involved in this negative regulation. The m2-muscarinic-acetylcholine-receptor-mediated inhibition of the forskolin-stimulated accumulation of cAMP was also attenuated by treatment with CTX and PMA. The ablation of α2AAR-mediated inhibition was not observed when α2AAR was expressed in Rat2 fibroblast cells, suggesting that this negative regulation is not dependent on the receptor type but is instead a phenomenon common to Gi-coupled receptors in CHO cells. Reverse-transcriptase-mediated PCR and Northern blot analysis showed that the expression of GOS8/RGS2 mRNA, which is a member of the regulator of G-protein signalling (RGS) group of proteins, was considerably increased by pretreatment with CTX. These results indicate a novel regulatory pathway, whereby a cytosolic factor induced by the elevation of cellular cAMP levels negatively regulates Gi signalling in a protein-kinase-C-dependent manner.

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