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PubMed1996

Activation of stable DNA replication in rapidly growing Escherichia coli at the time of entry to stationary phase.

The conditions are described in which DNA replication can occur, in the absence of protein synthesis, in wild-type Escherichia coli cells. Chromosome replication, which is normally inhibited by addition of chloramphenicol, becomes resistant to this drug after nutritional shiftup, e.g. from minimal medium to Luria broth. This replication activity appears transiently when nutritionally upshifted cells enter stationary phase. The activity strictly requires recA+, but it is independent of recB+ and dnaA+. It can occur in the absence of concomitant transcription. Activation of the replication does not result from induction of the SOS response. As the characteristics of this DNA replication resemble those of the previously characterized stable DNA replication, it is termed nutritional shiftup-activatable stable DNA replication, nSDR. Possible mechanisms of the activation of nSDR in rapidly growing cells at the time of entry to stationary phase are discussed.

Bacterial Proteins↗
PubMed1981

Use of whole-cell fixation to visualize replicating and maturing simian virus 40: identification of new viral gene product.

Formaldehyde fixation of simian virus 40 (SV40)-infected CV-1 cells at appropriate times after infection permits us to isolate crosslinked complexes of SV40 minichromosomes during the time of DNA replication and during packaging with viral proteins. Such crosslinked complexes can be separated on the basis of density on CsCl/guanidine . HCl density gradients. During the course of these studies we observed the presence of a low molecular weight protein in a region of the gradient much enriched with viral nucleoproteins. This protein is present only in infected cells and has a molecular weight and amino acid composition consistent with it being the product of the so-called SV40 agnogene.

Amino Acid Sequence↗
PubMed1972

Deoxyribonucleic acid-deoxyribonucleic acid hybridization assay for replication origin deoxyribonucleic acid of Escherichia coli.

Deoxyribonucleic acid (DNA)-DNA hybridization on nitrocellulose filters can be used to assay for replication origin DNA from Escherichia coli if the DNA attached to the filters is enriched for the replication origin sequences. Such DNA can be readily isolated from very rapidly growing cells. When low amounts of this DNA were attached to filters, radioactively labeled DNA from the replication origin hybridized 1.7 times as well as radioactive replication terminus DNA. Under identical conditions, radioactively labeled DNA from exponentially growing cells hybridized only 1.3 times as well as radioactive replication terminus DNA. The replication origin, replication terminus, and randomly labeled DNA hybridized with similar efficiencies to filters containing DNA isolated from cells incubated in the absence of required amino acids. This DNA appeared to have all sequences present at equal frequencies. The hybridization assay was used to demonstrate that the DNA synthesized shortly after the addition of amino acids to cells previously deprived of required amino acids was primarily from the replication origin and then rapidly became similar to DNA synthesized by exponentially growing cells.

Amino Acid Sequence↗
PubMed1998

An initiation zone of chromosomal DNA replication at the chicken lysozyme gene locus.

The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated. Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5' border of this zone is probably located downstream of the lysozyme 5' nuclear matrix attachment region. Preferred initiation occurs in a 3'-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.

Animals↗
PubMed1992

RecA protein of Escherichia coli and chromosome partitioning.

Escherichia coli cells deficient in RecA protein frequently contain an abnormal number of chromosomes after completion of ongoing rounds of DNA replication. This suggests that RecA protein may be required for correct timing of initiation of DNA replication; however, we show here that initiation of DNA replication is properly timed in recA mutants. We also find that more than 10% of recA mutant cells contain no DNA. These anucleate cells appear to arise from partitioning of all the DNA into one daughter cell and no DNA into the other daughter cell. Based on these and previously published results, we propose that RecA protein is required for equal partitioning of chromosomes into the two daughter cells.

Cell Cycle↗
PubMed1989

Sequence of centromere separation: kinetochore formation and DNA replication in dicentric chromosomes showing premature centromere separation in rat cerebral cells.

A subpopulation of rat cerebral endothelial cells, designated B1, exhibits an array of multicentric chromosomes. Because of the formation of bridges at anaphase, this cell population produced new types of multicentrics at every cell division. These chromosomes showed kinetochore proteins at every centromeric site and all centromeric regions replicated their DNA at the end of the S phase, more or less simultaneously. A new subpopulation of cells, designated B2, obtained from the original sample frozen at Wayne State University displayed several dicentrics. In contrast to B1 these chromosomes exhibit premature centromere separation as reported for mouse and human cell lines. These B2 dicentrics show only one site of kinetochore protein deposition. The timing of DNA replication around the centric region of prematurely separating centromere is also changed similar to the earlier reported premature DNA synthesis for mouse dicentrics. These observations suggest a universality of relationship between premature centromere separation, a lack of kinetochore formation, and early replication of the centric/pericentric DNA associated with these centromeres. The cause of sudden change from activity to inactivity of these chromosomes, though interesting, is not clear.

Animals↗
PubMed1995

The effects of follicle-stimulating hormone treatment on early meiotic oocytes of Podarcis sicula (Lacertilia).

The effects of follicle-stimulating hormone (FSH) on early meiotic oocytes were studied by cytological, autoradiographic, and photometric techniques. In addition to regulating oogonial proliferation, oogenesis, and folliculogenesis, the hormone influenced germ cell number and the time course of early meiosis. FSH did not affect the timing of DNA replication and amplification and did not change the amount of rDNA accumulated in the nucleus by amplification. A genetic control mechanism for these processes is suggested.

Animals↗
PubMed1997

Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors.

We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation.

Cell Nucleus↗
PubMed1982

The activity of 3-methyladenine DNA glycosylate in animal tissues in relation to carcinogenesis.

3-Methyladenine is one of the major products formed by reaction of a large number of environmental methylating agents with DNA in vivo and in vitro. In spite of the rapid spontaneous depurination of this base an enzyme, 3-methyladenine DNA glycosylase, has been shown to catalyse its excision. The relevance of this enzyme in carcinogenesis induced by alkylating agents was studied. Acute or chronic treatment of rats with diethylnitrosamine or with N-acetylaminofluorene caused a slight increase in glycosylase activity in liver. Experiments with liver regenerating after partial hepatectomy showed a similar increase to occur at the time of DNA replication. It could be that the increase found after treatment with carcinogens was related to the accompanying increase in cell replication, rather than being the result of a specific induction by the carcinogen. Glycosylase activity was found to be higher in the liver of the rabbit and cat than in rat or hamster liver. Organ differences (liver, kidney and brain of the rabbit) were smaller than the species differences found for enzyme activity in liver.

Animals↗
PubMed1997

Polyadenylation of telomerase RNA in budding yeast.

Telomerase RNA is a subunit of a stable ribonucleoprotein particle required for telomere replication. We find that, at steady state, 5-10% of the telomerase RNA in Saccharomyces cerevisiae and Kluyveromyces lactis contains a poly(A) tail of about 80 nt. In S. cerevisiae, the poly(A)+ fraction quickly disappeared when a conditional pap1 or rna15 mutant was shifted to the nonpermissive temperature, indicating that polyadenylation is accomplished by the same machinery that polyadenylates mRNAs. Potential cis-acting polyadenylation elements were identified in the telomerase RNA sequence; when they were mutagenized, the polyadenylation pattern shifted, but was not eliminated. The corresponding mutants displayed wild-type growth. By putting the RNA under the control of an inducible promoter, we were able to show that synthesis of the poly(A)+ RNA precedes that of the poly(A)- fraction. This supports, but does not prove, a model in which all telomerase RNA is first polyadenylated and then rapidly processed to give the stable poly(A)-form. Cell cycle arrest experiments showed an increase in the poly(A)+ form between G1 and S phase, consistent with an induction of telomerase RNA transcription at the time of DNA replication.

Base Sequence↗
PubMed1995

Cyclin B/p34cdc2 triggers phosphorylation of DNA ligase I during Xenopus laevis oocyte maturation.

Phosphorylation of DNA ligase I has been analyzed during Xenopus laevis early development. The enzyme, which is involved in DNA replication and DNA repair events, is accumulated during oogenesis to reach a maximum in the stage VI oocyte, and remains at a constant level during maturation. When maturation of the oocyte is induced (in vivo or in vitro), this leads to a post-translational modification of the protein. In stage VI oocytes, a DNA ligase I of apparent molecular mass 180 kDa is detected immunologically whereas a 190-kDa form is found in unfertilized eggs and persists until the tadpole stage. This modification is due to phosphorylation performed by a protein kinase that is turned on 3-4 h after induction of the maturation. Activation of the kinase requires protein synthesis, and appearance of phosphorylated DNA ligase coincides with activation of histone H1 kinase activity. Induction of DNA ligase I modification and maturation are induced in the absence of protein synthesis following injection of maturation promoting factor into oocytes. Immunoprecipitated oocyte DNA ligase I is phosphorylated and its molecular mass modified by purified cyclin B/p34cdc2 in vitro. DNA ligase I phosphorylation is not induced in oocyte extract where only mitogen-activated-protein kinase is induced. Phosphorylation of DNA ligase I induced by cdc2 kinase occurs at the time new DNA replication and recombination activities appear in eggs.

Animals↗
PubMedDate not supplied

Virus synthesis and replication: reovirus vs. vaccinia virus.

The strategies with which two viral genomes that consist of double-stranded nucleic acid express themselves in infected cells are compared. The reovirus genome comprises ten segments of double-stranded RNA, each of which is, in essence, a gene. Each is transcribed into plus-stranded RNA which has two functions: to serve as messenger RNA for the synthesis of the ten reovirus "primary" proteins, and to serve as template for the synthesis of minus-strands with which they remain associated, thereby giving rise to progeny double-stranded RNA. One of the most fascinating unsolved features of the reovirus multiplication cycle is the nature of the mechanism that ensures that each progeny virus particle contains a complete set of the ten individual genome RNA segments. The vaccinia virus genome is a linear molecule of double-stranded DNA which possesses sizable terminal redundancies (up to 7 percent, depending on the strain). The vaccinia virus multiplication cycle can be divided into a well-defined early and late period. During the early period, infecting virus particles are first uncoated to cores within which some 40-50 percent of the viral genome is transcribed. These cores are then uncoated further to naked viral DNA, a process that is mediated by protein(s) translated from the "core" messenger RNA. The overall transcription pattern in highly complex and is regulated both at the transcriptional as well as at the translational level. The most profound program changes occur at the time when DNA replication begins, when the transcription of "early" messenger RNAs, some of which are translated into "early" enzymes, gives way to that of "late" messenger RNAs, most of which are translated into structural virus components.

DNA, Viral↗
PubMed2001

Triggering ubiquitination of a CDK inhibitor at origins of DNA replication.

To ensure proper timing of the G1-S transition in the cell cycle, the cyclin E-Cdk2 complex, which is responsible for the initiation of DNA replication, is restrained by the p21(Cip1)/p27(Kip1)/p57(Kip2) family of CDK (cyclin-dependent kinase) inhibitors in humans and by the related p27(Xic1) protein in Xenopus. Activation of cyclin E-Cdk2 is linked to the ubiquitination of human p27(Kip1) or Xenopus p27(Xic1) by SCF (for Skp1-Cullin-F-box protein) ubiquitin ligases. For human p27(Kip1), ubiquitination requires direct phosphorylation by cyclin E-Cdk2. We show here that Xic1 ubiquitination does not require phosphorylation by cyclin E-Cdk2, but it does require nuclear accumulation of the Xic1-cyclin E-Cdk2 complex and recruitment of this complex to chromatin by the origin-recognition complex together with Cdc6 replication preinitiation factors; it also requires an activation step necessitating cyclin E-Cdk2-kinase and SCF ubiquitin-ligase activity, and additional factors associated with mini-chromosome maintenance proteins, including the inactivation of geminin. Components of the SCF ubiquitin-ligase complex, including Skp1 and Cul1, are also recruited to chromatin through cyclin E-Cdk2 and the preinitiation complex. Thus, activation of the cyclin E-Cdk2 kinase and ubiquitin-dependent destruction of its inhibitor are spatially constrained to the site of a properly assembled preinitiation complex.

Animals↗
PubMed1992

Increase of cytokeratin D during liver regeneration: association with the nuclear matrix.

An increase of a 45 kD protein (p45) in the nuclear matrix has been observed when rat liver cells were proliferatively activated in vivo by a partial hepatectomy. The maximal levels of the association of p45 with the nuclear matrix have been detected 24 hr after hepatectomy just at the time when DNA replication is also maximal. By amino acid sequence analysis, immunoblotting and immunocytochemical methods, it has been demonstrated that p45 is identical to rat cytokeratin D. Immunogold staining of nuclear matrix-intermediate filament preparations from cultured hepatocytes indicated that p45 is associated with cytoskeletal filaments that are strongly interconnected to the lamina, whereas no intranuclear localization of the protein has been detected. With an overlay assay a specific binding of labeled p45 to two nonidentified high-molecular weight proteins and also to lamin B has been observed. Northern blot analysis revealed a biphasic pattern of expression of the messenger RNA for cytokeratin D during liver regeneration. A sharp increase in the messenger RNA levels occurred in the prereplicative phase of liver regeneration a few hours before the accumulation of the protein in the nuclear matrix fraction, and a second peak occurred 48 hr after partial hepatectomy.

Amino Acid Sequence↗
PubMed1976

[Feeding rhythms and the diurnal rhythm of cell proliferation in pharmacologically induced liver growth (author's transl)].

Stimulation of hepatic DNA synthesis can be achieved in the intact rat by alpha-hexachlorocyclohexane (alpha-HCH = alpha-benzene hexachloride). The extent of stimulation is high in the morning and low in the evening. These rhythmic variations in the rate of DNA synthesis are synchronized indirectly by the light-dark rhythm, but directly by the animal's feeding habits: Rats eat preferentially during the night. If the diurnal rhythm of food intake is abolished, the rhythmic fluctuations in the rate of DNA synthesis are no longer detectable; if rats are adapted to daily feeding periods of only 5 h, these fluctuations are pronounced and almost synchronized. Further experiments show that the time of feeding determines the time of DNA replication. It is concluded that food intake provides a "2nd stimulus" or permissive factor, which is required for the induction of DNA synthesis in a certain critical stage of the prereplicative phase. Labelling experiments with orotic acid suggest that foot intake initially induces an increase of RNA synthesis. The results indicate that controlled feeding schedules provide the possibility to synchronize, in the living animal, a proliferating population of hepatocytes. A hypothesis is derived which offers an explanation for the generation of the diurnal rhythm of cell proliferation in the liver.

Animals↗
PubMed1996

GFP tagging of budding yeast chromosomes reveals that protein-protein interactions can mediate sister chromatid cohesion.

BACKGROUND: Precise control of sister chromatid separation is essential for the accurate transmission of genetic information. Sister chromatids must remain linked to each other from the time of DNA replication until the onset of chromosome segregation, when the linkage must be promptly dissolved. Recent studies suggest that the machinery that is responsible for the destruction of mitotic cyclins also degrades proteins that play a role in maintaining sister chromatid linkage, and that this machinery is regulated by the spindle-assembly checkpoint. Studies on these problems in budding yeast are hampered by the inability to resolve its chromosomes by light or electron microscopy. RESULTS: We have developed a novel method for visualizing specific DNA sequences in fixed and living budding yeast cells. A tandem array of 256 copies of the Lac operator is integrated at the desired site in the genome and detected by the binding of a green fluorescent protein (GFP)-Lac repressor fusion expressed from the HIS3 promoter. Using this method, we show that sister chromatid segregation precedes the destruction of cyclin B. In mad or bub cells, which lack the spindle-assembly checkpoint, sister chromatid separation can occur in the absence of microtubules. The expression of a tetramerizing form of the GFP-Lac repressor, which can bind Lac operators on two different DNA molecules, can hold sister chromatids together under conditions in which they would normally separate. CONCLUSIONS: We conclude that sister chromatid separation in budding yeast can occur in the absence of microtubule-dependent forces, and that protein complexes that can bind two different DNA molecules are capable of holding sister chromatids together.

Bacterial Proteins↗
PubMed1996

End of the line: proteolytic degradation of cyclin-dependent kinase inhibitors.

Cyclin-dependent kinase inhibitors (CKIs) are crucial regulators of cell-cycle progression. The CKI Sic1 controls the timing of DNA replication by inhibiting Clb-Cdc28 kinase. Phosphorylation of Sic1 by CIn-Cdc28 kinase alleviates this inhibition by targeting Sic1 for degradation through the ubiquitin-mediated proteolytic pathway.

Cell Cycle↗
PubMed2000

Coordinating development with the cell cycle in Caulobacter.

During the Caulobacter life cycle, the timing of DNA replication, cell division and development is precisely coordinated. Recent work has begun to unravel the complex regulatory networks that couple these processes. A key aspect of these regulatory networks is the dynamic localization of multiple histidine protein kinases that control a master response regulator, thus driving downstream pathways.

Bacterial Proteins↗