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PubMed1979

Maintenance of replication patterns in human-mouse hybrids retaining only one human chromosome.

The time of termination of DNA replication of human chromosomes in human-mouse hybrids retaining only one human chromosome was analyzed. Hybrids between SV40-transformed human skin fibroblasts and mouse peritoneal macrophages were used for these studies. Data obtained from hybrids containing only human chromosome 7 or 17 were compared with data from related hybrids containing additional human chromosomes. When either human chromosome 7 or 17 was present alone, it terminated replication at the same stage of the S phase as in hybrids in which other human chromosomes were present (relative to the time of termination of replication of the mouse chromosomes). In comparing the hybrids containing single human chromosomes, it was found that chromosome 17 terminated replication much earlier than chromosome 7. Therefore, the relationship between the replication times of these chromosomes normally observed in human cells was maintained in the hybrids in the absence of all other human chromosomes. The results also indicate that the presence of SV40 gene sequences in chromosomes 7 and 17 did not alter the relative times of termination of replication of those chromosomes.

Animals↗
PubMed1993

Sequence of centromere separation. Minor satellite DNA does not influence separation of inactive centromeres in transformed cells of mouse.

Neoplastic cells may carry inactive centromeres on some multicentric, yet stable, chromosomes. We report that some inactive centromeres in L929 mouse cells do not contain minor satellite DNA, the DNA fraction which has been suggested to constitute the centromere. We compared the sequence of separation of inactive centromeres carrying the minor satellite with those lacking this fraction. The sequence of separation appears to be independent of whether or not the inactive centromeres carry the minor satellite DNA. The timing of replication of the inactive centromeres is also independent of this DNA. Hence, minor satellite of mouse is not a factor in holding together the subunits of inactive centromeres. Extension of these results to active centromeres might suggest that the minor satellite DNA is not a factor responsible for adhesion of the two centromere sub-units up until late meta-anaphase.

Animals↗
PubMed1970

The dampening of DNA replication cycles after x-irradiation or ultraviolet irradiation.

Escherichia coli strains 15T(-) (555-7) and B/r were grown in the presence of thymine-(14)C to label all DNA. The ability of these parental DNA's to undergo cycles of replication subsequent to cellular irradiation with either X-ray or ultraviolet light (UV) was followed with density labels. Exposed cells were shifted into the density medium at times which were approximately multiples of normal rounds of DNA replication. A portion of the parental DNA, replicated semiconservatively once during an initial cycle following UV or X-irradiation in E. coli, failed to replicate again within the time studied. The time course of semiconservative parental DNA replication is altered.

Carbon Isotopes↗
PubMed1975

Cell cycle-dependent induction of mutations along a yeast chromosome.

The relation between DNA replication and the action of the mutagen N-methyl-N'-nitro-N-nitroso-guanidine has been studied in Saccharomyces cerevisiae. The frequenceis of reversion to prototrophy of six auxotrophic markers located along one arm of chromosome VII were examined as a function of the vegetative cell cycle. Exponentially growing cells were treated with nitrosoguanidine and then separated by zonal rotor centrifugation into fractions equivalent to stages in the cell cycle. The frequency of reversion for five of the six markers is greatest during the period of DNA replication. Each marker has a single point of maximum reversion, approximately 10-fold greater than the frequency observed at other points in the cell cycle. For any one marker the effect of nitrosoguanidine is restricted to an interval shorter than the period of DNA replication. The two markers most distant from each other, ade5 and leul, both have their highest reversion frequency early during DNA replication. The peak reversion frequency for lys5 is somewhat later, while the peaks for tyr3 and trp5 occur near the end of DNA replication. The results indicate that nitrosoguanidine acts primarily during DNA replication and that different markers appear to be affected at different intervals during the DNA biosynthetic period. If nitrosoguanidine does act at the growing point of DNA replication, these observations indicate that the initiation of DNA replication occurs at specific times during the period of DNA synthesis and at specific initiation sites. Further, there must be more than one point of initiation of DNA replication on one arm of chromosome VII.

Cell Division↗
PubMed1973

Gene expression during the development of Bacillus subtilis bacteriophage phi 29. I. Analysis of viral-specific transcription by deoxyribonucleic acid-ribonucleic acid competition hybridization.

The ribonucleic acid (RNA) specified by bacteriophage phi29 was analyzed to determine its composition at various times in the viral lytic cycle. Although viral-specific RNA was detected immediately after infection, a large increase in the rate was observed at 10 min when DNA synthesis began. phi29 was found to resemble other viruses in that gene expression occurred in two stages which could be defined temporally as "early" and "late." Early RNA appeared before the onset of viral deoxyribonucleic acid (DNA) replication and accounted for approximately 40% of the viral genetic potential. This RNA was also present late in the infectious cycle because of the slow turnover rate of phi29-specific RNA (approximately 10 min half-life) and the continued synthesis of much early viral RNA throughout infection. Late RNA was first detected at approximately the same time as viral DNA replication, although late transcription was not dependent upon DNA synthesis. This RNA was only partially displaced by early RNA in the appropriate competition experiments, suggesting that it contained sequences not present in the early class. Expression of viral genes was sensitive to rifamycin throughout the lytic cycle, the sensitivity resulting from a dependence upon the rifamycin phenotype of the host RNA polymerase.

Bacillus subtilis↗
PubMed1993

CLB5 and CLB6, a new pair of B cyclins involved in DNA replication in Saccharomyces cerevisiae.

The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes.

Amino Acid Sequence↗
PubMed2003

Replicate real-time PCR testing of DNA in maternal plasma increases the sensitivity of non-invasive fetal sex determination.

BACKGROUND: We determined fetal sex in pregnancies referred for invasive prenatal diagnosis procedures by analysis of DNA in maternal plasma. METHODS: Twelve pregnancies at risk of X-linked haemophilia and 32 pregnancies at risk of chromosomal aneuploidies at a gestational age ranging from 10 to 18 weeks recruited before chorionic villus sampling or amniocentesis were involved in the study. Male fetal DNA in maternal plasma was detected by using real-time polymerase chain reaction with the SRY gene as a marker. RESULTS: The specificity of the system reached 100% (no Y signal was detected in 17 women pregnant with a female fetus) and the sensitivity reached 100% (SRY amplification in 27 examined samples). CONCLUSIONS: Amplification of free fetal DNA in maternal plasma is a valid and rapid technique for predicting fetal sex in first- and second-trimester pregnancies and could allow the restriction of invasive sampling procedures to male fetuses at risk of X-linked disorders.

Aneuploidy↗
PubMed1978

Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3.

Virus-specific DNA fragments that are shorter than the full-length viral genomes have been isolated from HeLa cells productively infected with adenovirus type 3. A number of predominant size classes could be detected by gel electrophoresis and hybridization, and the array of sizes was similar or identical to the selection in DNA purified from incomplete particles of this serotype (E. Daniell, J. Virol. 19:685-708, 1976). A large fraction of these short DNA molecules contained long inverted terminal repetitions, as did DNA molecules from incomplete particles. Restriction analysis showed that these subgenomic molecules consist of sequences from the two molecular ends of the normal genome. These results suggest that the predominance of left-hand end fragments seen in packaged incomplete DNAs results from selective packaging, whereas the predominance of certain size classes of intracellular viral DNA is a function of prepackaging events. The incomplete DNAs were generated at all times during viral DNA replication, and the yield relative to complete DNA did not seem to vary significantly with time or multiplicity of infection or when the virus was propagated on different human cell types.

Adenoviruses, Human↗
PubMed1992

Role of the host cell nucleus in the replication of African swine fever virus DNA.

An examination by autoradiography of African swine fever virus-infected alveolar macrophages pulse labeled with [3H]thymidine showed that, at early times of viral DNA replication, the grains were localized exclusively in the nucleus in 20% of the cells, while in 45% the label was found in the cytoplasm. In the remaining 35%, newly synthesized DNA was detected in both the nucleus and the cytoplasm. At later times, the percentage of cells with grains in the nucleus decreased considerably. Pulse-chase experiments indicated that the DNA synthesized in the nucleus is then transported to the cytoplasm. The presence of virus-specific DNA sequences in the nucleus was confirmed by in situ hybridization of infected macrophages. Similar hybridization experiments with African swine fever virus-infected VERO cells followed by confocal microscopy also indicated the existence of a nuclear stage in the localization of the viral DNA. These results suggest a mechanism for African swine fever virus DNA replication with an initial stage in the nucleus followed by a cytoplasmic phase. Specific nuclear forms associated with the hybridization signal have been observed in African swine fever virus-infected macrophages and VERO cells. The nuclear forms seen in macrophages are consistent with a mechanism for the egress of the viral DNA from the nucleus that involves initial budding at the nuclear membrane.

African Swine Fever Virus↗
PubMed1999

Allele-specific late replication and fragility of the most active common fragile site, FRA3B.

FRA3B at 3p14.2 is the most active of the common fragile sites in the human genome and is expressed when cells are exposed to the DNA replication inhibitor, aphidicolin. Several lines of evidence suggest that fragile sites are regions of late replication. To elucidate the relationship between the timing of replication across the FRA3B region and its corresponding fragility, we labeled cells with 5-bromo-2'-deoxyuridine (BrdU) and adopted an immunofluorescent procedure to visualize late replicating DNA (BrdU-substituted DNA) in metaphase chromosomes. We also chose 21 markers along the FRA3B region and analyzed the timing of replication using BrdU-labeled DNA from different stages of the cell cycle sorted by flow cytometry. Our results show that there are two distinct alleles that replicate at different stages in the cell cycle and that breaks/gaps preferentially occurred on the chromosome 3 with the late replication allele. These results provide direct evidence that allele-specific late replication is involved in the fragility of the most active common fragile site, FRA3B.

Acid Anhydride Hydrolases↗
PubMed1993

Identification of genomic regions required for DNA replication during Drosophila embryogenesis.

A collection of Drosophila deficiency stocks was examined by bromodeoxyuridine (BrdU) labeling of embryos to analyze the DNA replication patterns in late embryogenesis. This permitted us to screen 34% of the genome for genes that when absent in homozygous deficiencies affect the cell cycle or DNA replication. We found three genomic intervals that when deleted result in cessation of DNA replication in the embryo, 39D2-3;E2-F1, 51E and 75C5-7;F1. Embryos deleted for the 75C5-7;F1 region stop DNA replication at the time in embryogenesis when a G1 phase is added to the mitotic cell cycle and the larval tissues begin to become polytene. Thus, this interval may contain a gene controlling these cell cycle transitions. DNA replication arrests earlier in embryos homozygous for deletions for the other two regions. Analysis of the effects of deletions in the 39D2-3;E2-F1 region on DNA replication showed that the block to DNA replication correlates with deletion of the histone genes. We were able to identify a single, lethal complementation group in 51E, l(2)51Ec, that is responsible for the cessation of replication observed in this interval. Deficiencies that removed one of the Drosophila cdc2 genes and the cyclin A gene had no effect on replication during embryogenesis. Additionally, our analysis identified a gene, pimples, that is required for the proper completion of mitosis in the post-blastoderm divisions of the embryo.

Animals↗
PubMed1986

Mitomycin-induced chromatid breaks in HeLa cells: a consequence of incomplete DNA replication.

The formation of chromosome aberrations induced by alkylating agents such as mitomycin C has been shown to require the passage of the treated cell through S phase. However, the exact mechanisms by which mitomycin C-induced DNA lesions are translated into chromosome aberrations during S phase are not known. The purpose of these studies was to better understand the molecular basis of chromosome aberration formation after mitomycin C treatment. The morphology of metaphases of cells treated in G1 phase with mitomycin C resembled that of prematurely condensed chromosomes of S-phase cells. Consequently we postulated that chromosome aberrations resulted from cells reaching mitosis without completing DNA replication. This was tested by treating HeLa cells in G1 phase with mitomycin C and then analyzing these cells at mitosis for residual DNA damage and DNA content. Utilizing the DNA alkaline elution assay for DNA damage, we showed that HeLa cells progress through S phase into mitosis with intact DNA-DNA interstrand cross-links. These cross-links, originally induced into parental DNA, were associated equally with parental and newly replicated DNA at the time the cells reached mitosis. This suggests that recombinational events had taken place during the DNA replication process. Cells that were treated in G1 phase and allowed to proceed to mitosis in the presence of bromodeoxyuridine to density label newly replicated DNA were analyzed with cesium chloride density sedimentation. Unreplicated DNA was present in the mitotic cells of the treated populations but not in the untreated control cells. Further, flow cytometric measurements, made under hypotonic conditions in order to reduce chromatin condensation effects, demonstrated that the mitotic cells from the mitomycin C-treated populations contained 10-20% less DNA than untreated mitotic controls. These results indicate that chromosome breaks induced by mitomycin C are the result of cells reaching mitosis without having fully completed DNA replication.

Chromatids↗
PubMedDate not supplied

FISH-detected delay in replication timing of mutated FMR1 alleles on both active and inactive X-chromosomes.

X-chromosome inactivation and the size of the CGG repeat number are assumed to play a role in the clinical, physical, and behavioral phenotype of female carriers of a mutated FMR1 allele. In view of the tight relationship between replication timing and the expression of a given DNA sequence, we have examined the replication timing of FMR1 alleles on active and inactive X-chromosomes in cell samples (lymphocytes or amniocytes) of 25 females: 17 heterozygous for a mutated FMR1 allele with a trinucleotide repeat number varying from 58 to a few hundred, and eight homozygous for a wild-type allele. We have applied two-color fluorescence in situ hybridization (FISH) with FMR1 and X-chromosome alpha-satellite probes to interphase cells of the various genotypes: the alpha-satellite probe was used to distinguish between early replicating (active) and late replicating (inactive) X-chromosomes, and the FMR1 probe revealed the replication pattern of this locus. All samples, except one with a large trinucleotide expansion, showed an early replicating FMR1 allele on the active X-chromosome and a late replicating allele on the inactive X-chromosome. In samples of mutation carriers, both the early and the late alleles showed delayed replication compared with normal alleles, regardless of repeat size. We conclude therefore that: (1) the FMR1 locus is subjected to X-inactivation; (2) mutated FMR1 alleles, regardless of repeat size, replicate later than wild-type alleles on both the active and inactive X-chromosomes; and (3) the delaying effect of the trinucleotide expansion, even with a low repeat size, is superimposed on the delay in replication associated with X-inactivation.

Adolescent↗
PubMed1984

Effect of delaying DNA replication on blastocyst formation in the mouse.

Differentiation in the early mouse embryo involves cellular responses to both spatial and temporal signals. The temporal signals that trigger blastocyst formation, the first differentiative event, are not yet understood, but it has been suggested that the numbers of DNA replications undergone since fertilization might act as the timing mechanism. Preimplantation mouse embryos were treated with aphidicolin, an inhibitor of eukaryotic DNA polymerase alpha, for eight hours during the S phase of the fourth cleavage division. This treatment produced a delay in cell division but the morphologic event of cavitation, which signals the onset of blastocyst formation, was not delayed. Treated embryos actually cavitated a few hours ahead of control embryos at approximately half the cell number. This result indicates that blastocyst formation is not timed by the number of DNA replicative cycles completed since fertilization, but by some other intrinsic cellular clock.

Animals↗
PubMed2000

Alterations of H19 imprinting and IGF2 replication timing are infrequent in Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is an overgrowth disorder resulting from dysregulation of multiple imprinted genes through a variety of distinct mechanisms. A frequent alteration in BWS involves changes in the imprinting status of the coordinately regulated IGF2 and H19 genes on 11p15. Patients have been categorized according to alterations in the imprinted expression, allele-specific methylation, and regional replication timing of these genes. In this work, IGF2/H19 expression, H19 DNA methylation, and IGF2 regional replication timing were studied in nine karyotypically normal BWS fibroblasts and two BWS patients with maternally inherited 11p15 chromosomal rearrangements. Informative patients (9/9) maintained normal monoallelic H19 expression/methylation, despite biallelic IGF2 expression in 6/9. Replication timing studies revealed no changes in the pattern of asynchronous replication timing for both a patient with biallelic IGF2 expression and a patient carrying an 11p15 inversion. In contrast, a patient with a chromosome 11;22 translocation and normal H19 expression/methylation exhibited partial loss of asynchrony and a shift toward earlier replication times. These results indicate that in BWS, (1) H19 imprinting alterations are less frequent than previously estimated, (2) IGF2 imprinting and H19 imprinting are not necessarily coordinated, and (3) alterations in regional replication timing are generally not correlated with either chromosomal rearrangements or the imprinting status of IGF2 and H19.

Beckwith-Wiedemann Syndrome↗
PubMed1986

Inhibition of DNA polymerase alpha by gossypol.

Our earlier studies have shown that gossypol is a specific inhibitor of DNA synthesis in cultured cells at low doses. In an attempt to determine the mechanism for the inhibition of DNA synthesis by gossypol we observed that gossypol does not interact with DNA per se but may affect some of the enzymes involved in DNA replication. These studies indicated that gossypol inhibits both in vivo and in vitro the activity of DNA polymerase alpha (EC 2.7.7.7), a major enzyme involved in DNA replication, in a time- and dose-dependent manner. Kinetic analysis revealed that gossypol acts as a noncompetitive inhibitor of DNA polymerase alpha with respect to all four deoxynucleotide triphosphates and to the activated DNA template. Inhibition of DNA polymerase alpha does not appear to be due to either metal chelation or reduction of sulfhydryl groups on the enzyme. Gossypol also inhibited HeLa DNA polymerase beta in a dose-dependent manner, but had no effect on DNA polymerase gamma. These results suggest that inhibition of DNA polymerase alpha may account in part for the inhibition of DNA synthesis and the S-phase block caused by gossypol. The data also raise the possibility that gossypol may interfere with DNA repair processes as well.

DNA Polymerase I↗
PubMed2003

Puralpha is essential for postnatal brain development and developmentally coupled cellular proliferation as revealed by genetic inactivation in the mouse.

The single-stranded DNA- and RNA-binding protein, Puralpha, has been implicated in many biological processes, including control of transcription of multiple genes, initiation of DNA replication, and RNA transport and translation. Deletions of the PURA gene are frequent in acute myeloid leukemia. Mice with targeted disruption of the PURA gene in both alleles appear normal at birth, but at 2 weeks of age, they develop neurological problems manifest by severe tremor and spontaneous seizures and they die by 4 weeks. There are severely lower numbers of neurons in regions of the hippocampus and cerebellum of PURA(-/-) mice versus those of age-matched +/+ littermates, and lamination of these regions is aberrant at time of death. Immunohistochemical analysis of MCM7, a protein marker for DNA replication, reveals a lack of proliferation of precursor cells in these regions in the PURA(-/-) mice. Levels of proliferation were also absent or low in several other tissues of the PURA(-/-) mice, including those of myeloid lineage, whereas those of PURA(+/-) mice were intermediate. Evaluation of brain sections indicates a reduction in myelin and glial fibrillary acidic protein labeling in oligodendrocytes and astrocytes, respectively, indicating pathological development of these cells. At postnatal day 5, a critical time for cerebellar development, Puralpha and Cdk5 were both at peak levels in bodies and dendrites of Purkinje cells of PURA(+/+) mice, but both were absent in dendrites of PURA(-/-) mice. Puralpha and Cdk5 can be coimmunoprecipitated from brain lysates of PURA(+/+) mice. Immunohistochemical studies reveal a dramatic reduction in the level of both phosphorylated and nonphosphorylated neurofilaments in dendrites of the Purkinje cell layer and of synapse formation in the hippocampus. Overall results are consistent with a role for Puralpha in developmentally timed DNA replication in specific cell types and also point to a newly emerging role in compartmentalized RNA transport and translation in neuronal dendrites.

Animals↗
PubMed1998

Cell reproduction and morphological changes in Mycoplasma capricolum.

The cell reproduction of Mycoplasma capricolum was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of Escherichia coli. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0. M. capricolum cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.

Bacterial Proteins↗