Anticoagulation, centrifugation time, and sample replicate number in the microhematocrit method for avian blood.
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Animals↗
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The juxtaposition of ABL with BCR and risk for fusion may come at the time of BCR replication in late S-phase.
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Acute Disease↗
Origins of replication and gene regulation.
Eukaryotic chromosomes appear to consist of many replicons, the time of replication of which is probably controlled by specific origins. However, plasmids without specific eukaryotic origins may also replicate in some cells when injected into nuclei or transferred during transformation. The efficiency and the mechanisms of their initiation are still uncertain. A number of reports are cited which indicate that natural eukaryotic DNAs initiate their replication from specific origins. The nature of these origins are known in only a few instances and no general conclusions can yet be given about the nucleotide sequences involved. Short dispersed repeats of the Alu type appear to function as origins since they enhance the efficiency of replication of vector plasmids in Xenopus eggs. Certain sequences from a variety of eukaryotic DNAs also enhance the replicative potential of plasmids in yeast cells. The common features of such initiators or enhancers is uncertain. If dispersed repeats are origins in mammalian chromosomes, the number appears to be excessive. Either only a subset are functional, or the functional ones are only suborigins in larger replicons in which master origins (not yet isolated) function in the regulation of the timing of replication. Evidence is cited which indicates that the regulation of the time of replication of a gene or gene cluster is part of a regulatory system that makes the DNA available for transcription or leaves it in an inactive state. About one-half the DNA in mammalian cells is replicated in the first half of S phase (SE). After a brief pause in mid-S phase, the remainder of the DNA is replicated in what is designated late S (SL). The fractions replicated in SE and SL may vary in other phylogenetic groups, but wherever division of differentiated cells occurs such fractions are likely to be found. The following hypothesis is proposed. The DNA replicated in SL is suppressed in transcription, if it has the appropriate promoter regions, because the newly replicated DNA is complexed with proteins that suppress transcription. These proteins are only available during SL. Those genes replicated in SE are complexed with a different set of proteins which leave the promoter regions open for transcription when the appropriate regulatory molecules are available. In this way an inactive state or potentially active state can be transmitted from one cell generation to the next. Evidence is cited which indicates that genes which are active in all cells at some stage in the cell cycle are replicated in SE.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Strand specific quantitative real-time PCR to study replication of hepatitis C virus genome.
Qualitative detection of negative hepatitis C virus (HCV) RNA has been used widely to demonstrate HCV replication. However, relative quantitation of both positive and negative HCV RNA strands has never been reported for studying viral genome replication. A strand specific real-time PCR carried out in the highly conserved 5'-non-coding region of HCV genome and monitored either by the DNA binding dye SYBR Green I or by molecular beacons is described. Using these techniques, it was found that negative HCV RNA strand was a 100-1000 times less abundant than the positive strand in the liver of HCV infected patients.
5' Untranslated Regions↗
Control of F'lac replication in Escherichia coli B-r.
The timing of replication of an F'lac plasmid during the division cycle of Escherichia coli B/r lac(-)/F'lac was examined in relation to the timing of initiation of chromosome replication. This was accomplished by measuring the induction of beta-galactosidase and the incorporation of radioactive thymidine into cells at different ages in cultures growing exponentially at various rates. In cells growing with interdivision times of 27, 36, and 55 min, the F'lac replicated at various stages in the division cycle but always at approximately the same time as initiation of chromosome replication. In cells growing with an interdivision time of 85 min, the F'lac episome replicated midway through the division cycle, whereas chromosome replication initiated at the start of the cycle. Measurements of absorbance at 450 nm per cell suggested that the F'lac replicated when the cells reached a mass which was a constant multiple of the number of episomes per cell at each growth rate. In contrast, the mass per cell at initiation of chromosome replication in cells with an 85-min interdivision time was significantly lower than this constant value. A possible explanation for the apparent coupling between F'lac replication and initiation of chromosome replication at the higher growth rates, and the lack of coupling at the lowest growth rate, is discussed.
Carbon Isotopes↗
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↗
Time-delay discrimination training: replication with different stimuli and different populations.
Two time-delay conditions for teaching complex visual discriminations to normal preschoolers and children with mild and moderate intellectual handicaps were compared. One condition involved spatially separating the distinctive components from the redundant parts of both stimuli (multiple dynamic distinctive-feature prompts). The other condition involved adding a colored field to the correct stimulus (single static nondistinctive-feature prompt). The effect of the latter condition was assessed with unlearned and learned tasks. The study consisted of four experiments. In one experiment, children were also required to use the prompts for self-monitoring responses given before prompting had occurred. The results indicated that for all populations and stimuli (a) time delay of multiple dynamic distinctive-feature prompts consistently produced learning, and (b) time delay of the single static nondistinctive-feature prompt almost never produced learning and frequently led to a complete loss of discriminative performance on previously learned tasks. The resistance to disruption was a function of the training history (i.e., with or without time delay) and IQ level. Self-monitoring increased the efficacy of time delay when multiple dynamic distinctive-feature prompts were used but not when the single static nondistinctive-feature prompt was used.
Adolescent↗
Do Chinese and English speakers think about time differently? Failure of replicating Boroditsky (2001).
English uses the horizontal spatial metaphors to express time (e.g., the good days ahead of us). Chinese also uses the vertical metaphors (e.g., 'the month above' to mean last month). Do Chinese speakers, then, think about time in a different way than English speakers? Boroditsky [Boroditsky, L. (2001). Does language shape thought? Mandarin and English speakers' conceptions of time. Cognitive Psychology, 43(1), 1-22] claimed that they do, and went on to conclude that 'language is a powerful tool in shaping habitual thought about abstract domains' (such as time). By estimating the frequency of usage, we found that Chinese speakers actually use the horizontal spatial metaphors more often than the vertical metaphors. This offered no logical ground for Boroditsky's claim. We were also unable to replicate her experiments in four different attempts. We conclude that Chinese speakers do not think about time in a different way than English speakers just because Chinese also uses the vertical spatial metaphors to express time.
Asian People↗
The beta-major and beta-minor globin genes in murine erythroleukemia cells replicate during the same early interval of the S phase.
The time of replication in S phase was determined for the 7.3 kb EcoRI segment containing the beta-major globin gene and the 14 kb EcoRI segment containing the beta-minor globin gene in a murine Friend erythroleukemia virus transformed (MEL) cell line. Cells were obtained from different intervals of S phase by centrifugal elutriation to avoid artifacts of chemical synchronization. Newly synthesized DNA from different parts of S phase were obtained by isolation of 5-bromouracil (BU) labelled DNA from these cells. The BU-DNA synthesized during four different intervals of S was transferred to diazobenzyloxymethyl (DBM) paper and hybridized with a beta-globin cDNA probe. Quantitation showed that both beta-globin segments were replicated in the first quarter of the S phase with no significant difference in their time of replication in this MEL cell line.
Animals↗
Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.
A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.
Bacterial Proteins↗
The efficiency and timing of plasmid DNA replication in Xenopus eggs: correlations to the extent of prior chromatin assembly.
Injection of the circular plasmid FV1 (derived from type I bovine papilloma virus) into Xenopus eggs before the start of the first cell cycle dramatically increases the efficiency of plasmid replication once eggs are chemically activated. We call this the preloading effect and report kinetic and quantitative characterization of this phenomenon here. The timing and the amount of FV1 synthesis were measured by both BrdUTP density labelling and an optimized method of selective enzymatic digestion of replicated and unreplicated molecules using the three methyladenosine-sensitive isoschizomers, DpnI, MboI and Sau3a. DpnI in 100 mM NaCl proved particularly useful for distinguishing and quantitating unreplicated, once-replicated, and repeatedly replicated molecules accumulated over several cell cycles. Our results reveal that both the amount of DNA replicated and the timing of synthesis during the first S-phase correlate with the length of the preloading period. Longer preloading leads to larger amounts of DNA being replicated sooner. In fact, up to 30-50% of 1 ng injected plasmid can replicate in a semiconservative cell cycle-dependent manner during the first S-phase. But such high levels of synthesis during the first cell cycle appear to limit the egg's ability to rereplicate this material in subsequent cell cycles. The preloading effect does not depend on synthesis of either viral or egg proteins, but does appear to correlate with the extent of plasmid assembly into chromatin before the start of the cell cycle. We postulate that each plasmid molecule must achieve a critical degree of chromatin assembly before it can proceed along the replication pathway. These observations illuminate some of the difficulties inherent in building a vector for gene insertion into Xenopus embryos, but also suggest an experimental strategy toward this aim.
Animals↗
Neural induction promotes large-scale chromatin reorganisation of the Mash1 locus.
Determining how genes are epigenetically regulated to ensure their correct spatial and temporal expression during development is key to our understanding of cell lineage commitment. Here we examined epigenetic changes at an important proneural regulator gene Mash1 (Ascl1), as embryonic stem (ES) cells commit to the neural lineage. In ES cells where the Mash1 gene is transcriptionally repressed, the locus replicated late in S phase and was preferentially positioned at the nuclear periphery with other late-replicating genes (Neurod, Sprr2a). This peripheral location was coupled with low levels of histone H3K9 acetylation at the Mash1 promoter and enhanced H3K27 methylation but surprisingly location was not affected by removal of the Ezh2/Eed HMTase complex or several other chromatin-silencing candidates (G9a, SuV39h-1, Dnmt-1, Dnmt-3a and Dnmt-3b). Upon neural induction however, Mash1 transcription was upregulated (>100-fold), switched its time of replication from late to early in S phase and relocated towards the interior of the nucleus. This spatial repositioning was selective for neural commitment because Mash1 was peripheral in ES-derived mesoderm and other non-neural cell types. A bidirectional analysis of replication timing across a 2 Mb region flanking the Mash1 locus showed that chromatin changes were focused at Mash1. These results suggest that Mash1 is regulated by changes in chromatin structure and location and implicate the nuclear periphery as an important environment for maintaining the undifferentiated state of ES cells.
Animals↗
Pathogenesis of HSV-1/2 induced vaginitis/vulvitis of the mouse: dependence of lesions on genetic properties of the virus and analysis of pathohistology.
A scoring system for herpes simplex virus (HSV) induced vaginitis/vulvitis in Balb/c mice was delineated from vaginal infections. Four degrees of vaginitis/vulvitis could be distinguished after infection with suitable strains of HSV despite nearly identical replication rates. The time course of replication, inflammation and pathohistology was compared further. Grade 0 was defined by lack of symptoms despite presence of strong replication, which was detectable at days 3-6. Focal necrotic lesions of the epithelial layer were present containing HSV-specific antigens. DNA could be detected by hybridization only in the outer zone of these areas. At day 6 these zones began to be re-epithelialized. In the vaginal lumen abundant detached epithelial cells and granulocytes were already present by day 2. Grade 1 was macroscopically characterized by a slight inflammation commencing on days 5-6. Replication and antigens in the epithelium were found on days 2-6. HSV-antigens were only detected above the basal membrane, and some infiltration with granulocytes and lymphocytes was observed below the basal membrane at day 4. Grade 2 showed strong redness and inflammation as well as hyperemia. Cellular infiltrates were present in the large antigen containing epithelial lesions and below the basal membrane. From day 4 on, neurons were HSV-antigen and DNA positive and macrophages in the stroma contained antigen. The vulva was also shown to be involved. Grade 3 exhibited prolonged severe hyperemia, and destruction of the epithelium and the stroma with necrosis and infiltration, especially of the vulva. This grading system was shown to depend on certain unknown genetic properties of HSV-strains. Neither thymidine-kinase activity, replication in macrophages, fusion activity of strains nor presence or absence of the Hpa I P-fragment were shown to be of importance for severity of vaginitis/vulvitis. Vaginitis/vulvitis was shown to be an all or none response to HSV independent of the rate of replication. The set of virus genes responsible for neuroinvasiveness after vaginal or i.p. inoculation was found to be different. The time course of replication (mainly days 3-6) and inflammation (days 5-10) indicates that inflammation seems to be a secondary immunological phenomenon induced later by the replication phase of HSV. Our system could be useful for separately testing drugs with antiviral and anti-inflammatory properties.
Animals↗
A computational model of mitochondrial deoxynucleotide metabolism and DNA replication.
We present a computational model of mitochondrial deoxynucleotide metabolism and mitochondrial DNA (mtDNA) synthesis. The model includes the transport of deoxynucleosides and deoxynucleotides into the mitochondrial matrix space, as well as their phosphorylation and polymerization into mtDNA. Different simulated cell types (cancer, rapidly dividing, slowly dividing, and postmitotic cells) are represented in this model by different cytoplasmic deoxynucleotide concentrations. We calculated the changes in deoxynucleotide concentrations within the mitochondrion during the course of a mtDNA replication event and the time required for mtDNA replication in the different cell types. On the basis of the model, we define three steady states of mitochondrial deoxynucleotide metabolism: the phosphorylating state (the net import of deoxynucleosides and export of phosphorylated deoxynucleotides), the desphosphorylating state (the reverse of the phosphorylating state), and the efficient state (the net import of both deoxynucleosides and deoxynucleotides). We present five testable hypotheses based on this simulation. First, the deoxynucleotide pools within a mitochondrion are sufficient to support only a small fraction of even a single mtDNA replication event. Second, the mtDNA replication time in postmitotic cells is much longer than that in rapidly dividing cells. Third, mitochondria in dividing cells are net sinks of cytoplasmic deoxynucleotides, while mitochondria in postmitotic cells are net sources. Fourth, the deoxynucleotide carrier exerts the most control over the mtDNA replication rate in rapidly dividing cells, but in postmitotic cells, the NDPK and TK2 enzymes have the most control. Fifth, following from the previous hypothesis, rapidly dividing cells derive almost all of their mtDNA precursors from the cytoplasmic deoxynucleotides, not from phosphorylation within the mitochondrion.
Animals↗
Rapid determination of adenoviral vector titers by quantitative real-time PCR.
Replication defective adenoviruses have been used as vectors in a variety of settings including gene transfer, gene manipulation, and functionality studies. A quantitative real-time PCR-based assay is described for rapid determination of physical titers of recombinant adenovirus vectors. This method is based on amplification of a 77 bp fragment located near the left end of the adenovirus type 5 genome. Evaluation of this method demonstrated that it is simple, sensitive and reproducible, and has a dynamic range of quantitation over 5 logs. This assay is applicable to purified adenovirus as well as vectors prepared by simple cell lysis procedure, requiring only a small amount of starting material. The simplicity and short turn-around time of this assay should facilitate rapid titer determination for a large collection of adenoviral vectors.
Adenoviridae↗
Centromere separation. Early replication of repetitive DNA associated with inactive centromeres.
Four types of stable dicentric and one octacentric chromosomes from mouse brain tumor cells and L-929 cells were analyzed for the timing of replication of repetitive deoxyribonucleic acid (DNA) located in the centric and pericentric regions associated with active versus inactive centromeres. The repetitive DNA present in the heterochromatin blocks of inactive centromeres replicates much earlier than similar DNA associated with the active centromeres. The former appears to replicate during early to mid S when several euchromatic segments are still replicating. There seems to be little or no overlap in the timing of replication of the repetitive DNA present in the vicinity of prematurely separating centromeres (which are accessory and nonfunctional) and those that separate at meta-anaphase junction (which are the functional centromeres). In the absence of any information about the mechanism(s) controlling initiation and completion of DNA synthesis in the two types of heterochromatic blocks, the differential timing of replication of the DNA with similar base composition remains an enigma.
Animals↗
Separation vs. replication of inactive and active centromeres in neoplastic cells.
The inactive centromeres in neoplastic and transformed cells exhibit premature separation at prophase or pro-metaphase. The factor(s) that control this behavior are not known. Using a human breast cancer cell line, MDA 435, and a transformed mouse cell line (L929), we studied the relationship between the sequence of centromere separation and the replication of centromeric region associated with the active and inactive centromeres. Whereas the inactive centromeres in L929 cells replicate their pericentric heterochromatin earlier than that associated with the active centromeres, those in MDA 435 cells exhibited no strong correlation between early separation and replication. A comparison between the intragenomic patterns of separation with replication of only active centromeres showed that the former is not dependent upon the latter in either L929 cells or MDA 435 cells. These studies indicate that, whereas inactive centromeres in neoplastic cells separate prematurely in different species, there is no uniformity in the control for replication nor does the timing of separation depend upon the timing of replication of the centric region.
Animals↗
Adaptation to juvenile rheumatic disease: a controlled evaluation of functional disability with a one-year follow-up.
Compared the adaptation of 165 patients with juvenile rheumatic disease (JRD) to that of their healthy siblings. Patients were divided into those with mild functional disability and those with moderate/severe disability. Adaptation in several domains was assessed by parents and children on two occasions 1 year apart. The adjustment difficulties of the JRD children were limited primarily to social functioning but appeared also in the psychological and family problems domains. Compared to "mild" patients, "moderate/severe" patients had more adjustment difficulties; in some areas, mild patients functioned as well as their healthy siblings. Some Time 1 differences were replicated at Time 2. The results help to delineate (a) the specific domains in which children with chronic disease have adjustment difficulties and (b) the factors that put children at risk for developing adjustment problems.
Activities of Daily Living↗