Time of replication of genes responsible for a temperature-sensitive function in a cell cycle-specific ts mutant from a hamster cell line.
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Bromodeoxyuridine↗
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Selection and timing of replication of plasmids R1drd-19 and F'lac in Escherichia coli.
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Cell Count↗
The structure of chromatin replicated in vitro.
Nuclei from concanavalin-A-activated lymphocytes were used to study the replication of chromatin in vitro. Micrococcal nuclease was employed to obtain information about the structure of the replicated chromatin. The nuclease digestion products were examined by sucrose gradient sedimentation and by gel electrophoresis. Experiments are presented which indicate that DNA replicated in vitro is organized into chromatin whose structure is similar to that of bulk chromatin. This conclusion is based on the following observations: (a) DNA replicated in vitro is associated with typical chromatin subunits (nucleosomes) even after short replication times, when the newly replicated DNA consists almost entirely of Okazaki fragments; (b) the length of internucleosomal spacer DNA in part of the replicated chromatin corresponds to that in bulk chromatin. Evidence which suggests that the structure of nucleosomes is transiently altered in the vicinity of the replication fork is presented.
Animals↗
Time of replication of ARS elements along yeast chromosome III.
The replication of putative replication origins (ARS elements) was examined for 200 kilobases of chromosome III of Saccharomyces cerevisiae. By using synchronous cultures and transfers from dense to light isotope medium, the temporal pattern of mitotic DNA replication of eight fragments that contain ARSs was determined. ARS elements near the telomeres replicated late in S phase, while internal ARS elements replicated in the first half of S phase. The results suggest that some ARS elements in the chromosome may be inactive as replication origins. The actively expressed mating type locus, MAT, replicated early in S phase, while the silent cassettes, HML and HMR, replicated late. Unexpectedly, chromosome III sequences were found to replicate late in G1 at the arrest induced by the temperature-sensitive cdc7 allele.
Chromosome Mapping↗
Dimensions of sexual behaviour in homosexual men: replicability across time and country.
Data on sexual practices in the past two months were obtained from 176 Australian and 159 New Zealand homosexually active men. Factor analysis yielded three major underlying dimensions of homosexual behaviour in each sample, accounting for 53.4 and 54.0% of total variance at baseline and 55.1 and 62.1% at follow-up, respectively. These three dimensions represented unsafe sex involving predominantly anal activity, safe (oral and mutual masturbatory) sex without condoms, and safe anal sex with condoms and withdrawal. Comparison of the four matrices (two countries by two times), using Cattell's S index, indicated high stability of the factor structure across countries and times. These three dimensions of homosexual behaviour appear to represent consistent behavioural clusters across samples and times. They suggest that use of these dimensions in further research on homosexually active men is warranted and that homosexual activities cluster into a number of limited dimensions on which sexual behaviours are interrelated.
Acquired Immunodeficiency Syndrome↗
Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.
The historic arguments for the participation of eukaryotic DNA replication in the control of gene expression are reconsidered along with more recent evidence. An earlier view in which gene commitment was achieved with stable chromatin structures which required DNA replication to reset expression potential (D. D. Brown, Cell 37:359-365, 1984) is further considered. The participation of nonspecific stable repressor of gene activity (histones and other chromatin proteins), as previously proposed, is reexamined. The possible function of positive trans-acting factors is now further developed by considering evidence from DNA virus models. It is proposed that these positive factors act to control the initiation of replicon-specific DNA synthesis in the S phase (early or late replication timing). Stable chromatin assembles during replication into potentially active (early S) or inactive (late S) states with prevailing trans-acting factors (early) or repressing factors (late) and may asymmetrically commit daughter templates. This suggests logical schemes for programming differentiation based on replicons and trans-acting initiators. This proposal requires that DNA replication precede major changes in gene commitment. Prior evidence against a role for DNA replication during terminal differentiation is reexamined along with other results from terminal differentiation of lower eukaryotes. This leads to a proposal that DNA replication may yet underlie terminal gene commitment, but that for it to do so there must exist two distinct modes of replication control. In one mode (mitotic replication) replicon initiation is tightly linked to the cell cycle, whereas the other mode (terminal replication) initiation is not cell cycle restricted, is replicon specific, and can lead to a terminally differentiated state. Aberrant control of mitotic and terminal modes of DNA replication may underlie the transformed state. Implications of a replicon basis for chromatin structure-function and the evolution of metazoan organisms are considered.
Animals↗
Sex chromosome-autosome translocations in the leaf-nosed bats, family Phyllostomidae. I. Mitotic analyses of the subfamilies Stenodermatinae and Phyllostominae.
Mitotic analyses using RBA- and C-banding were performed on Stenodermatine bats with X-autosome (XY1Y2) and X- and Y- autosome (neo-XY) translocations. RBA-banded metaphases of females revealed differential replication of the inactive X chromosome. An early replicating band comprises the short arm of the X, and an intermediate replicating band is located interstitially on the long arm. The early replicating short arm has a homologous counterpart either in the form of a free autosome (the Y2) or as part of the Y. Both the "autosomal" short arm of the X and its homologue fused to the Y are C-band negative and behave autonomously from the remainder of the sex chromosomes. They are separated from X and Y chromatin by centromeric heterochromatin which presumably acts as a barrier. The intermediate replicating region of the long arm of the X is also present in the subfamily Phyllostominae. In both subfamilies this region lacks a homologous counterpart. However, it may also represent a translocated autosome which, unlike the short arm of the X, is not separated from the inactive X by centromeric heterochromatin. Its intermediate replication time may represent a retarded replication due to its juxtaposition to late replicating X chromatin. These data are discussed in light of the theory of the evolution of sex chromosome heteromorphism, specifically as it applies to mammals.
Animals↗
Evidence that both G + C rich and G + C poor isochores are replicated early and late in the cell cycle.
Since the G + C content of a gene is correlated to that of the isochore in which it resides, and early replicating isochores are thought to be relatively G + C rich, early replicating genes should also be rich in G + C. This hypothesis is tested on a sample of 44 mammalian genes for which replication time data and sequence information are available. Early replicating genes do not appear to be more G + C rich than late replicating genes, instead there is considerable variation in the G + C content of genes replicated during both halves of S phase. These results show that both G + C rich and poor fractions of the genome are replicated early and late in the cell cycle, and suggest that isochores are not maintained by the replication of DNA sequences in compositionally biased free nucleotide pools.
Animals↗
DNA replication fork progression rate and temporal organization of S phase in normal epidermis and in basal cell carcinoma.
The double-pulse labeling technique for DNA fiber autoradiography was applied to epidermal cells from normal human skin and from human basal cell carcinoma (BCC). We aimed to measure the size and replication rate of the replication unit (RU) for both types of cell and to account, from these results, for our previous observation of a near doubling of S-phase duration in BCC, compared with normal skin. The mean RU size was 76 +/- 4 micron in BCC, not significantly different from the 68 +/- 6 micron value found in normal skin, so the mean of those two values (i.e., 72 micron), was used in further calculations. The rate of replication fork progression was 0.59 +/- 0.005 micron/min in the normal epidermis and 0.33 +/- 0.03 micron/min in BCC, corresponding to a replication time of the average RU equal to 61 min and 109 min, respectively. Thus, with an unchanged RU size in BCC, the observed 1.8-fold decrease in the rate of fork progression in the tumor can account entirely for our previous observation of a 1.8-fold increase in S-phase duration in this tumor, without requiring the assumption of any change in the temporal organization of DNA synthesis in the malignant cells. Considering S phase as an ordered process in which a major part, if not all, of the genome replicates at genetically determined times, we suggest that the clusters of replication units are, in turn, organized into temporally defined "sets". These sets are composed of all the clusters (whatever their chromosomal location) that are programmed to initiate replication during the same fraction of the S period. This hypothesis implies that DNA synthesis in a given set is triggered by some event coupled to progression of replication in the immediately preceding set. Based on a S-phase duration of 10.2 hours in normal skin and of 19.2 hours in BCC (our previous data), and assuming perfect synchrony and homogeneity of the clusters within each set and of each cluster's constitutive RUs, the minimum number of sequentially replicating sets, in both instances, can be estimated as roughly equal to 10.
Basal Cell Carcinoma↗
Relationship between chromosome replication and cell division in a thymineless mutant of Escherichia coli B/r.
The relationship between chromosome replication and cell division was investigated in a thymineless mutant of Escherichia coli B/r. Examination of the changes in average cell mass and DNA content of exponential cultures resulting from changes in the thymine concentration in the growth medium suggested that as the replication time (C) is increased there is a decrease in the period between termination of a round of replication and the subsequent cell division (D). Observations on the pattern of DNA synthesis during the division cycle were consistent with this relationship. Nevertheless, the kinetics of transition of exponential cultures moving between steady states of growth with differing replication velocities provided evidence to support the view that the time of cell division is determined by termination of rounds of replication under steady-state conditions.
Cell Division↗
Unidirectional replication as visualized by two-dimensional agarose gel electrophoresis.
Two-dimensional (2D) agarose gel electrophoresis is progressively replacing electron microscopy as the technique of choice to map the initiation and termination sites for DNA replication. Two different versions were originally developed to analyze the replication of the yeast 2 microns plasmid. Neutral/Neutral (N/N) 2D agarose gel electrophoresis has subsequently been used to study the replication of other eukaryotic plasmids, viruses and chromosomal DNAs. In some cases, however, the results do not conform to the expected 2D gel patterns. In order to better understand this technique, we employed it to study the replication of the colE1-like plasmid, pBR322. This was the first time replicative intermediates from a unidirectionally replicated plasmid have been analyzed by means of N/N 2D agarose gel electrophoresis. The patterns obtained were significantly different from those obtained in the case of bidirectional replication. We showed that identification of a complete are corresponding to molecules containing an internal bubble is not sufficient to distinguish a symmetrically located bidirectional origin from an asymmetrically located unidirectional origin. We also showed that unidirectionally replicated fragments containing a stalled fork can produce a pattern with an inflection point. Finally, replication appeared to initiate at only some of the potential origins in each multimer of pBR322 DNA.
DNA Replication↗
The nuclear membrane determines the timing of DNA replication in Xenopus egg extracts.
We have exploited a property of chicken erythrocyte nuclei to analyze the regulation of DNA replication in a cell-free system from Xenopus eggs. Many individual demembranated nuclei added to the extract often became enclosed within a common nuclear membrane. Nuclei within such a "multinuclear aggregate" lacked individual membranes but shared the perimeter membrane of the aggregate. Individual nuclei that were excluded from the aggregates initiated DNA synthesis at different times over a 10-12-h period, as judged by incorporation of biotinylated dUTP into discrete replication foci at early times, followed by uniformly intense incorporation at later times. Replication forks were clustered in spots, rings, and horseshoe-shaped structures similar to those described in cultured cells. In contrast to the asynchronous replication seen between individual nuclei, replication within multinuclear aggregates was synchronous. There was a uniform distribution and similar fluorescent intensity of the replication foci throughout all the nuclei enclosed within the same membrane. However, different multinuclear aggregates replicated out of synchrony with each other indicating that each membrane-bound aggregate acts as an individual unit of replication. These data indicate that the nuclear membrane defines the unit of DNA replication and determines the timing of DNA synthesis in egg extract resulting in highly coordinated triggering of DNA replication on the DNA it encloses.
Animals↗
Modified rapid radiometric method for detection of Mycobacterium tuberculosis from sputum samples.
A modified, simple and radiometric method for early detection of M. tuberculosis from sputum samples has been developed using a biphasic vial system for detection of 14C-CO2 produced by the metabolism of 14C-U-acetate on glycerol-free Lowenstein-Jensen medium (LJM). Of the 84 smear positive sputum samples examined, 85.7% and 86.9% were scored positive by radiometric and visual methods respectively. The detection rates at 1st, 2nd and 3rd week of the test were 53.3%, 60.7% and 82.1% by radiometry and 1.2%, 11.9% and 54.8% by visual methods respectively. The mean detection time was 10.7 days by the radiometric and 21.0 days by the visual method. An average replication time of primary culture from 54 sputum samples was 25.58 +/- 6.92 h (range 10.0-39.1 h).
Acetates↗
Replication of the heterogeneous heterochromatin of the sex chromosomes of Microtus cabrerae.
We used bromosubstitution to investigate the mode of replication of different types of heterochromatin located in the sex chromosomes of Microtus cabrerae. Our results clearly show that, although the heterochromatin is late replicating, the replication timing of different types of constitutive heterochromatin is related to their banding properties: R-type heterochromatin replicates before G-type heterochromatin, and this replication is asynchronous in both sex chromosomes. Furthermore, the late replication behaviour of the inactive X may spread to its constitutive heterochromatin. In some cells, a region of the constitutive heterochromatin of the late replicating X spontaneously switches to early replication, which may be related to transcriptional activity. Replication behaviour of the constitutive heterochromatin in the Y chromosome is similar to that of the late replicating X.
Animals↗
Nucleic acid synthesis in Bacillus subtilis infected with bacteriophage beta-22.
Changes in the synthesis of host and phage nucleic acid after infection of Bacillus subtilis with virulent bacteriophage beta22 were analyzed by deoxyribonucleic acid (DNA)-DNA and ribonucleic acid (RNA)-DNA hybridization. Host DNA replication continued during the first third of the 55-min latent period and then ceased at approximately the time replication of the phage genome was initiated. Host-specific RNA was synthesized concurrently with phage RNA during the first half of the latent period but was repressed late in the infection. For much of the latent period, the population of phage-specific RNA changed continually as new species were transcribed and earlier species were repressed; detectable changes ceased coincidentally with the appearance of intracellular phage. Control over transcription of phage DNA was to some degree an intrinsic property of the interaction of B. subtilis DNA-dependent RNA polymerase and the phage genome, since only the early species of phage RNA were synthesized in vitro by B. subtilis polymerase and pure beta22 DNA. In vitro transcription of late functions was demonstrated by using the endogenous RNA polymerase activity of the nucleoprotein complex (nuclear fraction) from infected cells.
Bacillus subtilis↗
[Genetic activity of the G- and R-band DNA of human mitotic chromosomes].
The concept of genetic inactivity of G-band DNA had been reinvestigated using the modified approach of Korenberg et al (1978). Coefficients of correlation and partial correlation between the relative gene density (g'), the relative G-band material richness (kH/C) and the relative chromosome size (s') were calculated. The kH/C was calculated as the ratio of brightness of fluorescence of chromosomes stained by Hoechst 33258 (Hi) and by chromomycin A3(Ci). The kH/C is the characteristics of G-band chromosome richness, because G-bands become bright after Hoechst 33258 staining and R-bands are bright after chromomycin A3 staining, while no significant C-bands in chromosomes which may be stained by these fluorochromes are discovered. For the kH/C determination the flow cytometry data of Langlois et al (1982) were used. The relative size of chromosomes was determined, based on the flow cytometry data of Young et al (1979). According to Korenberg, the "gene density" (g') in a chromosome was calculated as a ratio of the number of genes located in the chromosome before 1984 (Human Gene Mapping 7) to the relative size of this chromosome. Correlation between the "gene density" and the G-band richness was rs = -0.65. Out of 107 genes located in either G- or R-bands (Human Gene Mapping 7), 90 were mapped in the R-band and only 17 were ascribed to the G-band in metaphase chromosomes. The data on gene replication time show that all genes of the general cell activity and a portion of tissue-specific genes replicate during the early S-phase, together with R-band materials. These three independent lines of evidence are consistent with the notion that the R-band DNA is more genetically active than G-band DNA. The nature of "junk" DNA of G-bands is discussed.
Cell Line↗
OriC plasmids do not affect timing of chromosome replication in Escherichia coli K12.
The variability of the time interval between successive rounds of chromosome replication was estimated by density-shift experiments, by measuring the conversion of heavy DNA to hybrid density and light DNAs upon transfer of a steady-state culture growing in medium with [13C]glucose and 15NH4Cl to medium with light isotopes. The coefficient of variation (CV%) for the interreplication time of the Escherichia coli K12 chromosome was found to be 17%, i.e. similar to that for interdivision time. The presence of additional copies of oriC in the cell on a high copy number plasmid did not increase the CV of interreplication time. It is concluded that a single rate-limiting event is unlikely to time the initiation of chromosome replication. The regulation of initiation at oriC and the coordination with cell division is discussed.
Chromosomes, Bacterial↗
Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans.
The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.
DNA, Bacterial↗