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PubMed1987

Role of replication time in the control of tissue-specific gene expression.

Late-replicating chromatin in vertebrates is repressed. Housekeeping (constitutively active) genes always replicate early and are in the early-replicating R-bands. Tissue-specific genes are usually in the late-replicating G-bands and therein almost always replicate late. Within the G-bands, however, a tissue-specific gene does replicate early in those cell types that express that particular gene. While the condition of late replication may simply be coincident with gene repression, we review evidence suggesting that late replication may actively determine repression. As mammals utilize a developmental program to Lyonize (facultatively heterochromatinize) whole X chromosomes to a late-replicating and somatically heritable repressed state, similarly another program seems to Lyonize individual replicons. In frogs, all genes begin embryogenesis by replicating during a very short interval. As the developmental potency of embryonic cells becomes restricted, late-replicating DNA gradually appears. This addition to the repertoire of gene control--i.e., repression via Lyonization of individual replicons--seems to have evolved in vertebrates with G-bands being a manifestation of the mechanism.

Animals↗
PubMed1986

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv.

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37 degrees C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13-18 times slower than that of E. coli.

Chloramphenicol↗
PubMed1991

Origins of replication: timing and chromosomal position.

Several new methods have been used to localize replication initiation sites in mammalian chromosomes. The results of these studies argue strongly for the presence of defined sequence elements that function much like the origins in the genomes of simple microorganisms. However, relatively disparate results from in vivo and in vitro studies suggest that initiation reactions in mammalian chromosomes may have unique features, possibly related to a more complicated chromosomal architecture.

Animals↗
PubMed1989

Replication timing of 10 developmentally regulated genes in Physarum polycephalum.

We have tested the hypothesis which stipulates that only early-replicating genes are capable of expression. Within one cell type of Physarum - the plasmodium - we defined the temporal order of replication of 10 genes which were known to be variably expressed in 4 different developmental stages of the Physarum life cycle. Southern analysis of density-labeled, bromodesoxyuridine-substituted DNA reveals that 4 genes presumably inactive within the plasmodium, were not restricted to any temporal compartment of S-phase: 1 is replicated in early S-phase, 2 in mid S-phase and 1 in late S-phase. On the other hand, 4 out of 6 active genes analysed are duplicated early, with the first 30% of the genome. Surprisingly, the two others active genes are replicated late in S-phase. By gene-dosage analysis, based on quantitation of hybridization signals from early and late replicating genes throughout S-phase, we could pinpoint the replication of one of these two genes at a stage where 80-85% of the genome has duplicated. Our results demonstrate that late replication during S-phase does not preclude gene activity.

Blotting, Southern↗
PubMed1991

Chromosome condensation from prophase to late metaphase: relationship to chromosome bands and their replication time.

As chromosomes condense during early mitosis, their subbands fuse in a highly coordinated fashion. Subband fusion occurs when two large subbands flanking one minor subband come together to form one band, which takes on the cytological characteristics of the original flanking subbands. Using four different banding techniques--GTG (G-bands obtained with trypsin and Giemsa), GBG (G-bands obtained with BrdU and Giemsa), RHG (R-bands obtained by heating and Giemsa), and RBG (R-bands obtained with BrdU and Giemsa)--we studied subband fusion from prophase (1,250 bands per haploid set) to late metaphase (300 bands). To quantify the condensation process, a fusion index was established. We found that chromosomes contain preferential zones of condensation. From prophase to late metaphase, the early replicating subbands (R-subbands) fuse more readily with each other than do the late-replicating subbands (G-subbands). R-bands usually replicate early and condense late independently of the adjacent G-bands, which replicate late but condense early. Therefore, chromosome bands can undergo DNA replication and chromatin condensation relatively autonomously. Our data suggest that (1) chromosome replication and condensation are closely connected in time, (2) the metaphase bands represent independent units of chromatin condensation, and (3) the condensation process is an important feature of chromosome organization.

Cells, Cultured↗
PubMed1976

Levoamphetamine vs dextroamphetamine in minimal brain dysfunction. Replication, time response, and differential effect by diagnostic group and family rating.

Double-blind crossover randomized Latin square comparison of placebo, dextroamphetamine, and levoamphetamine in 31 consecutively diagnosed children with minimal brain dysfunction (MBD) replicated a smaller nonrandom study. Both isomers showed significantly more benefit than placebo but were not significantly different from each other. Dextroamphetamine showed a nonsignificant trend of superiority over levoamphetamine. Of 25 subjects who responded well to drugs, three responded only to levoamphetamine, five only to dextroamphetamine, and 17 to both. This study seems to confirm the efficacy of levoamphetamine in MBD. An unsocialized aggressive subgroup (308.4) showed a nonsignificant trend for levoamphetamine superiority, in contrast to the hyperkinetic (308.0) and overanxious (308.2) subgroups. Those who responded best to levoamphetamine tended (not significantly) to be from poorer functioning families. Parents' ratings, but not teachers' or psychiatrists' ratings, showed significant placebo effect.

Amphetamine↗
PubMed1988

Time of replication of yeast centromeres and telomeres.

The time of replication of centromeres and telomeres of the yeast S. cerevisiae was determined by performing Meselson-Stahl experiments with synchronized cells. The nine centromeres examined become hybrid in density early in S phase, eliminating the possibility that a delay in the replication of centromeres until mitosis is responsible for sister chromatid adherence and proper chromosome segregation at anaphase. The conserved sequence element Y', present at most telomeres, replicates late in S phase, as do the unique sequences adjacent to five specific telomeres. The early and late replication times of these structural elements may be either essential for their proper function or a consequence of some architectural feature of the chromosome.

Centromere↗
PubMed1990

Position effects on the timing of replication of chromosomally integrated simian virus 40 molecules in Chinese hamster cells.

Simian virus 40 (SV40) DNA molecules chromosomally integrated at different sites in three Chinese hamster lung fibroblast lines replicated during the middle portion of S phase but not precisely at the same time in all three cell lines. The time of replication was unrelated to the presence of T antigen or to its relative activity in promoting SV40 replication. SV40 sequences and chromosomal DNA sequences adjacent to the SV40 insert in one cell line expressing a temperature-sensitive T antigen showed a T-antigen-independent difference in replication timing from the homologous, allelic locus not linked to SV40. Our results indicate that the timing of replication of these integrated SV40 molecules is dependent upon the site of integration and is not determined by the level of T antigen replication-promoting activity.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1987

5-aza-C-induced changes in the time of replication of the X chromosomes of Microtus agrestis are followed by non-random reversion to a late pattern of replication.

Treatment with 5-azacytidine (5-aza-C) causes an advance in the time of replication and enhances the DNase-I sensitivity of the inactive X chromosome in Gerbillus gerbilllus fibroblasts. We found that these changes were not stably inherited and upon removal of the drug the cells reverted to the original state of one active and one inactive X chromosome. In order to determine whether this reversion was random, we used a cell line of female Microtus agrestis fibroblasts in which the two X chromosomes are morphologically distinguishable. In this work we show that the reversion to a late pattern of replication is not random, and the originally late replicating X chromosome is preferentially "reinactivated", suggesting an imprinting-like marking of one or both X chromosomes. The changes in the replication pattern of the X chromosome were associated with changes in total DNA methylation. Double treatment of cells with 5-aza-C did not alter this pattern of euchromatin activation and reinactivation. A dramatic advance in the time of replication of the entire X linked constitutive heterochromatin (XCH) region was however, observed in the doubly treated cells. This change in the replication timing of the XCH occurred in both X chromosomes and was independent of the changes observed in the euchromatic region. These observations suggest the existence of at least two independent regulatory sites which control the timing of replication of two large chromosomal regions.

Animals↗
PubMed1986

Insertion of an R1 plasmid into the origin of replication of the E. coli chromosome: random timing of replication of the hybrid chromosome.

A 16 bp BgI II fragment was deleted in vitro from the minimal origin of replication of the Escherichia coli chromosome, oriC, and was replaced by a 10 kb R1 miniplasmid, pKN1562, containing the basic R1 replicon and a kanamycin resistance gene. The deletion-insertion was transferred by homologous recombination into the chromosome of a dnaA(ts) strain. P1 transduction separated the origin "mutation" from the dnaA46 allele. Integration of mini-R1 into oriC was verified by Southern blotting and by analysis of the R1 incompatibility phenotype. It was possible to isolate normal R1 miniplasmids from the integrated R1. Chromosome replication was initiated at random times after a short delay. The constructed strains grew 20%-30% slower than the wild type and showed more heterogeneous cell sizes.

Chromosomes, Bacterial↗
PubMed1986

5-Azacytidine-induced conversion to cadmium resistance correlates with early S phase replication of inactive metallothionein genes in synchronized CHO cells.

Previous studies have shown both hypermethylation and late replication of DNA sequences to be associated with gene inactivity. To determine whether there is a causal relationship between patterns of DNA methylation and replication timing during S phase, we have examined the timing of replication of the inactive, hypermethylated metallothionein (MT) I and II genes in synchronized, cadmium-sensitive (Cds) CHO cells. The time of S-phase replication of the MT genes was ascertained by determining the period of S phase wherein cadmium-resistant (Cdr) cells could be induced with highest frequency by pulse treatment of synchronized Cds cells with the hypomethylating drug 5-azacytidine (5-aza-CR), and by analyzing Southern blots of density fractionated DNAs isolated from synchronized cells pulse-labeled with BrdU during different intervals after release from hydroxyurea blockade. Southern filter hybridization analyses demonstrated replication of both MTI and II gene sequences within the first half of S phase. Consistent with this result, phenotypic conversion of Cds to Cdr was maximal immediately after hydroxyurea release and decreased abruptly within three hours. The replication of inactive hypermethylated MT genes in early S phase argues that transcriptional inactivity and gene-specific hypermethylation are not sufficient conditions for late DNA replication.

Animals↗
PubMed1975

Retardation time measurementson replicating bacillus subtilis chromosomes: effect of EDTA concentration.

We have found that high concentrations of EDTA (greater than 0.024 M) are necessary to produce large, constant numbers of intact replicating Bacillus subtilis chromosomes in lysates of log phase cells. The retardation time of replicating chromosomes in log phase cell lysates is about double that for chromosomes in stationary phase cell lysates, thus making measurement of retardation time a sensitive way to detect and study replicating chromosomes. A theory is developed to predict retardation times for many possible models of DNA replication. The retardation time data on log phase cells is sufficient to eliminate many replication models, but many possibilities remain.

Bacillus subtilis↗