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PubMed1997

Replication initiation and elongation fork rates within a differentially expressed human multicopy locus in early S phase.

Replication of the 400 copies of the 43 kb human ribosomal RNA (rDNA) locus spans most of the S phase. To examine the basis for the unusual pattern of rDNA replication, a sensitive strategy was developed to map origins of DNA replication and measure apparent rates of fork progression within a chromosomal locus. This technique, termed differential intragenomic replication timing, revealed that initiation within the actively transcribed rDNA occurred in early S within a 10.7 kb region spanning the promoter and 5' external transcribed spacer. Forks emanating from this early bidirectional origin progressed at an apparent slow rate with the sense and anti-sense forks moving at 0.32 and 0.23 kb/min. Using a photochemical-based technique, the chromatin status of the rDNA repeats was assayed throughout the S phase. Approximately 85% of the rDNA repeats were in a transcriptionally active chromatin structure at the start of S phase. A progressive decrease in the transcription state of the rDNA loci was observed, reaching a minimum between 3 and 6 h in mid S phase. Altogether, the data suggest a link between RNA polymerase I mediated transcription and site-specific initiation of DNA replication within the rDNA multicopy locus.

Chromatin↗
PubMed1990

DNA methylation and late replication probably aid cell memory, and type I DNA reeling could aid chromosome folding and enhancer function.

DNA methylation in mammals is reviewed, and it is concluded that one role of methylation is to aid cell memory, which is defined as the ability of mitotically derived progeny cells to remember and re-establish their proper cellular identity. Methylation of X-linked CpG-rich islands probably stabilizes X-chromosome inactivation, but other mechanisms appear to be involved. Late replication is discussed as a key ancestral mechanism for X inactivation, and it is emphasized that early and late replication domains may each be self perpetuating. Therefore, early-late replication timing becomes another strong candidate mechanism for cell memory. A chromosome-loop folding enigma is discussed, and it is concluded that special mechanisms are needed to explain the formation and maintenance of specific looped domains. DNA reeling, such as done by type I restriction-modification enzymes, is proposed to provide this special mechanism for folding. DNA reeling mechanisms can help to explain the cis-spreading of X-chromosome inactivation as well as long-range action by enhancers.

Animals↗
PubMed2002

Mutation screening and imprinting analysis of four candidate genes for autism in the 7q32 region.

Genetic studies indicate that chromosome 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate gene approach to identify the relevant gene and report the analysis of four adjacent genes localised to a 800 kb region in 7q32 that contains an imprinted domain: PEG1/MEST, COPG2, CPA1 and CPA5-a previously uncharacterised member of the carboxypeptidase gene family. Screening these genes for DNA changes and association analysis using intragenic single nucleotide polymorphisms (SNPs) provided no evidence for an etiological role in IMGSAC families. We also searched for imprinting mutations potentially implicated in autism: analysis of both DNA methylation and replication timing indicated a normal imprinting regulation of the PEG1/COPG2 domain in blood lymphocytes of all patients tested. The analysis of these four genes strongly suggests that they do not play a major role in autism aetiology, and delineates our strategy to screen additional candidate genes in the AUTS1 locus.

Amino Acid Sequence↗
PubMed1999

Abnormal methylation does not prevent X inactivation in ICF patients.

DNA undermethylation is a characteristic feature of ICF syndrome and has been implicated in the formation of the juxtacentromeric chromosomal abnormalities of this rare syndrome. We have previously shown that in female ICF patients the inactive X chromosome (Xi) is also undermethylated. This result was unexpected since female ICF patients are not more severely affected than male patients. Here we show that CpG island methylation is abnormal in some ICF patients but in other ICF patients, the difference in methylation pattern between Xi and Xa (active X) is maintained. The consequences of Xi undermethylation on gene expression were investigated by enzyme assays. They showed that significant gene expression did not correlate with CpG island methylation status. The widespread Xi undermethylation does not affect overall Xi replication timing and does not prevent Barr body formation suggesting that a normal methylation pattern is not required for normal chromatin organization of Xi. Molecular investigation of some X-chromosome intron regions showed that the methylation changes in ICF female patients extend to non CpG islands sequences. Our results suggest that the genetic alteration of DNA methylation in ICF syndrome has little consequence on X chromosome gene expression and chromatin organization.

Centromere↗
PubMed1995

[In situ quantitative analysis of Drosophila histone gene in S-phase].

We used a novel multiparametric microfluorometry analytic system to determine the replication timing of Drosophila histone gene DNA by in situ quantitative analysis of the gene in S-phase under a fluorescent microscope. There are 110 copies of histone genes per genome and each one of them is 5 kb in size. Primary cultured embryo cells were used to make preparations for microscopic analysis. Cells were first stained with DAPI and the total nuclear DNA contents in each nucleus reflected on the fluorescent intensity. We collected data of the fluorescent intensity from 400 of the cells in S-phase (Fig. 4). Then, the very same preparation was subjected to FISH (fluorescent in situ hybridization) using biotinylated DNA probes and FITC, and the fluorescent intensity of the hybridization signals were quantitatively detected from the same 400 cells and in the same order. This data showed the relative quantity of the signals representing the histone genes. From the correlation of fluorescent intensity of DAPI and that of FITC of the cells in S-phase, we found that the histone gene DNA completed its replication during early stage in S-phase (Fig. 5). The method we introduced here is considered to be able to use in many other cases of quantitative analysis directly in cells.

Animals↗
PubMed2001

Marek's disease virus infection in the brain: virus replication, cellular infiltration, and major histocompatibility complex antigen expression.

Marek's disease virus (MDV) infection in the brain was studied chronologically after inoculating 3-week-old chickens of two genetic lines with two strains of serotype I MDV representing two pathotypes (v and vv+). Viral replication in the brain was strongly associated with the development of lesions. Three viral antigens (pp38, gB, and meq) were detected in the brain of infected chickens. Marked differences between v and vv+ pathotypes of MDV were identified for level of virus replication, time course of brain lesions, and expression of major histocompatibility complex (MHC) antigens. Two pathologic phenomena (inflammatory and proliferative) were detected in the brain of chickens inoculated with vv+MDV, but only inflammatory lesions were observed in those inoculated with vMDV. Inflammatory lesions, mainly composed of macrophages, CD4+ T cells, and CD8+ T cells, started at 6-10 days postinoculation (dpi) and were transient. Proliferative lesions, characterized by severe infiltrates of CD4+CD8- T cells (blasts), started at 19-26 dpi and persisted. Expression of MHC antigens in endothelial cells and infiltrating cells within the brain was influenced by MDV infection. Upregulation of MHC class II antigen occurred in all treatment groups, although it was more severe in those inoculated with vv+MDV. MHC class I antigen was downregulated only in those groups inoculated with vv+MDV. These results enhance our understanding of the nature and pattern of MDV infection in the brain and help to explain the neurovirulence associated with highly virulent MDV.

Animals↗
PubMed1995

Induction of feline acquired immune deficiency syndrome by feline leukemia virus: alteration in response to hormones in the hypothalamic-pituitary-gonadal system.

Male kittens who were infected with the feline leukemia virus (FeLV) were found at various times after exposure to contain a sequence of dysfunction in their hypothalamic-pituitary-gonadal (HPG) system. To understand whether the involved endocrine glands in this system were damaged by FeLV, the hypothalamus, pituitary, and testes were tested for hormonal responsiveness in vivo and in vitro. The infected cats were administered with luteinizing hormone-releasing hormone (LHRH) and human chorionic gonadotropin (hCG). Their response to the treatment was studied, and they were then compared with untreated control cats. Normal response to LHRH for the synthesis of follicle stimulating hormone (FSH), luteinizing hormone (LH), and testosterone were found in the infected cats prior to 10 weeks of infection. After 10 weeks, the response was reduced by 25%, 38%, and 42%, respectively. Twelve weeks after infection, the response to hCG for testosterone synthesis was drastically reduced. The control cats, however, demonstrated normal prolonged biphasic patterns of response to hCG. The in vivo administration of the tropic hormones had no effect on the titer of FeLV gs antigen in the blood of the infected cats. The medial basal hypothalamus (MBH) from the control cats and cats in their 13th week of infection were cultured in vitro, with the presence of high K+ ion (60 mM). The control MBH responded to K+ ion stimulation for LHRH release. The K(+)-stimulated release of LHRH in the control MBH was 99% higher than that of the infected MBH. In contrast, the amount of unreleased LHRH in the infected MBH was 74% higher than that of the control MBH. In in vitro culture, the control pituitary gland responded markedly higher to LHRH stimulation for the release of gonadotropins (FHS and LH) than that of the infected one (140% compared with 56% for FSH, and 70% compared with 28% for LH, respectively). Whereas, the amount of unreleased FSH and LH in the infected pituitary gland were 59% and 31%, higher than that of the control gland. These results suggest that (i) the progressive development of neuroendocrine glands' dysfunction is related to viral replication time; (ii) the in vivo and in vitro responses to tropic hormones in the infected endocrine glands are drastically reduced; and (iii) this reduction in hormonal response may be caused by defective regulation of peptide hormonal secretion.

Animals↗
PubMed1988

Relationship between the timing of DNA replication and the developmental competence in Acanthamoeba castellanii.

In Acanthamoeba, two different cell types are known. Trophozoites are generated in the mitotic division cycle, whereas cells committed at late G2 phase of the cell cycle develop into cysts in response to starvation. In this paper we study the role of timing of DNA replication in regulating development. The investigation was performed with cultures growing in a non-defined medium (ND cells) that show a high encystation competence and with cultures that have been growing in a chemically defined medium (D cells) for several years and show a low encystation competence. Bivariate DNA/BrdUrd distributions show that ND cells progress through a cycle in which the short replication phase occurs immediately and exclusively after prior completion of mitosis. These cells arrest at late G2 phase of the cell cycle during the stationary stage. In D cells, DNA replication and mitosis seem to be uncoupled, since replication takes place before as well as after mitosis. These cells arrest within their replication phase during the stationary stage. These findings indicate that D cells do not progress into late G2 phase of the cell cycle and hence do not have the competence for commitment. The alternate timing of DNA replication and the low encystation competence of D cells can be reversed by cultivation of these cells in ND medium. Synchronization experiments reveal that late G2 phase ND cells exhibit a low capacity for BrdUrd incorporation and growth after transfer into D medium, whereas ND cells of earlier phases of the cell cycle show premitotic incorporation of BrdUrd into nuclear DNA and growth. These findings suggest on the one hand that premitotic DNA synthesis is a prerequisite for growth of cells in D medium, and that there is a dependence of the induction of premitotic DNA synthesis on the cell cycle, and on the other hand that a reciprocal relationship exists between the capacity of premitotic DNA synthesis and commitment to differentiation.

Acanthamoeba↗
PubMed2001

5-Aza-2'-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation.

5-Aza-2'-deoxycytidine (5-azadC) is widely used as a potent inhibitor of DNA methyltransferase. Cells treated with this drug show various phenomena such as the reactivation of repressed genes, change in replication timing, and decondensation of heterochromatin. A number of studies using this drug have been reported so far but it is still controversial whether such changes are due to 5-azadC-induced demethylation itself or the side effects of the drug. Here we report that 5-azadC treatment induces histone hyperacetylation in mouse centromeric heterochromatin which normally contains methylated DNA and hypoacetylated histones. Treatment also affects the intranuclear distribution of histone deacetylase 2 (HDAC2). However, histone hyperacetylation was not observed in DNA methyltransferase 1-deficient cells with a reduced level of genomic DNA methylation. Our results suggest that 5-azadC-induced histone hyperacetylation is independent of DNA demethylation and that DNA methylation is not essential for the maintenance of the histone hypoacetylated state in centromeric heterochromatin.

Acetylation↗
PubMed1997

A numerical study of the critical line of Kauffman networks.

Kauffman networks were introduced in 1969 as a model of genetic regulatory systems. One of the most striking successes of this model is its ability to reproduce, for a critical value of its parameters, the observed scaling laws of the average cell replication time and of the average number of cell types in a given organism vs. the number of genes. Yet, the numerical evidence for such scaling laws in the model is still unsatisfactory, and restricted to a particular critical point, while we expect that the scaling behaviour is universal along the critical line. In this paper we try to sharpen the evidence for the scaling behaviour of critical systems, carrying on a detailed numerical investigation of their properties. We measure the length of the cycles (which in the model represents the period of cell cycles) and their number (which represents the number of cell types) for a point of the critical line different from the only one previously studied. Our results seem to confirm that such quantities scale as radicalN for all critical systems, at least for lengths and numbers small enough. On the other hand, we found that their probability distributions are very broad (power-law like) and become broader with system size. This means that there is an effective scale of the length and of the number of cycles that increases much faster than radicalN, and in the infinite size limit the biological analogy found by Kauffman may be lost. A numerical study of the modular structure of critical networks supports this conclusion. The implications of this fact for the biological interpretation of the model are briefly discussed. Finally, we found that the typical weight of the attraction basins tends to zero as a power law in the infinite size limit, with an exponent which seems to be universal along the whole critical line.

Animals↗
PubMed1999

Making CENs of mammalian artificial chromosomes.

Mammalian artificial chromosomes (MACs) hold the promise of providing autonomous vectors for gene therapy in dividing cells. They would not require insertion into the genome and could include sufficient genomic sequences that surround the therapeutic gene to ensure proper tissue-specific and temporal regulation. Several groups have reported successful formation of MACs in human cells using transfection strategies that included alpha satellite DNA, the primary DNA found at normal human centromeres. These results, although extremely encouraging, have limitations such as unpredictable chromosome formation and success thus far in only one transformed human cell line. Examination of other cells where alpha satellite DNA has integrated into ectopic chromosomal locations, as well as naturally occurring dicentric and neocentromere-containing cell lines, suggests that alpha satellite DNA may not be necessary or sufficient for centromere formation. Overall, these results suggest that epigenetic modifications of centromeric DNA are required for efficient centromere formation. Models for this centromere-specific epigenetic modification include a specialized chromatin structure and differential replication timing of centromeric DNA. Thus, further investigation of these centromere-specific epigenetic modifications may suggest strategies for increasing the efficiency of generating human artificial chromosomes for use as gene therapy vectors.

Animals↗
PubMed2002

Global and gene-specific methylation patterns in cancer: aspects of tumor biology and clinical potential.

Heritable alterations of DNA that do not affect the base pair sequence itself but nevertheless regulate the predetermined activity of genes are referred to as epigenetic. Epigenetic mechanisms comprise diverse phenomena including stable feedback loops, nuclear compartmentalization, differential replication timing, heritable chromatin structures, and, foremost, DNA cytosine methylation (1-3). DNA cytosine methylation has recently gained major attention in the field of basic molecular biology as well as in studies of human diseases including cancer. Changes in DNA methylation patterns in human malignancies have been shown to contribute to carcinogenesis in multiple ways. Both hypo- and hypermethylation events have been described in various neoplasias leading to chromosomal instability and transcriptional gene silencing. DNA methylation research has entered the clinical arena and methylation patterns have become a major focus of clinicians seeking novel prognostic factors and therapeutic targets. The following minireview covers aspects of the basic molecular biology of DNA methylation and summarizes its importance in human cancers.

Apoptosis↗
PubMed1990

Characterization of the heterochromatin in moose (Alces alces) chromosomes.

The karyotype of moose (2n = 68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analyzed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating. The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-Trichness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.

Animals↗
PubMed1993

Silent substitutions in mammalian genomes and their evolutionary implications.

An analysis of silent substitutions in pairwise comparisons of homologous genes from different mammals has shown that, in spite of individual fluctuations, their frequencies (which are very strongly correlated with the frequency of substitutions per synonymous site calculated according to Li et al. 1985) do not vary, on the average, with the GC levels of silent positions. This holds in the general case, in which silent positions of pairs of homologous genes share the same composition, namely in the human/other primates, human/artiodactyls, and in the mouse/rat pairs, as well as in the special cases in which the composition of silent positions are different, namely in the human/rabbit and the human/rat (or human/mouse) pairs. A slightly lower frequency found for low GC values in the human/bovine and human/pig pairs seems to be due to the specific gene samples used. These results contradict the previously claimed existence of differences in mutation rates and of mutational biases in third codon positions of coding sequences located in different isochores of mammalian genomes. They also imply that the variations in nucleotide precursor pools through the cell cycle and the differences in replication timing, or in repair efficiency, which were reported for different isochores, do not lead, as claimed, to differences in mutation rates, not in mutational biases in mammals. The differences claimed appear to be due to using small gene samples when individual fluctuations from gene to gene are relatively large.

Animals↗
PubMed1990

High-resolution dynamic and morphological G-bandings (GBG and GTG): a comparative study.

A high-resolution replication banding technique, dynamic GBG banding (G-bands after 5'-bromodeoxyuridine [BrdUrd] and Giemsa), showed that, at a resolution of 850 bands/genome, GBG banding and GTG banding (G-bands after trypsin and Giemsa) produce almost identical patterns. RBG band (R-bands after BrdUrd and Giemsa) and RHG band (R-bands after heat denaturation and Giemsa) patterns were previously shown to be only 75%-85% coincident; thus GTG banding more accurately reflects replication patterns than does RHG banding. BrdUrd synchronization uses high concentrations of BrdUrd both to substitute early replicating DNA and to arrest cells before the late bands replicate. Release from the block is via a low thymidine concentration. The banding is revealed by the fluorochrome-photolysis-Giemsa (FPG) technique and produces the GBG banding that includes concomitant staining of constitutive heterochromatin. As opposed to other replication G-banding procedures, BrdUrd synchronization and GBG banding produces a reproducible replication band pattern. The discordance between homologs after GBG banding is similar to that after GTG banding and no lateral asymmetry of the constitutive heterochromatin has been observed. Also, BrdUrd synchronization neither significantly depresses the mitotic index, nor induces chromosome breaks. Thus, GBG banding seems as clinically useful as GTG banding and provides important information regarding replication time.

Adult↗
PubMed1989

Ambulatory blood pressure monitoring during sustained treatment with conventional and extended-release felodipine in mild-to-moderate hypertension.

To assess the duration of the antihypertensive effect of the dihydropiridine calcium antagonist felodipine in conventional (C-F) and slow-release (ER-F) formulations, 12 patients with essential hypertension underwent ambulatory blood pressure monitoring (ABPM) at the end of a 2-week treatment period with C-F 5 mg b.d., ER-F 10 mg once daily (o.d.) and placebo. C-F, ER-F and placebo were given in a double-blind 3 x 3 latin square design 4 times replicated. There was no systematic change in the ABP profile over the three study periods regardless of the treatment. In comparison to placebo, the mean 24-h systolic and diastolic blood pressures showed a significant and similar reduction after both formulations of F. Compared to placebo, C-F and ER-F induced a significant reduction in systolic blood pressure for 15 and 21 h, respectively, and of diastolic blood pressure for 16 and 21 h, respectively. Three patients complained of headache (mild in 2, moderately severe in 1), and two patients of nocturia, with either formulation of F.

Adult↗
PubMed1993

Bi- and multivariate analyses of diallel crosses: a tool for the genetic dissection of neurobehavioral phenotypes.

The genetic-correlational approach provides a very powerful tool for the analysis of causal relationships between phenotypes. It appears to be particularly appropriate for investigating the functional organization of behavior and/or causal relationships between brain and behavior. A method for the bivariate analysis of diallel crosses that permits the estimation of correlations due to environmental effects, additive-genetic effects, and/or dominance deviations is described, together with a worked-out example stemming from a five times replicated 4 x 4 diallel cross between inbred mouse strains. The phenotypes chosen to illustrate the analysis were locomotor activity and rearing frequency in an open field. Large, positive additive-genetic and dominance correlations between these two phenotypes were obtained. This finding was replicated in another, independently executed, diallel cross.

Alleles↗
PubMed1990

Inheritance of species-specific behaviors in the paradise fish (Macropodus opercularis): a diallel study.

Species-specific elements of the paradise fish's ethogram were recorded in one familiar and three different unfamiliar environments, which were designed to model certain features of this species' natural habitat: (1) a densely vegetated home range, (2) a novel open field, (3) a small novel place, and (4) a small novel place with a predator. The inheritance of the behavioral elements was investigated employing a five-times-replicated diallel cross among three inbred strains. A detailed Hayman analysis of variance and a variance-covariance analysis were performed to uncover the genetic architectures of these phenotypes. Additive genetic effects and/or ambidirectional dominance was found to be characteristic of most species-specific behavioral elements studied, suggesting an evolutionary history of stabilizing selection.

Animals↗