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PubMed2000

Cloning and kinetics of expression of Brucella abortus heat shock proteins by baculovirus recombinants.

In an effort to develop genetically engineered Brucella abortus (BA) vaccines, the genes encoding heat shock proteins (HSPs) GroEL, GroES, and HtrA were cloned and expressed in the BAC-TO-BAC Baculovirus System, and the kinetics of protein expression were analyzed using various insect cell lines in suspension cultures, different cell densities in suspension cultures, multiplicities of infection and recombinant virus replication times. Trichoplusia ni cells expressed only BA HtrA, but Spodoptera frugiperda (Sf9) cells expressed all three recombinant proteins. The best GroEL expression was achieved by infecting 2x10(6) Sf9 cells/ml with an MOI 10 of recombinant virus and harvesting the cells after 96h of virus replication. GroES and HtrA were best expressed when infecting 2x10(6) Sf9 cells/ml with an MOI 1 of recombinant viruses and harvesting the cells after 120h of virus replications. Under these conditions BA recombinant HSPs were expressed as follows: GroEL at 16% of the total cellular proteins (105microg/ml concentration); GroES 2% (15.25microg/ml); and HtrA 8% (84.48microg/ml). This is the first report of cloning and expression of BA genes in the baculovirus system.

Animals↗
PubMed1990

The probability of G1 cells to enter into S increases with their size while S length decreases with cell enlargement in Allium cepa.

The distribution of cell surface area projection (cell size) has been measured at birth and at initiation of DNA synthesis in steady-state populations of Allium cepa root meristems. The conditional probability, P(I/G1), that initiation occurs given that the event of being in G1 also occurs has been estimated from these data. P(I/G1) was found to increase when cells became larger. The distribution of G1 duration has been constructed from indicated cell size distributions. The absolute frequencies of G1 times showed a maximum in the zone of cells with short G1 periods; about 14% of cells appear to enter into S with G1 congruent to 1 h. These results suggest that the increase of P(I/G1) was due to cell enlargement and not to cell aging. By comparing the cell size distribution at initiation of S and at the end of this period, a drastic reduction of cell size variability during DNA replication was observed and both curves were seen as rather similar in shape although they obviously had different modal points. These observations support that there is a negative correlation between the initiation size and the duration of genome duplication, and that cells which initiate DNA synthesis with the same size have a similar replication time. From this hypothesis, a plot of S duration versus cell size at initiation of this period was constructed by comparing the distributions of cell size at start and end of replication; this plot was also consistent with the existence of a negative correlation between cell initiation size and S length.

Allium↗
PubMed1986

Replication and segregation of eukaryotic chromosomal DNA.

Transmission of the genetic information from the parental to the two daughter cells resulting from each cell division requires this genetic information to be duplicated prior to cell division. This ensures that each daughter cell will inherit an exact and complete copy of the information contained in the parental cell. The duplication of the genetic information, which is codified in deoxyribonucleic acid (DNA), is called "DNA replication". During the last 33 years, since the structure of DNA was discovered by Watson and Crick (1953), DNA replication has been investigated using genetical, cytological, and molecular techniques. The present review is an attempt to summarize most of the knowledge that has been accumulated throughout these years on eukaryotic DNA replication, with special emphasis on the methodologies that have been employed. Besides, some of the current "hot spots" in this subject, such as the controversy concerning whether or not there are specific DNA sequences which are recognized as replication origins, the existence of a close association between replication timing and gene expression, and the mechanisms operating at the time of nascent replicon maturation and segregation of sibling DNA molecules, are discussed with special deference.

Animals↗
PubMed2000

Modification in the inherent mode of allelic replication in lymphocytes of patients suffering from renal cell carcinoma: a novel genetic alteration associated with malignancy.

Using fluorescence in situ hybridization (FISH) to interphase nuclei, we examined the replication timing of 1 allele relative to its counterpart in PHA-stimulated peripheral blood lymphocytes of normal subjects and patients suffering from a solid tumor (renal cell carcinoma). In the FISH assay, an unreplicated DNA sequence is identified by a single dot-like hybridization signal, whereas a replicated region gives rise to a duplicated, bipartite signal. Accordingly, lymphocytes of normal individuals show 2 patterns of allelic replication: (i) synchronized replication of allelic counterparts, as exemplified by the biallelically expressed loci TP53 and D21S55; and (ii) non-synchronized replication of allelic partners, as exemplified by the early and late replicating alleles of GABRB3, an imprinted locus subjected to monoallelic expression. However, when present in lymphocytes of the cancer patients, all 3 loci change their replication mode: alleles of TP53 and D21S55 become asynchronous, whereas the early replicating allele of GABRB3 delays replication, leading to relaxation in the imprinted mode of replication. Based on the tight relationship between temporal order of allelic replication and allelic mode of expression, the modified order of allelic replication observed in nonmalignant cells of individuals diagnosed with cancer represents a novel genetic alteration associated with malignancy. This alteration detected by simple cytogenetic means, applied to peripheral blood lymphocytes, offers a potential test for cancer identification. Genes Chromosomes Cancer 27:270-277, 2000.

Aged↗
PubMed1992

Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe.

In the budding yeast, S. cerevisiae, two-dimensional (2D) gel electrophoresis techniques permit mapping of DNA replication origins to short stretches of DNA (+/- 300 bp). In contrast, in mammalian cells and Drosophila, 2D gel techniques do not permit precise origin localization; the results have been interpreted to suggest that replication initiates in broad zones (several kbp or more). However, alternative techniques (replication timing, nascent strand polarity analysis, nascent strand size analysis) suggest that mammalian origins can be mapped to short DNA stretches, just like S. cerevisiae origins. Because the fission yeast, Schizosaccharomyces pombe, resembles higher organisms in several ways to a greater extent than does S. cerevisiae, we thought that S. pombe replication origins might prove to resemble--and thus be helpful models for--animal cell origins. An attempt to test this possibility using 2D gel techniques resulted in identification of a replication origin near the ura4 gene on chromosome III of S. pombe. The 2D gel patterns produced by this S. pombe origin indeed resemble the patterns produced by animal cell origins and show that the S. pombe origin cannot be precisely located. The data suggest an initiation zone of 3-5 kbp. Some aspects of the 2D gel patterns detected at the S. pombe origin cannot be explained by the rationale of initiation in broad zones, suggesting that future biochemical and genetic studies of this complex origin are likely to provide information useful in helping to understand the apparent conflict between the 2D gel mapping techniques and other mapping techniques at animal cell origins.

Chromosomes, Fungal↗
PubMed2001

DNA replication forks pause at silent origins near the HML locus in budding yeast.

Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed.

DNA Replication↗
PubMedDate not supplied

Telomere control of replicative lifespan.

The replicative capacity of cells may limit the lifespan of key systems in the body. It has long been known that normal human cells have a finite lifespan when placed in cell culture, and their lifespan is dependent on the age of the individual donor. The mechanism of the genetic program that times this process has been elusive. The telomere hypothesis of cell aging proposes that the length of the telomeric repeat array at chromosomal termini can time replication number and signal cell cycle arrest when critical telomere lengths are obtained. The erosion of telomeric DNA in normal tissues appears to be due to the lack of expression of components of the telomere maintenance system. Telomerase, the key enzyme involved in telomere replication, is not expressed in somatic tissues, but is expressed in germ cells, where telomere length is stably maintained, so that viable chromosomes can be transmitted to the next generation. Evidence is reviewed that correlates telomere length, telomerase activity, and the manipulation of telomere length with cell replicative capacity and cellular immortalization. Strong circumstantial evidence exists that indicates a role for telomere biology in the control of replicative capacity and in tumorigenesis.

Animals↗
PubMed2000

Chromatin assembly at kinetochores is uncoupled from DNA replication.

The specification of metazoan centromeres does not depend strictly on centromeric DNA sequences, but also requires epigenetic factors. The mechanistic basis for establishing a centromeric "state" on the DNA remains unclear. In this work, we have directly examined replication timing of the prekinetochore domain of human chromosomes. Kinetochores were labeled by expression of epitope-tagged CENP-A, which stably marks prekinetochore domains in human cells. By immunoprecipitating CENP-A mononucleosomes from synchronized cells pulsed with [(3)H]thymidine we demonstrate that CENP-A-associated DNA is replicated in mid-to-late S phase. Cytological analysis of DNA replication further demonstrated that centromeres replicate asynchronously in parallel with numerous other genomic regions. In contrast, quantitative Western blot analysis demonstrates that CENP-A protein synthesis occurs later, in G2. Quantitative fluorescence microscopy and transient transfection in the presence of aphidicolin, an inhibitor of DNA replication, show that CENP-A can assemble into centromeres in the absence of DNA replication. Thus, unlike most genomic chromatin, histone synthesis and assembly are uncoupled from DNA replication at the kinetochore. Uncoupling DNA replication from CENP-A synthesis suggests that regulated chromatin assembly or remodeling could play a role in epigenetic centromere propagation.

Autoantigens↗
PubMed1998

A polymer model for the structural organization of chromatin loops and minibands in interphase chromosomes.

A quantitative model of interphase chromosome higher-order structure is presented based on the isochore model of the genome and results obtained in the field of copolymer research. G1 chromosomes are approximated in the model as multiblock copolymers of the 30-nm chromatin fiber, which alternately contain two types of 0.5- to 1-Mbp blocks (R and G minibands) differing in GC content and DNA-bound proteins. A G1 chromosome forms a single-chain string of loop clusters (micelles), with each loop approximately 1-2 Mbp in size. The number of approximately 20 loops per micelle was estimated from the dependence of geometrical versus genomic distances between two points on a G1 chromosome. The greater degree of chromatin extension in R versus G minibands and a difference in the replication time for these minibands (early S phase for R versus late S phase for G) are explained in this model as a result of the location of R minibands at micelle cores and G minibands at loop apices. The estimated number of micelles per nucleus is close to the observed number of replication clusters at the onset of S phase. A relationship between chromosomal and nuclear sizes for several types of higher eukaryotic cells (insects, plants, and mammals) is well described through the micelle structure of interphase chromosomes. For yeast cells, this relationship is described by a linear coil configuration of chromosomes.

Animals↗
PubMed1998

The S phase: beginning, middle, and end: a perspective.

Events in the S phase of the cell cycle have been investigated to a relatively limited extent in comparison with those in G1 and M phases. Four aspects of S are briefly discussed in this report: (1) the final biochemical step permitting initiation of DNA synthesis, (2) determination of replication timing of individual genes and its mechanism, (3) S phase processes that lead to the onset of M phase, and (4) resetting the S-phase machinery.

Animals↗
PubMed1981

The initiation of chromosome replication in a dnaAts46 and a dnaA+ strain at various temperatures.

The regulation of chromosome replication initiation was studied at various temperatures with an E. coli dnaA46 strain and its dnaA+ parent. We find that, in both strains, the "initiation mass" varies depending upon growth temperature while the replication time remains constant relative to the cell doubling time. In the permissive temperature range, the initiation mass of the dnaA46 mutant strain is larger by a constant factor than that for a dnaA+ strain. We conclude that, even at temperatures permissive for growth of the dnaA46 strain, the activity of the dnaA46 product is lower than that of the wild-type protein. The dnaA gene product, therefore, plays an important role in regulating initiation.

DNA Replication↗
PubMed1998

A multivariate quantitative-genetic analysis of behavioral development in mice.

The present experiment attempted a behavior-genetic dissection of early behavioral development in laboratory mice. To this end, we used a full, replicated diallel cross to uncover the genetical architecture as well as the multivariate genetic structure underlying early behavioral ontogeny. A number of standard sensorimotor tests were administered on postnatal Days 3, 5, 8, 10, 13, 17, and 22 to a total of 622 pups from 120 litters (4-6 pups per litter) from a four times replicated complete diallel cross between five inbred mouse strains. The first day on which an animal showed adult performance was taken as its score on that test. MANOVA did not show any effects of the pup's sex on the speed of development. Hayman's analysis of variance for diallel tables indicated no or only weak additive-genetic effects. Dominance was absent in almost all cases, except for the auricular startle response, where weak directional dominance for fast development was found. These results are in accordance with an evolutionary past of directional selection for well-canalized development. Factor analyses of the phenotypic and additive-genetic correlation matrices indicate that at least two factors are necessary to describe the behavioral variation.

Adaptation, Psychological↗
PubMed1989

Increased exercise tolerance and reduced electrocardiographic ischaemia 3 and 12 hours after oral felodipine in effort angina.

The antianginal properties and the duration of action of two doses of felodipine, a dihydropyridine calcium antagonist with a vascular:myocardial potency ratio approximating 100:1, were investigated in 15 patients suffering from disabling effort angina pectoris with reproducible exercise tolerance. Felodipine (5 mg, 10 mg) and placebo were administered once in the morning on three different days, with a 24 h interval between them, according to a double-blind 3 x 3 latin square design, 5 times replicated. Symptom-limited cycloergometric exercise tests were performed 3 and 12 h after administration. Duration of exercise to ST segment depression of 1 mm and to peak exercise was increased (all P less than 0.01) by both doses of felodipine in comparison with placebo. Twelve hours after administration, the 10-mg dose induced a significant improvement in the exercise time and a smaller ST segment depression (all P less than 0.01) in comparison with the 5-mg dose. The relationship between ST segment depression and the pressure-rate product during exercise was favourably influenced by the 10-mg dose at 3 and 12 h after intake, and by the 5-mg dose only at 3 h after intake. These findings suggest an increase in coronary blood flow induced by felodipine. Apart from mild headache there were no other unwanted effects. In conclusion, felodipine improves exercise tolerance and reduces electrocardiographic ischaemia for up to 12 h after single oral administration in patients with effort angina. Increasing the dose from 5 mg to 10 mg produces a more prolonged effect, with increased exercise tolerance 12 h after intake.

Administration, Oral↗
PubMed1993

Regional base composition variation along yeast chromosome III: evolution of chromosome primary structure.

The recent determination of the complete sequence of chromosome III from the yeast Saccharomyces cerevisiae allows, for the first time, the investigation of the long range primary structure of a eukaryotic chromosome. We have found that, against a background G+C level of about 35%, there are two regions (one in each chromosome arm) in which G+C values rise to over 50%. This effect is seen in silent sites within genes, but not in noncoding intergenic sequences. The variation in G+C content is not related to differential selection of synonymous codons, and probably reflects mutational biases. That the intergenic regions do not exhibit the same phenomenon is particularly interesting, and suggests that they are under substantial constraint. The yeast chromosome may be a model of the structure of the human genome, since there is evidence that it is also a mosaic of long regions of different base compositions, reflected in wide variation of G+C content at silent sites among genes. Two possible causes of this regional effect, replication timing, and recombination frequency, are discussed.

Animals↗
PubMed1981

Evidence of diurnal variation of human epidermal cell proliferation. II. Duration of epidermal DNA synthesis.

In the epidermis of six healthy male volunteers the DNA synthesis time was measured by in vitro double labeling technique in a longitudinal study (48 h) with taking skin punch biopsies every 6 h. The external conditions of the experiments and one group of volunteers were identical to those of a previous study on LI of the epidermal keratinocytes. 14C-thymidine and 3H-thymidine were used for double labeling the DNA-synthesizing nuclei of keratinocytes. The duration of DNA replication time proved to be nearly constant, with minor fluctuation, during the entire time span of experiments (mean 6.19 +/- 0.3 h). Thus, the present results are in accordance with the conception that a periodically changing entrance rate of G1-cells into the S-phase of the generation cycle of keratinocytes is predominantly responsible for circadian variations in the number of DNA-synthesizing epidermal keratinocytes.

Adult↗
PubMed1981

Vitamin B12-dependent replication of L1210 mouse leukemia cells. A model system for cobalamin-folate inter-relationships.

L1210 mouse leukemia cells were made cobalamin-deficient by propagation in a medium from which cyanocobalamin was omitted and fetal bovine serum (containing protein-bound cobalamins) was replaced by bovine serum albumin. These cobalamin-deficient cells exhibited a normal replication time of 12 h, provided that the medium contained excess folate or 5-formyltetrahydrofolate. The cells responded poorly, however, to 5-methyltetrahydrofolate unless exogenous cobalamin was added. A cobalamin dependency was also observed when low levels of folate or 5-formyltetrahydrofolate were used. With 5-methyltetrahydrofolate, optimal stimulation of growth was observed with free and transcobalamin-II-bound cobalamin at 4,000 pM and 2 pM, respectively. Under cobalamin-replete conditions, cells contained 2,000 to 4,000 molecules of cobalamin/cell, and in the deficient state, this value declined to less than 10 molecules/cell; optimal replication on 5-methyltetrahydrofolate required approximately 180 molecules/cell. Cobalamin-deficient cells cultured in the absence of folate reached an arrested state from which limited replication could be induced by the addition of aquacobalamin; normal replication was induced by aquacobalamin plus 5-methyltetrahydrofolate. Results of this investigation are interpreted in terms of the requirement for tetrahydrofolate in cell replication and the production of this compound from folate and 5-formyltetrahydrofolate (via cobalamin-independent pathways) and from 5-methyltetrahydrofolate (via the cobalamin-dependent methionine synthetase).

Animals↗
PubMedDate not supplied

C-myc transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment.

In rats, partial hepatectomy induces reasonably synchronized DNA replication in the remaining liver after approximately 20 h. Events occurring during the earlier stages of liver regeneration are of interest because they may tell us how cells in vivo respond when they move from a differentiated resting state (G0 phase) to a proliferative state. We report here that the expression of the c-myc oncogene is increased up to 10-15-fold of the normal level within 1-3 h after partial hepatectomy. This expression begins to decrease rapidly after 4 h and has returned to less than double the normal level after 8 h, at which time replicative DNA synthesis has still not begun. A still larger increase in c-myc transcription (approximately 600-fold) is observed in the liver when protein synthesis is inhibited by an injection of cycloheximide. These findings suggest the existence of a short-lived protein that is synthesized soon after partial hepatectomy, and which suppresses the expression of c-myc.

Animals↗
PubMed1993

Immunoglobulin heavy chain enhancer is located near or in an initiation zone of chromosomal DNA replication.

In several animal viruses, enhancers have been implicated in both DNA replication and transcriptional activation. The linkage of the two mechanisms appears intimate, in that common DNA binding factors can be shared. The immunoglobulin heavy chain (Igh) intronic [heavy chain joining region (JH)-mu chain constant region (C mu)] enhancer (E mu) is required for tissue-specific transcription of Igh genes and is essential for somatic recombination of diversity (D) and J segments. We show here that E mu is located at or near an origin of chromosomal DNA replication, which is more active in B lymphocytes than fibroblasts. E mu does not fulfill two criteria demonstrated for some cellular origins. E mu can initiate but not maintain autonomous replicating activity in B cells. E mu is unable to impart early replication timing to a transfected VDJ-C mu Igh locus in B cells. Instead we propose that E mu-associated ori activity contributes to tissue-specific Igh expression through local effects on chromatin structure leading to subsequent accessibility of transcription and/or recombination factors for the enhancer.

3T3 Cells↗