Extra DNA Synthesis Research Articles

Chromosome methylation and measurement of faithful, once and only once per cell cycle chromosome replication in Caulobacter crescentus.

Caulobacter crescentus exhibits cell-type-specific control of chromosome replication and DNA methylation. Asymmetric cell division yields a replicating stalked cell and a nonreplicating swarmer cell. The motile swarmer cell must differentiate into a sessile stalked cell in order to replicate and execute asymmetric cell division. This program of cell division implies that chromosome replication initiates in the stalked cell only once per cell cycle. DNA methylation is restricted to the predivisional cell stage, and since DNA synthesis produces an unmethylated nascent strand, late DNA methylation also implies that DNA near the replication origin remains hemimethylated longer than DNA located further away. In this report, both assumptions are tested with an engineered Tn5-based transposon, Tn5Omega-MP. This allows a sensitive Southern blot assay that measures fully methylated, hemimethylated, and unmethylated DNA duplexes. Tn5Omega-MP was placed at 11 sites around the chromosome and it was clearly demonstrated that Tn5Omega-MP DNA near the replication origin remained hemimethylated longer than DNA located further away. One Tn5Omega-MP placed near the replication origin revealed small but detectable amounts of unmethylated duplex DNA in replicating stalked cells. Extra DNA synthesis produces a second unmethylated nascent strand. Therefore, measurement of unmethylated DNA is a critical test of the "once and only once per cell cycle" rule of chromosome replication in C. crescentus. Fewer than 1 in 1,000 stalked cells prematurely initiate a second round of chromosome replication. The implications for very precise negative control of chromosome replication are discussed with respect to the bacterial cell cycle.

Physiological Aspects and Experimental Reversion of Flowering in Fargesia murieliae (Poaceae, Bambusoideae)

From about 1993 to the present (1998), millions of plants of Fargesia murieliae, an ornamental bamboo, were flowering monocarpically in western Europe. All plants being ramets of a single genet, introduced in Europe about 80 years ago, the simultaneous flowering of all these ramets constitutes a single giant compound inflorescence. Flowering plants were subjected to different conditions of light intensity and temperature yielding different modes of development of flowering. Under low light intensity and high ambient temperatures (22-250C), reversion offlowering is ultimate- ly observed by development of vegetative shoots from basal buds offlowering culms. Under conditions that induce oxidative stress (full sun and drought) flowering and senescence proceed rapidly. The process of senescence is linked to oxidative stress as measured in elevated activities of superoxide dismutase and ascorbate peroxidase in the semelauctant inflorescences. By strong photoreduction of catalase activity, high light intensity is directly responsible for inactivation of one of the major defence mechanisms of the cells, thereby effectively accelerating flowering and senescence. In blades of spathes subtending the semelauctant inflorescences (short paracladial zone), DNA replication as well as loss of DNA from nuclei is observed. As this phenomenon is observed in green spathes prior to the development of the terminal inflorescence, DNA replication is a very early marker for senescence. A role for extra DNA synthesis prior to senescence is suggested in cytokinin biosynthesis.

Differential repair activity of human chromosomes

The frequency of radioactive label (3H-thymidine) incorporation in human lymphocyte chromosomes in the course of repair (extra) synthesis of DNA was assessed. Chromosomes 7, 12, and 21 were found to have a lower, and chromosome 22 a higher repair activity than was expected when a uniform incorporation of the label along the genome was hypothesized. Segments were detected that incorporated an increased number of labels in the course of extra DNA synthesis.

A cytophotometric analysis of the structure of hypothalamic cell populations in the frog, Rana temporaria (L.), with special reference to seasonal changes in chromatin status.

We used cytophotometry after the Feulgen reaction and UV cytophotometry to measure the DNA content of quiescent cells of the hypothalamic preoptic region (HPR) of adult and juvenile frogs (Rana temporaria) that had been caught in their natural habitat in winter, spring and summer. The histone-to-DNA ratio in cell nuclei was cytophotometrically determined using a combined Feulgen, heparine and alcian-blue staining procedure. The vast majority of HPR cells studied had nuclei with a diploid DNA content. However, we observed great variability in the Feulgen-DNA content of the HPR cell population, which was not detected in the diploid standard (hepatocytes). This heterogeneity in the diploid sample of the HPR cell populations was always greater in prespawning frogs and may have been due to differences in the chromatin arrangement in nuclei. About 1% of cells had a DNA content either ranging between diploid and tetraploid levels (H2C cells) or at the tetraploid level (4C and 2C x 2 cells). The proportion of these cells was not affected by the age of the animals or the annual cycle, thus suggesting that there is no age-related increase in the mean DNA content in the frog HPR. The mean DNA contents of H2C and 4C cells were much higher than those in the standard (hepatocytes). This cannot be simply attributed to the presence of different amounts of nuclear proteins, but rather indicates that at least a certain proportion of the highest DNA contents may be due to a real extra-DNA synthesis.

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Cell dedifferentiation was induced inVicia faba root tissues by removing the whole root meristem (decapitation) and the behaviour of the nuclear DNA in the dedifferentiating cells was studied by means of cytophotometric and autoradiographic analyses. Cytophotometric determination after Feulgen-staining showed that: 1. the vast majority of nuclei in differentiated cells were in the DNA postsynthetic phase, but their Feulgen absorption was lower than that of DNA postsynthetic nuclei (G2, 4 C) in the meristem; 2. such a Feulgen absorption was detected in certain nuclei after root decapitation; 3. all the mitoses in the dedifferentiating tissues were diploid, fully matching the Feulgen absorption of mitoses in the meristem.

A mechanism for rich-medium inhibition of the repair of daughter-strand gaps in the deoxyribonucleic acid of UV-irradiated Escherichia coli K12 uvrA

Ultraviolet-irradiated Escherichia coli K12 uvrA(B,C) cells show higher survival if plated on minimal growth medium (MM) rather than on rich growth medium (RM). This phenomenon has been referred to as ‘minimal medium recovery’ (MMR). UV-irradiated (4 J/m 2) uvrA cells showed a similar rate of protein synthesis, whether incubated in MM or RM, however, they showed a severe depression in DNA synthesis when incubated in MM that lasted for about 30 min, and the normal rate of DNA synthesis was not reestablished until about 60 min after irradiation. When a sample of these same cells was switched to RM immediately after UV-irradiation, there was only a slight slowing of DNA synthesis, and the normal rate of synthesis was reestablished by 60 min. An additional mmrA mutation or growth retardation by valine blocked both this extra DNA synthesis in RM, and the inhibitory effect of RM on survival. These findings suggest that the absence of a marked delay in DNA synthesis observed in RM may be responsible for the inhibitory effect of RM on the survival of UV-irradiated excision-deficient cells. Two hypotheses, which are not mutually exclusive, are propose and supported by data to explain why a fast rate of DNA synthesis after UV-irradiation partially inhibits postreplication repair and enhances cell lethality.

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological, autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after 3H-thymidine and 3H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. 3H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). 3H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.

Extra DNA replication and cell proliferation in wing imaginal discs of aDrosophila species hybrid.

Species hybrids obtained from corsses betweenD. azteca andD. athabasca display characteristic growth anomalies. These are restricted to hybrid males while hybrid females resemble the pure species. The ratio of the wing length to body length is 1 in pure species, 1.3 in hybrid males from the crossD. azteca x D. athabasca and 0.65 in hybrid males from the reciprocal cross. This indicates a disproportionate enlargement and diminuation, respectively, restricted to some tissues. Whereas the number of ommatidia in the compound eye is normal, the number of wing cells is approximately twice as high in hybrid giant males as in hybrid females and pure species. Microdeterminations of the DNA content of wing discs show that an extra DNA replication step occurs during the early pupal stage of hybrid giant males; the pupal stage of these males lasts about 58 h longer than that of the pure species. In eye-antenna discs no extra DNA synthesis has been found. Incorporation experiments reveal a delayed uptake of radioactively labeled thymidine into the DNA of wing discs in late third instar larvae and early pupae of hybrid giant males. The results of this study are interpreted to indicate that a disturbed balance between sex-linked and autosomal genes in hybrid males is responsible for the modified rhythm of DNA synthesis and of cell proliferation in certain cell types.

The Relationship between the Endomitotic Cell Cycle and the Enhanced Capacity for Protein Synthesis in Leguminosae Embryogeny

Summary The idea that the presence of multiple genomes per cell leads to an enhanced level of gene product in developing Leguminosae cotyledon cells is critically discussed. The gene dosage concept originally preconceived by Scharpe and van Parijs (1973) is further elucidated and the importance of a number of assumptions which were not always or even incompletely fulfilled are discussed. The results obtained with other Leguminosae species by different authors are compared to the results obtained with the garden pea. A critical analysis of all available data leads to the conclusion, although at present some of the important parameters are scarcely under control, that the gene dosage concept is a realistic approach. The question whether the gene dosis phenomenon is also underlying the development of the Leguminosae embryo suspensor and the controversial question whether the suspensor nuclei may undergo an extra DNA synthesis besides the phenomenon of endoreplication have been discussed since recently new data are available.

Extra DNA synthesis in embryo suspensor cells ofPhaseolus coccineus

DNA synthesis in the embryo suspensor cells ofPhaseolus coccineus has been studied by autoradiography after 30 minutes and 12 hours feedings with3H-thymidine (3H-TdR). It has been observed that two different DNA syntheses occur in cell nuclei of the suspensor, one concerned with the process of chromosome endoreduplication, the other with the process of extra DNA synthesis in several heterochromatic regions of the genome. Characteristics of the extra DNA synthesis in our material are: i) a greater3H-TdR uptake occurs during extra DNA synthesis than during DNA synthesis involved in chromosome endoreduplication; ii) extra DNA synthesis is not concomitant with the process of chromosome endoreduplication; iii) only some of the DNA strands in one and the same polytene chromosome region seem to be involved in the phenomenon of extra DNA synthesis; iv) extra DNA synthesis occurs in both chromosome regions known to carry ribosomal cistrons and in other regions of the genome.

Extrachromosomal DNA and the origin of oocytes in the telotrophic-meroistic ovary ofCreophilus maxillosus (L.) (Staphylinidae, Coleoptera-Polyphaga).

The development of the telotrophic ovary in the Staphylinid beetle,Creophilus maxillosus was examined. Cells, termed chordoblasts were identified in the germarium of 1-day-old pupae. Each of the chordoblasts undergoes a series of synchronous mitoses. Owing to the precise control of the cleavage plane, which is vertical to the long axis of the ovariole, each of the chordoblasts gives rise to a linear chain of sibling chordocytes. Extra DNA synthesis within each sibling string is usually limited to the most posterior chordocyte only, this being an oocyte progenitor.Divisions of the oocyte progenitor are differential mitoses in which the extra DNA material is transported preferentially towards the posterior pole of the spindle. As extra DNA synthesis and preferential segregation of this material result in gradual increase of this DNA in the nuclei of oocyte progenitors, cytokinesis of these cells becomes highly unequal, the larger of the two cells produced at each differential mitosis being as a rule the posterior cell, i.e. the oocyte progenitor of the next cell generation. As a resul of the series of differential mitoses each chordoblast gives rise to a number of nurse cells and only one definitive oocyte.It is suggested that somatic prefollicular tissue plays a decisive role in oocyte determination in the Coleopteran telotrophic ovary.

Complete and incomplete extra DNA reduplication during spermatogenesis of Carausius morosus Br. (Insecta, Phasmida)

Cytological investigations and cytophotometric DNA determinations were carried out on Feulgen stained squash preparations of male germ cells of Carausius morosus. During zytogene an extra DNA synthesis begins in all spermatocytes. In part of the cysts a complete reduplication of the nuclear DNA takes place. Following this “zygoreduplication phase” the chromosomes of the spermatocytes are present in “tetrapachytene” form and spermatogenesis then proceeds with twice the somatic number of chromosomes. In the other cysts DNA synthesis is blocked after a ten per cent increase. This DNA could be traced through succeeding stages (pachytene, diffuse stage, and diplotene), but neither its structural basis nor its fate is known. The two types of spermatogenesis are described up to and including diplotene.

Nuclear fragmentation in dedifferentiating cells of Nicotiana glauca pith tissue grown in vitro

Callus formation of Nicotiana glauca pith tissue grown in vitro was studied with cytological and autoradiographic methods during the first 144 hr of culture. A high frequency of prospective proliferating cells showed nuclear fragmentation and nucleolar extrusion. Experiments with tritiated thymidine showed that DNA was synthesized in cells with fragmented nuclei and that fragmentation could be considered a step in the formation of multinucleate cells appearing at 144 hr of culture. No metaphase accumulation was induced at any time with colchicine indicating that mitoses could not be accounted for the rapid onset of cellularization. Evidences of extra DNA synthesis at the nucleolar region and of the presence of cytoplasmic DNA at earlier times of culture are reported.

An Autoradiographic Study on the Development of the Female Gametophyte inGinkgo Biloba

Abstract Methyl green-pyronin staining and autoradiographic techniques after incorporation of tritiated thymidine, uridine, lysine and tryptophan are used to study the female gametophyte of Ginkgo biloba. In one mature gametophyte four distinct regions are distinguished: 1) the outer region (region a in fig. 1); 2) the region between archegonia (region b in fig. 2); 3) the very limited region (one single cell layer) surrounding each archegonium (region c in fig. 1); 4) the inner region, which makes up the largest part of the gametophyte (region d in fig. 1). The following observations are made: i) cells of region a. These cells are characterized by an uniform cell size but by an heterogeneous nuclear size. Nuclei of celles of region a incorporate tritiated thymidine even after mitotic divisions have ceased. Yet the pattern of incorporation is not that typical of chromosome endore-duplication; it better conforms an extra DNA synthesis of some regions of the genome. Labelling of cytoplasm after tritiated th...

Pattern of labelling after H3-thymidine incorporation in the nucleus of hypocotyl cells of Sinapis alba L.

DNA-synthesis in the hypocotyls of Sinapis alba L. was studied with H3-thymidine labelling. Cells from hypocotyl segments were stained by the Feulgen-method and squash preparations were made. The following labelling patterns were observed: 1. Labelling of the chromocentres only. 2. Nuclear area evenly labelled. 3. No radioactivity in the chromocentres. This pattern was rarely seen. — The frequency of the first two types in different tissue segments is not equal. In segments with more differentiated cells there was an increase in the percentage of nuclei with radioactivity only in the chromocentres. This could be due to a prolongation of the phase of synthesis in the chromocentres in this tissue. — The total number of labelled nuclei decreases basipetally as well as with the age of the hypocotyl. In hypocotyls of seedlings older than 52 hrs radioactivity appeared only sporadically in the nuclei. The decrease in the number of labelled nuclei is faster than the decline of the corresponding measurable total DNA synthesis in the hypocotyl. This can either be due to extra nuclear DNA synthesis or depend on an increase in DNA synthesis in the later replicating heterochromatic region of the nucleus.

Alcuni Aspetti Citologici Dell'Endosperma diPhaseolus CoccineusL.

Abstract Some cytological aspects of Phaseolus coccineus L. endosperm. — Cytological observations were made on the endosperm of Phaseolus coccineus, in a limited stage of development of the seed, using different dyes and labelling with 3H-thymidine and 3H-uridine. The results seem to indicate that, contrary to suspensor cells, in the endosperm of Phaseolus, at least in the considered stage, extra DNA synthesis is not present. Chromosomes in these nuclei, however, undergo several endoreduplication cycles. Nucleoli may be either one or more in a cell, and show a characteristic structure. They are often eccentric in the nucleus and extrusions of nucleolar content in the nucleoplasm or cytoplasm are seen. An apparent alternate activity of RNA synthesis in the nucleoli of the suspensor and in those of endosperm cells is discussed.

Cytochemical and Autoradiographic Analyses on the Embryo Suspensor Cells ofPhaseolus Coccineos

SUMMARYSpecific staining methods (Feulgen, methyl green-pyronin, toluidine blue molybdate, acridine orange), digestion tests (DNase and RNase) and autoradiographic techniques (incorporation of tritiated thymidine, uridine, lysine, tryptophan; DNA detection by means of tritiated actinomycin D) have been used to study the embryo suspensor of Phaseolus coccineus. Most of material studied consisted of developing seeds in which the cotyledons of the embryo occupied one third to one half of the endospermatic cavity. In the typically club-shaped suspensor, both the « handle » portion (which contains cells with low to medium degree of endopolyploidy) and the « knob » portion (which contains polytene chromosome cells) have been analyzed.Indications have been found that the cells with low and medium degree of endopolyploidy undergo extra DNA synthesis (gene amplification) on many heterochromatic chromosome regions during early stages of embryogenesis. Later on, the extra DNA synthesized seems to be released, from t...