Metabolic DNA in the hepatocyte nuclei in newborn rats.

The behaviour in time of labelled nuclear DNA in the hepatocytes of newborn rats was studied using autoradiographic and biochemical techniques in two groups of experiments. In the first group H-3-thymidine was injected to the mothers at the 16th day of pregnancy and the amount of labelled DNA was evaluated in the newborns after delivery. In the second group H-3-thymidine was injected to the newborns two hours after birth and the labelled DNA was studied at the same time intervals as the first group. The amount of labelled thymidine incorporated into the first group of animals remains constant for the first three days of life, thereafter a reduction in specific activity of DNA is observed concomitant with an increase of the percentage of labelled nuclei and a decrease of the number of grains per nucleus. These results show that mitotic divisions, which are absent during the first three days of life, take place between the third and sixth days of life. The decrease of the specific activity is therefore due to dilution and not to loss of labelled DNA. In the second group of experiments the DNA labelled with H-3-thymidine shows a decrease by about 30--40% per day during the first three days of life accompanied by a decrease in the number of grains per nucleus without changes in the percentage of labelled nuclei. These data show that DNA synthesized during the first day after birth is metabolically unstable, unlike that synthesized during foetal life.     

Cytophotometric analysis of the cell population composition of the the preoptic area of the hypothalamus in the common frog

DNA contents in squashed cells of the adult frog hypothalamic preoptic region (HPR) were measured using the Feulgen and UV cytophotometry techniques. The histone-DNA ratio in the cell nucleus was determined by means of a combined Feulgen-heparin-Alcian blue staining procedure. The nuclei of the vast majority of HPR cells have a diploid DNA content. However, in cells of this group the mean values of DNA amount and the distribution range were always higher than those in hepatocytes used as a diploid standard. Such a heterogeneity in DNA content in the diploid part of HPR cell population could apparently suggest some differences in the nuclear chromatin arrangement to be always higher in spring before the frog spawning, and it seems to be characteristic of this type of cells. About 1 per cent of cells with hyperdiploid surplus of DNA (H2c cells) as well as of tetraploid cells (4c and 2c X 2 cells) is found in HPR in frogs sacrificed both in winter and in summer. The quota of these cells has no reference either to the frog's age or to the annual cycle. The fact that the mean DNA values in H2c and 4c cells are much higher than in the standard cannot be explained by the presence of different amounts of nuclear proteins only. It is suggested that at least some part of the highest DNA values may be due to an actual extra DNA synthesis in a small constantly existing pool of HPR cell population.     

Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

The solution to the cytological paradox of isomorphy

Cells with polyploid nuclei are generally larger than cells of the same organism or species with nonpolyploid nuclei. However, no such change of cell size with ploidy level is observed in those red algae which alternate isomorphic haploid with diploid generations. The results of this investigation reveal the explanation. Nuclear DNA content and other parameters were measured in cells of the filamentous red alga Griffithsia pacifica. Nuclei of the diploid generation contain twice the DNA content of those of the haploid generation. However, all cells except newly formed reproductive cells are multinucleate. The nuclei are arranged in a nearly perfect hexagonal array just beneath the cell surface. When homologous cells of the two generations are compared, although the cell size is nearly identical, each nucleus of the diploid cell is surrounded by a region of cytoplasm (a "domain") nearly twice that surrounding a haploid nucleus. Cytoplasmic domains associated with a diploid nucleus contain twice the number of plastids, and consequently twice the amount of plastid DNA, than is associated with the domain of a haploid nucleus. Thus, doubling of ploidy is reflected in doubling of the size and organelle content of the domain associated with each nucleus. However, cell size does not differ between homologous cells of the two generations, because total nuclear DNA (sum of the DNA in all nuclei in a cell) per cell does not differ. This is the solution to the cytological paradox of isomorphy.     

Adipose tissue of Drosophila melanogaster: VII. Distribution of nuclear DNA amounts along the anterior-posterior axis in the larval fat body

The DNA content of fat body nuclei was measured cytophotometrically as a function of position along an anterior-posterior (A-P) axis of the tissue throughout larval development. There was a dramatic 55-fold increase in the average amount of DNA per nucleus during the first 3 days of this period, but there was no further increase during the final day. However, the rate of increase had regional specificity. The amount of DNA per nucleus correlated significantly with its position along the A-P axis of the tissue: nuclei in a posterior direction contain gradually increasing amounts of DNA. There was up to a seven-fold difference between the smallest anterior and largest posterior nucleus. In addition, for three of the ages studied there was a subgradient in the posterior region with a slope that was considerably steeper than that of the overall tissue gradient. The tissue has three characteristic morphological regions, anterior, medial, and posterior, which can be recognized early in development and which are maintained throughout the larval period. The distribution of nuclear DNA classes determined for cells in each region for the final 2 days of larval life became fixed before the final day of development. The significance of the DNA gradient in terms of a protein storage gradient is discussed.     

Cytophotometric and electron microscopic study of chronic human skin lymphomas. I. A study of skin manifestations

Electron microscopic and cytophotometric studies of low-grade malignant cutaneous lymphomas and non-malignant skin diseases revealed substantial differences between these groups of diseases. In the latter case, the DNA content per nucleus is diploid but in the former an atypical distribution of DNA content per nucleus (more than 5% aneuploid and polyploid nuclei) is observed in addition to a significant excess of the mean DNA content per nucleus above the diploid standard level. In lymphomas electron microscopy reveals clusters of atypical lymphoid cells with irregularly shaped nuclei, nuclear pockets, nuclear extrusions, network of cytoplasmic microfilaments. These features never occur with skin diseases. The data obtained can be helpful in the diagnostics of the low-grade malignant cutaneous lymphomas.     

In vitro culture of Aloe barbadensis Mill.: Morphogenetic ability and nuclear DNA content

In vitro culture of Aloe barbadensis is very difficult for both callus Induction and plant regeneration. By contrast, micropropagation from shoot meristems is easily achieved. A DNA microdensitometric study was performed on different organs of A. barbadensis and during in vitro culture of different explants. It was shown that morphogenetic ability is correlated to nuclear DNA content in the explant and/or in the callus. In particular, plant regeneration (or micropropagation) is obtained only from calli (or from explants) in which the amount of DNA per nucleus falls strictly within the diploid limits (2C and 4C) of the species.     

A cytophotometric measurement of DNA in murine hepatocytic nuclei during cytomegalovirus infection

Summary The amount of Feulgen-DNA in the nuclei of normal murine hepatocytes as well as those from livers on the third and fifth day after lethal, cytomegalovirus infection was assessed by scanning cytophotometry. The technique enabled the measurement of the increasing content of DNA in hepatocytic nuclei during the course of the disease. The results suggest that although some of this increase may be due to cellular DNA synthesis, most of it is due to viral DNA replication. Lastly, the megalohepatocytes which characterise this disease reflect viral replication in predominantly diploid hepatocytes.     

A cytophotometric and karyometric study of the cardiac muscle cells of young rats exposed to intermittent high altitude hypoxia

Thirty-day-old rats were exposed to intermittent high altitude in barochamber (7000 m, 4 h per day, 4 exposures) and number of mono- and binucleated myocytes as well as DNA content per nucleus were determined in the right ventricular myocardium. The data indicate enhancement of mitotic division of mono- and binuclear diploid cells in hypoxic animals. Simultaneously, an increase in mononuclear and binuclear tetraploid cells occurred; the latter is probably due to DNA synthesis not followed by mitosis of some binuclear diploid cells.     

A cytophotometric measurement of DNA in murine hepatocytic nuclei during cytomegalovirus infection

The amount of Feulgen-DNA in the nuclei of normal murine hepatocytes as well as those from livers on the third and fifth day after lethal, cytomegalovirus infection was assessed by scanning cytophotometry. The technique enabled the measurement of the increasing content of DNA in hepatocytic nuclei during the course of the disease. The results suggest that although some of this increase may be due to cellular DNA synthesis, most of it is due to viral DNA replication. Lastly, the megalohepatocytes which characterise this disease reflect viral replication in predominantly diploid hepatocytes.     

Patterns of genome size in the copepoda

Adult somatic nuclear DNA contents are reported for eleven cyclopoid species (Megacyclops latipes, Mesocyclops edax, M. longisetus, M. ruttneri, M. leuckarti, M. woutersi, Macrocyclops albidus, Cyclops strenuus, Acanthocyclops robustus, Diothona oculata, Thermocyclops crassus) and for the harpacticoid Tigriopus californicus and range from 0.50 to 4.1 pg DNA per nucleus. These diploid genome sizes are consistent with previously published values for four Cyclops species (0.28–1.8 pg DNA per nucleus), but are strikingly smaller than those reported for marine calanoids (4.32–24.92 pg DNA per nucleus). We discuss three explanations, none of them exclusive of another, to account for the smaller size and range of cyclopoid genome sizes relative to calanoid genome sizes: (1) higher prevalence of chromatin diminution in the Cyclopoida, (2) phylogenetic structure or older age of the Calanoida relative to Cyclopoida and (3) nucleotypic selection that may influence life history variation and fitness. Measurements of genome size were made on Feulgen stained, somatic cell nuclei, using scanning microdensitometry which is well suited to the sparse and heterogeneous populations of copepod nuclei. The importance of measuring large numbers of nuclei per specimen, possible sources of variation associated with cytophotometric measurements, and appropriate use of internal reference standards and stoichiometry of the Feulgen stained nuclei are discussed.     

Transcription of Simian Virus 40 II. Hybridization of RNA Extracted from Different Lines of Transformed Cells to the Separated Strands of Simian Virus 40 DNA

The amount of simian virus 40 (SV40) DNA present in various SV40-transformed mouse cell lines and “revertants” isolated from them was determined. The number of viral DNA copies in the different cell lines ranged from 1.35 to 8.75 copies per diploid quantity of mouse cell DNA and from 2.2 to 14 copies per cell. The revertants had the same number of viral DNA copies per diploid quantity of mouse cell DNA as their parental cell lines. (However, they showed an increased number of viral DNA copies per cell due to their increased amount of DNA.) By using separated strands of SV40 DNA, the extent of each DNA strand transcribed into stable RNA species was determined for the transformed and “revertant” cell lines. From 30 to 80% of the “early” strand and from 0 to 20% of the “late” strand was present as stable RNA species in the cell lines tested. There was no alteration in the pattern of the stable viral RNA species present in three concanavalin A-selected revertants, whereas in a fluorodeoxyuridine-selected revertant there appeared to be less viral-specific RNA present in the cells.     

Separation of intact rat hepatocytes and rat liver nuclei into ploidy classes by velocity sedimentation at unit gravity

A system is described which permits the separation of isolated hepatocytes and isolated rat liver nuclei belonging to different ploidy classes by velocity sedimentation at unit gravity.The problem of obtaining single cells suspensions is discussed and preparations were obtained that contained 96% single hepatocytes.By improving the sedimentation method, it took 2.5 h to separate rat liver nuclei on sucrose gradients into diploid and tetraploid ploidy classes. Recoveries were generally over 95%. The diploid band was 99% pure. DNA and protein content of the ploidy classes were measured. After partial hepatectomy and [³H]thymidine injection it was found that the label moved largely into the tetraploid compartment.Isolated hepatocytes were fractionated in 1 h on Ficoll gradients. Erythrocytes were separated from small nucleated cells and the population of hepatocytes was clearly separated from these two cell populations. Diploid hepatocytes were 80% and tetraploid hepatocytes were 99% pure. Viability was about 80% after fractionation.The gene dosage of NADPH cytochrome c reductase, succinate dehydrogenase and lactate dehydrogenase was estimated in diploid and tetraploid hepatocytes. Gene dosage was equal in diploid and tetraploid hepatocytes for succinate dehydrogenase and NADPH cytochrome c reductase. It is suggested, after correcting for non-viable tetraploid hepatocytes, that the gene dosage of lactate dehydrogenase was significantly lower in diploid than in tetraploid hepatocytes.     

Cytophotometric identification of tetraploid Purkinje cells in young and aged rats.

DNA content of cerebellar basket and Purkinje cells of four month, one year, and two year old albino rats was determined by Feulgen cytophotometry. DNA content per nucleus remained essentially constant during aging although there was a slight shift to lower Feulgen-DNA values occurring in the one year age group. Purkinje cells of all three age groups were found to contain tetraploid amounts of DNA as compared to diploid basket cells.     

Cytophotometric identification of tetraploid purkinje cells in young and aged rats

DNA content of cerebellar basket and Purkinje cells of four month, one year, and two year old albino rats was determined by Feulgen cytophotometry. DNA content per nucleus remained essentially constant during aging although there was a slight shift to lower Feulgen-DNA values occurring in the one year age group. Purkinje cells of all three age groups were found to contain tetraploid amounts of DNA as compared to diploid basket cells.     

Determination of the nuclear DNA content of Saccharomyces cerevisiae and implications for the organization of DNA in yeast chromosomes.

The DNA content of the nucleus of the yeast Saccharomyces cerevisiae has been determined by both renaturation kinetics and DNA per cell measurements. Renaturation kinetics experiments were performed by following the decrease of optical hyperchromicity at 260 nm and by hydroxyapatite chromatography. DNA per cell measurements were made by the diaminobenzoic acid method and by the ethidium bromide method of Klotz &; Zimm (1972b). The conclusion from the above experiments is that the S. cerevisiae nucleus contains 9 × 10⁹ ± 2 × 10⁹ daltons of DNA. Previously we (Lauer &; Klotz, 1975) had measured the molecular weight of the largest piece of DNA in the yeast nucleus to be 2 × 10⁹ ± 0.2 × 10⁹. Here we extend this work by using a more highly protein-denaturing buffer system and conclude that the largest piece of DNA in the S. cerevisiae nucleus contains 1.5 × 10⁹ to 2.2 × 10⁹ daltons of DNA in both haploid and diploid cell lysates. From genetics, the largest yeast chromosome should contain 13% of the genome, or 0.9 × 10⁹ to 1.5 × 10⁹ daltons of DNA (using our DNA per cell range). Thus, the large DNA we measure contains from one to two times the amount of the DNA predicted from genetics to be in the largest chromosome. In light of these new data, viscoelastic measurements on yeast DNA are now consistent with the idea that each chromosome contains one piece of DNA.     

Nuclear variation withinAvenae

The amount of DNA per nucleus and the dry mass of isolated nuclei does not vary significantly between the six diploid species investigated. No differences were found either between tetraploid species or between hexaploids. Between the diploids, tetraploids and hexaploids taken as groups there was respectively a 1, 2 and 3 ratio maintained for the amount of DNA. For chromosome volume significant differences were found between the tetraploids but not in 2x or 6x series. Correlations are shown between chromosome volume, nuclear DNA content and nuclear dry mass. In polyploids the nuclear dry mass is less than expected from the values obtained for the diploids.Contribution No. 20 from the Canada Agriculture Research Station, Ste Foy, P.Q.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.