CHANGES IN THE DNA CONTENT OF ADRENAL MEDULLA NUCLEI OF RATS INTERMITTENTLY EXPOSED TO COLD

In the adrenal medulla of rats exposed intermittently to cold (+4°C) for 100 and 300 hours, a considerable decrease (24 to 40 per cent) of the DNA content per nucleus was observed, followed by restoration to normal or above normal values within 10 days after the withdrawal of the stimulus. The findings were obtained with a scanning integrating histophotometer, and confirmed by microinterferometric investigations (on the basis of the measurement of total dry mass of nuclei isolated in aqueous medium before and after treatment with DNase) and by microchemical determinations, combined with the count of the nuclei in the homogenates. The observed decrease of DNA content cannot be attributed to errors of the methods used, nor to consequences of cellular degeneration. The available evidence seems to indicate a real decrease rather than a change in the state of a part of DNA in the nucleus in vivo whereby it becomes extractable by aqueous solutions. The restoration cannot be due to mitotic processes, which were actually never detected even with the use of colchicine, since the adrenal medulla cells in the adult rat are known to be irreversible, postmitotic cells. A correlation between the functional activity of the adrenal medulla cells and the content or state of DNA in their nuclei is demonstrated.     

II. DIFFERENCES IN ADRENAL MEDULLA NUCLEAR DNA CONTENT AMONG RATS OF DIFFERENT STRAINS FOLLOWING INTERMITTENT EXPOSURE TO COLD

The amount of DNA per nucleus in the adrenal medulla cells of four different strains of rats (Wistar, Sprague-Dawley, Long-Evans, and Italico) is determined both under control conditions and after 300 hr of intermittent exposure to cold. The adrenal medulla nuclei of the four strains of rats contain the same amount of DNA; however, the loss of DNA observed after the same experimental treatment differs markedly in the different strains. The loss is small in Wistar and Sprague-Dawley rats (8–13%), larger in Long-Evans rats (20%) and still larger in Italico rats (45%). The DNA loss in Wistar rats increases if the animals are fed the same diet as the Italico rats, and the DNA loss in Italico rats is reduced if the animals are fed the same diet as the Wistar rats. The different behavior of the four strains is discussed in terms of turnover of DNA.     

III. DECREASE OF LABELED DNA IN CELLS OF THE ADRENAL MEDULLA AFTER INTERMITTENT EXPOSURE TO COLD

Italico rats were injected with thymidine-³H 6 hr after the end of 300 hr of intermittent cold treatment. This plan of experiment ensured replacement in the adrenal medulla of lost DNA which is specifically sensitive to cold treatment and has a labeling index sufficiently high for statistical evaluation. The labeling index in the adrenal medulla decreases to one-half of the initial value within 10 days in animals subjected to further intermittent cold treatment and within 32 days in animals kept at room temperature. The very low mitotic index and the absence of doubling of the labeling index show that the observed labeling cannot be ascribed to pre-mitotic DNA synthesis. The concept of metabolic DNA adequately explains the findings.     

DNA Turnover in Adrenal Medullary Cells of Long Evans Rats

The nuclei of adrenal medullae of Long Evans rats, exposed intermittently to cold for a total of 300 h, showed a 20% decrease of their DNA content. This decrease is less than that found in similarly treated Italico rats (45%).
When ³H-thymidine was injected into Long Evans rats 6 h after the end of 300 h of intermittent cold exposure, incorporation of the label in the nuclei was approximately half of that found in Italico rats subjected to the same treatment. If the rats were re-exposed to cold, a decrease of labelling with a biological half-life of 17 days was observed; in animals kept at room temperature the half-life was 27 days.
Analysis of the DNA turnover in the adrenal medulla of Long Evans rats compared with Italico rats, shows that the smaller loss induced by cold exposure in Long Evans rats can be attributed to a reduced response to cold rather than to a slower DNA turnover under normal conditions or to a lower level of synthesis.     

AN ANALYSIS OF DNA LOSS AND SYNTHESIS IN THE RAT ADRENAL MEDULLA NUCLEI UPON COLD STIMULATION

The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at +4°C followed by 9 hr at room temperature) have been further studied with the aid of Feulgen histophotometry and H₃-thymidine radioautography. The amount of DNA decreases progressively with increasing length of cold exposure until 300 hr (-32%). Later a rapid change takes place, whereby DNA content per nucleus returns to values which are slightly, but consistently lower than normal. At termination of a period of cumulative exposure to cold, an analysis of a whole-day experimental cycle shows that the DNA decrease is due to loss of DNA during cold exposure and that DNA synthesis occurs upon return to room temperature. The balance between these two processes can be divided into three stages: (a) loss of DNA up to 300 hr of cumulative cold exposure; (b) marked increase in DNA by 350 hr; (c) oscillation around zero or slightly negative at 400 hr and beyond. These variations are due to: (1) the extension of DNA synthesis into the period of cold exposure as clearly demonstrated by radioautography (stage b), and (2) a later still greater DNA loss (stage c) which partly offsets the increased synthesis. A complex pattern of adaptation of the adrenal medulla cells, as regards DNA content, to the repetitive cold stimulus is thus demonstrated.     

CONSTANCY OF DNA CONTENT IN ADRENAL MEDULLA NUCLEI OF COLD-TREATED RATS

Reports of changes in DNA content of certain types of cells following exposure to conditions of stress has led to the suggestion that two kinds of DNA may be present. One is genetic DNA, and the other is called "metabolic" DNA. In a further attempt to investigate the possibility of this phenomenon, determinations of DNA content were made on Feulgen-stained nuclei of adrenal glands and kidneys in cold-treated rats. Feulgen-stained nuclei were measured by two-wavelength microspectrophotometry. Particular attention was given to the handling of the smears in hydrolysis and staining. Mean values of Feulgen-DNA contents in a total of 720 nuclei demonstrated (a) a constancy of DNA content within 2% in individual nuclei both in adrenal medulla and kidney cortex, (b) no more than an average of 2% difference in DNA content between control and experimental nuclei, and (c) no more than an average of 1.5% difference in DNA content between normal kidney cortex nuclei and normal adrenal medulla nuclei. These results confirm the view that the more precise the measurement, the more accurately the constancy rule is obeyed. Moreover, there is no support for the concept of a metabolic DNA in the rat adrenal medulla.     

Density dependent inhibition of cell growth in cultures of primary and established lines of cells

The effect of cell crowding on DNA synthesis (incorporation of ³HTdR and ³²PO₄) was studied by an improved method in monolayers of secondary cells and established cell lines, either normal or transformed by viruses or carcinogens. The method was based mainly on pulse labeling of cultures of cells a few hours after their seeding in equal numbers onto areas of different size in identical dishes, a condition which ensured equal physiological conditions and different degrees of crowding of cells. DNA synthesis was hardly inhibited in crowded monolayers of secondary chick, mouse and hamster embryo cells. The incorporation of radioactive thymidine and phosphate into DNA of cell lines such as BHK 21, 3T3/SV40 and L929 was strongly inhibited. An SV40-transformed line of hamster kidney cells (HKT7) synthetized DNA equally well in sparse as in crowded monolayers. In lines of human amnion (FL) and BHK 21 cells which were more extensively studied the degree of inhibition of DNA synthesis was inversely proportional to their density. Autoradiography after ³HTdR pulse-labeling indicated that the same proportion of cell nuclei were labeled in sparse and in crowded cultures. The extent of labeling (number of grains per nucleus) was lower in crowded cultures of those cells that also showed inhibition of incorporation of this label as measured by scintillation. The inhibition is thus expressed in retardation of DNA synthesis in cells in S phase rather than arresting it in a larger percentage of cells.     

Dynamics of DNA replication: an ultrastructural study

DNA replication in cells takes place in domains scattered throughout the nucleoplasm. We have characterized the dynamics of DNA synthesis in synchronized mid-S-phase HeLa cells. Saponin-permeabilized cells were allowed to elongate nascent DNA chains in presence of biotin-dUTP for 5, 15, and 30 min (a pulse experiment), or for 5 min followed by an incubation with unlabeled precursors for 10 or 25 min (a pulse-and-chase experiment). The replication foci were then identified in ultrathin sections using immunogold labeling of the incorporated biotin. Total number of particles per nucleus, total scanned area of the nucleus, size, shape, and gold particle number of each labeled cluster, and the density of clusters per nucleus were evaluated. We have demonstrated that as replication proceeds, the labeled sites increase in size up to 240 nm (30 min incorporation) while maintaining a broadly round shape. In pulse-and-chase experiments the labeled DNA was shown to spread to occupy DNA foci of approximately 400 nm in diameter. These results demonstrate that DNA replication is compartmentalized within cell nuclei at the level of DNA foci and support the view that the synthetic centers are spatially constrained while the chromatin loops are dynamic during DNA synthesis.     

H-thymidine incorporation into nuclear DNA of leaf cells

The incorporation of ³H-thymidine into nuclear DNA of leaf cells of Nanthium pennsylvanicum was studied as a function of concentration and specific activity of the radioisotope. From the assessment of the average number of grains per nucleus and the percent of labeled nuclei, it was concluded that the incorporation was a linear function of concentration of the exogenous radioisotopic solution and a logarithmic function of the incubation time. Ten microcuries per milliliter on the average yielded 20% of labeled nuclei with 18 grains per nucleus. Seven-fold increase in concentration only doubled the amount of ³H-thymidine incorporated. The lamina regions near the vein incorporated a significantly greater amount of the radioisotope than the lamina region at some distance from the vein. The specific activities of 2, 3.35, 6.7 and 15.3 c/mmole had no effect upon the amount of ³H-thymidine incorporated, if the amount of microcuries of the incubation solution was the same in each activity. Considering the total number of molecules, the estimated rates of incorporation indicated that at the activity of 2 c/mmole, the system operated with about 7 times higher rates as compared with the activity of 15.3 c/mmole.     

Is there "Metabolic" DNA in the Mouse Seminal Vesicle?

This study was designed to answer the question: Is H³-thymidine uptake by nuclei of the mouse seminal vesicle evidence for DNA synthesis and mitosis, or does it signify some "metabolic" function of DNA unrelated to chromosome duplication? Mice were given an intraperitoneal injection of H³-thymidine. Six hours later Feulgen squashes of the seminal vesicle epithelium were made and covered with autoradiographic stripping film. The silver grains above labeled nuclei were counted, and the Feulgen dye contents of these same nuclei were determined photometrically after removal of the grains from the emulsion. Unlabeled nuclei were also measured. The dye contents of non-radioactive nuclei form a unimodal distribution, indicating that polyploidy is absent from this tissue. The radioactive nuclei fall into two groups. In the first, the average dye content is the same as that of the cold nuclei (2C). In the second, the values range from 2C to 4C. In the 2C to 4C group the grain count is proportional to the dye content, showing that incorporation is correlated with synthesis. The radioactive 2C nuclei arose by mitosis during the course of the experiment. This is shown by the following facts: (1) They frequently occur in pairs. (2) They average smaller than unlabeled 2C nuclei. (3) Their average grain count is approximately half that of the 4C nuclei. (4) Labeled division figures are found. (5) A mitotic rate estimated from the number of labeled 2C nuclei accords reasonably well with one based on the number of observed mitoses. Since the incorporation of thymidine accompanies DNA synthesis and precedes mitosis, there is no reason to postulate a special "metabolic" DNA in this tissue.     

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.     

Macromolecular synthesis in the livers of aging mice as revealed by electron microscopic radioautography

For the purpose of studying the aging changes of macromolecular synthesis in animal cells, we studied many groups of aging mice during development and aging from fetal day 19 to postnatal newborn, juvenile, young adults, aged and senescent adults up to 12 and month 24 (2 years). They were injected with ³H-thymidine, ³H-uridine or ³H-leucine, precursors for DNA, RNA and proteins, as well as ³H-glucose, ³H-glucosamine, ³⁵S-sulfuric acid, or ³H-glycerol for glucide and lipid precursors, respectively, then sacrificed and the liver tissues were taken out, fixed and processed for light and electron microscopic radioautography. On many radioautograms the localization of silver grains demonstrating DNA, RNA and proteins in hepatocytes in respective aging groups were analyzed qualitatively. The number of silver grains and the number of cell organelles in each cell of each animal in respective aging groups were analyzed quantitatively in relation to the aging of individual animals. The results revealed that the localization of respective precursors as well as the number of silver grains in cell nuclei, cell organelles, changed with the aging of animals. The numbers of labeled nuclei and cell organelles, as well as the numbers of silver grains in nuclei and cell organelles changed due to aging of individual animals. The number of mitochondria, the number of labeled mitochondria and the mitochondrial labeling index labeled with silver grains were counted in each hepatocyte. It was demonstrated that the numbers of mitochondria, the numbers of labeled mitochondria and the labeling indices showing DNA, RNA and protein synthesis at various ages from embryonic day 19 to postnatal newborn day 1, 3, 9, 14, adult month 1, 2 and 6, reaching the maxima, then decreased to senile year 1 to 2, indicating the aging changes. The results indicated that mitochondria in hepatocytes synthesized nucleic acids and proteins independently from the nuclei, but their synthetic activities were affected from the aging of the individual animals.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary To determine the time and duration of the first and second DNA synthetic phases in fertilized egg cells and central cells of rice, a total of 753 ovules were sampled at 2 h intervals during the first 30 h after pollination and exposed to ³H-thymidine for 2 h at 25 °C. Autoradiographic observation of labeled nuclei was made for fertilized egg cells, as well as for central and antipodal cells. The first and second DNA synthetic phases in fertilized egg cells were found 8–12 h and 21–25 h after pollination, respectively. The durations of each cell-cycle phase in the egg cell were estimated to be 4–6 h for G₁, 4 h vor S and for G₂, and 2 h for M. In the central cell, the first DNA synthesis took place at 3–4 h after pollination, i.e., immediately after fertilization, followed by the formation of the primary endosperm nucleus. Antipodal cells also showed labeled nuclei in the early stages after fertilization. The first divisions of fertilized egg cell and primary endosperm nucleus were observed at 16–18h and at 4–6 h after pollination, respectively. The present observations suggest that sperm and egg nuclei participate in fertilization with haploid amount (1C) of DNA and fertilized egg cell originates thus in 2C state.     

AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-³H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.     

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.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

RADIOAUTOGRAPHIC LOCALIZATION OF DEOXYTHYMIDINE TRIPHOSPHATE IN TRADESCANTIA POLLEN GRAINS DURING DNA SYNTHESIS

Tradescantia pollen grains, isolated during the period of DNA synthesis in the generative cell, accumulate deoxythymidine triphosphate (dTTP)-³H after incubation with thymidine-³H in the presence of millimolar deoxyadenosine. Most of this dTTP-³H was found to resist extraction by the fixative, cold ethanol-acetic acid, and its location was investigated by radioautography with thin, dry emulsion. Substantial binding of dTTP-³H occurred as an artifact; but when nuclei were isolated from the fixed pollen grains by sonication, it was found that they were differentially labeled: generative nuclei contained dTTP-³H, vegetative nuclei did not. This observation is discussed and is interpreted as evidence supporting the idea that thymidine is phosphorylated only in the generative cell of the pollen grain.     

DNA TURNOVER IN ADRENAL MEDULLARY CELLS OF DIFFERENT STRAINS OF RATS AND ITS ENHANCEMENT AFTER INTERMITTENT EXPOSURE TO COLD

In Italico and Wistar rats maintained at room temperature 1% of nuclei of cells in the adrenal medulla incorporate ³H-thymidine. Following intermittent exposure to cold, the numbers incorporating increase to 10% and 7% in Italico and Wistar strains respectively. Only in the Wistar strain does the increase occur during actual exposure.
Assuming S to be 6–8 hr the labelling indices actually determined indicate a turnover time of DNA of 21–29 days. Since mitotic indices in experimental and control animals were found to be 0.004% without colchicine treatment and 0.009% after 3 hr of colchicine treatment, the turnover times of cells were calculated to be 1388–3125 days. To correlate mitotic indices and labelling indices, an S period of 400 hr would have to be assumed. It is concluded therefore that: (i) most of the observed labelling of DNA is due to metabolic turnover of DNA and only a small proportion represents pre-mitotic synthesis, (ii) differences in the rates of DNA synthesis found during exposure to cold in Italico and Wistar rats respectively are sufficient to account for the differences in reduction of DNA previously found between the two strains under these experimental conditions.     

Extra DNA synthesis in the dedifferentiating cells ofVicia faba roots

Summary 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.After³H-thymidine (³H-T) feeding of the decapitated roots and autoradiography, the following results were obtained: 1. two populations of labeled nuclei, characterized by two different levels of scattered labeling occurred in dedifferentiating tissues, slightly labeled nuclei being much more numerous than heavily labeled nuclei; 2. the percentage of labeled nuclei was much greater than that of DNA presynthetic nuclei in the root tissues; 3. almost all the mitoses were labeled after a 16-hour³H-T feeding; 4. the percentage of slightly labeled nuclei paralleled that of dedifferentiating cells; 5. the duration of the DNA synthesis phase and that of the gap between completion of DNA synthesis and mitosis differed in heavily and slightly labeled nuclei; 6. all nuclei which entered DNA synthesis also entered mitosis.These results are interpreted to mean that: 1. after decapitation, two different DNA syntheses occur in the dedifferentiating root tissues ofV. faba: DNA reduplication in cells which dedifferentiate starting from a DNA presynthetic nuclear condition (heavily labeled nuclei) and extra DNA synthesis in cells which dedifferentiate starting from a DNA postsynthetic nuclear condition (slightly labeled nuclei); 2. extra DNA synthesis is required in these dedifferentiating cells for entry into mitosis.