Specific Activity Of DNA Research Articles

Onset of Cell Proliferation in the Shortened Gut

Cytokinetics in colonic mucosa were studied after ileal resection in young female rats. The RNA content of the transverse colon was increased 32% after 7 days; after 14 days the increase was present throughout the colon. The DNA content of the ascending colon increased 29% after 10 days. Specific activity of DNA labeled with [3H-methyl]thymidine was elevated 29% in the ascending colon and elevated 49% in the descending colon on the 7th postoperative day. Colonic cell proliferation is stimulated within 1 week of ileal resection.

Fractional incorporation of [3H]thymidine and DNA specific activity as assays of inhibition of tumour growth.

The Fractional Incorporation (FI) of [3H] thymidine ([3H]TdR) has been examined in small lung tumours after cyclophosphamide (CY) treatment in vivo and compared to the DNA specific activity (SA) at different times after treatment. FI was found to correlate with the incidence of labelled cells after treatment, whereas SA did not, due to the loss of DNA from drug-killed cells 72 h after treatment. The FI is independent of the precursor concentration in the tissue, and therefore may give a better index of DNA synthesis in irregularly perfused tissues than SA. Following either CY or 60Co radiation treatment, the time necessary for FI to reach the pretreatment level is quite similar to the growth delay measured for the FI depression 45 h after treatment and growth delay has been established in the Lewis lung tumour, which would allow the prediction of growth delay induced by another agent to be made within 2 days of treatment.

Alteration of DNA by 5-(3-methyl-1- triazeno)imidazole-4-carboxamide (NSC-407347)

5-(3-Methyl-1-triazeno)imidazole-4-carboxamide (NSC-407347, MTIC), an active intermediate in the metabolism of 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (NSC-45388, DTIC), was investigated for its effect on DNA. When [ 3H]MTIC was added to an aqueous solution of calf thymus DNA, it was rapidly hydrolyzed, and less than 0.5 per cent of the added 3H was recovered in DNA as methyl groups attached to its base and phosphate constituents. It was estimated that the hydrolysis of phosphotriesters in methylated DNA released 0.5 per cent of total bound 3H of DNA in the experiment in vitro, and 5.7 per cent in vivo. When MTIC was added to tissue culture cells which had been prelabeled with [ 3H]thymidine, there was a large and immediate increase in acid-soluble radioactivity of both the cell media and the cells. At the same time, there was an accelerated drop in the specific activity of DNA, indicating that degeneration of DNA had occurred; MTIC at 10 −3 M had a greater effect than at 10 −5 M. Paper chromatographic studies showed that the acid-soluble radioactivity was predominantly in the form of thymidine. MTIC at 10 −4 M induced repair synthesis of DNA but inhibited semi-conservative replication. The extent of repair synthesis was greater at 30 min than after 5 min. Treatment of cells with MTIC at 10 −3 M irreversibly reduced the sedimentation of DNA in alkaline sucrose gradient. At 10 −5 M, the reduction was reversible, and a normal pattern was restored in 30 min. Comparison of these results with those of sedimentation analysis of DNA in formamide gradient at neutral pH indicated that single strand breaks which were apparent in alkaline sucrose were probably the result of exposure of alkali-labile lesions in DNA to high pH.

Mast cell activation and tissue cell proliferation.

The effect of mast cell activation and degranulation on the proliferation in the intact mesentery was studied in Sprague-Dawley rats. Mast cell activation was achieved by a single intraperitoneal injection of Compound 48/80. The proliferation was studied using three independent methods for estimation of cell production and DNA synthesis: 1. the mitotic index, 2. the relative number of cells having a DNA content in the S and G2 regions, by Feulgen photometric measurement in individual cells, and 3. the specific DNA activity, employing a method which combines a liquid scintillation technique after an intravenous injection of 3H-thymidine and Feulgen photometric determination of the DNA content per membrane preparation. It was found that the proliferation of the normal mesenchymal cells adjacent to the activated and degranulated mast cells in the mesentery was significantly increased within 24 and 32 h, the maximum increase being more than 20-fold compared to untreated controls. The results suggest that the common type of mast cell may have a pathophysiological function related to stimulation of local cell proliferation.

Decreased unscheduled DNA synthesis in nondividing aged WI38 cells

The ability of cells from young and old cultures of WI38 human fibroblasts to undergo unscheduled DNA synthesis was studied. Following inhibition of semi-conservative synthesis in media lacking arginine and containing a hydroxyurea block, cells were irradiated with u.v. light and repair replication was measured by determination of specific activity of DNA and by autoradiography. Unscheduled DNA synthetic capacity was significantly reduced in old cultures. Quantitation of silver grains revealed that this reduction was a result of decreased repair activity by individual cells, which correlated well with a similar decrease in the proportion of dividing cells in the population. This observation that unscheduled DNA synthesis is reduced in older cells is in apparent contradiction to some earlier reports, and the failure of other investigators to identify aging changes in repair capacity is discussed in relation to differences in experimental procedure.

Biosynthesis of DNA and RNA in neuronal and glial cells from various regions of developing rat brain

Slices of cerebral hemispheres, brain stem, and cerebellum from rats 5-30 days old were used for in vitro incorporation of [methyl-3H]thymidine and [6-14C]orotic acid into DNA and RNA, respectively. The rates of DNA and RNA synthesis decreased markedly during development, with the most marked decrease observed for DNA. The different brain regions showed specific patterns of decline of DNA and RNA synthesis. Following incubation of slices, the tissues were fractionated to obtain fractions enriched in neuronal cells and in glial cells. In cerebellum, the granule neurons were separated from the Purkinje neurons. The glial:neuronal ratio of DNA specific activity was different in the three regions examined: in cortex it decreased from 6 at 10 days to 3 at 20-30 days; in brain stem it was 3 throughout 10-30 days; in the cerebellum (glia:granule neuron ratio) it was also 3 at 30 days but only 0.3 at 10 days. Concerning the RNA incorporation, small differences were found between neuronal and glial cells.

Biosynthetic events of hydrid regeneration : I. The role of DNA synthesis during tentacle elaboration.

The results of a combined morphological and biochemical study of the role of DNA synthesis during distal regeneration inHydra oligactis revealed that a burst of3H-thymidine incorporation into DNA preceded the elaboration of each of the initial three tentacles. In addition, the relative level of each burst of precursor incorporation relfected the number of tentacles formed at that time. Cytological localization of concentrated amounts of labeled material in nuclei of the hypostome and tentacle regions provided corroborative evidence for the biochemical findings.Evidence that the increased DNA specific activity levels described above are associated with tentacle initiation derived from studies in which regenerating hydra were cultured in hydroxyurea and studies in which hydra regenerated proximally rather than distally. Hydra regenerating in 8 mg/ml (0.105 M) hydroxyurea developed morphologically recognizable hypostomes but no tentacles, and incorporated3H-thymidine into DNA at a level distinctly below that exhibited by uncut, untreated animals. Similarly, hydra regenerated a normal, functional basal disc in the absence of any increased DNA specific activity. Therefore, it is suggested that tentacle initiation inH. oligactis requires concomitant DNA synthesis and, as such, represents an epimorphic phenomenon.

Partial purification of endonuclease activity from human lymphoblasts Separation of activities for depurinated DNA and DNA irradiated with ultraviolet light

Crude extracts of cultured human lymphoblasts (CCRF-CEM) contain endonuclease activity that cleaves ultraviolet-irradiated DNA in preference to untreated DNA. Purification of this activity was carried out using ultraviolet-irradiated PM2 phage DNA (5000 ergs/mm 2) as substrate in the enzyme assay. Since endonuclease specific for depurinated or depyrimidinated DNA might account for the apparent ultraviolet-irradiated DNA-specific activity, fractions derived during purification were also assayed with partially depurinated DNA. Chromatography of a 45–60% (NH 4) 2SO 4 fraction on a Sephadex G-100 column yielded a peak of activity (35 000 daltons) highly active against depurinated DNA but also active for ultraviolet-irradiated DNA. Further purification by DEAE-cellulose chromatography resolved two activities. One was highly specific for depurinated DNA with only minor activity for ultraviolet-irradiated DNA, and was strongly stimulated by Mg 2+. The other was non-specifically active against ultraviolet-irradiated or untreated DNA and was independent of Mg 2+. Additional studies suggest that neither of these two activities but a third enzyme is responsible for the ultraviolet-irradiated DNA-specific endonuclease activity observed in crude cell extracts.

Nucleic acid metabolism in adult tissues of Gromphadorhina in response to ecdysterone: Quantitative effects

Tissues of two species of adult Madagascar cockroaches, Gromphadorhina portentosa and G. bruneri, were examined to determine their competence to respond to ecdysterone at the transcriptional level. The tissues surveyed were fat body, testes, and male accessory glands. Hormone treatment was carried out either in vivo, by injection, or in vitro using the isolated tissues. The tissues were then exposed to radioactive adenine to label newly formed nucleic acids. In quantitative experiments, nucleic acids and ATP were extracted by acid precipitation of the tissue homogenate. ATP was assayed using crude firefly luciferase, whereas RNA and DNA were quantitated colorimetrically. Radioactivity was determined by liquid scintillation spectrometry. Differences in specific activity between experimental and control groups were expressed as percentages of control values. In vivo experiments with both male and female fat body showed an increase in ATP and decrease in RNA and DNA specific activities following 1 hr of treatment, and a decrease in ATP and increase in RNA and DNA specific activities after 8 hr of treatment. Sequential injections over an 8 hr period yielded the same pattern of incorporation as that described for 1 hr. Hydrocortisone treatment was ineffective, as was in vitro treatment with either hormone. Experiments with testes, both in vivo and in vitro, were largely negative, although some changes in specific activities were noted. With male accessory glands, there was an increase in ATP, RNA, and DNA specific activities at 1 hr of treatment as well as following sequential administration of hormone over 8 hr. It is concluded that adult Gromphadorhina fat body is competent to respond to ecdysterone on the level of nucleic acid metabolism in vivo, but that in vitro conditions for optimal response remain to be defined. Male adult accessory glands are competent to respond on this level both in vivo and in vitro. In general, it appears that an early effect of the hormone is to bring about an increase in the ATP pool, whereas a later effect is to stimulate the synthesis of nucleic acids.

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.

Regulators of Cell Division in Plant Tissues. XXII* Physiological Aspects of Cytokinin-induced Radish Cotyledon Growth

The cytokinin 6-benzylaminopurine (BAP) markedly stimulated the lateral expansion of excised immature radish cotyledons after a lag period of about 10 h. This growth occurred principally by cell enlargement, especially in the light which enhanced the response. However, a marked response 'to cytokinin occurred in the complete absence of red light during germination, cotyledon excision and incubation. Contact with BAP for 5 h significantly stimulated growth, but a maximum response required more than 24 h of contact; potassium chloride also promoted cotyledon expansion and acted synergistically with cytokinin. The response to cytokinin did not appear to be mediated by ethylene, gibberellins, polyamines or cyclic nucleotides. Growth induction did not alter the respiration rate and appeared to be inde- pendent of chloroplast function. Inhibitors of DNA and RNA synthesis and of protein synthesis on cytoplasmic ribosomes almost completely abolished BAP-induced growth, control growth being less markedly affected. There were, however, no significant BAP-induced increases in total DNA or RNA levels or specific activity before the initiation of growth stimulation. Similarly, BAP had no effect on any individual RNA species until after the lag period, when there was a small enhancement of uridine incorporation into RNA species with similar electrophoretic mobility to rRNA. Although total protein levels were not affected by BAP, the cytokinin enhanced amino acid incorporation into protein within the lag period, an effect which persisted when transcription was strongly inhibited by actinomycin D. Phosphorylation of total protein was stimulated by BAP only well after the onset of cytokinin-induced growth. Protein methylation, however, was stimulated by BAP during the lag period, and the effect was at least as early as the BAP-enhanced incorporation of methionine into protein. The possible role of translational control in the mechanism of cytokinin action is discussed.

Evidence for reproductive death of dividing cells in thymus and spleen following whole-body gamma-irradiation of mice.

Mice were given whole-body gamma-irradiation with 300 and 800 rad 1 hour or 1 day after the administration of 3H-thymidine. The time-course of the specific activity of DNA in thymus and spleen and the release of radioactivity into the fraction of DNA soluble in 0-14 N NaCl were determined for 28 hours after exposure. The results indicate that on average the loss of mitotically-active cells was delayed compared with the loss of a representative fraction of non-dividing cells. This suggested that different mechanisms might be implicated in the dying process of the dividing and non-dividing lymphoid cells. Using a dose-fractionation method, the total activity of DNA was determined in thymus and spleen at 24 hours after the first exposure and 25 hours after the in vivo labelling of DNA. A parallelism has been found between the results of this experiment and the recovery effect of the Elkind-Sutton type. It may be concluded that the dying process responsible for the loss of the dividing cells from thymus and spleen after irradiation probably involves reproductive death.

Distribution and Dosimetry of Tritium in Newborn Rats after in Utero Exposure to 3 H-TdR

In order to explore the suitability of continuous3 H- TdR infusion into pregnant rats for radiotoxicity studies in the offspring, the incorporation of tritium into the developing animals has been studied by biochemical means. In the offspring, the total incorporated activity (μCi/g wet weight) and the specific activity of DNA (μCi/mg DNA) show a direct relationship to the tritium activity applied to mothers. The distribution of activity between DNA and acid soluble fraction is organ specific, but independent of the activity applied. The slopes of the regression lines for DNA specific activity are organ specific. These facts have to be taken into consideration when dose calculations are attempted. On the other hand, the direct relationship between activity applied to mothers and the incorporation into the offspring establishes the system of continuous3 H- TdR infusion into pregnant rats as a suitable model to study3 H- TdR toxicity in mammalian cells under conditions in which all cell nuclei are exposed to...

Effect of cAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV-1).

AbstractThe effect of cAMP on a monkey kidney cell line (CV‐1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of 3H‐thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA.

Stimulation of DNA synthesis in mouse pancreas by triiodothyronine and glucagon.

An injection of triiodothyronine and glucagon stimulates the growth of several mouse tissues. The greatest increase in DNA specific activity occurred in the cells of the exocrine pancreas after a lag period of about 30 hr. There was also an increase in cell proliferation in the liver. Cellular hypertrophy, as measured by the ratio of RNA to DNA was increased in liver and kidney. This system should prove to be particularly useful for studying the factors controlling pancreatic growth. (Endocrinology 94: 1157, 1974)

The alteration of the regenerative activity and the cell cycle of partially hepatectomized rat liver following administration of D-galactosamine.

ABSTRACTA single injection of d‐galactosamine given to rats at different times after partial hepatectomy (PH) changes the pattern of regenerative proliferation. When administered during the pre‐replicative phase of regeneration, the onset of DNA synthesis and the increase in labelling index after injection of 3H‐thymidine are delayed by about 12 hr. The injection of d‐galactosamine at 24 hr after PH inhibits the drop in DNA synthesis occurring normally during the following 12 hr period. This was detected by a high labelling index and by an increased specific activity of DNA. The findings indicate a lengthening of the S phase, while G2 and M remain normal. Two modes of action of d‐galactosamine on the cell cycle are discussed.

Estimation of DNA, RNA and 125I- and 3H-labelled DNA in the same sample

Estimation of DNA, RNA, and the specific activity of DNA after labelling with [ 3H]thymidine and/or [ 125I]iodeoxyuridine has been accomplished using a recently developed procedure for the estimation of DNA with p-nitrophenylhydrazine ( pNPH). Samples of the pNPH reaction mixture are used for RNA estimation by th orcinol procedure and for 125I and tritium measurement. Correction for 125I contribution to the tritium measurement in double labelling experiments is accomplished either by use of a simple calibration curve (for high 3 H 125 I ratios) or by removal of 125I by hydrolysis and precipitation as AgI; in the latter procedure the efficiency of removal of 125I was greater than 99%.

The in vitro incorporation of [ 3H]thymidine into DNA and of 32P into phospholipids and rna in the aorta of rhesus monkeys during early atherogenesis

We measured the in vitro incorporation of [ 3H]thymidine into the DNA of segments of abdominal aorta from Rhesus monkeys fed atherogenic diets containing either peanut oil or corn oil for 15, 30 and 60 days. In all groups the serum cholesterol was significantly elevated but no differences in the aortic DNA specific activities could be detected. The incorporation of 32P into phospholipids and RNA was measured in the abdominal, distal and proximal thoracic segments of aorta from monkeys fed atherogenic diets for 30 days. In all 3 aortic segments there was a similar pattern of incorporation of 32P into the different classes of phospholipids. Maximal specific activities were found in the phosphatidyl inositol fraction. Apart from a decreased incorporation of 32P into the phosphatidyl choline and RNA of the distal thoracic aorta of peanut oil fed animals, no consistent differences could be observed in the phospholipids and RNA specific activities from the aortic segments of the control animals and of the animals fed the atherogenic diets. The phospholipid and RNA specific activities of the abdominal aortas in all groups were significantly lower than those of the distal and proximal thoracic segments.

DNA labelling and its assay in yeast

In yeast and other fungi the absence of thymidine kinase makes the specific labelling of DNA difficult. Until now, specific labellings of DNA have resulted in very low incorporation. On the other hand, after non-specific labelling of nucleic acids, DNA assay by modifications of Schmidt and Thannhauser's technique have provided a precision of only ± 30 %. We present a modification of this technique, which we have extensively used and which permits an estimate of the activity incorporated into DNA with an error of ± 2.5 %. All critical steps are described, including labelling of the culture. This method allows DNA of high specific activity to be obtained, and at the same time avoids the cell selection and DNA fragilization due to autoirradiation. The kinetics of incorporation of label into RNA and DNA have been studied. RNA, which represents 96–98 % of the total nucleic acids in yeast is labelled more rapidly than DNA. DNA specific activity increases considerably during the first generation in the labelled medium, before reaching an equilibrium. This situation makes obtaining DNA pulse labelling of high specific activity difficult. A method of “pseudo-pulse” experiment has been devised by comparing two cultures with different specific activities of neosynthesized DNA.

Effect of different conditions of light on the nucleic acid fractions in cotyledons ofChenopodium rubrutn L.

The nucleic acid fractions in cotyledons of young Chenopodium rubrum plants exposed to continuous light, continuous darkness and short (8 h) day, respectively and labelled with32P 24 h prior to harvesting were studied by means of chromatography on MAK columns. Some parameters of cotyledon growth (dry weight, cotyledon area, occurrence of mitoses) were also investigated. The changes in the nucleic acid fractions agreed with the dynamics of cotyledon growth. In continuous light the content of all fractions increased. The radioactivity of DNA and s-RNA did not undergo any great changes and only r-RNA increased. The specific activity of r-RNA increased slightly, that of soluble RNA and DNA was reduced. In continuous darkness the content of all the fractions did not undergo any great changes. The radioactivity as well as the specific activity of all fractions decreased. In short day the content of the nucleic acid fractions did not change conspicuously. Only the specific activity of s-RNA increased in a noticeable way while the radioactivity of r-RNA and the specific activity of DNA decreased and this of r-RNA did not change. The changes in nucleic acid metabolism were partially connected with changes in32P uptake which depended upon light conditions but they were not merely a consequence of this fact. Obviously, there also exists a more direct relationship between nucleic acid synthesis and growth.