Effect of prostaglandins on H-thymidine uptake into human epidermal cells

Prostaglandins A₂, B₁, E₁, E₂, F_1α and F_2α were added to cultures of human epidermal cells (keratinocytes) for 24 hours at 37°C, and the effects on ³H-thymidine uptake into DNA was measured. At 70 μg/ml all prostaglandins tested except PGF_2α inhibited the uptake of ³H-thymidine greater than 50%. However, at 35 μg/ml, PGA₂ and PGB₁ were the only two prostaglandins to show significant inhibition, 96% and 51% respectively. At 17.5 μg/ml only PGA₂ caused substantial inhibition, 68%. In order to determine if the PGA₂ action was mediated by membrane receptors propranolol, phentolamine, metiamide and prostynoic acid were added in conjunction with PGA₂. None of the above receptor antagonists were able to reduce the PGA₂-induced inhibition of ³H-thymidine uptake. These results indicate that the pre-incubation of human keratinocytes with prostaglandins for 24 hours results in a decrease of ³H-thymidine incorporation in DNA. The precise mechanism of action is unknown at this time.     

DIURNAL VARIATION IN THE LABELING INDEX OF MOUSE EPIDERMIS: A DOUBLE ISOTOPE AUTORADIOGRAPHIC DEMONSTRATION OF CHANGING FLOW RATES

A diurnal rhythmicity in the labeling index was observed in the epidermis of hairless mice, injected with either ¹⁴C- or ³H-thymidine, at different times during a 24 hr period. A modified autoradiographic technique, using ¹⁴C- and ³H-thymidine and two overlying emulsion layers, makes it possible to clearly differentiate synthesizing cells which are singly labeled with either carbon-14 or tritium, and cells labeled with both isotopes. At various times during a 24 hr period, hairless mice were injected with thymidine-2-¹⁴C and colcemid, followed at 2 or 3 hr by a second injection of ³H-thymidine. The labeling indices were calculated for the ¹⁴C- and ³H-thymidine injection times. These labeling indices were consistent with the control, single isotope, labeling indices and exhibited the same diurnal rhythm. Cells singly labeled with ³H- or ¹⁴C-thymidine have either started or completed DNA synthesis during the interval between the two injections. Flow rates into and out of DNA synthesis, throughout the 24 hr period, can be calculated from these singly labeled cells. The flow rates varied rhythmically throughout the day and paralleled changes in the labeling indices. The influx and efflux flow rates, at all times measured, were not equal. The influx flow rate was reflected in the efflux rate at a time later equal to the duration of S. By means of these flow rates, the per cent of cells in DNA synthesis was calculated for each hour during a 24 hr period. The resulting labeling index curve matches the observed 24 hr diurnal rhythm in labeling indices. By extension of these flow rates through mitosis, the resulting mitotic index curve is comparable to the reported 24 hr diurnal rhythm in mitotic indices.     

Differential reactivity in mammalian chromosomes

Leucocyte cultures were treated with both ³H-thymidine and low temperature. Leucocyte cells were pulse labeled with ³H-thymidine for 15 to 20 minutes, and then placed in nonisotopic medium for 0, 1, 2, 3 and 4 hours respectively. Each culture was immediately treated with low temperature at 0–3° C for 24 hours. No metaphase chromosome were labeled at 0 and 1 hour after reincubation. Labeled metaphases were first observed after 2 hours of reincubation (3.9%); they increased after 3 hours (57%) and 4 hours of reincubation (39%). Labeled anaphases or telophases were also detectable in increasing proportions after 4 hours. Cell division proceeds very slowly through metaphase at low temperature. After labeling in the final 15 to 20 minutes of the S-period, one X-chromosome usually showed the late-replicating pattern. Label was found in the special segments of the X-chromosomes, X_E–a, X_L–a and X_L–b. Late-replicating regions in autosomes coincide more or less with the special segments. Differential reactivity in human chromosomes by low temperature was suggested to take place during the final part of G₂ after DNA synthesis.     

Hemocytoblastoid reaction of thymic subcapsular cells to diphtheria toxoid

Subcapsular cells lining the thymic stroma vary from low to high forms, while others have a hemocytoblastoid aspect. The purpose of the present study was to establish whether the transformation of the low forms into hemocytoblastoid subcapsular cells can be induced by an antigen. Rats given 10 Lf of diphtheria toxoid intramediastinally were killed at periods ranging from 3 to 24 hours later. Other rats were injected with ³H-thymidine at various intervals after the toxoid injection, and were killed one hour later. The observations revealed a rapid hemocytoblastoid transformation of subcapsular cells following administration of the toxoid. The transformation is detectable as early as three hours after the injection and can be completed after nine hours. Radioautography revealed that DNA duplication is initiated rapidly in the transforming subcapsular cells, since it can be completed 9 to 12 hours after the toxoid injection. Other observations suggested the transformation of reactive perithymic fibroblasts into subcapsular cells as well as the transformation of hemocytoblastoid fibroblasts and subcapsular cells into free hemocytoblastoid cells.     

GROWTH OF THE CHICKEN EMBRYONIC LENS TRANSPLANTED ONTO THE CHORIO-ALLANTOIC MEMBRANE

The influence of neural retina on the growth of chicken embryonic lens was studied by comparing the growth pattern of the lens transplanted onto chorio-allantoic membrane (CAM) with that of the normal lens. The lens from 6-day embryo, transplanted onto CAM after labeled with ³H-thymidine, continued to grow in the absence of neural retina at least for 12 days of incubation, although its growth rate was reduced. In the transplanted lens, no ³H-labeled epithelial cell differentiated into fiber at least for 2 days of incubation and ³H-labeled nuclei first appeared in the fiber cells on the fourth day of incubation, while, in the normal lens of 6-day embryo labeled with ³H-thymidine in situ, ³H-labeled epithelial cells differentiated into fibers within 24 hours. On the other hand, the fiber cells differentiated before transplantation maintained the nearly normal growth rate on CAM. The neural retina transplanted onto CAM together with lens induced the new fibers from the lens epithelium. These observations suggest that the neural retina initiates and promotes the fiber differentiation in the chicken lens, but its continued influence is not always necessary for the successive differentiation of epithelial cell into fiber and especially for the growth of the differentiated fiber cells.     

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with ³H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying ³H-thymidine (10^–6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10^–8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10^–6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.     

In vitro studies of the effect of intermittent compressive forces on cartilage cell proliferation.

Intermittent compressive (IC) forces (96 mm Hg, 0.3 Hz) inhibit by 35–60% the serum stimulated increase in ornithine decarboxylase activity (ODC) in chick embryo epiphyseal cartilage cells and rat chondrosarcoma cells. IC had no effect on mouse fibroblast L-cells ODC. The dose-response pattern of the IC effect indicated an all-or-none response with a threshold at 80 mm Hg, a pressure roughly equivalent to the in vivo weight bearing force. The k_m of the cartilage cell ODC, measured at four hours, was about 0.1 mM and was not affected by IC. The V_max, on the other hand, was significantly reduced by IC which is consistent with less enzyme or non-competitive inhibition. IC also produced a significant increase in cAMP levels in both cartilage explants and isolated cells in the presence and absence of serum and a significant reduction in ³H-thymidine incorporation into DNA. The findings show that cellular cAMP, on one hand, and ODC and DNA synthesis, on the other hand, change in opposite directions following exposure to serum and/or IC. Investigation of the IC effect on DNA synthesis in serum-deprived synchronized cartilage cells revealed that IC reduced the number of cells going into S but did not lengthen the G₁ phase. Exposure to IC early in G₁ (0–13 hours) produced the full effect, whereas IC application between 13 to 24 hours (pre S) had no effect. IC had no effect on ³H-thymidine incorporation in L-cells.     

The effect of photosensitizing dyes on the 3H-thymidine incorporation of cells grown in vitro

The photodynamic inactivation of ³H-thymidine incorporation in mouse embryo (ME) and mouse L cells by acridine orange (AO), methylene blue (MB) or neutral red (NR) has been studied by estimating the number of nuclei capable of incorporating ³H-thymidine during a 24 h period following light exposure. In the dark NR and AO reduced the number of ME-nuclei incorporating ³H-thymidine but MB caused an increase in non-scheduled DNA synthesis. The dark effect on L cells was less but the photoinactivation of thymidine uptake was proportionally greater in these cells. Polyoma virus was shown to be capable of growing in cells whose thymidine uptake was reduced or completely stopped by photoinactivation with NR. However, if the NR damage was very great, or when AO was used to photosensitize cells, the synthesis of viral DNA was interfered with.     

Differentiation of lymphocytes in mouse bone marrow. II. Kinetics of maturation and renewal of antiglobulin-binding cells studied by double labeling

DNA labeling by ³H-thymidine in vitro and antiglobulin-¹³¹I binding in vitro were used to determine the development and turnover of immunoglobulin-bearing lymphocytes in mouse bone marrow.Bone marrow cells from CBA mice previously injected repeatedly with ³H-thymidine for 1–84 hr were exposed to ¹³¹I-labeled rabbit-antimouse globulin for 30 min at 0 °C, and examined radioautographically. The antiglobulin-binding cells in bone marrow were predominantly (97–98%) nondividing small lymphocytes. Some plasmacytoid and monocytoid cells, but not the proliferating large lymphoid cells, also bound antiglobulin. The ³H-thymidine labeling index of the small lymphocyte population showed a rapid exponential increase (50% in 32 hr). The first small lymphocytes to show ³H-thymidine labeling were those lacking antiglobulin-binding capacity, reaching approximately 90% ³H-thymidine labeling after 2 days. Small lymphocytes which bound antiglobulin-¹³¹I at a concentration of 1.0 μg/ml became labeled with ³H-thymidine only after a lag of approximately 1.5 days. More avid antiglobulinbinding cells were delayed a further 12 hr in ³H-thymidine labeling. During in vitro culture the proportion of antiglobulin-binding small lymphocytes increased progressively in bone marrow but decreased in spleen cell suspensions.The results demonstrate a continuous, rapid renewal of immunoglobulin-bearing small lymphocytes in adult mouse bone marrow. Surface immunoglobulin molecules are not detectable when marrow small lymphocytes are first formed, but they appear and increase progressively in density as the cells mature.     

The fine structure of the proliferative cells of the mouse intestine as revealed by electron microscopic autoradiography with 3H-thymidine

Summary The duodenal and colonic epithelia in mice were observed with electron microscopic autoradiography 2, 5 and 24 hours after a single injection of ³H-thymidine. After 2 hours, in the duodenum, silver grains are found in many undifferentiated cells, in a few young goblet cells, in some crystal-containing cells, and in some lymphocytes. In the colon after 2 hours silver grains are seen in some undifferentiated cells, and in many young goblet cells. Undifferentiated cells are characterized by a few short microvilli, poorly developed rough-surfaced endoplasmic reticulum, abundant free ribosomes, and a few apical moderately dense granules. In normal animals, absorptive cells seem to arise from undifferentiated cells, and goblet cells — from younger goblet cells. Undifferentiated cells could also become young goblet cells. Crystal-containing cells, which may not be of epithelial origin, proliferate in the epithelium in the adult animal.     

Inhibition of basal protein degradation in rat embryo fibroblasts by cycloheximide: Correlation with activities of lysosomal proteases

Rat embryo fibroblasts were grown in medium containing ¹⁴C-leucine and ³H-thymidine. After a 24-hour chase in nonlabeled medium, cultures were placed in either fresh growth medium or medium containing 10–20 μg/ml cycloheximide. Cell monolayers were processed at daily intervals for three days. Four hours prior to processing, cultures were placed in fresh medium and the accumulation rate of trichloracetic acid soluble ¹⁴C in the media assayed. Cycloheximide effects a progressive decrease in the fractional degradation rate of the labeled cell protein, primarily during the first 24 hours. The specific activities of cathepsin D, cathepsin B, and neutral protease correlate closely with the fractional degradation rate. Other lysosomal hydrolases show little change during this period. The activities of the lysosomal proteases approach a new steady state which is correlated with the new steady state leve of protein synthesis. A model is proposed which relates the rate of protein break-down in the cell to the level of protein synthesis. The data also suggests the possibility that subpopulations of high turnover and low turnover cells exist in these cultures.     

Untersuchungen zur Aussagefähigkeit der Histoautoradiographie mit 3H-Thymidin am Dünndarmepithel

Zusammenfassung Es wurden die mittleren Silberkornzahlen über Krypten-und/oder Zottenepithelzellkernen in Duodenum, Jejunum und Ileum von NMRI-Mäusen 3/4, 12, 24 und 36 Std nach ³H-Thymidin-Injektionen bestimmt.Die tritium-markierten Kerne der differenzierten Zottenepithelzellen zeigen nach 12, 24 und 36 Std annähernd die Hälfte der mittleren Silberkornzahl der Kerne der Regenerations population in den Krypten 45 min nach ³H-Thymidin-Injektion. Diese Ergebnisse werden dahingehend interpretiert, daß durch die hier angewendete Methode der Histoautoradio graphie mit ³H-Thymidin und bei Auswertung von Zellkollektiven lediglich eine Markierung der Kern-DNS erfaßt wird, die während der prämitotischen Ruhephase (S-Phase) stattfindet. Ein Anhalt für den Nachweis eines Funktionsstoffwechsels der Kern-DNS (metabolische DNS) ließ sich nicht gewinnen.

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Investigation on the significance of histoautoradiographic results with ³H-thymidine at the epithelium of small intestine

Summary The average silver grain numbers over nuclei of the epithelium in duodenum, jejunum and ileum of NMRI-mice were determined 3/4, 12, 24 and 36 hours after injection of ³H-thymidine.After 12, 24 and 36 hours the labeled nuclei of the differentiated cells on the villi show nearly half the average silver grain count of the nuclei of the regenerative population in the crypts 45 minutes after ³H-thymidine administration. In interpreting these results, the incorporation of tritiated thymidine into nuclear DNA is only indicated during premitotic DNA-synthesis (S-phase) by the applied method of histoautoradiography with ³H-thymidine and evaluation of cell-collectives of intestinal epithelium. There is no evidence here of the functional metabolism of nuclear DNA (metabolic DNA).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.     

Effect of cell physiological state on infection by rat virus

Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with ³H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated ³H-thymidine at the time of infection.     

Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus

Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-³H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed.     

Effect of thiopyrimidine ribonucleosides on DNA and RNA synthesis in synchronized mammalian cell cultures

The uptake of ³H-uridine into RNA and of ³H-thymidine into DNA was investigated in synchronized Chinese hamster cells which had been exposed to thiopyrimidine ribonucleosides. The cells were synchronized at metaphase by reversal of colcemid inhibition; these cells were then labeled with either ³H-thymidine or ³H-uridine at selected times, and analyzed in autoradiographs. Incorporation of ³H-thymidine into DNA was not inhibited by administration to the cells of 2-thiouridine or 4-thiouridine (4 × 10⁻³ M). Exposure of the cells to the anti-metabolites for over 15 h significantly reduced the incorporation of ³H-uridine into nuclear RNA and completely blocked the labeling of cytoplasmic RNA. This finding is interpreted as an indication that RNA synthesis was inhibited in cells which continued to synthesize DNA. The inhibition of RNA synthesis hindered cell division and decreased cell viability. This lethal effect is similar to the “unbalanced growth” induced by inhibitors of DNA synthesis. The thiopyrimidine ribonucleosides, however, killed mammalian cells without inhibiting DNA synthesis.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

LYMPHOCYTE POPULATIONS IN MOUSE BONE MARROW: QUANTITATIVE KINETIC STUDIES IN YOUNG, PUBERTAL AND ADULT C3H MICE

Continuous ³H-thymidine infusion was used to characterize two kinetic subpopula-tions of small lymphocytes in mouse bone marrow during normal growth and development. Young (4 wk), pubertal (8 wk) and mature (16 wk) C3H mice were infused subcutaneously with ³H-thymidine for periods up to 10 days. Femoral marrow was then examined in radioautographic smears. During the first 3 days the proportion of marrow small lymphocytes labelled by ³H-thymidine showed a rapid exponential increase to 93%, 81% and 72% in 4 wk, 8 wk and 16 wk mice respectively. The rate of appearance of labelled small lymphocytes then declined markedly but remained higher in younger than in older animals. The labelling curves were found to represent the summation of two exponential curves from which the proportions and renewal rate of corresponding cell populations were calculated. Most marrow small lymphocytes comprised a rapidly renewing population but in mice of increasing age the relative incidence of these cells fell (93-3% at 4 wk; 88-0% at 8 wk; 78-5% at 16 wk) and their half-renewal time (T_½) lengthened (14 hr at 4 wk; 18 hr at 8 wk; 24 hr at 16 wk). The remaining small lymphocytes were slowly renewing with mean T_½ of 4, 7 and 14 days in 4, 8 and 16 wk mice, respectively. Some heavily labelled small lymphocytes persisted in the marrow up to 10 wk after fourteen daily ³H-thymidine injections in 10–12 wk mice. The numbers of rapidly renewing cells decreased from 604 times 10³ to 228 times 10³ per mm³ of marrow from 4 wk to 16 wk, respectively, while slowly renewing cells increased from 44 times 10³ to 61 times 10³ per mm³. The total number of nucleated marrow cells per femur increased from 4 wk to 16 wk but the rapidly renewing small lymphocytes per femur fell in numbers by 36% and in renewal rate by 63%. The results demonstrate a selective change in bone marrow small lymphocytes with age; rapidly renewing cells decline in number and renewal rate while the number of slowly renewing cells increases. The concept of bone marrow as a primary lymphoid organ is discussed.     

Application and efficiency of scintillation autoradiography for Drosophila polytene chromosomes

Summary A rapid method of autoradiography using the scintillation cocktail (Toluene and scintillation fluid, Omnifluor) has been described earlier. Its application and efficiency have been tested using both ³H-thymidine and ³H-uridine. The optimum time required for processing the autoradiograms has been found to be 24 h dry exposure followed by 48 h in the scintillation mixture. Detailed analysis of the autoradiograms with ³H-uridine reveals that with the rapid method the 100% level of labelling index is reached by 48 h while with the conventional method the same level is reached by 10 to 12 days of dry exposure. The maximum grain density is reached by 16 to 17 days by the conventional method. While by the rapid method, the maximum grain density is approximately 80% of the control, this grain density is reached by 48 h (plus 24 h of dry exposure) and thereafter forms a plateau. With Toluene alone the grain density never exceeds 20%. The background is also relatively low and less variable in the O-T-processed autoradiograms, as compared to the two controls. These results support that the scintillation fluid plays the key role in augmenting the labelling. Furthermore, although the maximum grain density by the rapid technique is 80% of the control, the grain density obtained by the rapid method gives less coincidence and superimposition of grains.On the other hand, with ³H-thymidine, although all labelling patterns could be resolved, the labelling index (i.e., percent of labelled cells) is about 40% at 48 h (plus 24 h) and about 79.5% at 96 h with the rapid method, as compared to about 30% and 44% with the conventional method at the two time points, respectively. Only with 16–17 days' dry exposure the ³H-thymidine labelling index increases to 67%. The frequency of the initial patterns (DD-2C) which are usually less frequent, has been found to have increased with the rapid method. No difference in grain density of labelling of ³H-thymidine could be detected between the rapid method and the conventional method. The resolution of grains also seems to be better by the rapid method, due probably to smaller size and lack of superimposition of grains. Other applications, advantages and limitations have been discussed.     

Autoradiographic detection of the uptake of the label from bacterial3H-DNA in relation to the kinetics of the mitotic cycle in barley embryos

Extirped barley embryos were pre-cultivated in aerated liquid nutrient solution for 24 h and then cultivated for 6 h in nutrient solution containing either³H-DNA fromBacillus subtilis or³H-thymidine. After this treatment the embryos were thoroughly washed and transferred to the fresh nutrient medium. Samples were fixed at different intervals up to 24 h. Feulgen squashes were made and covered with autoradiographic emulsion. Microautodiagrams of different parts of the embryos (root meristem, shoot apex plus meristem of the third leaf, second leaf meristem, coleoptile, scutelum) were observed. Labelling of the nuclei after the application of both³H-DNA and³H-thymidine was found in the proliferating parts of the embryos but no label was found in the scutelum. The labelling index values were almost similar in different embryo organs after the treatment with³H-DNA and³H-thymidine. Labelling index and the fraction of labelled mitoses at different intervals after the application of the labelled substances were almost similar after treatment with³H-DNA and³H-thymidine, except some variations due to irrelevant differences in the kinetics of the mitotic cycle. No disappearance of the activity of³H-DNA was observed at different intervals after removal from the labelled solutions during cultivation for other 24 h in non-labelled nutrient medium either containing DNA fromBacillus subtilis or without it. The embryos which were immersed into 0.2% NaCl solution with either one of the labelled compounds did not show any initiation of the S phase nor uptake of³H-DNA. All these results demonstrate that the label from³H-DNA is localized in those cell nuclei which were in the S phase during treatment but they do not yet distinguish unambiguously between the adsorbtion of polymerous DNA or its degradation and reutilization of low-molecular weight products.     

AN AUTORADIOGRAPHIC STUDY OF THE 3H-URIDINE AND 3H-THYMIDINE INCORPORATION IN THE REGENERATING MOUSE LIVER

An autoradiographic study was made of the ³H-uridine incorporation into RNA and DNA in nucleus and cytoplasm of parenchymal cells in the regenerating liver of the mouse after a pulse time of 2 hr. After a decreased uptake of precursor into the parenchymal nucleus during the first 6 hr compared with the normal value, incorporation increased and was maximal at 36 hr; normal values were restored at 72 hr. The cytoplasmic labelling, after an initial small decrease, reached a maximum at 12 hr; this changed to normal 48 hr after hepatectomy. RNase-digestion of the liver sections left a small incorporation in both nucleus and cytoplasm: presumably DNA. This incorporation is maximal at 12 hr over the nucleus and at 24 hr over the cytoplasm. After a 2 hr pulse of ³H-thymidine, there was a marked uptake of the precursor into DNA about 24 hr after hepatectomy. This was maximal at 48 hr and reached normal values at 72 hr. A small amount of incorporation of ³H-thymidine into DNA was seen immediately after the operation, and this population of weakly labelled nuclei was still rather large 72 hr later.