INCORPORATION OF TRITIATED THYMIDINE INTO THE SKIN AND HAIR FOLLICLES

The incorporation of tritiated thymidine into the skin has been studied after the initiation of new hair growth by plucking. Techniques are described whereby the incorporation into hair follicles and into the basal layer of the epidermis can be studied independently. Fluctuations are observed in the levels of incorporation in both cell populations through the hair growth cycle. These fluctuations show great regularity. the early fluctuations have been attributed to synchronous progression of cells through the cell division cycle. the significance of the fluctuations is discussed with particular reference to (I) possible persistence of cell cycle synchrony, (II) regulatory feedback mechanisms controlling DNA synthesis, perhaps through specific mitotic inhibitors, (III) the existence of G_o, G₁ and G₂ cell sub-populations.     

INCORPORATION OF TRITIATED THYMIDINE INTO THE SKIN AND HAIR FOLLICLES

Diurnal fluctuations in incorporation of tritiated uridine and tritiated thymidine are reported for full thickness skin and its two proliferative populations, the epidermal basal layer and the hair follicle germ or matrix. Recently developed techniques whereby these two populations can be studied independently were utilized. Resting (unplucked) skin and skin in the middle of the hair growth cycle (10 days after plucking) was studied. the diurnal fluctuations differed at these two stages of the hair cycle. Generally a single minimum level of activity was observed shortly after the beginning of the dark period of the day, i.e. after 18.00 hours.     

INCORPORATION OF TRITIATED THYMIDINE BY EUCARYOTIC MICROALGAE1

The uptake and incorporation of tritiated thymidine (³H-TdR) by axenic laboratory cultures of marine diatoms and dinoflagellates was measured. ³H-TdR was incorporated into nucleic acids by all four algae examined during a two to six hour period prior to cytokinesis and not during other times of the cell cycle. Between 90-95% of the ³H label incorporated into (cold trichloroacetic acid insoluble) nucleic acids was recovered from DNA. Incorporation of ³H-TdR appears to accurately indicate the timing of DNA synthesis. The incorporation of ³H-TdR by eucaryotic algae during long term (24 h) incubations does not generally preclude using ³H-TdR uptake to estimate bacterial production and growth during short term incubations.     

THE EFFECTS OF TRITIATED THYMIDINE INCORPORATION ON SECONDARY ROOT PRODUCTION BY PISUM SATIVUM

Hummon , Margaret R. (Montana State U., Missoula.) The effects of tritiated thymidine incorporation on secondary root production by Pisum sativum. Amer. Jour. Bot. 49(10): 1038–1046. Illus. 1962.—Most studies of effects of radiation on plants have involved a general exposure of all the cells and tissues of an organ or entire plant. Tritiated thymidine offers a tool for selective irradiation of the nucleus with little effect on the cytoplasm of a cell. Furthermore, differential incorporation due to variation in the pattern of DNA synthesis permits selective irradiation of cells and tissues. In this study, developing primary roots of Pisum sativum were submerged for brief periods in a solution of tritiated thymidine. This resulted in an alteration of the lateral root pattern. In the area corresponding to the region of elongation during treatment, subsequent lateral root production was suppressed. This correlated with the portion of the root in which there was incorporation of tritiated thymidine into a high percentage of pericycle nuclei. Abnormal development of vascular tissues also occurred, with evidence of altered polarity in the xylem. Although incorporation also occurred in the apical meristem, the latter was not affected at this level of exposure. Thus, differential sensitivity as well as differential incorporation may have been involved in producing the temporary alteration of lateral root production.     

TEMPORARY LOCALIZED DEPRESSION OF TRITIATED THYMIDINE INCORPORATION IN MOUSE TISSUES AFTER PULSE LABELLING

Approximately two to six in every 100 mice injected with ³H-TdR appear not to incorporate the labelled precursor into the DNA. The tritium activity appears to be distributed throughout these poor utilizers of thymidine. The lack of incorporation of the precursor is not always general to the whole animal but may be restricted to a given tissue. The effect does not appear to be permanent but varies with time.     

EFFECT OF TRITIATED THYMIDINE ON THE KINETICS AND VIABILITY OF 9L CELLS IN VITRO

The colony-forming efficiency of 9L rat gliosarcoma cells was unaffected by treatment with 0.1 μCi/ml of [³H]TdR. However, when cells were treated with 1 or 10 μCi/ml of [³H]Tdr, cell growth was reduced and cell survival decreased. When monolayer 9L cells were treated with 1 μCi/ml of [³H]TdR for up to 72 hr, approximately 5% survived, which is closely related to the percentage of non-cycling cells in this system. When cells were treated with 10 μCi/ml of [³H]TdR for 72 hr, less survival was observed. the additional cell kill observed may be induced by [³H]TdR released from doomed cells into petri dishes during the incubation period of the colony-formation assay.     

INCORPORATION OF TRITIATED THYMIDINE DURING VARIOUS STAGES OF LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

³H-thymidine was incorporated into leaf tissue of Xanthium pennsylvanicum during the stage of active cell division, during cellular differentiation, and into mature cells. Incorporation into nuclear DNA was high in the early stages of development. No nuclear incorporation was found after cessation of cell division. However, significant incorporation could be demonstrated in cytoplasm of differentiating and mature cells. Depending upon the time of growth in the radioisotope and the time of growth after treatment, ³H-thymidine, or its metabolized fraction, was incorporated into the secondary wall depositions of epidermal cells, mesophyll parenchyma cells, xylem cells, and chloroplasts. Autoradiographic technique and liquid scintillation spectrometry were used in these studies. The significance of ³H-thymidine incorporation into various organelles is discussed in relation to cell metabolism and its regulation during leaf development.     

LOW LEVEL INCORPORATION OF TRITIATED THYMIDINE INTO THE NUCLEAR DNA OF PURKINJE NEURONS OF ADULT MICE

Adult mice were pulse labeled with tritiated thymidine [³H]TdR and killed 9 hr later. A low level incorporation of [³H]TdR into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

EARLY AND LATE INCORPORATION OF TRITIATED THYMIDINE INTO SKIN CELLS AND THE PRESENCE OF A LONG-LIVED G0-SPECIFIC PRECURSOR POOL

40 min after a single injection of 50 µCi of tritiated thymidine a 3 mm punch of DBA-1 mouse skin contains about 1000 dpm. This value remains constant for at least 48 hr after injection. 50 hair follicles contain about 40 dpm, and from these values the activity calculated to reside in the basal layer of a 3 mm punch of skin is 760 dpm. These values also remain constant with time after injection. Fresh punches of skin contain much more activity. The fixative-soluble fraction (the difference between fresh and fixed values) decays slowly with time. The values for DBA-2 mice are similar. Plucking the hair from the follicles appears immediately to increase the size of the fixative-soluble fraction and decrease the fixed tissue values to about 500 dpm per punch for whole skin and about 1 dpm per 50 follicles for DBA-1. Thus almost all the activity is restricted to the epidermis. The fixative-soluble fraction returns approximately to the unplucked value between 24 and 48 hr after plucking. However, during this period the fixed tissue values are rising rapidly as stimulated cells enter S. It appears that in both strains labeled material remains available for incorporation into stimulated cells for at least 48 hr after a single injection. The amount persisting appears to decrease with time. The whole-fixed skin, the hair follicles, and the epidermis all contain cells that are capable of becoming labeled after stimulation 8–48 hr after an injection. The label in question does not become incorporated into normal cycling skin or hair follicle cells. It is concluded that the DNA precursor pool is possibly connected with G₀ cells and that both the hair follicle and the basal layer of the epidermis contain these resting cells.     

OBSERVATIONS ON THE UPTAKE OF TRITIATED THYMIDINE IN THE PRONUCLEI OF FERTILIZED SAND DOLLAR EMBRYOS

Following fertilization of the egg of the sand dollar Echinarachnius parma, tritiated thymidine (H³TDR) was taken up independently by the male and female pronuclei beginning within about 15 to 20 minutes, and the labeled pronuclei fused at about 30 to 40 minutes. At cleavage 90 minutes later the labeled nuclear material was distributed to both daughter cells. Unfertilized eggs and sperm exposed to H³TDR did not show nuclear localization of thymidine. DNA replication, thus, is initiated in the haploid pronuclei shortly after fertilization and prior to fusion. The major portion of DNA synthesis, as evidenced by thymidine uptake, appears to be during a 20 to 30 minute period after fertilization. Fertilization is associated with the activation of a mechanism which initiates early and independent replication of DNA in both the male and female pronuclei.     

THE USE OF TRITIATED THYMIDINE IN THE STUDY OF TISSUE ACTIVATION DURING GERMINATION IN ZEA MAYS

Stein, O. L. (Montana State U., Missoula), and H. Quastler. The use of tritiated thymidinein the study of tissue activation during germination in Zea mays. Amer. Jour. Bot. 50(10): 1006-1011. Illus. 1963.—Corn embryos were exposed to H³thymidine at various times during the first 80 hr of germination. An analysis of labeled nuclei was made from autoradiographs, and the number and position of mitoses were recorded. Those tissues which approach maturity during embryogeny (root cap, coleorhiza, scutellur node) are first to resume DNA synthesis (30 hr after soaking). No mitoses were observed in these tissues. In shoot and root, mitoses usually precede DNA synthesis, indicating that the nuclei of the dormant embryo have a DNA value of 4C(twice the diploid DNA) or more. The shoot begins its activity much later than the root (50 hr). The shoot apex was the last region to boeomo active, some 70 hr after initiation of the soaking treatment.     

EFFECTS OF BORON DEFICIENCY ON MITOSIS AND INCORPORATION OF TRITIATED THYMIDINE INTO NUCLEI OF SUNFLOWER ROOT TIPS

Boron deprivation has multiple effects upon root growth within 6 hr after this essential micronutrient is withheld. Root elongation is inhibited and this response has been attributed to a cessation of mitosis and DNA synthesis. Our preliminary results using an autoradiographic analysis of sunflower roots labeled with [³H]-thymidine demonstrated no difference in label distribution between +/-B root tips. We found that mitosis in inhibited in -B roots but does not completely cease. Scintillation counting of whole root tips shows that boron-deficient roots up to 72 hr of treatment incorporate radioactive label at a level comparable to that of the controls. Because mitosis and presumably DNA synthesis are affected by prolonged boron deficiency, these results may be brought about by a change in membrane integrity or permeability. We propose that effects of boron deprivation on DNA synthesis and mitosis in sunflower are secondary and that primary events involve alterations in cellular membranes.     

COMPUTER SUBTRACTION OF BACKGROUND IN AUTORADIOGRAPHY WITH TRITIATED THYMIDINE

An iterative method is suggested to estimate true labelling index even when background and experimental (background + true labelling) grain count distributions appear to be incompatible, generally a result of unavoidably insufficient counting in routine work. A computer program applying the iterative method has been written in Fortran IV for an IBM 1130 machine. This program, available on request, displays grain count distributions before as well as after correction and computes the labelling index and average grain count per labelled cell, with respective variance and standard errors.     

DIFFERENT ACTION OF SUICIDAL DOSES OF TRITIATED THYMIDINE AND HYDROXYUREA ON MURINE HAEMOPOIETIC CELLS

In order to gather information on the factors that cause the different action of suicidal doses of tritiated thymidine (³H-TdR) and of hydroxyurea on murine stem cells, the incorporation of ³H-TdR into DNA of bone marrow and spleen cells has been studied. Continuous death of labelled cells after suicidal ³H-TdR is indicated by a more pronounced decline of total DNA-bound radioactivity in bone marrow and spleen cells compared to that in control animals which had received tracer doses of ³H-TdR. Extensive and rapid loss of DNA-bound radioactivity occurred in ³H-TdR labelled animals after hydroxyurea treatment indicating an instantaneous and highly effective killing of labelled cells. After double labelling of DNA with ³H-TdR and ¹²⁵iodo-deoxyuridine (¹²⁵I-UdR), the decline of the ratio of DNA-bound ¹²⁵I to DNA-bound ³H after suicidal ³H-TdR indicates prolonged tritium reutilization. Following hydroxyurea, reutilization was completed within the first 12 hr after drug administration. These findings explain in part the slow recovery of different stem cell compartments after suicidal ³H-TdR on the basis of protracted tritium reutilization as compared to the fast recovery which follows the rapid action of hydroxyurea.     

A COMPARISON OF METAPHASE ARRESTING AGENTS AND TRITIATED THYMIDINE AUTORADIOGRAPHY IN MEASUREMENT OF THE RATE OF ENTRY OF CELLS INTO MITOSIS IN THE CRYPTS OF LIEBERKÜHN OF THE RAT

Three methods of estimating cell production rate were used: the rate of accumulation of metaphases blocked by Colcemid, or by vinblastine, and the rate of increase of labelled nuclei after administration of tritiated thymidine. These rates were determined for three sites in the small intestine by counts made on whole micro-dissected crypts, fixed at various times after the administration of the agents.
Within the limits of error of the methods, the cell production rate per crypt was the same when measured by each method (35/hr), and showed a slight fall from the proximal to the distal end of the small intestine (36/hr to 33/hr). the advantages and limitations of each method are discussed.     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

IN VITRO AND IN VIVO LABELLING OF ANIMAL TUMOURS WITH TRITIATED THYMIDINE

The labelling indices obtained by incubating tumour specimens with tritiated thymidine in vitro under hyperbaric oxygen have been compared with those obtained by labelling a matched tumour in vivo. The correspondence between these individual labelling indices is sometimes very poor; however, the average labelling index derived from groups of tumours labelled in the two ways does not differ significantly. There was a large variation from field to field within any tumour, and considerable variation from one tumour to another within each tumour type. The mitotic index was also compared in the matched tumour preparations; the mitotic index in vitro was almost always considerably lower than the values observed in vivo.     

TIMING OF INCORPORATION OF TRITIATED NUCLEOSIDES INTO DNA AND RNA OF EMBRYONIC CELLS OF RANA PIPIENS

The incorporation of tritiated nucleosides into DNA and RNA has been examined in partially synchronized cells of Rana pipiens embryos at the neurula and tailbud stages. Tritiated thymidine and deoxyguanosine are incorporated into the DNA in two maxima, or waves, during the S phase at both stages. More DNA replicates in the early maximum at the neurula stage than at the tailbud stage. A comparison of the degree of incorporation of labelled deoxyguanosine to labelled thymidine into DNA suggests that earlier replicating DNA at both stages may be GC-rich compared to later replicating DNA. The incorporation of tritiated uridine into RNA during the S phase also differs between the neurula and tailbud stages. Pulse and continuous label experiments indicate that at the neurula stage the highest rate of RNA synthesis occurs late in the S phase whereas at the tailbud stage the higher rate of RNA synthesis has shifted to an interval earlier in the S phase.     

THE EFFECT OF THYMIDINE ON THE DURATION OF G1 IN CHINESE HAMSTER CELLS

The generation time of a Chinese hamster cell line was varied by the use of different lots of sera in the culture media. Analysis of the division waves following thymidine synchronization showed that lengthening of the generation time was a result of an increase in duration of the G₁ phase and that thymidine treatment reduced the duration of G₁ back to its minimum value.