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DNA Synthesis and Turnover in the Bullfrog Tadpole during Metamorphosis*

deoxyuridine (IdUrd) has been used to estimate the turnover of DNA in liver, tail, and hind limb during spontaneous and triiodothyronine-induced metamorphosis. It was found that the total amount of liver DNA remained constant and there was no significant loss of the label from the liver DNA, which would be expected if there was an increase in DNA turnover during metamorphosis. Also, the change in specific activity of liver DNA parallels that of tail DNA during spontaneous metamorphosis. These data suggest that metamorphic transitions in the tadpole liver do not involve significant changes in DNA turnover. It was observed that the incorporation of label into hind limb DNA showed a high variability among individual animals as compared to liver and tail tissue. The data presented suggest that the observed variability is not a random phenomenon but related directly to the rate at which animals will metamorphose.

From the Department of Chemistry, Florida State Uniuersity, Tallahassee, Florida 32306 1Z51-labeled deoxyuridine (IdUrd) has been used to estimate the turnover of DNA in liver, tail, and hind limb during spontaneous and triiodothyronine-induced metamorphosis.
It was found that the total amount of liver DNA remained constant and there was no significant loss of the label from the liver DNA, which would be expected if there was an increase in DNA turnover during metamorphosis.
Also, the change in specific activity of liver DNA parallels that of tail DNA during spontaneous metamorphosis. These data suggest that metamorphic transitions in the tadpole liver do not involve significant changes in DNA turnover. It was observed that the incorporation of label into hind limb DNA showed a high variability among individual animals as compared to liver and tail tissue. The data presented suggest that the observed variability is not a random phenomenon but related directly to the rate at which animals will metamorphose.
The regulation of cell replication and differentiation involves many areas of research. The precocious metamorphosis of anurans in response to thyroid hormones is an example (1). It has been shown that the urea cycle enzymes necessary for the transition of ammonia-excreting larvae to urea-excreting adults are synthesized by the liver of thyroid hormone-treated animals in a manner similar to those undergoing spontaneous metamorphosis (2). Reports describing the effects of thyroid hormones on nucleic acids and proteins and on the ultrastructural reorganization in liver cells harbor conflicting assumptions regarding the extent of liver cell synthesis and/or turnover during thyroid hormone-induced metamorphosis and spontaneous metamorphosis (3)(4)(5)(6)(7). It has been assumed that the biochemical changes in the liver of tadpoles undergoing metamorphosis occurred in a "fixed population of cells" (1,2). This would preclude any significant change in total liver DNA or DNA turnover during metamorphosis.
However, there have been several reports which suggested that the metabolism of DNA was also involved in the biochemical changes in the liver during metamorphic transitions. Elias  Atkinson et al. (3) reported an increase in labeled thymidine incorporation into liver DNA in uiuo during spontaneous and triiodothyronine-induced metamorphosis. These observations led Atkinson (11) to postulate that the thyroid hormones induced macromolecular changes in the liver which could not occur in a "fixed population of cells." The "fixed cell uersus nonfixed cell" controversy is thus a critical impasse to our further understanding of the action of thyroid hormone in tadpole liver. We have tried to approach this question in a different way by using the thymidine analog ['251]IdUrd to estimate the rate of DNA biosynthesis and turnover. It has been shown that after the incorporation of IdUrd into DNA, the iodine moiety (label) remains covalently bound to the DNA until cell death (12)(13)(14). When the labeled cell dies, rapid deiodination and excretion limits the reutilization of radioactivity by other cells (15), allowing the estimation of the rate of cell death. Cytological investigations of tadpole liver cells show no evidence of polyploidy at anytime during thyroid hormone-induced or spontaneous metamorphosis (3,4,7). Since liver is a discrete integral tissue, we can assume that the total liver DNA is directly proportional to the number of liver cells. Therefore, the rate of new cell synthesis and/or cell death during metamorphosis can be estimated on the basis of the metabolism of ['Y]IdUrd and the total DNA content of the liver.  We observed essentially no change during metamorphosis in the specific activity of liver DNA (Fig. 2) or in total liver DNA (cpm/specific activity from Table I and Fig. 2). The amount of label in the tail diminishes as shown in Table  I It is interesting to note (Table I) that the specific activity of the hind limbs decreases sharply (animals 1 through 5) and then remains essentially constant (animals 5 through 12). The sharp decrease was predictable due to the increase in total DNA. However, there is also an overall increase in hind limb DNA from animal 5 through animal 12 with no ,predicted decrease in specific activity (Fig. 2, Table I). Furthermore, as shown in Table I, there was an increase in labeling of the hind limbs as a function of stage of development. This may appear to be inconsistent with the contention that, due to chemical breakdown of the analog in vivo, label cannot be incorporated into DNA after the initial labeling period. However, it appears that the apparent "labeling" of the hind limbs during the 14-day period shown in Table I actually reflects increased  incorporation of [1*61]IdUrd during the first few hours of labeling into limb tissue of animals which will metamorphose more rapidly. This would account for the leveling off of the specific activity of hind limb DNA. This interpretation is also suggested by the labeling of hind limbs during the first 24-hour period (Table I) where the percentage of injected counts per min (3.1 i 1.4) covers a range of values similar to the range shown for the 14-day animals (animals 1 to 12, Table I).

MATERIALS
Triiodothyronine-induced Metamorphosis-The amount of DNA in the liver proved to be uniform (*5'S within a given group of animals) with or without thyroid hormone in agreement with the reports of Paik and co-workers (5, 6) and Kistler et al. (7). Differences in whole tissue weight induced by triiodothyronine were observed after 1 to 2 weeks of triiodothyronine treatment. These differences (triiodothyronine/control, 1.3/2.4) varied with experimental groups, suggesting the need to express DNA content on a "per liver" basis rather than the per gram of tissue notation used by Finamore and Frieden (19). It has also been shown that total tail muscle DNA remains essentially constant (mg of DNA/animal = 485 (+22)) during triiodothyronine-induced metamorphosis (7). As described in Table II, tadpoles were labeled prior to triiodothyronine treatment.
If there was triiodothyronineinduced synthesis of DNA, in tail and liver, there should be a decrease in prelabeled DNA. As shown in Table II