Nucleotides and Amino Acids in Planaria

Neither intact nor regenerating planaria incorporated any signiiicant amount of tritiated thymidine, tritiated L-leucine, or C14-Lleucine from free solution even over long periods of exposure. Tritiated thymidine was incorporated into regenerating rat liver and fed to planaria, but tritium hydrogen exchange rendered this method useless. A large incorporation of C14-uracil and C14-Lleucine into planaria was obtained by growing baker's yeast in a defined medium containing the labeled compound and then feeding the acetone dried yeast to the planaria. C14 assay of the amino acid components of planaria fed on Cl*-L-leucine labeled yeast showed CI4 in only the L-leucine component and yielded a minimum estimate for protein turnover of 46% in seven weeks. Radioautographs of planaria fed on C14-uracil labeled yeast exhibited specific incorporation into ribonucleic acid. Previous radioisotopic studies on planaria are evaluated and criticized. The amino acid composition of planarian protein was determined. Although planaria have been used extensively for studies on regeneration and morphogenesis (Brondsted, '55; Wolff and Dubois, '47; Worn, '47, '61, '62; Dubois, '49, '50; Flickinger, '59, '64) and on the mechanisms of learning and memory (McConnell et al., 59, '62; Best and Rubinstein, '62; Corning and John, '61; Jacobson, '63; Barnes and Katzung, '63; Westerman, '63; Van Deventer and Ratner, '64; Halas et al., '62) only limited information has been available on the underlying cytochemical mechanisms. Lindh ('57) and Clement ('44) have studied differences in nuclei acid composition and content during regeneration of planaria. Pedersen ('59) has conducted histochemical studies on them. Henderson and Eakin ('61) have studied the effect of purine analogues on differentiation. A salient gap in the information needed to interpret the results of these many studies is that there has been no really satisfactory method of estimating the rates of renewal and transformation of the various kinds of cells in planaria, nor the rates of turnover of such constituents as ribonucleic acid (RNA) and protein in these cells. High resolution radioautographic procedures (e.g., Everett et al., '60; Cronkite et al., '60) using tritium or C" for labeling of appropriate precursor moleJ. EXP. WL., 159: 397404. cules would seem to provide a suitable experimental method to furnish this needed information. Utilization of these methods assumes as a necessary condition that one can obtain sufficient incorporation of the isotopically tagged precursor to obtain a radioautograph. There are technical difficulties in obtaining this incorporation. The present study is concerned with the development of a successful method of obtaining such isotopic incorporation and subsequent high resolution radioautographs.