Continuous exposure of embryonic explants to thymidine-H3 was used to determine patterns of late-replicating DNA synthesis, i.e. DNA synthesized during the last quarter of the S period, in chromosomes from different germ layers of developing frog embryos (Rana pipiens). Autoradiographic localization of silver grains over chromosomes 1, 7 and 8, as well as unkaryotyped chromosomes, revealed that patterns of late-replicating DNA are similar i n determined cells that will undergo different pathways of differentiation. In undetermined early gastrula cells and in differentiated tailbud dorsal axial cells (nerve tube, somites and notochord) more sites of DNA synthesis were late-replicating in chromosomes 1, 7 and 8 and in unkaryotyped chromosomes than in cells of the neural plate-dorsal mesoderm of neurulae where differentiation is occurring and in neurula endoderm where determination is taking place.

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Variations in the rates of DNA and RNA synthesis during the DNA synthetic ( S ) period of the cell cycle have been determined in explants of early Rana pipiens embryos.Cells of dorsal axial ectoderm-mesoderm regions and belly endoderm regions of the embryo were partially synchronized using 5-fluorodeoxyuridine. The partially synchronized explants were incubated continuously or at intervals during the S period with radioactive nucleic acid precursors. Autoradiographic and biochemical evidence indicates that at the gastrula, neurula and tailbud stages the DNA is replicated discontinuously in two maxima with a slowing in the synthetic rate in the middle of the S period. There is an increase in the proportion of the DNA which replicates late as development proceeds from gastrulation. At the neurula and tailbud stages the proportion of late replicating DNA is greater in the belly endoderm than in the dorsal axial ectoderm-mesoderm.Experiments utilizing H3-5-uridine indicate that RNA is synthesized discontinuously in two maxima during the S period in both dorsal axial and belly regions at the earlier neurula stage. By the tailbud stage, however, a significant decrease in the second maximum of RNA transcription occurs. RNA extraction experiments indicate that these changes can be attributed, in part, to changes in the synthesis of DNA-like RNA. These findings are discussed in relation to cell determination.Gastrulation in developing amphibian embryos marks the time when the germ layers are formed and the fates of cells begin to be fixed. Cells of the early gastrula are undetermined and are competent to differentiate into various cell types. During gastrulation dorsal ectoderm and mesoderm cells are determined to form neural tissue, and somites and notochord, respectively (Holtfreter and Hamburger, '55). The endoderm cells are determined to form gut derivatives later during neurulation (Balinsky, '61 ). These processes of determination are paralleled by a gradual restriction in the competence of cells for various kinds of differentiation.A marked change in the synthetic activity of cell nuclei also occurs during gastrulation in that the length of the S period like RNA (D-RNA) and soluble RNA procedes that of ribosomal RNA which begins to be transcribed later during gastrulation and neurulation (Brown and Littna, '64). As cells of the germ layers become determinted, however, the rate of D-RNA synthesis decreases (Woodland and Gurdon, '68), and the number of different kinds of D-RNA molecules transcribed from redundant DNA sequences is reduced considerably (Flickinger, '70 a,b). These experiments indicate that genetic activity decreases as development proceeds from the neurula to the larval stage. Over the same period of time, more kinds of D-RNA molecules appear to accumulate in the cytoplasm (Denis, '66; Greene and Flickinger, '70).This investigation is aimed at examining the cause of the progressive restriction in

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DNA fiber autoradiography has shown an increase in size of replicons during early development of the frog embryo. Replicons of endoderm cells were considerably larger than those of dorsal ectoderm and mesoderm cells in tailbud embryos. Late replicating DNA in partially synchronized tailbuds has a more rapid rate of replicon elongation than does early replicating DNA.

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FOUR FIGURESThe importance of the yolk platelets f o r the development of the amphibian embryo is highlighted by the fact that they contain 72% of the protein of the embryo (Laurence, Needham, Miall and Shen, '44) ; furthermore the nitrogen studies of Qregg and Ballentine ('46) indicated that cytoplasmic proteins are synthesized at the expense of yolk proteins. By means of the microbiological assay method Kutsky, Eakin, Berg and Kavanau ('53) have demonstrated only a small rise in free and non-protein amino acids through neurulation, but since their study does not cover the period around hatching when much of the yolk is being utilized, it is difficult to know if cytoplasmic proteins arise from free amino acids, peptides or modified yolk proteins. However, Holtfreter 's ( '50) chromatographic results indicated the larval amino acid pool of the frog to be no greater that that of the egg and also Schechtman ('47) demonstrated the presence of the yolk protein livetin in the early chick embryo by serological means. Bragg ( '39) has found that during the differentiation period yolk platelet decomposition is first seen in the regions of the embryo which will differentiate earliest, e.g., the nervous system. It has been found by Flickinger ('49) that amino