DEPENDENCE OF PERIODIC DNA SYNTHESIS ON PROTEIN SYNTHESIS: DELAY OF DNA SYNTHESIS IN THE EARLY CLEAVAGE STAGE OF SEA URCHIN EMBRYOS TREATED WITH CYCLOHEXIMIDE AND COLCHICINE

The effects of cycloheximide and colchicine on cleavage and syntheses of DNA and proteins in cleaving embryos of the sea urchin, Hemicentrotus pulcherrimus , were examined. Cycloheximide caused delay of cell division with prolongation of the streak stage. Both inhibitors also caused delay in initiation of DNA synthesis. The decrease in the rate and prolongation of the period of DNA synthesis caused by these inhibitors varied with their concentrations and the time of administration. Initiation of DNA synthesis was delayed when cycloheximide was added to suspensions of embryos between the time after preceding DNA synthesis terminated and a definite time before the predicted time of initiation of the next synthesis of DNA, except at the stage of pronuclear fusion. However, when the inhibitor was added after initiation of the synthesis, the latter proceeded normally. Addition of 10 m m cycloheximide immediately after fertilization or 2 m m cycloheximide 60 min before fertilization also delayed DNA synthesis at the stage of pronuclear fusion, indicating that synthesis at this stage also required prior protein synthesis. Colchicine had less inhibitory effect on protein synthesis, but greatly delayed initiation of DNA synthesis and prolonged its duration. These facts suggest that a definite amount of a particular protein must be synthesized and accumulated in each synthetic cycle before initiation of DNA synthesis.     

Emergence of the Extrachromosomal Circular DNA Complexes as One of the Earliest Signals of Cellular Differentiation in the Early Development of Mouse Embryo

A small number of mouse embryos and embryonal carcinoma cells were pressed by mica sheet; then the extruded DNA complexes were adsorbed to mica and processed for electron microscopy. Extrachromosomal circular DNA complexes longer than 1 μm emerged during the compaction process of mouse embryos and during the differentiation of embryonal carcinoma cells induced with retinoic acid. These DNA molecules are discussed as possible products of developmental gene rearrangements occurring in the chromosomal DNA.     

SORBITOL IN THE EGGS OF THE SILKWORM, BOMBYX MORI

Sorbitol was found to be a normal and sole monosaccharide demonstrable in the late ovarian and in the early embryonic stages of the silkworm, Bombyx mori. This observation was first made on a line of non-diapausing polyvoltine Katsumata strain, but was later confirmed by the analysis of two other Katsumata lines and on several polyvoltine or bivoltine strains. The identification of the sugar extracted from the diapause eggs of a bivoltine strain and from the non-diapausing eggs of a Katsumata strain was based on analyses involving paper chromatography, paper electrophoresis and sorbitol dehydrogenase.     

Effects of β-Mercaptoethanol-Containing Medium and Feeder Cells on the Differentiation of Embryonal Carcinoma Cells

Pluripotent, feeder-dependent teratocarcinoma cell lines were cultured without a feeder layer in a medium containing 10 ^−;4 M β-mercaptoethanol (β-medium) and compared for the development of early markers with cells cultured with a feeder layer. The cells cultured in β-medium lost the PNA (peanut agglutinin) receptor typically at low density. This change was accompanied with enhanced secretion of plasminogen activator and the loss of sensitivity to anti-F9 serum, indicating the stem cell differentiation. In contrast, the cells cultured on a feeder layer did not show any marker changes, thus indicating the lack of differentiation. These results indicate that while the presence of feeder cells inhibits cell differentiation, cultivation in β–medium permits the differentiation of pluripotent teratocarcinoma cells.     

POLYSOMES OF THE SEA URCHIN EMBRYO: AN IMPROVED METHOD FOR EXTRACTION OF INTEGRATED POLYSOMES

Suitable conditions for extracting integrated polysomes from embryos of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus were investigated.
Integrated polysomes could not be extracted under the conditions reported by other investigators. It was found, however, that use of 5 m m MgCl₂, 0.30 m KCl, 0.5 m m EDTA and 2 m m cycloheximide was effective for maintaining the integrity of polysomes. At higher concentrations of Mg²⁺, and even at higher concentrations of K⁺, monosomes and polysomes aggregated to form polysome-like particles which had sedimentation patterns with a small amount of nascent peptide. Thus, a medium consisting of 0.05 m Tris-HCl buffer, pH 7.5, 0.30 m KCl. 5 m m MgCl₂, 0.5 m m EDTA-2K, 2 m m cycloheximide, 5 m m mercaptoethanol and 0.5% (v/v) Nonidet P-40 is concluded to be the most suitable for extraction of sea urchin polysomes. Under the conditions used EDTA did not suppress polysome degradation completely and their degradation was linear with time.     

NATURE OF THE INHIBITION OF PROTEIN SYNTHESIS BY ETHIONINE AND AMINO ACID UPTAKE IN EARLY SEA URCHIN EMBRYOS

The inhibition of protein synthesis by ethionine reported previously was found to be apparent, and ethionine inhibited only amino acid uptake like other usual amino acids. Even under such strong inhibition of the uptake, the syntheses of protein and DNA remained almost undiminished. The uptake of amino acid mixture by sea urchin embryos in the early cleavage stage was found to be carried out by active transport, since it was temperature-sensitive and was inhibited by 2,4-dinitrophenol. The uptake of an amino acid mixture or of single amino acids, e.g., valine, leucine and phenylalanine, was inhibited nonspecifically by an excess amount of other single amino acids added exogenously. Reflecting the inhibition of amino acid uptake, in vivo incorporation of amino acids into the protein fraction was apparently inhibited by excess amounts of other amino acids. As far as tested, the inhibition seems to be nonspecific and competitive for all amino acid species. The uptakes of leucine and phenylalanine were inhibited mutually by competition, with almost the same Km and Ki.     

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS

Aphidicolin at 2 μg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 μg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 μg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 μg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase β or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.     

POLYSOMES OF THE SEA URCHIN EMBRYO: NATURE OF SO-CALLED "CYCLIC AMINO ACID INCORPORATION" AND CONDITIONS TO PREPARE CELL-FREE SYSTEMS FOR PROTEIN SYNTHESIS

Cyclic formation of active polysomes in a cell-free system, as reported previously, could not be demonstrated under the conditions reported. However, cyclic fluctuation was observed in incorporation of amino acids into the hot TCA-insoluble fraction. Failure to demonstrate polysome formation seemed to be due to the lack of GTP and to the necessity to protect SH groups during preparation of the system and its assay. Addition of exogenous GTP and ME effectively restored polysomal activity in the cell-free system. The previously reported cyclic incorporation of amino acids into the hot TCA-insoluble fraction in cell-free systems in the absence of these reagents might be due to cyclic variation in some partial reactions or some phenomenon other than protein synthesis.     

Tea leaf peroxidase--Isolation and partial purification

Tea leaf peroxidase occurred as the insoluble form in a homogenate.A large part of the peroxidase from tea leaves was solubilizedin a homogenate by adding Tween-80. After fractionation by 0.3saturation with (NH₄)₂SO₄ and filtration of the homogenate througha Sephadex G-25 column, specific activity of the peroxidasein the effluent increased about 25 times. Crude peroxidase wasfractionated into six components; A-l, A-2, B-l, B-2, B-3 andC, by DEAE- and CM-cellulose column chromatography. The fractionsshowed different mobilities on disc gel electrophoregram atpH 4.2. An isoelectric focusing experiment indicated that pivalues of the A-l and A-2 components are 5.4 and 5.0, repsectively. ¹ Present address: National Institute of Agricultural Science,Ministry of Agriculture and Forestry, Nishigahara, Kita-ku,Tokyo, Japan. (Received October 19, 1970; )