Eduardo Scarano

Thin-layer chromatography of nucleotides, nucleosides and nucleic acid bases

Abstract Thin-layer chromatographic separations on a new layer, microcrystalline cellulose “Avicel”, and on DEAE-cellulose are presented. They include: 1. 1. R F values of 15 bases and methylated analogues of the bases in six different solvents, of 11 nucleosides in 5 different solvents, and of 8 nucleotides in two solvents. 2. 2. Separation of mixtures of deoxynucleoside and ribonucleoside, mono-, di-, and tri-phosphates of the adenine, guanine, uracil, and cytosine series. 3. 3. Separation of mixtures of the four ribonucleoside 2′,3′-monophosphates, of the four ribonucleosides 5′-monophosphates, and of the four deoxyribonucleoside 5′-monophosphates. The advantages of microcrystalline cellulose over other materials for thin-layer chromatography are discussed.

Correlation between synthesis and methylation of DNA in HeLa cells

Abstract For the whole cell cycle the methylation of DNA was studied in synchronized HeLa cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA synthesis is over. No DNA minor thymine is found in the isolated nuclei.

The enzymatic deamination of 5'-deoxycytidylic acid and of 5-methyl-5'-deoxycytidylic acid in the developing sea urchin embryo.

Abstract The importance of DNA synthesis during the early development of the sea urchin egg is discussed, as compared with the constancy of many other cellular constituents. The amount of enzymes which deaminate dCMP and MedCMP has been determined in different stages of development of the sea urchin embryo. A striking decrease in the two deaminases was observed, see Fig. 6. The following hypotheses have been discussed. (1) A cycle of conversion of the pyrimidine deoxynucleotides may exist in multiplying cells. In this way an appropriate assortment of pyrimidine deoxynucleotides is produced for DNA synthesis. (2) The deamination of dCMP to dUMP may have a pacemaker function for the DNA synthesis.

Ribonucleic acids in the early embryonic development of the sea urchin: I. Quantitative variations and 32P orthophosphate incorporation studies of the RNA of subcellular fractions

Quantitative determinations of the total RNA and of the RNA of the subcellular fractions of the developing sea urchin embryo are reported. By differential centrifugation a nuclear fraction, a mitochondrial fraction, two microsomal fractions and a supernatant fraction have been prepared. It has been found that in the early embryonic development the total RNA of the embryo does not change, whereas an increase of the nuclear RNA and an equivalent decrease of the cytoplasmic RNA occurs. Pulse experiments with 32P orthophosphate at several stages of the early embryonic development are reported. At all stages of development the RNA of the subcellular fractions incorporate the 32P orthophosphate in a way peculiar to each fraction.

Enzymatic DNA modifications in isolated nuclei from developing sea urchin embryos

Abstract Nuclei were prepared from developing sea urchin embryos. The isolated nuclei incubated in vitro in the presence of 14 C-methyl S -adenosylmethionine methylate their own DNA. The DNA methylase activity is lost by heating the nuclei at 65°C for 10 min. Some kinetic properties of the reaction are described. 14 C-formate and 3 H-deoxythymidine are not incorporated into DNA of the isolated nuclei. 5-Methylcytosine and thymine are the only products of the DNA methylation by the isolated nuclei. DNA adenine and guanine are not labelled under the same conditions. A mechanism of synthesis at the polymer level of the two DNA ‘minor’ bases by the action of specific DNA cytosine methylases and DNA-5 methylcytosine aminohydrolases is suggested. This mechanism is discussed as the possible chemical basis of the synchron model of cell differentiation.

On the regulatory properties of deoxycytidylate aminohydrolase

Abstract In a previous paper we suggested the occurrence of at least one regulatory site ( Scarano et al., 1962 , Scarano et al., 1963 ) on dCMP aminohydrolase. The present paper reports further experiments that support this hypothesis.

Thin-layer electrophoresis of nucleic acid derivatives on microcrystalline cellulose

Abstract Separation of nucleic acid derivatives by thin-layer electrophoresis on a microcrystalline cellulose, Avicel, is reported. The present paper describes: 1. 1. Separation of the four 2′, 3′-ribonucleotides from an RNA alkaline hydrolysate. 2. 2. Separation of the four deoxynucleotides and the four deoxynucleosides. 3. 3. Separation of deoxynucleoside mono-, di- and triphosphates. 4. 4. Separation of ten dinucleotides from a deoxyribonuclease digest. 5. 5. Two-dimensional separation of 13 bases occurring in nucleic acids.

Effect of trypsin on DNA methylation in isolated nuclei from developing sea urchin embryos.

Abstract Nuclei isolated from the developing sea urchin embryo Paracentrotus lividus and incubated in the presence of [3H-methyl] S-adenosylmethionine methylate their own DNA. Addition of small amounts of trypsin produces a 20-fold increase in DNA methylation. The time kinetics and the specificity of the trypsin activation of DNA methylation are described. The only products of the reaction are 5-methylcytosine and thymine. DNA adenine, guanine and cytosine are not labeled. The distribution of the counts between 5-methyl-cytosine and thymine is variable. While 5-methylcytosine originates by enzymatic methylation of DNA cytosines, the origin of the labeled thymine cannot be inferred with certainty.

Deoxycytidylate aminohydrolase during embryonic development of Rana esculenta

Abstract 1. The amount of deCMP-deaminase (deoxycytidylate aminohydrolase) per embryo has been determined in different stages of development of Rana esculenta. An increase in the content of enzyme per embryo was observed during development. The enzymic content increases approximately twice during the period from cleavage to the end of gastrulation, is relatively constant during neurulation until after hatching, and finally at stage 27 increases to a level twice that of the previous stage. 2. Experiments were made with two batches of eggs at two temperatures (15° and 18°). The development is more rapid at the higher temperature and the levels of activity are generally higher at the higher temperature, but there were no significant changes in the enzymic pattern. 3. Comparisons of gynogenetic haploids with normal diploids were made. No significant difference was observed between haploid and diploid patterns of activity.

AFFINITY CHROMATOGRAPHY AND CONFORMATIONAL ISOMERS OF dCMP-AMINOHYDROLASE

ABSTRACT Matrices containing ligands which interact with catalytic or allosteric sites have been successfuly used for the purification of the allosteric enzyme dCMP-aminohydrolase. This enzyme is eluted specifically by a competitive inhibitor from a matrix coated with ligands which interact with the catalytic sites of the enzyme. The allosteric activator elutes the enzyme from a matrix coated with hydrazide-UTP which behaves as an allosteric inhibitor exploiting the allosteric conformational changes of dCMP-aminohydrolase.