Erwin Chargaff

Evidence of common regularities in the composition of pentose nucleic acids.

Abstract 1. 1. The results of a survey of the nucleotide composition of pentose nucleic acids (PNA) have been presented. The material analyzed included whole tissues and various subcellular fractions derived from beef, rat and frog liver and kidney, from sea urchin eggs and embryos, and from several microorganisms. 2. 2. It appears characteristics of all preparations studied that the bases with 6-amino groups (adenine, cytosine) and those with 6-keto groups (guanine, uracil) occur in approximately equal number. 3. 3. This regularity was found only when the total PNA of the sample was analyzed. 4. 4. Some of the structural implications of the findings are discussed.

Studies on the nucleotide arrangement in deoxyribonucleic acids IV. Patterns of nucleotide sequence in the deoxyribonucleic acid of rye germ and its fractions

Abstract The procedure of the differential analysis of pyrimidine nucleotide distribution has been applied to the total deoxyribonucleis acid of rye germ and to a complete series of seven DNA fractions prepared by the fractional dissociation of rye germ nucleoprotein. protein. The fractions exhibited the previously observed gradation in composition with gradually rising adenine and thymine contents and similarly diminishing concentrations of guanine, cytosine and methylcytosine. The general distribution of cytosine and methylcytosin in the fractions was definitely uneven, with a relatively higher proportion of the methyl derivative occurring in the first fractions. Even greater differences were observed in the relative distribution of solitary pyrimidine nucleotides, that of thymidylic and methylcytidylic acids showing a remarkable similarity and exceeding by far the corresponding contribution of cytidylic acid to thr solitary units. The survey of solitary units was supplemented by a study of the distribution of many of the pyrimidine dinucleotide sequences that are flanked by purine nucleotides in the polymer chains. Significant differences in the composition of the fractions were observed, but in all preparations more than two thirds of the pyrimidines occurred as tracts consisting of three or more pyrimidines in a row. In no case was the pattern of nucleotide distribution that to be expected from a random arrangement of constituents. After the discussion of certain outstanding features of the nucleotide arrangement, including the evidence for a preferential association of methylcytidylic and guanylic acids, the bearing of the findings on schemes of DNA replication is considered.

Induced formation of ribonucleic acids and plastid protein in Euglena gracilis under the influence of light

Abstract 1. 1. Euglena cells grown in the light, which possess the photosynthetic apparatus, contain considerably more protein and RNA than the cells grown in the dark. 2. 2. The additional protein of the green cells is localized in the plastid fraction and appears to be part of the chloroplast structure. The additional RNA is distributed among the plastid, microsome and soluble fractions. 3. 3. During chloroplast formation in resting colorless cells exposed to light, the protein of the plastid fraction increases rapidly after a considerable lag period. The RNA of both the plastid and microsome fractions increases without apparent lag; its synthesis ceases before that of the plastid protein. 4. 4. The RNA produced during chloroplast formation may represent specific ribosomal material necessary for the synthesis of the plastid proteins.

Studies on the structure of ribonucleic acids.

Abstract The course of action of crystalline ribonuclease on the ribonucleic acids from yeast and from pig liver was studied with the use of chromatographic and spectroscopic procedures. The findings formed the basis for the development of a number of conceptions as to the structure of the macromolecular pentose nucleic acids. About 60 to 70 mole per cent of the nucleotides present in the initial substrates were liberated by enzymatic action as rapidly dialyzable nucleotides of low molecular weight. This fraction consisted of free cytidylic and uridylic acids (comprising a high proportion of the total pyrimidine nucleotides) and of combined purine and pyrimidine nucleotides. All nucleic acid preparations yielded a nondialyzable residue, resistant to enzymatic attack, which was found to consist to about two thirds of guanylic acid and of varying amounts of the other nucleotides. The exposure of pentose nucleic acids to weak alkali results ultimately, as is known, in their complete hydrolysis to mononucleotides. In the case of yeast ribonucleic acid, it could be shown that this rapid hydrolysis does not proceed at random, but that the several mononucleotides are released at widely differing rates, uridylic acid being the first and adenylic acid the last to be detached. The spectroscopic observations centered around a comparison of the ultraviolet extinctions of the intact nucleic acids with those of the enzymatic breakdown products and of the mononucleotide mixtures to which they gave rise on alkaline hydrolysis. At 260 mμ, the extinctions of the intact compounds and of their nondialyzable cores, produced by ribonuclease, were very considerably lower (25 to 37%) than those of the corresponding mononucleotide mixtures. The ultraviolet absorption spectra of the fractions rendered dialyzable by the enzyme showed, however, no such extinction decrease. The implications of these findings for the problems of ribonuclease specificity and ribonucleic acid structure are discussed and possible patterns of nucleotide sequence in backbone and sidechains of a branched macromolecule are considered.

Changse in protein and ribonucleic acid during the formation of chloroplasts in Euglena gracilis

Abstract Etiolated Euglena gracilis cells can form chloroplasts in the absence of growth and without the net synthesis of protein and ribonucleic acid. During this process, approximately 17% of the cellular proteins appear in the chlorplasts fraction. Only the supernatant fraction shows a significant decrease, and it is tentatively concluded that the chloroplastic proteins originate in the latter fraction. The ribonucleic acid distribution in the cell is little changed. The development of chloroplasts appears to be linked with a stimulation of the uptake of adenine by the ribonucleic acid and of leucine by the protein. These stimulations are apparent in all fractions of the cells. It is concluded that no preferential synthesis of protein and RNA occurs in the chloroplasts during their final development. The formation of specific ribonucleic acid molecules during the process is indicated.

Studies on the nucleoside arrangement in deoxyribonucleic acids I. The relationship between the production of pyrimidine nucleoside 3′5′-diphosphates and specific features of nucleotide sequence

Abstract Procedures are described for the isolation from an enzymic digest of calf thymus deoxyribonucleic acid of the series of dinucleotides all comprising cytidylic acid as one of the components. Some of the properties of these dinucleotides are reported. The content in sequential isomers has been determined in the case of adenylic-cytidylic and thymidylic-cytidylic. When the dinucleotides adenylic-cytidylic, cytidylic-cytidylic, and thymidylic-cytidylic are subjected to acid hydrolysis, they undergo decomposition at different rates, but all yield the corresponding pyrimidine nucleoside 3′,5′-diphosphate as a major product. These results, in conjunction with studies on the kinetics of acid hydrolysis of pyrimidine nucleosides and their phosphoric acid esters, permit the formulation of a mechanism of nucleoside diphosphate production from oligonucleotide precursors which is applied to the study of DNA polymers in the following paper.

Studies on the nucleotide arrangement in deoxyribonucleic acids. II. Differential analysis of pyrimidine nucleotide distribution as a method of characterization.

Abstract This paper describes a procedure for the classification and unique characterization of DNA specimens, including those that cannot be distinguished by base analysis. It is founded on a quantitative and standardized method for the determination of the 3′,5′-diphosphates of deoxycytidine and thymidine released at three defined stages of acid degradation. A larger fragment, namely the isomer mixture of cytidine-thymidine triphosphates, also was estimated quantitatively in these hydrolysates. Other fragments were likewise identified; they were all derivatives of pyrimidine nucleotides, each unit carrying esterified phosphoric acid at both ends. The evidence is considered in detail that prompts the conclusion that the nature and rate of formation of these fragments are characteristic of the arrangement of the pyrimidine nucleotides within the DNA polymer chain and reflect the length of all-pyrimidine tracts that are flanked by purines. Ten DNA specimens, both total preparations and fractions, of different origin were subjected to differential distribution analysis. It is shown that these preparations varied widely in several aspects of the distribution of pyrimidine nucleotides (and therefore also of purine nucleotides); and that the general plan of DNA structure appears to include a higher proportion of polypyrimidine and polypurine regions than could have been expeected from polynucleotide sequences formed through a random arrangement of monomers.

Studies on the nucleotide arrangement in deoxyribonucleic acids VIII. A comparison of procedures for the determination of the frequency of pyrimidine nucleotide runs

Abstract Two DNA preparations of mammalian origin, viz ., the DNA from calf thymus and the DNA from human spleen, were employed in a comparative study of different methods suitable for the investigation of the arrangement of pyrimidine nucleotides in the polymer chain. The conditions under comparison were (a) the degradation of DNA with dilute sulfuric acid; (b) the degradation of DNA with formic acid-diphenylamine; (c) the degradation of apurinic acid with dilute alkali. The released pyrimidine nucleotide runs of length 1 to 6 were determined directly by two-dimensional paper chromatography. The range and validity of the several procedures described here and in the literature are considered in detail and the bearing of the findings on the general problem of the nucleotide sequence in DNA is discussed.

On the distribution and biological significance of the nucleoside phosphotransferases

Abstract Nucleoside phosphotransferases were found in a large variety of plant and animal tissues. In plants and bacteria, only 5′-nucleotides are formed, while in animals small amounts of the 3′-isomers are also produced. The rates of phosphate transfer from phenylphosphate and 5′-inosinic acid are of the same order of magnitude in plants, bacteria and protozoa, but in the mammals transfer from the nucllotide is much slower. The enzyme is unevenly distributed in the organs of the rat, with the highest activities in liver, spleen and kidney, and the lowest in muscle. The bulk of the enzymic activity is localized in the cytoplasmic particles. Considerable increases in the activity of the enzyme occur during germination of wheat, regeneration of rabbit muscle, and bacterial growth. The results point to an active role of the nucleoside phosphotransferases in nucleotide biosynthesis.

Relation of ribonucleic acid to the photosynthetic apparatus in Euglena gracilis

Abstract The ribonucleic acids of green and etiolated Euglena gracilis cells show small but definite differences in their nucleotide distribution. The green organisms have a higher proportion of adenylic and uridylic acids and a lower content of cytidylic and guanylic acids. Permanently bleached cells resemble the etiolated organisms in their ribonucleic acid composition. The differences in the ribonucleic acids of the green and etiolated oragnisms are probably related to the differenced in structure and metabolism of the two types of cells. Two additional nucleotides are present in small amounts in the ribonucleic acid of Euglena gracilis. One of them is probably identical with a nucleotide recently found in the ribonucleic acids from other organisms and the second one is unknown.