Herman S. Shapiro

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.

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.

Studies on the nucleotide arrangement in deoxyribonucleic acids: X. Frequency and composition of pyrimidine isostichs in microbial deoxyribonucleic acids and in the DNA of E. coli phage T 3

Abstract DNA preparations from Bacillus cereus, Escherichia coli, Salmonella typhimurium and typhosa, Serratia marcescens, and Rhodopseudomonas spheroides and also from bacteriophage T 3 have been studied with respect to the distribution and the composition of the pyrimidine isostichs released by mild acid hydrolysis. Two analytical procedures were employed in these investigations, namely the direct two-dimensional chromatography of the hydrolysate and the fractionation of the fragments on DEAE-cellulose. These DNA specimens showed several common characteristics: (a) The frequencies of the total pyrimidine isostichs of length 1 to 8 are similar in all preparations and correspond to the distribution predicted for a random polynucleotide. (b) The relative frequencies of individual oligonucleotide runs liberated by the various AT- and GC-types of DNA vary greatly from each other and deviate from the calculated random frequencies. (c) There exists a disparity in the relative contribution of cytosine and thymine to the isostichs of length 1 to 5: the shorter sequences are relatively richer in the first pyrimidine, the longer ones in the second. (d) Polycytidylic acid runs are comparatively very rare, whereas the frequency of polythymidylic acid sequences often exceeds random predictions. (e) DNA from genetically related species (E. coli, S. typhimurium and S. typhosa) exhibit very similar patterns of pyrimidine arrangement. The DNA of phage T 3 resembles, also in the frequency of many of its individual pyrimidine sequences, a randomly constructed polynucleotide. Though it has almost the same base composition as the DNA of its host species, E. coli, it differs from the latter in sequence characteristics.

STUDIES ON THE NUCLEOTIDE ARRANGEMENT IN DEOXYRIBONUCLEIC ACIDS. VII. DIRECT ESTIMATION OF PYRIMIDINE NUCLEOTIDE RUNS.

Abstract A two-dimensional paper-chromatographic system is described permitting the direct separation of the pyrimidine oligonucleotide runs of length 1–6. The procedure is applicable to as little as 1 mg of DNA hydrolasate. The characteristics of the ultra-violet absorption and chromatographic mobility of the various pyrimidine nucleotide clusters are presented; and the method is exemplified through the quantitative distribution analysis of the DNA of human spleen.

A Direct Test of Antiparallelism in Complementary Sequences of Calf Thymus Deoxyribonucleic Acid

A Direct Test of Antiparallelism in Complementary Sequences of Calf Thymus Deoxyribonucleic Acid

Studies on the nucleotide arrangement in deoxyribonucleic acids III. Identification of methylcytidine derivatives among the acid degradation products of rye germ DNA

Abstract This paper reports the isolation from an acid hydrolysate of rye germ deoxyribonucleic acid of three derivatives of methylcytidine, viz. , the 3′,5t-diphosphate and the two dinucleoside triphosphates that this pyrimidine nucleoside shares with cytidine and thymidine, respectively. Some of the properties of these compounds are also described.

Maintenance of DNA integrity during murine erythroleukemia cell differentiation induced by ethionine and other hypomethylation agents.

The extent of single-strand nicks in DNA from murine erythroleukemia cells induced to differentiate to hemoglobin synthesis in the presence of the hypomethylating agent ethionine was estimated and compared to those levels in uninduced cells and from cells induced to differentiate upon exposure to dimethylsulfoxide or butyrate ion. Although ethionine has been shown to cause more extensive hypomethylation in the DNA of induced cells than that caused by dimethylsulfoxide or butyrate ion, the frequency of detected single-strand breaks in the DNA of uninduced, control cells was not significantly different from that of cells exposed to any of these inducing chemicals. This data indicates that no correlation exists between DNA hypomethylation and DNA single-strand breaks and that unmethylated CpG loci likely do not operate as specific endonuclease recognition sites or as potential origins of transcription in these mammalian cells.