Nucleotide Arrangement Research Articles

Studies on the nucleotide arrangement in deoxyribonucleic acids IX. Selective degradation of pyrimidine deoxyribonucleotides

The study of the sequence of purine nucleotides in deoxyribonucleic acids requires the availability of suitable preparations of apyrimidinic acid, i.e., of polydeoxyribophosphate chains retaining the purines of the parent DNA in unchanged sequence and proportions. For an understanding of the degradation of DNA a detailed knowledge and a complete balance of the hydrazinolysis of pyrimidine deoxyribomononucleotides were required. The course and the rate of the degradation by hydrazine of thymidylic and deoxycytidylic acids were studied under a variety of conditions. These nucleotides, when treated with anhydrous hydrazine, give rise to stoichiometric amounts of (a) urea; (b) the hydrazone of 2-deoxyribophosphate; and (c) a heterocyclic compound. The latter is 4-methyl-5-pyrazolone in the case of thymidylic acid, and 3(5)-amino-pyrazole in the case of deoxycytidylic acid. The hydrazone of the sugar phosphate is cleaved quantitatively with benzaldehyde to yield 2-deoxyribophosphate and benzalazine. The various breakdown products were identified by comparison with authentic preparations. The first-order reaction of the degradation with anhydrous hydrazine is completed at 60° within 1.5 h for deoxycytidylic acid and within 4 h for thymidylic acid. At 40°, the time required for completion is about 4 times as long. Other orienting experiments demonstrated that purine deoxyribonucleotides withstand the treatment with hydrazine; that aqueous hydrazine solutions at various pH values effect a slower and irregular degradation; and that the replacement of hydrazine by monosubstituted hydrazine derivatives, such as phenyl- or methylhydrazine does not yield the desired results.

Nucleotide composition and arrangement of soluble RNA in posterior silkgland

1. 1. The nucleotide composition and nucleotide arrangement of soluble RNA in the posterior silkgland have been compared for two kinds of silkworm, which produce two different kinds of fibroin which differ in their amino acid composition. 2. 2. The s-RNA's from the two kinds of worm are similar to each other in their nucleotide composition and arrangement. 3. 3. Some differences have been observed in the nucleotide arrangements when compared with yeast s-RNA. A few minor components including pseudo-uridylic acid are also detected in silkgland s-RNA. 4. 4. The composition of the ribosomal RNA is similar in the two kinds of worm.

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

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.

FIBRES OF GUANINE NUCLEOSIDES AND NUCLEOTIDES.

IT has been shown by Gellert, Lipsett and Davies1 that guanylic acid will form a gel under certain pH conditions and they were able to draw fibres from gels of the disodium salt of 3′-guanylic acid and of the free acid, 5′-guanosine monophosphate. From X-ray diffraction studies of these fibres the foregoing workers concluded that they consisted of a helical arrangement of the ribose nucleotides.

Studies on the nucleotide arrangement in deoxyribonucleic acids VI. Pyrimidine nucleotide clusters: Frequency and distribution in several species of the AT-type☆

The pyrimidine oligonucleotide clusters released by the hydrolysis of DNA specimens from calf thymus, rye germ, and wheat germ have been studied. Sequences of the general structure (pyrimidine deoxyriboside)n(phosphorric acid residue)n+1 with n of 1–8, have been separated quantitatively and their distribution in the three DNA preparations has been compared. Clusters of 9 or more pyrimidines in a row account for 6–8 % of the pyrimidines. In all cases, the results deviate considerably from what would be expected from a random arrangement of clusters. Almost all oligonucleotide units, comprised in the fractions with n of 1–4, have been separated, characterized, and estimated. Whereas the totol abundance of the various oligonucleotide classes was rather similar in the three DNA specimens, which were all of the AT-type, calf-thymus DNA differed widely from the specimens from rye and wheat germ in the amounts of individual oligonucleotides isolated. Some pyrimidine oligonucleotides which could have been expected were not encountered, conspicuously those consisting of 4 or 5 cytosine units in a row, whereas the corresponding thymine oligonucleotides were observed invariably. Previous observations showing that in the plant nucleic acid 5-methylcytosine and cytosine do not share the same pattern of arrangement within the polymer have been confirmed. The bearing of some of the findings on the general problem of DNA structure is considered.

Studies on the nucleotide arrangement in deoxyribonucleic acids. V. Pyrimidine nucleotide clusters: isolation and characterization.

Abstract The isolation and characterization of the pyrimidine oligonucleotide clusters occuring in DNA are described. The method is based on the fractionation of acidic hydrolysates of DNA on DEAE-cellulose and on the subsequent two-dimensional paper chromatography of these fractions. Pyrimidine oligonucleotide clusters of up to nine pyrimidine units in length, and individual pyrimidine oligonucleotides up to the pentanucleotide level, have been isolated and characterized with the use of their distinctive spectral ratios and, in many instances, their composition. The fractionation method accounts for nearly all of the original DNA phosphorus.

Studies on the nucleotide arrangement in deoxyribonucleic acids VI. Pyrimidine nucleotide clusters: Frequency and distribution in several species of the AT-type

The pyrimidine oligonucleotide clusters released by the hydrolysis of DNA specimens from calf thymus, rye germ, and wheat germ have been studied. Sequences of the general structure (pyrimidine deoxyriboside) n (phosphorric acid residue) n+1 with n of 1–8, have been separated quantitatively and their distribution in the three DNA preparations has been compared. Clusters of 9 or more pyrimidines in a row account for 6–8 % of the pyrimidines. In all cases, the results deviate considerably from what would be expected from a random arrangement of clusters. Almost all oligonucleotide units, comprised in the fractions with n of 1–4, have been separated, characterized, and estimated. Whereas the totol abundance of the various oligonucleotide classes was rather similar in the three DNA specimens, which were all of the AT-type, calf-thymus DNA differed widely from the specimens from rye and wheat germ in the amounts of individual oligonucleotides isolated. Some pyrimidine oligonucleotides which could have been expected were not encountered, conspicuously those consisting of 4 or 5 cytosine units in a row, whereas the corresponding thymine oligonucleotides were observed invariably. Previous observations showing that in the plant nucleic acid 5-methylcytosine and cytosine do not share the same pattern of arrangement within the polymer have been confirmed. The bearing of some of the findings on the general problem of DNA structure is considered.

Studies on the nucleotide arrangement in deoxyribonucleic acids VII. Direct estimation of pyrimidine nucleotide runs

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.

Studies on the nucleotide arrangement in deoxyribonucleic acids V. Pyrimidine nucleotide clusters: Isolation and characterization

The isolation and characterization of the pyrimidine oligonucleotide clusters occuring in DNA are described. The method is based on the fractionation of acidic hydrolysates of DNA on DEAE-cellulose and on the subsequent two-dimensional paper chromatography of these fractions. Pyrimidine oligonucleotide clusters of up to nine pyrimidine units in length, and individual pyrimidine oligonucleotides up to the pentanucleotide level, have been isolated and characterized with the use of their distinctive spectral ratios and, in many instances, their composition. The fractionation method accounts for nearly all of the original DNA phosphorus.

Studies on the nucleotide arrangement in deoxyribonucleic acids VII. Direct estimation of pyrimidine nucleotide runs

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.

Studies on the nucleotide arrangement in deoxyribonucleic acids VII. Direct estimation of pyrimidine nucleotide runs

Studies on the nucleotide arrangement in deoxyribonucleic acids VII. Direct estimation of pyrimidine nucleotide runs

An investigation of the fractionation and sequential characterization of the ribonucleic acids of rat liver☆

The isolation of RNA from rat liver by phenol extraction was followed by its separation into two principal fractions: one insoluble (I-RNA), on soluble (S-RNA), in 10 % NaCl solution. I-RNA differed substantially in its composition from S-RNA, not only in adenylic acid and guanylic acid values, but more strikingly in the proportion of pseudouridylic acid which was much higher in S-RNA. The further fractionation of both RNA species was brought about through the stepwise extraction of the respective histone nucleates with increasing concentrations of NaBr in the presence of chloroform. In this manner, I-RNA was divided into 7 or 8 fractions, S-RNA into 5; but with one exception these fractions differed little from each other in the distribution of the major nucleotide components, though the pseudouridylic acid contents fluctuated characteristically. The additional characterization of the preparations made use of their enzymic degradation by pancreatic ribonuclease (EC 2.7.7.16). The pattern of mono- and oligonucleotides thus released revealed certain distinctive differences in the nucleotide arrangement prevailing in the various preparations. The results are considered in the general context of the distribution of solitary purine nucleotides and of pyrimidine nucleotide clusters. Certain neighbor preferences are also suggested.

An investigation of the fractionation and sequential characterization of the ribonucleic acids of rat liver

The isolation of RNA from rat liver by phenol extraction was followed by its separation into two principal fractions: one insoluble (I-RNA), on soluble (S-RNA), in 10 % NaCl solution. I-RNA differed substantially in its composition from S-RNA, not only in adenylic acid and guanylic acid values, but more strikingly in the proportion of pseudouridylic acid which was much higher in S-RNA. The further fractionation of both RNA species was brought about through the stepwise extraction of the respective histone nucleates with increasing concentrations of NaBr in the presence of chloroform. In this manner, I-RNA was divided into 7 or 8 fractions, S-RNA into 5; but with one exception these fractions differed little from each other in the distribution of the major nucleotide components, though the pseudouridylic acid contents fluctuated characteristically. The additional characterization of the preparations made use of their enzymic degradation by pancreatic ribonuclease (EC 2.7.7.16). The pattern of mono- and oligonucleotides thus released revealed certain distinctive differences in the nucleotide arrangement prevailing in the various preparations. The results are considered in the general context of the distribution of solitary purine nucleotides and of pyrimidine nucleotide clusters. Certain neighbor preferences are also suggested.

An investigation of the fractionation and sequential characterization of the ribonucleic acids of rat liver

The isolation of RNA from rat liver by phenol extraction was followed by its separation into two principal fractions: one insoluble (I-RNA), on soluble (S-RNA), in 10 % NaCl solution. I-RNA differed substantially in its composition from S-RNA, not only in adenylic acid and guanylic acid values, but more strikingly in the proportion of pseudouridylic acid which was much higher in S-RNA. The further fractionation of both RNA species was brought about through the stepwise extraction of the respective histone nucleates with increasing concentrations of NaBr in the presence of chloroform. In this manner, I-RNA was divided into 7 or 8 fractions, S-RNA into 5; but with one exception these fractions differed little from each other in the distribution of the major nucleotide components, though the pseudouridylic acid contents fluctuated characteristically. The additional characterization of the preparations made use of their enzymic degradation by pancreatic ribonuclease (EC 2.7.7.16). The pattern of mono- and oligonucleotides thus released revealed certain distinctive differences in the nucleotide arrangement prevailing in the various preparations. The results are considered in the general context of the distribution of solitary purine nucleotides and of pyrimidine nucleotide clusters. Certain neighbor preferences are also suggested.

Distribution of guanylic acid residues in tobacco mosaic virus nucleic acid.

THE mode of arrangement of purine and pyrimidine nucleotides in tobacco mosaic virus nucleic acid was studied using the actions of pancreatic ribonuclease and micrococcal phosphodiesterase. This work showed that in tobacco mosaic virus nucleic acid the purine nucleotides and pyrimidine nucleotides occur in ‘clusters’ of two or more and also ‘singly’ alternating with each other. The extension of these studies to nucleic acids of three viral strains (TMV, HR and M) revealed for the first time significant differences in the arrangement of nucleotides in each of their molecules1–5. Additional information on the distribution of guanylic acid residues in tobacco mosaic virus nucleic acid, obtained by the action of an enzyme specific to secondary phosphate esters of guanosine 3′-phosphates, is given in the present communication.

PRODUCTS OBTAINED BY DIGESTION OF THE NUCLEIC ACIDS OF SOME STRAINS OF TOBACCO MOSAIC VIRUS WITH PANCREATIC RIBONUCLEASE

Paper electrophoresis and paper chromatography were recently combined under conditions which resulted in a two-dimensional procedure for the separation and determination of oligonucleotides.1 This system provides the means to extend previous chemical studies2-4 on mutant strains of tobacco mosaic virus (TMV). The proposed approach is to subject the ribonucleic acid (RNA) of each strain to the action of pancreatic ribonuclease (RNase), and then to separate and analyze the nucleotide and oligonucleotide fragments. Owing to the known specificity of action of RNase, the nature and quantity of the various products obtained by its action provide a clue to the sequential arrangement of nucleotides in the original polynucleotide chains. A similar approach has already been used with strains of TMV,3 4 but the higher resolution of the mapping procedure applied in the present instance allows the comparison of strains to be made in considerably greater detail. We are reporting here the results of such extensive analyses of the RNase digests of the nucleic acids from ordinary TMV and from the M and HR strains.

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

This paper reports the isolation from an acid hydrolysate of rye germ deoxyribonucleic acid of three derivatives of methylcytidine, viz., the 3′,5t́-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.

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

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 nucleoproteins VI. The deoxyribonucleoprotein and the deoxyribonucleic acid of bovine tubercle bacilli (BCG)

The preparation of a deoxyribonucleoprotein from the BCG variant of bovine type tubercle bacilli is described. The principal steps involved precipitation at pH 4.2, removal of contaminating materials at half-saturation with ammonium sulfate, and fractionation with ethanol at a low temperature. The purified product was shown to contain about 0.4 amino acid residue for one nucleotide residue. An approximate estimation of the principal amino acids contained in the nucleo-protein revealed a preponderance of dicarboxylic over basic amino acids. The deoxyribonucleic acid was freed of its protein component by tryptic digestion and examined with respect to the distribution of purines and pyrimidines. It belongs to the extreme “GC type”. More detailed aspects of the arrangement of nucleotides in the nucleic acid chain were studied through the application of the method of differential distribution analysis, making use of the results of the stepwise acid hydrolysis. The sugar moiety of the deoxypentose nucleic acid was identified tentatively as deoxyribose. The nucleotide composition of the pentose nucleic acid of BCG also was determined.

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

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.