Showing papers on "Base pair published in 1969"

Determination of stability of the DNA double helix in an aqueous medium

Abstract: The possibility of determining the free energy of stabilization ΔG0 of native DNA structure with the help of calorimetric data on heats ΔH of transition from the native to denaturated state is considered. Results of microcalorimetric measurements of heats of denaturation of T2 phage DNA at, different values of pH and ionic strength of solution are given. Values of free energy of stabilization of the DNA native structure ΔG0 under various conditions have been obtained. It is shown that under conditions close to physiological ΔG0 approaches 1200 cal/mole per base pair.

Interaction of the RNA Polymerase Inhibitor Chromomycin with DNA

Abstract: Formation of a complex between chromomycin and DNA has been shown to depend on the guanine content and on base pairing of the polydeoxynucleotides. A limit of association is reached when one chromomycin molecule is bound per four nucleotide base pairs. The rate of complex formation is independent of the size of the sugar side chains of the antibiotic and of the nature and base composition of DNA. In contrast the rate of complex dissociation increases with a decrease in size of the sugar side chains. The inhibition by chromomycin and its derivatives of DNA and RNA polymerase reactions exhibits similar features. Probably the dissociation rate of the complex determines the inhibitory activity. Chromomycin by complex formation protects guanine rich segments of DNA very efficiently against the attack of nucleases. After enzymatic hydrolysis chromomycin containing base paired oligonucleotide fragments remain in the reaction mixture and can be isolated by electrophoresis. The antibiotic is not covalently bound to these fragments and can be removed by thorough extraction with ether.

Fluorescence of proflavine--DNA complexes: heterogeneity of binding sites.

Abstract: Measurements of the relative quantum yield of fluorescence of proflavine bound to DNA as a function of the number of bound dyes per nucleotide and the ionic strength allow the determination of the binding constants and respective number of the two types of sites previously postulated. It is demonstrated that 2–3% of the base pairs form sites where the dye is strongly bound and fluoresces normally while in the other set of sites the binding constant is 3–4 times weaker and the fluorescence completely quenched. Comparison with complexes of Pro with double stranded polynucleotides poly (A + U), poly (I + C), poly(G + C), confirm that the strong binding sites correspond to A-T-rich regions of the DNA while the quenched sites seem to require the presence of a neighboring guanine. The role of charge transfer in quenching of fluorescence and mutagnic action is considered. An original method for the determination of free dye and bound dye, based upon the use of an external quencher is described in the Appendix.

Nature of the hisD3018 Frameshift Mutation in Salmonella typhimurium

Abstract: Histidinol dehydrogenase from three differing revertants of ICR-191A-induced frameshift hisD3018 has been purified and examined for amino acid replacements. The enzyme from one spontaneously arising revertant, R7, contains an extra proline residue, whereas that from another, R5, contains an extensive frameshifted sequence, four amino acid residues of which have been identified to date. The amino acid replacement data are in agreement with the in vitro code word assignments and allow the characterization of the hisD3018 frameshift as an addition of one nucleotide pair, most likely guanine plus cytosine. Enzymatic data for those ICR-191A-induced revertants of hisD3018 arising within the hisD gene indicate that the enzyme is wild type and, therefore, that ICR-191A can cause deletions as well as additions of single base pairs. The wild-type amino acid sequence is restored in enzyme from an N-methyl-N'-nitro-N-nitrosoguanidine (NG)-induced revertant, R29, suggesting that NG is a base-deleting as well as a base-substituting mutagen. The unusual response of hisD3018 to external suppressors is considered in terms of reinitiation of protein synthesis out of phase, coupled with suppression of a nonpermissive missense codon so generated, and of an alternative hypothesis invoking a true frameshift suppressor transfer ribonucleic acid with an extended or deleted anticodon.

Salt effects on the denaturation of DNA

Abstract: When DNA's of differing GC:AT base ratios, e.g. synthetic poly dAT, T4 DNA,calf thymus DNA, E. coli DNA, and M. lysodeikticus DNA, are heat-denatured at neutral pH in increasing concentrations of N(a)(2)SO(4) or C(s)(2)SO(4) as supporting electrolytes,the variation of melting temperature with average base composition, dT(m)/dX(G)(C), changes from 45°C (in 0.002M Na) to ll°C (in 4.5M Na) and from 42°C (in 0.002M Cs) to 3°C(in 4.5M Cs). The decrease of dT(m)/dX(G)(C) is a monotonic function of decreasing water activity in the salt solutions. We interpret this decreased composition dependence of the thermal stability of the various DNA's as being due to a destabilization of the GC base pairs relative to the AT base pairs by the concentrated salt media. A simple quantitative treatment shows that k = 8GC/SAT decreases from a value of 4.14 (in 0.01MN(a)) to 1.86 (in 3M Na) and from 4.18 (in 0.01M Cs) to 1.42 (in 3M Cs). SAT is the equilibrium constant for the formation of a hydrogen-bonded AT base pair from a pair of unbonded bases at the junction between a helical region and a denatured region and SGC is the like constant for the formation of a GC base pair. These results corroborate our previous findings of a strongly reduced composition dependence of the negative logarithm of the methylmercuric hydroxide concentration necessary to produce 50% denaturation when the helix-coil transition of DNA is studied in concentrated Cs(s)SO(4)(ultracentrifugation) instead of in dilute N(a)(2)SO(4) (ultraviolet spectrophotometry).

Nucleic acids and polynucleotides

Abstract: The conformation of native double helical DNA is well-known, but it is possible that small regions occur within native DNA, undetectable by X-ray diffraction methods, which have different conformations. Model structures are the synthetic deoxypolynucleotides of defined sequence. Under the conditions used, DNA, poly d(A-T) • poly d(A-T), and poly d(T-G) • poly d(C-A) can all give similar X-ray diffraction patterns, whereas poly dA • poly dT, poly dI • poly dC, poly dG • poly dC, and poly d(T-C) • poly d(G-A) clearly differ from DNA. This led to the tentative hypothesis that those DNA's in which all purines are in one strand and all pyrimidines in the other differ in structure from those (such as native DNA) in which purines and pyrimidines alternate or are irregular. We now find that poly d(I-C) • poly d(I-C) does not fit the hypothesis and is a most unusual structure, having seven or eight base pairs per turn. Both molecular model building and circular dichroism studies suggest that it is a left-handed helix. A number of purified tRNA's have been crystallized. We have obtained, from unfractionated tRNA, crystalline “powder” X-ray diffraction patterns showing rings and spots to about 20 A resolution. It is not clear whether cocrystallization has occurred, or whether there is fractional crystallization, though preliminary evidence favors the latter. Determination of the structure of crystalline tRNA has many features in common with protein crystallography, but there are a number of distinct differences.

Conformation of ribosomal RNA of E. coli: an infrared analysis.

Abstract: The concentration of A–U and G–C base pairs in RNA can be measured by an infrared method, which has been applied to E. coli ribosomal RNA. Results are compared with those of other spectroscopic methods.

Hybridization of chromosomal RNA to native DNA

Abstract: Chromosomal RNA, which is associated with chromosomal proteins in the chromosomes of higher organisms, possess the ability to hybridize to homologous native DNA. The proportion of native DNA thus hybridized is similar to the proportion of denatured DNA which hybridizes with chromosomal RNA, and both are similar to the proportions of chromosomal RNA and DNA in native chromatin.

Forces involved in the Conformational Stability of Nucleic Acids

Abstract: ONE of the most important problems in research on nucleic acid is the understanding of the forces which maintain the helical structure. After the elucidation of the double helix by Watson and Crick1, the helical secondary structure of DNA was thought to derive its stability from hydrogen bonding between the base pair. The discovery that polynucleotides could exist in single strand ordered helical conformation2–6 demonstrated that other types of forces are involved in the stability of this structure. These stacking forces between parallel bases were thought to be of the Van der Waals type. More recent work shows that the presence, absence or modification of the 2′ hydroxyl group plays an important part in the conformational stability of the oligo and poly-nucleotides7–12.

Studies on conformational changes in the DNA structure induced by protonation: Reversible and irreversible acid titrations and sedimentation measurements

Abstract: Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter asapp against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.

Studies on the secondary structure of ribosomal ribonucleic acid components of rabbit reticulocytes.

Abstract: The conformation in solution of fractionated 30 S and 19 S ribosomal RNA from rabbit reticulocytes has been studied by optical rotatory dispersion, analysis of thermal melting profiles and their derivatives, and spectrophotometric acid-base titration. From a consideration of the limitations of these methods, it has been possible to set limiting values on the degree of base-pairing and the lengths of the double helices: between 60 and 80% of the bases in 19 S and 30 S RNA are estimated to be paired. The paired segments are not shorter than 4 base pairs, and evidence from other sources is available which indicates that they are not longer than 8–16 base pairs. The spread of helix lengths is greater in the 30 S than in 19 S RNA; and other differences are noted. Several distinct populations of double helices, differing in their thermal stability, are present. Estimates are presented from spectrophotometric and titration data for the base compositions of the paired and unpaired regions.

Mechanism of dna chain growth, iii. equal annealing of t4 nascent short dna chains with the separated complementary strands of the phage dna

Abstract: Nascent short DNA chains isolated from T4-infected E. coli under a variety of conditions anneal equally to the separated complementary phage DNA strands. The samples examined include: pulse-labeled short chains isolated by alkaline sucrose gradient sedimentation from the T4D (wild type)-infected cells in both the early and late stages of phage DNA synthesis; nascent chains accumulated during ligase inhibition of T4 ts B20-infected cells; and the single-stranded nascent short chains isolated from T4D-infected cells by mild procedures involving no denaturation treatment. The results are consistent with the hypothesis that both strands of DNA are synthesized discontinuously.

Nucleotide distribution and functional orientation in the deoxyribonucleic acid of phage phi 80.

Abstract: The distribution of nucleotides in the deoxyribonucleic acid (DNA) of phi80 was determined by density analysis of molecular fragments of known length and origin. One half of the molecule includes a long, fairly homogeneous segment that contains 55% guanine plus cytosine (GC). DNA in the other half contains about 50% GC, except for a short stretch near the molecular end where the GC content is higher. Transcription of phi80 DNA was studied by ribonucleic acid-DNA hybridization tests with isolated molecular halves. At times early in the growth cycle, messenger is synthesized at a constant, relatively low rate and originates almost exclusively from DNA in the lower-GC half. At later times, messenger represents both halves and is synthesized at a greatly increased rate. The DNA of phi80 is closely analogous, physically and functionally, to lambda DNA. The similarity is most striking in the high-GC half, which is defined here as "left" since its molecular end contains a cohesive site homologous to that at lambda's left end. The left halves of the DNA of the two phages contain primarily late-functioning genes, and possess similar nucleotide distributions and some similar base sequences. The right halves of the two DNA molecules are less similar. Both contain the early genes, but they differ considerably in GC content (45% in lambda, 51% in phi80) and do not strongly cross-react in hybridization tests. The DNA of phi80 lacks the central 37%-GC segment found in lambda.

Infrared Spectrum of an Anticodon Fragment of a Transfer RNA

Abstract: WE have observed the infrared spectrum of a fragment of a transfer RNA (tRNA) in aqueous solution and obtained evidence for the existence of the hydrogen-bonded base pairs.

A new approach to the analysis of hybridization of bacterial nucleic acids. Analysis of ribosomal ribonucleic acids of Bacillus subtilis

Abstract: A new graphical analytical technique is described for the hybridization of bacterial RNA with denatured homologous DNA immobilized on cellulose nitrate membrane filters. To a constant amount of DNA, various amounts of bacterial RNA were added and the percentage of input RNA bound was plotted against the DNA/RNA weight ratio in a given experiment. When RNA samples were used that hybridize to denatured DNA as a single species, the resulting curves (RNA-hybridization-efficiency curves) could be analysed to show the percentage of the DNA capable of specifically binding the RNA and could also be used to detect the presence of minor RNA contaminants in a purified specimen. The method could also estimate the relative amounts of two species of RNA in a mixture when these were hybridized independently to different DNA cistrons or cistron groups. As an example of RNA that can be studied in this way, the 16s and 23s ribosomal RNA species of Bacillus subtilis were chosen. These each behave in DNA-RNA hybridization as a single species and bind independently to different groups of DNA cistrons. The results obtained from hybridization-efficiency curves were compared with those obtained by the more usual method of saturating the specific DNA regions with excess of ribosomal RNA (hybridization-saturation curves). It was confirmed by both approaches that 0.15 (+/-0.02)% of B. subtilis DNA would hybridize with 16s ribosomal RNA, 0.30 (+/-0.02)% would hybridize with 23s ribosomal RNA, and 0.46 (+/-0.02)% would hybridize with (16s+23s) ribosomal RNA. This agreement suggested that mass-action equilibria between hybridized and free RNA had a negligible effect on the hybridization curves over the range of DNA and RNA concentrations employed.

Studies of DNA‐dependent RNA polymerase

Abstract: DNA-dependent RNA polymerase reacts with a nucleic acid to form a binary complex, which can be isolated by density gradient centrifugation or by millipore filtration techniques. Studies of the complex of Escherichia coli RNA polymerase and phage lambda DNA show that temperature and ribonucloside triphosphates do not affect the extent of binding. The binary complex is very sensitive to ionic strength. Enzyme bound in the complex exchanges with free enzyme. In the presence of Mg2+, the lambda DNA becomes saturated with enzyme when 2.5 μg have been bound per microgram of DNA. Transfer RNA and synthetic polyribonucleotides also react with the enzyme at 0°C. In the presence of divalent cation at low ionic strength, the main complex of the enzyme and a polyribonucleotide has a sedimentation value of 15S. In the absence of divalent cation, or at higher ionic strength, the value is 13S. Polyuridylate and polycytidylate have a greater affinity for the enzyme than polyadenylate or transfer RNA. An interesting question regarding the specificity of interaction of RNA polymerase with native DNA is whether any of the RNA chains made have the sequence AUG, the principal N-formylmethionine codon, at the 5′ termini. Studies with T7 DNA show that, under conditions of high ionic strength, 24% of the RNA chains start August.