Nucleic Acid Complexes Research Articles

Mutagen-nucleic acid complexes at the polynucleotide duplex level in solution: intercalation of proflavine into poly(dA-dT) and the melting transition of the complex.

AbstractThe nmr chemical shifts and line widths of the nucleic acid base and sugar proton resonances and the proflavine ring protons can be monitored through the melting transition of the proflavine + poly(dA‐dT) complex, phosphate/dye (P/D) ratio = 24 and 8 in 1M salt solution. The nucleic acid and mutagen protons in the complex are in fast exchange between duplex and strand states with the midpoint of the melting transition monitored at the nucleic acid resonances increasing from 72.6°C for poly(dA‐dT) to 78.1°C for the P/D = 24 complex and 83.4°C for the P/D = 8 complex in 1M salt solution. The melting transition monitored by the proflavine resonances were 80.0°C for the P/D = 24 complex and 84.3°C for the P/D = 8 complex in 1M salt solution. Since the nucleic acid is in excess at high P/D ratios, the nucleic acid transitions are an average for the opening of mutagen‐free and mutagen‐bound base‐pair regions, while the proflavine transitions monitor the melting of mutagen‐bound base‐pair regions. The observed 0.75 to 0.95 ppm unfield shift at all four proflavine protons on formation of the complex with poly(dA‐dT) provides direct evidence for intercalation of the mutagen between base pairs of the nucleic acid duplex. We have deduced the approximate overlap geometry between the proflavine ring and nearest‐neighbor base pairs at the intercalation site from a comparison between experimental proflavine complexation shifts and those calculated for various stacking orientations. The experimental chemical shift of the poly(dA‐dT) adenine H‐2 resonance in the duplex state in the absence and presence of proflavine suggests that intercalation occurs preferentially at dT‐dA sites. The selective chemical shift changes at the sugar H‐2′,2″ and H‐3′ resonances of the poly(dA‐dT) duplex on complex formation demonstrates changes in the sugar pucker and/or torsion angles of the sugar phosphate backbone at the intercalation site.

Specific recognition of guanine bases in protein—nucleic acid complexes

Specific recognition of guanine bases in protein—nucleic acid complexes

A sedimentation velocity method for the separation of complementary strands of DNA

A method is described for the rapid separation of the complementary strands of homogenous DNA's. The method takes advantage of a difference in sedimentation coefficients between the complementary strands when these have been dissociated and complexed with poly(U-G). The separation procedure is readily carried out for preparative purposes by zone sedimentation in linear 5–20% (w/v) sucrose gradients containing 2 M NaCl. In analytical band sedimentation experiments, concentrated solutions of CsCl have been used to effect the separation of the complexed complementary strands. Sedimentation coefficients corresponding to corrected values at infinite dilution for the cesium salt of the nucleic acid complexes at 25°C ( S° 25, w)have been obtained for the complexed complementary strands of coliphage λb 2b 5c DNA and for the uncomplexed single strands of this DNA. The relative values of the parameters affecting S° 25, w have been determined. Under these conditions, for which preferential hydration of the sedimenting species is not involved, it was found that the more highly complexed strand has sustained a 24% increase in mass and a 28% decrease in frictional coefficient relative to uncomplexed DNA, while the strand binding the lesser amount of RNA has increased in mass by 11% and changed in frictional coefficient by an insignificant amount relative to the uncomplexed species. The differences in the partial specific volumes of the complexed species from that of the uncomplexed DNA do not contribute significantly to the differences in S° 25, w between the complexed complementary strands.

Water-Soluble Lysine-containing Polypeptides. II. The Interaction of Several Sequential Lysine–Glycine Polypeptides with DNA. A Circular Dichroism Study of DNA Conformation in Annealed Complexes

To serve as models of protein–nucleic acid complexes, annealed complexes of several sequential lysine–glycine polypeptides with DNA have been formed. Circular dichroic spectra of the DNA contained in the complexes indicates that several discrete conformations of DNA can exist when complexed.

Exciton-like splitting in acridine dye-nucleic acid complexes.

COMPLEXES between cationic acridine dyes and nucleic acids are of considerable interest because of the wide variety of biological effects in which the interaction between the dye and nucleic acids have been implicated1–3.

Thermal denaturation of nucleic acids in polyacrylamide gels

A system is described for measuring thermal denaturation of nucleic acid fractions directly in polyacrylamide gels. Total nucleic acids were fractionated by disc gel electrophoresis. The buffer within the gel was then exchanged for one commonly used in denaturation studies. Thermal denaturation profiles of DNA and ribosomal RNA in the gel were determined using a specially constructed Gel Carriage to position the appropriate fraction during spectrophotometric measurements. These profiles were compared with denaturation patterns obtained by classical methods in free solution; the two methods yielded similar patterns. Thermal denaturation profiles were also obtained for chloroplast light ribosomal RNA resolved by gel electrophoresis of total plant nucleic acids. Thus, denaturation patterns of individual, minor components present in complex nucleic acid mixtures can be directly measured in gels.

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm −1 to 900 cm −1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm −1 and at 813 cm −1 whereas the B and the C forms exhibit a band at 837 cm −1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.

The Nucleus in the Spores of Rhinosporangium seeberi

Rhinosporidiosis is a disease occurring usually in the tropics and is characterized by the production of nasal polyps supposedly caused by a fungus, Rhinosporangium seeberi, which was claimed after light microscopic studies to proliferate by “amitotic” division of the nucleus. The organism was studied electron microscopically and the spores were found to be bound by a non-collagenous wall enclosing a variety of vesicles, some containing smooth amorphous material, others coiled threads, and still others dark granules. Typically mitochondria, endoplasmic tubules, Golgi, and a membrane-bound nucleus were not seen although a few rudimentary membrane-bound spaces are present. These features as well as failure to culture the organism cast doubts about its taxonomic position, and it seemed the organism was essentially some nucleic acid complex organizing mucopolysaccharides in subepithelial zones in the nasal mucous membrane.

Optical rotatory dispersion properties of nucleic acid complexes with the oligopeptide antibiotics distamycin A and netropsin

ORD measurements of nucleic acids in the presence of the oligopeptides netropsin and distamycin A have indicated association of the antibiotics with DNA and strong conformational changes of the DNA structure with specificity to AT-rich helical regions. The RNA conformation is relatively unaffected by these antibiotics. The results are explained in terms of a perturbation of the DNA secondary structure as well as of the chromophore system of the oligopeptides.

Specificity of the three-stranded complex formation between double-stranded DNA and single-stranded RNA containing repeating nucleotide sequences

Three-stranded nucleic acid complexes have been shown to be formed from double-stranded DNA and single-stranded RNA polymers with restricted base sequences. Complex formation takes place only when the DNA contains all purine bases in one strand and all pyrimidine bases in the other strand and the RNA must be a polypyrimidine polymer. Hence, poly (dA)·(dT) forms a three-stranded complex with poly U, and poly d(T-C)·d(G-A) † † Abbreviations for the polynucleotides used in this study are those given in the Revised Tentative Rules (1965) of IUPAC-IUB in J. Biol. Chem. (1966) 241, 527. The abbreviation CH + indicates the protonated form of cytidylic acid. forms a three-stranded complex with poly (U-C) in acidic solutions. No other RNA's including the random co-polymer poly (U,C) interact with poly d(T-C)·dG-A) to give a stoichiometric three-stranded structure. Under no condition is a complex detected between poly d(T-G)·d(C-A) or poly d(A-T)·d(A-T) and any polyribonucleotide. The three-stranded poly d(T-C)·d(G-A)·(U-CH +) was characterized by cesium sulfate buoyant density studies, by continuous variations study, by ultraviolet spectral properties and by optical density-temperature profiles. Addition of poly (U-C) to poly d(T-C)·d(G-A) and addition of poly U to poly (dA)·(dT), prior to the addition of Escherichia coli RNA polymerase, effectively reduced the transcription rate of both strands of these DNA's. Other combinations of RNA's with DNA's showed essentially no inhibition; all of these RNA-DNA mixtures were also found to be inert as regards triplex formation. Possible biological roles of the triplexes are discussed.

The fractionation of quaternary ammonium complexes of nucleic acids. Evidence for heterogeneity of ribosomal ribonucleic acid.

The fractionation of quaternary ammonium complexes of nucleic acids. Evidence for heterogeneity of ribosomal ribonucleic acid.

Effects of Phosfon-S on Nucleic Acid Metabolism in Pisum sativum Alaska

Phosfon-S, a substance which inhibits stem elongation, alters nucleic acid metabolism in Pisum sativum Alaska. Methylated albumin kieselguhr (MAK) columns were used to fractionate (32)P-labeled nucleic acids. Phosfon-S treatment of the plants resulted in a decrease in soluble RNA and an increase in ribosomal RNA. Specific activities of the various nucleic acid fractions were lower as a result of treatment. The nucleic acids from treated tissues were more resistant to RNase degradation, and endogenous RNase activity was lower in treated tissues. When RNase treated nucleic acids were fractionated on MAK columns, the DNA-RNA fractions from treated plants had a higher specific activity than that of the control, which was not true before nuclease treatment. Spectrophotometric examination of this fraction revealed a difference in absorption spectra, possibly indicating a Phosfon-S nucleic acid complex. It is suggested that these alterations in nucleic acid metabolism could in turn alter a wide variety of metabolic processes, resulting in retarded growth.

Soluble complexes of nucleic acids with α-chymotrypsin and its derivatives

Evidence has been presented from kinetic and ultracentrifugation data that at pH 7–7.5 and in the absence of added salt, chymotrypsin combines with nucleic acid into a definite (stoichiometric) soluble complex containing about one chymotrypsin molecule for each three nucleotide units. Similar complexes are formed with the diisopropylphosphoryl derivative of α-chymotrypsin and with chymotrypsinogen. The kinetic data suggest that the reaction with chymotrypsin is irreversible in the absence of salt, but in the presence of KCl in concentrations of the order of 0.1 M or higher the complex dissociates into free enzyme and nucleic acid. The active center of chymotrypsin is not directly involved in the formation of the complex in which the enzyme seems to retain about 20% of its activity. Electron micrographs of the DNA complex of the diisopropylphosphoryl derivative of α-chymotrypsin revealed strands, of which the smallest and most frequently occurring diameter was of the order of 100 Å as compared to ≈ 20 Å for the DNA double helix. The strands showed less tendency to random association than those of the free nucleic acid. RNA-complexes also occur as distinct particles, but these have a less clearly defined shape. Some of the molecular properties of these complexes have been compared with those of a natural complex of about the same nucleic acid content (tobacco mosaic virus) and some speculations as to the finer structure of the chymotrypsin-complexes have been made.

Soluble complexes of nucleic acids with α-chymotrypsin and its derivatives

Evidence has been presented from kinetic and ultracentrifugation data that at pH 7–7.5 and in the absence of added salt, chymotrypsin combines with nucleic acid into a definite (stoichiometric) soluble complex containing about one chymotrypsin molecule for each three nucleotide units. Similar complexes are formed with the diisopropylphosphoryl derivative of α-chymotrypsin and with chymotrypsinogen. The kinetic data suggest that the reaction with chymotrypsin is irreversible in the absence of salt, but in the presence of KCl in concentrations of the order of 0.1 M or higher the complex dissociates into free enzyme and nucleic acid. The active center of chymotrypsin is not directly involved in the formation of the complex in which the enzyme seems to retain about 20% of its activity. Electron micrographs of the DNA complex of the diisopropylphosphoryl derivative of α-chymotrypsin revealed strands, of which the smallest and most frequently occurring diameter was of the order of 100 Å as compared to ≈ 20 A ̊ for the DNA double helix. The strands showed less tendency to random association than those of the free nucleic acid. RNA-complexes also occur as distinct particles, but these have a less clearly defined shape. Some of the molecular properties of these complexes have been compared with those of a natural complex of about the same nucleic acid content (tobacco mosaic virus) and some speculations as to the finer structure of the chymotrypsin-complexes have been made.

The biogenesis of anthocyanins: VII. The requirement for both purines and pyrimidines

Previous evidence that anthocyanin formation in Spirodela is dependent on the synthesis of a complex nucleotide or nucleic acid has been extended through a survey of the effects of a variety of purine and pyrimidine analogs. Almost all such compounds have been found to inhibit anthocyanin formation. The group includes analogs of all four bases which make up ribonucleic acid (RNA): 8-azaguanine, 8-azaadenine, 6-azauracil, and 2-thiocytosine, as well as Antrycide (2,6′-dimethyl-6,4′-pyrimidylamino-4, 2′-diaminoquinoline 1,1′-dimethosulfate), an inhibitor of purine incorporation into nucleic acid. The most effective inhibitor is still, as previously reported, 8-azaguanine. It decreases the anthocyanin formation by 50% at 3 × 10 −7 M. Subinhibitory concentrations of azaguanine, azaadenine and Antrycide, but not in general of the natural bases, clearly promote pigment formation. The inhibitions are reversed in each case by the corresponding purine or pyrimidine, or its riboside or ribotide. There is a specific requirement for a purine, not satisfiable by pyrimidines, and conversely a requirement for a pyrimidine, not wholly satisfied by purines. There is indication of a requirement for both uracil and cytosine. Experiments using simultaneously two types of inhibitors, namely, analogs of both purines and pyrimidines, confirm that both purine and pyrimidine cooperate in the synthesis of anthocyanin. It has been demonstrated that the reversing compounds function within the plant, rather than by preventing the absorption of the antagonist. It is concluded that an unstable nucleic acid, which must be continually resynthesized, is the catalyst or limiting factor in the synthesis of anthocyanin in Spirodela.

Back-mutation of a Neurospora crassa mutant by a nucleic acid complex from the wild strain

Back-mutation of a Neurospora crassa mutant by a nucleic acid complex from the wild strain

Estimation of metabolic reaction rates from a mathematical analysis of tracer experiments

The use of equations for the study of reaction rates of several metabolic schemes and the necessary conditions are discussed. In the case of one or two immediate precursors it is shown that under certain conditions the fractional rates can be derived from the slope and intercept of a line going through points that can be determined from the graphical integration of specific activity-time curves. A metabolic scheme based on the template hypothesis was postulated as a possible mechanism of polymer formation in cells, and a simple specific activity precursor-product relationship is derived. It is shown that “limiting fractional rates” can be evaluated in the steady state in the case that ( a) the immediate precursor is not known and the product is metabolically homogeneous, ( b) the immediate precursor is known but the product is a mixture of metabolically distinct substances, and ( c) the immediate precursor is not known and the product is metabolically heterogeneous. The possibility of having compartmentalization of a complex substance like protein or nucleic acid even in specific tissues is discussed.

Production of mitotic abnormalities by ethylene-diaminetetraacetic acid

Treatment of growing roots of onion with solutions of EDTA between 0.0004 and 0.002 M was found to modify the form and distribution of the chromosomes of the meristematic cells. Similar results were obtained with 0.002 M CaCl 2 or MgCl 2. The effects of EDTA are attributed to alterations in the general metabolism of the cell, rather than to direct breakage of chromosomes through chelation of divalent cations serving as bridges between macromolecular complexes of protein and nucleic acids.

A Self-reproducing Analogue

THE most striking peculiarity of living organisms is their property of self-reproduction. The most elementary forms, virus or phage particles, can reproduce themselves in favourable circumstances only, and this principle applies also to the multiplication of nucleic acid complexes in chromosomes. It is sometimes thought that the self-reproducing properties of nucleic acid depend upon its highly complex structure. Consequently, any mechanical analogue for self-reproduction would involve very intricate mechanisms. This does not seem to be so, and the device described here has the critical reproductive property although it is of the simplest character.

Modification of the release of cellular constituents by irradiated Escherichia coli

A release of cellular constituents occurred when x-irradiated (60,000 r) suspensions of Escherichia coli B/r were incubated in a phosphate buffer-glucose solution at 37 °C. ATP accounted for less than 10% of the total 260-mμ absorbing material released by such cells. The other constituents (showing maximum absorption at 260 mμ) lost from the exposed cells were nucleic acid fragments varying in size from free bases to more complex nucleic acid fragments. No release of peptides above that found with unirradiated cells under similar conditions was observed. The release of nucleic acid fragments was inhibited under each of the following conditions: ( a) the absence of phosphate in the suspending fluid; ( b) the absence of an exogenous metabolite; ( c) the presence of arsenate; ( d) a low temperature of incubation, i.e., in an ice bath. Ultraviolet-light-exposed cells, suspended in a glucose-phosphate solution at 37 °C., released 260-mμ absorbing material, and this release was dependent on the same conditions as those found for the x-ray exposed cells.