Showing papers on "Nucleic acid methods published in 1980"

Isolation and identification of cross-links from formaldehyde-treated nucleic acids.

Abstract: Cross-linked nucleosides have been isolated from formaldehyde-treated ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) by using reverse-phase high-pressure liquid chromatography. Methylene-bridged products containing cytosine, as well as the purines, have been isolated. A combination of ultraviolet and nuclear magnetic resonance (NMR) spectra, pKa values, hydrolysis, and reduction has been used to establish that the cross-links connect the amino groups of the nucleosides involved. For example, the 6-amino functions of two adenosine residues are linked by a methylene bridge to produce a compound which, when dissolved in deuterated dimethyl sulfoxide, displays 11 unsplit resonances in its proton-decoupled 13C NMR spectrum. The procedure reported here is more rapid and less laborious than an earlier one recommended by us for the isolation of cross-linked products from DNA [Dubelman, S., & Shapiro, R. (1977) Nucleic Acids Res. 4, 1815-1827]. This new approach may be of value in the study of other types of cross-linking reactions involving nucleic acids.

The experimental induction of antibodies to nucleic acids.

Abstract: Publisher Summary This chapter discusses the experimental induction of antibodies to nucleic acids Antibodies to nucleic acids have found many uses in the specific measurement of the naturally occurring or modified nucleic acids both in solution and in situ To obtain the required antibodies, it has been necessary to link nucleic acids or their components to carrier proteins or synthetic polypeptides to form immunizing complexes, because injection of purified nucleic acids alone into normal animals does not stimulate significant antibody production Once the antibodies are formed, they react with the nucleic acid in the absence of the carrier Three procedures for preparing such immunogens are described in detail in this chapter For ribonucleosides or ribonucleotides, the most convenient linkage involves periodate oxidation of the furanose ring bearing adjacent hydroxyl groups; this is followed by the condensation of the dialdehyde product of oxidation with lysine amino groups of protein Immunogens prepared by covalent linkage of the nucleic acid components to proteins are described in the chapter The preparation of immunogens, by periodate oxidation of ribonucleosides or nucleotides, is also discussed in this chapter

Antibodies specific for modified nucleosides: an immunochemical approach for the isolation and characterization of nucleic acids

Abstract: Publisher Summary The chapter discusses various antibodies specific for modified nucleosides that is an immunochemical approach for the isolation and characterization of nucleic acids. The rationale of an immunochemical approach for the isolation and characterization of nucleic acids is based on two important observations. First, most nucleic acids contain a limited number of modified nucleosides that are uniquely distributed throughout their structures. Second, antibodies that possess high degrees of affinity and specificity toward these modified nucleosides can be prepared, with minimal or no significant cross-reactivity, with the bulk of unmodified constituents present in nucleic acids. From the information presented in these sections, the feasibility of an immunochemical approach is demonstrated, by using specific anti-nucleoside antibodies: (a) to isolate hapten-containing oligonucleotides and ribonucleic acids (RNAs), (b) to assess the functional significance of the hapten component, and (c) to map the position of the hapten, within selected nucleic acid structures. The employment of the anti-nucleoside antibody is by no means restricted to the naturally occurring modified constituents of nucleic acids. Equally or perhaps more significant are their application in the detection, quantitation, isolation, and location of aberrant nucleoside adducts that appear in the nucleic acids of organisms exposed to various forms of radiation.

The ultraviolet photochemistry of nucleic acids and their components

Abstract: This review highlights recent developments in the photochemistry of nucleic acids and is based on material drawn from the scientific literature published between January, 1977, and December, 1979. The coverage is, of necessity, selective rather than comprehensive and emphasis has been placed on studies relating to the formation and characterization of photoproducts as opposed to those dealing with mechanistic aspects of photochemistry or the biological effects of UV light.

Organic chemical approach to photo-crosslinks of nucleic acids to proteins

Abstract: Various proteins are known to be linked covalently to nucleic acids under UV irradiation. The phOto-cross-links occurs from the interacting states between nucleic acids and proteins. The present approach to this problem involves the characterization and identification of photoproducts obtained from model systems using simple pyrimidine bases and amino acids as well as some mechanistic aspects. 5-Bromouracil or its derivatives undergoes selective photocoupling to tryptophan, indoles and some electron-rich aromatics under varthus irradiation conditions, where four modes of reactions were observed: involvement of (1) triplet bromouracil, (2) a double electron transfer in the presence of an electron carrier, (3) mixed aggregate formation in aqueous frozen solution, and (4) fluorescence quenching of the aromatics by bromouracfl. Other topics of the photochemistry of pyrimidine bases, including synthesis of fluorescent uracils using photo-cross-coupling with 5-chloroand 5-iodouracils, photoreactions between a 4-thiouracil and L—lysine and between thymine and L-tryptophan, and synthesis and behaviors of model compounds for stacking interactions between 5-bromouracil (or thymine) and tryptophan.

Labeling of Nucleic Acids With Psoralens

Abstract: Recently, useful applications of psoralens (furocoumarins) (FIGURE 1 ) as photochemical probes of chromatin structure,’-3 secondary structure in viral DNA and Drosophila ribosomal RNA,4.5 satellite DNA structure: bacterial and viral DNA repair mechanism^,'.^ and viral DNA-RNA hybrid structure’ have been reported. The photoreactivity of coumarins and furocoumarins has been correlated with their cycloaddition to the pyrimidine bases of DNA.’&’’ Musajo14 was the first to report on the photoreaction of psoralens with DNA. Apparently, psoralens interact in the dark with DNA in a manner similar to that of other polycyclic aromatic hydrocarbons (e.g., acridine orange, ethidium bromide, actinomycin). Upon subsequent irradiation with near-uv light, the intercalated psoralen molecule can form covalent bonds with nearby pyrimidine bases of the DNA. Studies by many workers have shown that both the fury1 and pyrone functional groups are involved in their photoreactions with nucleic acids. The most likely reaction involves the formation of interstrand cross-links in the DNA through the photocycloaddition of the 3,4and 4’J-double bonds of the furocoumarin m o l e ~ u l e s . ’ ~ ~ ’ ~ The photoreactivity of furocoumarins can be interpreted in terms of strong localization of the triplet excitation in the region of the C=C bond of the pyrone portion.”.” Recent theoretical and EPR/ODMR calculations also suggest that the triplet excitation is located in the pyrone C=C FIGURE 2 shows the energy level diagram for furocoumaryl compounds.” In all cases, the low-lying ’T,T* and 3 ~ , ~ * states represent the photoactivated species that are capable of reacting with substrates such as DNA bases. In this review, we will describe applications of furocoumarins plus near-uv light to the structural probing of nucleic acids, with emphasis on results from this laboratory. Details of experimental procedures are not included here, as they have already been described elsewhere by the present a ~ t h 0 r s . l ~

Photo‐Cross‐Linking Studies of Nucleic Acid Structure *

Abstract: In contrast to the wide variety of photoaffinity labeling studies of protein targets reviewed in this volume, there have been very few studies of photochemical labeling of nucleic acid targets. This is not because of a lack of interesting targets. Instead, it probably reflects the fact that, until very recently, it was relatively difficult to locate sites of covalent modification on large nucleic acids. As a result, most photoaffinity labeling studies in nucleic acid systems have involved either the direct exploitation of the photochemical reactivity of occasional strange bases’” or the use of strange bases or nucleic acid chain termini for the placement of photoactivatable groups.4.’ The nucleic acid was then used as a photoaffinity reagent to identify components of its binding site in nucleoprotein complexes. Often, these components were proteins, and, although occasional nucleic acid targets were found, there was no systematic attempt to look for them. A number of factors have sparked a renewed interest in nucleic acid photocross-linking and photoaffinity labeling. In one of the best-studied nucleoprotein systems, the ribosome system, it has become increasingly clear that the nucleic acid plays important structural and functional roles6 There are strong indications that particular nucleic acid structural features also play a role in various types of R N A processing in both prokaryotes and eukaryotes. There is a great need to prove the existence of these structures and show how they are recognized by various enzymes. Techniques for handling nucleic acids have advanced significantly in the past few years; these will greatly facilitate nucleic acid photoaffinity labeling. Recombinant DNA techniques allow us to isolate usable amounts of particular DNA sequences. The availability of a myriad of restriction nucleases and new, rapid nucleic acid sequencing techniques have revolutionized our ability to obtain primary structure information. Inevitably. these techniques will also catalyze great improvements in the identification of sites of chemical modification on nucleic acids. The electron microscope has proved to be a remarkably effective tool for low resolution location of the sites of attachment of proteins, cross-linkers, and annealed nucleic acid pieces.’.’ These systems and advances set the stage for the development of productive photoaffinity labeling studies on nucleic acids-the missing ingredient is a suitable

Photochemical cross-linking between proteins and nucleic acids as a probe of nucleoprotein interactions.

Abstract: The tendency of proteins and nucleic acids to cross-link covalently as a result of irradiation by ultraviolet (uv) light can be used as a probe of nucleoprotein structure. This “zero-length’’ cross-linking agent presumably joins together amino acid residues and nucleic acid constituents, thereby “freezing” existing interactions in the native complex. The major advantage of this approach is that photochemical cross-linking can be performed on naturally occurring nucleoprotein complexes under optimal conditions in which maximum binding or maximum stability of the native complex occurs. It does not require decomposition, further introduction of foreign reactive groups, and subsequent reconstitution of the complex’s components. The validity of conclusions derived from such an approach regarding proteinnucleic acid contacts is based on the assumption that the uv-induced covalent cross-links are formed specifically either between interacting regions on the macromolecules or between residues that are in close proximity in the native complex. Compliance with this condition is necessary if the photochemical cross-linking is to freeze existing contact points in the irradiated protein-nucleic acid complex. It is apparent, therefore, that the reliability of the photochemical approach depends on the ability of both purines and pyrimidines to form covalent adducts with a large number of amino acids and, at the same time, requires that specific covalent bonds be formed only between neighboring residues in the native structure. By analogy to photoalkylation reactions both of amino acids’ (FIGURE 1) and of nucleic acid constituents* (FIGURE 2), it could have been anticipated that covalent adducts of amino acids to the heterocyclic base constituents of nucleic acids would be a major product of irradiation of their mixtures. This, however, was not the case, since only trace amounts of such cross-links could be identified (unpublished observations). On the other hand, significant yields (up to 60%) of cross-linked products were obtained whenever a nucleoprotein complex with an appreciable binding constant (> lo5 M-’) was irradiated.’ These observations provided the initial clue of the specific involvement of neighboring residues in the photochemical cross-linking. On the basis of the model reactions ( FIGURFS 1 and 2), it can be assumed that, a t least in principle, all amino acid residues are capable of forming covalent adducts with the heterocyclic base constituents of the nucleic acids. Indeed, it has been reported that a large variety of amino acids are capable of forming covalent addition products with the pyrimidine constituents of DNA4; the involvement of purines in photochemical cross-linking to

Synthesis and interactions of poly-L-lysines containing nucleic acid bases.

Abstract: Poly-L-lysine derivatives containing nucleic acid bases were synthesized. Conformation of the obtained polymers was studied by CD and ORD. Formation of the polymer complex was studied and was related to the conformation of the polymers.

Nucleic Acid Sequence Determination

Abstract: It is often emphasised how closely the structure and function of biological materials are related to each other. This is certainly true for all biological macromolecules, but to some extent it is also true for any chemical substance: whether it takes part in a simple chemical reaction or in a complex biological process, the function of the molecule is determined by its structure. In the case of nucleic acids, however, the relationship of structure and function is unique: they are two aspects of the same basic property of the molecule; structure and function are, in fact, identical.

Recognition of nucleic acids and chemically-damaged DNA by peptides and protein.

Abstract: Recognition of nucleic acid base sequences or nucleic acid structures is a fundamental process at every step of genetic expression Very specific base sequences are recognized by operators, RNA polymerases, restriction endonucleases… Specific recognition of single stranded nucleic acids is achieved for example by helix destabilizing proteins which are involved in DNA replication, repair, and recombination The secondary and tertiary structure of ribonucleic acids certainly plays a key role in the recognition of tRNA’s by aminoacyl—tRNA synthetases, of ribosomal RNA’s by ribosomal proteins…

Interaction of the C1q component of complement and collagen with nucleic acids and polyanions

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