INTERACTIONS OF MITOMYCIN C WITH RNA.
Item
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Title
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INTERACTIONS OF MITOMYCIN C WITH RNA.
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Identifier
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AAI8103966
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identifier
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8103966
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Creator
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WEAVER, JANET LOUISE.
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Contributor
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Maria Tomasz
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Date
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1980
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Binding of reductively activated mitomycin C (MC) to RNA was studied at various MC:RNA reaction ratios, and binding curves were similar to those for DNA-MC complexes, with binding ratios reaching almost 1 mole MC/1 mole ribonucleotide in RNA when reactions were run in .01 M salt with high MC:RNA ratios. Results also paralleled those for DNA by exhibiting higher binding to single-stranded than to double-stranded RNA under identical reaction conditions. The RNA-MC bond was quite stable to heat, repeated gel exclusion chromatography, low pH, and 7 M urea, and it protected the complex from degradation by T(,1) and pancreatic ribonucleases.;Experiments with synthetic homopolymers of AMP, CMP, GMP and UMP, and with poly (U,G), indicated that the binding of MC was guanine-specific. A small amount of binding to polymers which did not contain guanine was observed in reactions run in low salt (0.01 M), but this was almost entirely suppressed when the salt concentration in the reaction mixture was raised to 0.20 M.;Reactions were carried out between MC and poly O('6)-methyl GMP, poly IMP, and poly GMP in which 40% of the guanine residues had been methylated at the N-7 position. Binding of MC was significantly inhibited only in the case of poly IMP, pointing to the 2-amino group of the guanine residues in nucleic acids as the probable major binding site of MC.;Formation of a covalent compound between mononucleotides, or GpC, and MC, to be used as a model for investigating the complex formed between MC and nucleic acids, was attempted by reductive activation of MC in the presence of the nucleotide under the conditions used for complex formation, but this was unsuccessful. RNA-MC complexes were hydrolyzed by 0.3 M KOH, and the digestion products separated by 2-dimensional thin layer chromatography or by paper electrophoresis, in another attempt to isolate a nucleotide-MC adduct, but no such adduct was isolated, and base ratios after hydrolysis and release of MC were identical to the base ratios of hydrolyzed control RNA.;Finally, poly G-MC and poly (U,G)-MC complexes were degraded by T(,1) and pancreatic ribonucleases, and the products analyzed. The MC-containing products isolated were a very small amount of a labile compound with the UV spectrum of a GMP-MC adduct, and a high molecular weight, water-insoluble complex containing UMP and GMP. The isolation of large, stable, enzyme-resistant segments of polynucleotide-MC complex with high binding ratios from the original complex, which had an overall binding ratio of 0.1, suggests that the binding may be cooperative. Covalent binding of MC to guanine may be stabilized by stacking interactions between the MC molecules. This type of noncovalent interaction may also account for the nonspecific binding observed when reactions are run in low salt.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
