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Title: Some structural and biochemical studies of nucleic acids
Author: Ward, Peter Frank Villiers
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1956
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In part I, the distribution of pyrimidine bases in ox spleen deoxyribonucleic acid (DNA) have been investigated by treating the freshly prepared nucleic acid with hydrochloric acid in the presence of ethyl mercaptan. By this means the purine bases ware cleaved and a polymeric mercaptalated deoxyribonucleic acid (MDNA) was obtained in which diethylmercaptal groups replaced the purines. The vicinal hydroxyl groups in acyclic mercaptalated residues rendered MDNA labile to alkaline scission. The controlled degradation of MDNA by alkali yielded a pyrimidine polynucleotide (PPN), mercaptalated monopyrimidine nucleosides and 2-deoxyribosediethylmercaptal phosphates. The examination of these products suggested that in the parent nucleic acid, pyrimidine nucleotides occurred together in tracts. Evidence was found for pyrimidine trinucleotides in which the average ratio thymine/cytosine was 1.4. In addition to adjacent pyrimidine nucleotides and possible adjacent purine nucleotides, evidence also indicated the existence of single pyrimidine nucleotides flanked by purine nucleotides. The fractionation of ox spleen deoxyribonucleic acid into adenine and thymine rich fractions and guanine and cytosine rich fractions was accomplished. The procedure involved the stepwise displacement of protein from the nucleoprotein with heparin, based on the highly insoluble complex formed between nucleohistone and heparin. The displacement is accompanied by the liberation of nucleic acid fractions. The guanine and cytosine rich fractions of the nucleic acid were more easily displaced from nucleoprotein than were the adenine and thymine rich fractions. The ratios (adenine and thymine/guanine and cytosine) varied progressively within the range 0.93 to 1.35 for individual fractions. In part II, the influence of nucleic acids on certain enzyme systems was investigated. The effect of herring roe and ox spleen deoxyribonucleic acids and ox liver ribonucleic acid on the rate of action of two systems and the stereospecificity of one system was examined, but no effect was detected. The systems studied were the hydrolysis of methyl(±)-mandelate using pig liver esterase and the digestion of horse haemoglobin using pepsin. The reported polynucleotidic nature of cysteinylglycinase from pig kidney (Binkley, F. (1952) Exp. Cell Res. Suppl.2 p. 145) was reinvestigated. An active dipeptidase which withstood exhaustive deproteinisation by mixtures of chloroform end amyl alcohol was prepared from pig kidney. This preparation gave negative tests for proteins (biuret, Folin's and Sakaguchi). The enzyme could be freeze dried without loss of activity and had a strong absorption peak at 260 mμ, but in its analytical composition and paper chromatographic behaviour it did not resemble a polynucleotide. The enzyme required activation by manganous ions and effected the hydrolysis of L-cysteinylglycine and L-leucylglycine. Activity was abolished when the enzyme was dialysed against distilled water and when it was heated to 98°C. The enzyme was unaffected by ribonuclease but was slowly inactivated by treatment with crystalline trypsin. Herring roe and ox spleen deoxyribonucleic acids and ox liver ribonucleic acid were tested for enzymic activity towards L-cysteinylglycine and L-leucylglycine but no hydrolysis of the dipeptides was observed. Comparison of the behaviour of the present enzyme with that of known dipeptidases suggests that the two are at least closely similar. Evidence is presented which indicates that the former is unlikely to be of a nucleic acid character.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available