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Title: Polyribonuceleotide synthesis by cytoplasmic enzymes
Author: Wykes, J. R.
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1965
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The possibility that the cell cytoplasm contains enzymes catalysing RNA biosynthesis was investigated in Landschutz ascites tumour cells. Enzyme fractions were obtained by differential centrifugation of disrupted cell homogenates. The incorporation of (alpha32p) UTP into polyribonucleotides catalysed by such fractions was investigated in reaction mixtures containing primer RNA, ATP, GTP and CTP to stimulate conditions for synthesis of RNA. The microsomal fraction was most active in incorporating (alpha32p) UTP into polyribonucleotides. Experiments in which the microsomal fraction was analysed by sucrose density gradient centrifugation confirmed the particulate nature of the enzyme. An acetone dried powder of the microsomal fraction was prepared and its properties further investigated. The incorporation of (alpha32p) UTP was stimulated by an ATP regenerating system but not by ATP, GTP and CTP in the presence of the latter. The incorporation of (alpha32p) UTP showed a requirement for RNA as a primer and an absolute requirement for Mg2+ ions while Mn2+ ions, spermine and putrescene inhibited the reaction. Alkaline hydrolysis of the reaction products after(alpha32p)UTP incorporation showed mainly UMP residues to be labelled irrespective of whether ATP, GTP and CTP were present in the reaction mixture indicating synthesis of polu U chains. 3H ATP, GTP and CTP were found to be incorporated into polyribonucleotides under the same conditions as (alpha32p) UTP, though individual nucleotides were 3H ATP at increasing substrate concentrations of GTP, CTP and UTP added to the reaction mixtures over the range of concentrations tested. These experiments indicate homopolyribonucleotide synthesis rather than the synthesis of RNA. Extraction of RNA from the reaction mixtures after 3H ribonucleoside 5'-triphosphate incorporation and analysis by sucrose density gradient centrifugation showed no labelling of the ribosomal RNA. The incorporated radioactivity appeared between the 4S region and the meniscus of the sucrose gradient. Determination of the chain length of the homopolyribonucleotide product showed only short sequences to be synthesised which is in agreement with the position of radioactive RNA on sucrose density gradients. Considerable phosphodiesterase activity was found to be associated with the microsomal fraction which could account for the lack of labelling of ribosomal RNA and the short chain lengths of homopolyribonucleotide synthesised. An attempt made to demonstrate homopolyribonucleotide occurance in vivo proved negative though the possibility could not be ruled out that short chain homopolyribobucleotides were present. The lack of ability of cytoplasmic fractions to catalyse in vivo the net synthesis if RNA is in agreement with in vivo experiments on the kinetics of nucleotide uptake into cells in the presence or absence of actinomycin D which indicate that synthesis of RNA occurs primarily in the nucleus on a DNA template. However limited homopolyribonucleotide synthesis does appear to be catalysed by the microsomal fraction, the possible significance of which is discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available