Aspects of ribonucleic acid biosynthesis in mammalian cells

Barclay, Margaret N. I (1968) Aspects of ribonucleic acid biosynthesis in mammalian cells. PhD thesis, University of Glasgow.

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Abstract

1. Experiments were carried out in vitro to characterise the RNA polymerase found in the nuclei of Landschutz ascites ttraiour cells. 2. The enzyme was found to be dependent on DM as a primer. The level of incorporation of ribonucleoside triphosphates was found to increase when all four ribonucleoside triphosphates were present. This incorporation was stimulated by the presence of up to 1 mumole/assay of Mn2 and to a lesser extent by Mg2. Ammonium sulphate was shown to raise the specific activity of the enzyme, the extent of the rise being peculiar to this salt. A requirement was also shown for 2-mercapto-ethanol. 3. Attempts were made to purify the fraction containing RNA polymerase. These were restricted owing to the aggregate form of the enzyme. The preparation was treated with DNase or micrococcal nuclease to destroy the native DNA present. The treated preparations were then fractionated on DEAE cellulose but subsequent recoveries of RNA polymerase were not reproducible. Treated and untreated polymerase preparations were fractionated by means of sucrose or glycerol gradients but no satisfactory separation was obtained. 4. Solutions of the aggregate preparation were made in various organic solvents. Sodium deoxycholate was found to give increased incorporation of ribonucleoside triphosphates and was therefore used in subsequent experiments. Several fractionation techniques were tested using the detergent treated preparation. Addition of streptomycin sulphate followed by removal of the resulting precipitate was found to reduce the concentration of DNA present while increasing the specific activity of the RNA polymerase. 5. The characteristics of the streptomycin sulphate supernatant were established and compared with those of the crude preparation. The purified enzyme maintains the same characteristics with respect to Mn2 Mg2 and ammonium sulphate, but no longer requires the presence of 2-mercapto-ethanol. RNA as well as DNA can be accepted by the streptomycin preparation as primer, although the addition of DNase to the reaction mixture, in the absence of added polynucleotide, still causes a lower rate of incorporation. 6. Incorporation of ribonucleoside triphosphates by the crude enzyme was found to take place in the presence and absence of ammonium sulphate. This incorporation as already noted, was stimulated, in the presence of ammonium sulphate, by the addition of the other three ribonucleotides. In the absence of ammonium sulphate no such stimulation was obtained, suggesting the formation of homopolymers. There was no incorporation of ribonucleotides by the purified preparation in the absence of ammonium sulphate, but in its presence addition of all four ribonucleotides caused an increase in enzyme activity. 7. In the presence of only one ribonucleoside 5'-triphosphate, the highest rate of incorporation by the crude enzyme was obtained with [3H]UTP, the lowest rate with [3H]GTP. Several experiments were performed using first [3H]UTP and later [

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J N Davidson
Keywords: Biochemistry
Date of Award: 1968
Depositing User: Enlighten Team
Unique ID: glathesis:1968-71991
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 24 May 2019 15:11
Last Modified: 24 May 2019 15:11
URI: http://theses.gla.ac.uk/id/eprint/71991

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