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Title: The action of oestradiol-17β on the RNA polymerases in the uterus of the immature rabbit
Author: Borthwick, Neil Matherson
ISNI:       0000 0001 3471 1490
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1974
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1. Several forms of DNA-dependent RNA polymerase have been purified from the uteri of immature rabbits. The isolation procedure involved the extraction of total uterine protein from a whole tissue homogenate using high salt concentrations. The RNA polymerases were partially purified by DEAE-cellulose chromatography and resolved into three species of enzyme which have been designated RNA polymerases A, B and C. These enzymes have been further purified by chromatography on phosphocellulose and by glycerol density gradient sedimentation. 2. The two major species of RNA polymerase, namely A and B, have been extensively characterised. Both enzymes sediment slightly faster than E. coli RNA polymerase in glycerol gradients suggesting a molecular weight in the range of 500,000 - 600,000. RNA polymerase A is more active in low concentrations of salt, although it can utilise both Mg++ and Mn++ efficiently, RNA B is more active in high concentrations of salt with, Mn++ rather than Mg++ present as the divalent cation. RNA polymerase A is insensitive to the action of the toxin amanitin which specifically inhibits RNA polymerase B at similar concentrations. However, RNA polymerase A is more susceptible to thermal treatment than is RNA polymerase B. The template specificities of both enzymes have also been investigated. 3. A third species of enzyme, designated RNA polymerase 0, has been partially purified and characterised. This enzyme may be cytoplasmic in origin or may be 'soluble' with the result that it is leached out readily from the nuclei. RNA polymerase C has some properties similar to those of enzymes A and B and some which are intermediate between the two major enzyme species. 4. Two RNA polymerase activities have been identified in isolated nuclei; one has been equated with RNA polymerase A while the other has been equated with RNA polymerase Bo In vitro incubation of oestradiol with uteri has shown that the stimulation of RNA polymerase activities in isolated nuclei is only slight when compared with the activities measured in nuclei obtained from uteri treated with oestradiol vivo. 6. When measuring the endogenous RNA polymerase activities of isolated nuclei, prior treatment of the rabbits with oestradiol had a profound effect on the transcriptional capacity. Within 30-45 min after hormone treatment, the activity of RMA polymerase B was considerably increased. This activity decreased towards control levels at 1-2h before exhibiting a second increase of activity at about 3h. From 1h after oestradiol treatment, RNA polymerase A activity in the isolated nuclei was also increased and reached a plateau by about 4h. Both activities have been shown to be sensitive to the action of actinomycin D. 7. Treatment of the animals with amanitin prior to oestradiol inhibited the hormone-induced stimulation of RNA polymerase A as well as totally inhibiting RNA polymerase B. However, when amanitin was administered after the early enhancement of RNA polymerase B, the ocstradiol-induced stimulation of RNA polymerase A was retained. Treatment of the animals with cycloheximide prior to oestradiol did not affect the stimulation of RNA polymerase B but prevented the oestradiol-induced enhancement of RNA polymerase A. However, when cycloheximide treatment was delayed until after the early stimulation of RNA polymerase B, the activity of RNA polymerase A was stimulated. This suggested that stimulation of RNA polymerase A activity was dependent on protein synthesis subsequent to the hormone-induced stimulation of RNA polymerase B. 9. Since some indications were obtained of an effect of cytoplasm from oestradiol-treated rabbit uteri on the MA polymerase activity in nuclei, attempts were made to concentrate any such components. It was found that a fraction of the cytoplasm isolated from uteri treated with oestradiol for 30 rain was capable of stimulating RMA polymerase A activity in nuclei isolated from control animal uteri. 10. The isolated RNA polymerases from immature rabbit uteri do not show any increase in activity in response to oestradiol irrespective of the time of treatment with hormone. No fractions from cytoplasm treated with hormone have been shown to possess any stimulatory activity for either RNA polymerase A or B. It is possible that the observations of increased RNA polymerase activities in isolated nuclei result from changes in the transcriptional machinery rather than being due to alterations in the RNA polymerases per se.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available