Changes in HTC cells' nuclear proteins associated with glucocorticoid stimulation

Edwards, R. J (1976) Changes in HTC cells' nuclear proteins associated with glucocorticoid stimulation. PhD thesis, University of Glasgow.

Abstract

This thesis describes an investigation of the effects of the synthetic glucocorticoid, dexaraethasone, on the nuclear non-histone chromosomal proteins of the Hepatoma Tissue Culture cell line. This cell line responds to dexamethasone by synthesising a small number of the proteins normally produced in the liver in response to glucocorticoids. Therefore it was hoped that a study of the non-histone chromosomal proteins in such a simplified system would provide some insight into the role of the chromosomal proteins in the hormonal control of transcription. The bulk non-histone chromosomal proteins were isolated by two methods. One method used mild denaturants, so minimising damage to the proteins, while the other used more extreme conditions, including acid extraction, so allowing quick isolation with the minimum opportunity for proteolytic degradation. A preparation of 0.35 M saline soluble proteins and a preparation of tightly bound proteins which remained bound to DNA in urea-guanidine HCl, were separately investigated. The isolated proteins were analysed by one and two dimensional polyacrylamide gel electrophoresis, The two dimensional gel system involved isoelectric focussing in the first dimension and SDS-urea polyacrylamide electrophoresis in the second dimension. The separated proteins were detected by staining and autoradiography or scintillation autoradiography, depending on the isotope used. The three fractions gave a different one dimensional electrophoresis pattern, although some of the bands appeared common to more than one fraction. The number of major bands present in the bulk non-histone chromosomal proteins, the saline soluble proteins and the proteins tightly bound to DNA were 25, 20 and 7 respectively. The glucocorticoids stimulate the induction of tyrosine amino transferase in the Hepatoma Tissue Culture cells. This enzyme was used as an indicator of the cells' responsiveness to the hormone and to obtain a time course of induction to compare with alterations in the nuclear chromosomal proteins. It was found that the level of the enzyme was above the control level by 1 hour and it continued to increase until 8-12 hours, thereafter remaining fully induced. The proteins were labelled with L-[3H]-tryptophan to monitor their turnover and with [32P]-orthophosphate to monitor phosphorylation. Dexamethasone (5 x 10.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J T Knowler
• Keywords: Cellular biology
• Date of Award: 1976
• Depositing User: Enlighten Team
• Unique ID: glathesis:1976-72281
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 24 May 2019 15:12
• Last Modified: 24 May 2019 15:12
• URI: https://theses.gla.ac.uk/id/eprint/72281