Studies on the transcription of mammalian DNA

Slimming, Thomas Kay (1970) Studies on the transcription of mammalian DNA. PhD thesis, University of Glasgow.

Abstract

The purpose of these studies was to establish the validity of a system for investigation of the manner in which transcription from mammalian DNA is regulated. Mammalian chromatin was isolated; its chemical composition was determined in terms of relative quantities of DNA, histone, acidic chromosomal protein and chromosomal RNA. A procedure employing binding of the dye bromsukphalein to protein was developed for estimation of protein in chromatin. Correlation between the relative quantities of DNa and histone and, to a lesser extent, between those of acidic chromosomal protein and chromosomal RNA, was noted. RNA transcribed from mammalian DNA was also studied. The best available procedure for characterisation of such RNA was considered to be DNA-RNA molecular hybrudisation. RNA was synthesized in vitro using purified bacterial DNA-dependent RNA polymerase. Procedures of purification from Micrococcus luteus (lysodeikticus) were compared with a view to obtaining both maximal yields and a product not contaminated by ribonuclease acitivity. Evidence that the enzyme catalysed the synthesis of RNA complementary to DNA template, that its acitivity was dependant on the presence of the latter and that DNS-RNA hybrid might be formed during RNA synthesis was obtained. Synthetic RNA was isolated by removal of all other componentsof RNA-synthesizing mixtures. Particular care was required to remove acid-soluble ribonucleotides. Otherwise, the fraction of synthetic RNA hybridisable to DNA appeared to be very small. Procedures for isolation of native DNA from mammalian cells were established, as were procedures for denaturation of DNA and for separation of denatured Landschutz ascites tumour cell DNA into "fast", "intermediate" and "slow" fractions by virtue of their relative rates of renaturation. DNA which had renatured was separated from that which had not by passage through hydroxyapatite. Under certain controlled conditions, the former, but not the latter, was retained by hydroxyapatite. Conditions for optimal binding of denaturated DNA to nitrocellulose and retention of it by nitrocellulose were established. DNA-nitrocellulose binding proved to be dependent on temperature, ionic strength and concentration of solvents which weaken hydrogen bonds, suggesting it is at least partly due to such bonding. Procedures and conditions for carrying out DNA-RNA hybridization between RNA in solution and DNA immobilised on nitrocellulose were established. RNA saturation curves and double reciprocal plots derived from them showed that RNA synthesized in vitro from native calf thymus DNA was hybridisable to approximately 20% of denatured calf thymus DNA under the conditions employed. This figure was the same after three different periods of incubation of an RNA-synthesizing mixture, but was greatly reduced when the materials used in such experiments were contaminated by ribonuclease activity. It was also markedly reduced on incubation of hybridization mixtures at room temperature in the presence of 30% v/v formamideinstead of at 67

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: J N Davidson
• Keywords: Biochemistry
• Date of Award: 1970
• Depositing User: Enlighten Team
• Unique ID: glathesis:1970-73915
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73915