Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.449437
Title: Characterization of repititive sequences in the nuclear DNA from baby hamster kidney (BHK-21/C13) cells
Author: Bell, A. J.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1978
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Abstract:
The physical properties of rapidly-renatured nuclear DNA from BHK-21/C13 cells were investigated in this study. Kenaturation products were isolated by rapid and selective adsorption to hydroxyapatite crystals, or, visualized by electron microscopy. "Instantaneously" renaturing BHK DNA sequences (i.e. those renaturing by C₀t lxlO⁻⁴Ms) were isolated by hydroxyapatite fractionation. Electron microscopic examination of the molecules fractionated by this procedure revealed that hydroxyapatite chromatography is not an ideal method for isolating these sequences prior to further physical characterization. In particular, foldback molecules with short duplex regions are not quantitatively adsorbed to hydroxyapatite in the renatured DNA fraction. Furthermore, single-stranded molecules contaminate the renatured DNA fraction eluted from hydroxyapatite. The adsorption of BHK DNA to hydroxyapatite at C₀t lxlO⁻⁴Ms as a function of single-stranded DNA length was studied to determine the interspersion of spontaneously annealing sequences. Adsorption values range from 2 to 28% for samples of average single-stranded length of 0.27 to 6lkb. The adsorption profile contains two linear phases. Taking into account the effects of random shearing of DNA containing spontaneously annealing sequences, it is proposed that two distinctive sets of spontaneously annealing sequences (R1 and R2) are present. There are similar numbers (approximately 5x10³) of R1 and R2 sequences in BHK DNA and they also occupy a similar proportion (approximately 1.9%) of the genome. The possible arrangements of sequences clustered within individual R1 and R2 sequence arrays are discussed. Electron-microscopic examination of BHK DNA annealed to C₀t lxlO⁻⁴Ms reveals the presence of bimolecular, looped-foldback, unlooped-foldback and bubbled-foldback renaturation products. The physical separation of these various renaturation products was not realised. Attempts to isolate highly-reiterated sequences by excision with a variety of restriction endonucleases were unsuccessful. Possible reasons for this outcome are discussed. Preliminary evidence for a thymidine-rich, high-density satellite DNA fraction, that resolves from main-band DNA on density gradient centrifugation of partly renatured BHK DNA is presented.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.449437  DOI: Not available
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