Studies on biochemical variants of BHK 21/C13 cells

Clements, G. B (1972) Studies on biochemical variants of BHK 21/C13 cells. PhD thesis, University of Glasgow.

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Abstract

Polyoma transformed BHK 21/C13 cells and some of their variants wore used during these investigations. The variants had previously been derived from BHK 21/C13 cells by selection for resistance to several purine and pyrimidine analogues in tissue culture. The behaviour of these cells was compered in several ways. The levels of resistance of the cells to some purine and pyrimidine analogues were determined. The variants when exposed to an analogue to which they were resistant continued to grow at more than one hundred times the concentration at which the majority of wild type cells were killed. The variants generally survived no better than the wild type in the presence of those purine and pyrimidine analogues for which resistance had not been selected. The growth curves of the wild typo and the variants under non-restrictive conditions were identical. The incorporation of radioactively labelled purines and pyrimidines from the growth medium was determined using both scintillation counting and autoradiography. Mid type cells incorporated each of the nucleic acid precursors used in large amounts. In most cases the variants were no longer capable of incorporating significant quantities of radioactive bases or nucleosides to whose analogues they were resistant. The phenomenon of metabolic co-operation is well established. It occurs between cells in contact at light microscopic level and causes an alteration in the phenotype of a co-operating variant cell to that of the cell with which it is in contact. Metabolic co-operation requires a donor-recipient relationship between at least two cells, with the cell exhibiting the enzyme activity being the donor. The technique of double label autoradiography was used to distinguish between two types of cell in a mixture after one bad been pre-labelled with 14.0 thymidine. Metabolic co-operation could be studied at the level of the individual cell. The effects of metabolic co-operation were investigated using cells growing under selective conditions. Under conditions in which the cells were autotrophic for a purine source metabolic co-operation influenced the selection. Variants were capable of growing when present as a mixture under conditions in which they would not be able to survive alone. This effect of metabolic co-operation has been termed "the kiss of life". Metabolic co-operation has implications both for the selection of variant cells in culture and also for the diagnosis and possible treatment of inborn errors of metabolism. The biochemical basis of metabolic co-operation was investigated. The phenotypic alteration was found to have a short half life which 10 compatible with there being an exchange of labelled material, possibly nucleotides, between cells. Metabolic co-operation may be suitable as an in vitro model of differentiating systems based on the setting up of concentration gradients of low molecular weight diffusible substances between cells. The reactivation of chicken erythrocyte nueicli was followed after fusion with the DM 21/013/P7Y cell variants. Sendai virus was grown up in embryonated eggs, inactivated within B-propriolactone and used to fuse the cells in suspension. The reactivation process followed the pattern described in other cells, the reactivating nucleus undergoing enlargement, synthesising DNA, MIA, forming a nucleolus and finally directing the synthesis of chick specified proteins. The process of reactivation progressed more rapidly within the hamster cells than the mouse A9 cells used by other workers. A variant selected for resistance to four purine analogues offered the possibility of following the reactivation of four enzyme activities simultaneously within a single type of cell. The time course of the appearance of all four was the came and correlated well with the appearance of a functional nucleolus within the reactivated erythrocyte nucleus.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J H Subak-Sharpe
Keywords: Biochemistry
Date of Award: 1972
Depositing User: Enlighten Team
Unique ID: glathesis:1972-73255
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: http://theses.gla.ac.uk/id/eprint/73255

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