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Studies on K12, a mammalian temperature-sensitive cell cycle mutant.

Money, Sarah Louise; (1992) Studies on K12, a mammalian temperature-sensitive cell cycle mutant. Doctoral thesis (Ph.D.), University College London. Green open access


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K12 is a temperature-sensitive Chinese hamster fibroblast cell cycle mutant which arrests at a point in mid-G1 when incubated at the nonpermissive temperature (npt) of 40°C. The effect of growth medium composition on the cell's viability at the npt was studied, using temperature shift experiments on both random and mitotically-synchronized cultures. Reducing the sodium bicarbonate level of the medium was found to significantly increase the survival time at 40°C, by allowing the execution point of the mutation to be passed for one or more extra cycles before eventual arrest at the block point. It would appear likely that bicarbonate, acting via its effects on the nutritional quality or pH of the medium, can, directly or indirectly, alter the activity of the K12 temperature-sensitive molecule. This observation implies that the activity of specific cell cycle events within this cell line, and possibly others, can be greatly influenced by the composition of the growth medium. In addition, the use of 7 mM NaHCO3 MCDB-302 medium allowed for the isolation of revertant K12 cells at a relatively high frequency which were capable of indefinite growth at 40°C, and which may have undergone amplification of the mutant gene. Attempts were also made to clone the K12 defective gene by transfection with various human cDNA and genomic libraries, in order to elucidate the nature of the gene product of interest. However, it seemed likely that the human homologue of the required sequence was unable to function in a Chinese hamster background, or was expressed in an inappropriate manner. An alternative route for the isolation of the gene by in vivo linkage of genomic DNA from the parental cell line, Wg-1A, and the selective marker pSV2neo was subsequently taken, but proved unlikely to result in its successful recovery.

Type: Thesis (Doctoral)
Qualification: Ph.D.
Title: Studies on K12, a mammalian temperature-sensitive cell cycle mutant.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by Proquest
URI: https://discovery.ucl.ac.uk/id/eprint/10108288
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