Maurer, Barry James (1996) Dihydrofolate reductase gene amplification in human cell lines VA2-B and hela BU25. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechETD:etd-10232007-082738
Mammalian gene amplification has been studied using as a model system the amplification of the endogenous Dihydrofolate Reductase (DHFR) gene in the human cell lines VA[subscript 2]-B and Hela BU25. Cell lines were stepwise-selected to high DHFR gene copy number using methotrexate, a DHFR enzyme inhibitor. Multiple cell lines and their derivatives were analyzed throughout the amplification process by karyotype, DHFR gene copy number, DHFR protein level, cell growth rate and, in some instances, by pulsed-field gel electrophoresis (PFGE).
Chromosomal fragmentation and rearrangements were observed in the initial stages of amplification in all cell lines examined. In subsequent stages, the formation of double minute chromosomes (DM) and Homogeneously and Abnormally Staining Regions (HSR/ASR) were observed in all cell lines except one. The order of appearance of DMs and HSRs varied among cell lines with HSRs becoming predominant at later stages under stable selection. In contrast to findings reported in other systems, DMs and HSRs were found to coexist within the same cell for extended periods of time in some cell lines.
A novel class of mammalian extrachromosomal, submicroscopic DNA elements was discovered in some cell lines through the use of PFGE. The amplified DHFR genes in one cell line, Hela 10B3, were found to reside solely in this class of DNA element ("amplisome"). Amplisomes may represent the initial, or an obligatory, molecular intermediate in the mammalian gene amplification process, at least in some cases.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||19 June 1995|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Imported from ETD-db|
|Deposited On:||23 Oct 2007|
|Last Modified:||26 Dec 2012 03:06|
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