DRUG RESISTANCE AND GENE AMPLICATION IN EUKARYOTES

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Studies in the past several years have shown that enzyme overproduction resulting from gene amplification is a common mechanism of resistance of eukaryotic cells to a wide range of antiproliferative agents or selective conditions. Since gene amplification occurs spontaneously at a high frequency, it can be a significant problem in the development of resistance of tumors to antineoplastic agents, and possibly lead to the overproduction of proteins which could cause neoplasia. Equally important, the study of how genes amplify enables one to delve into the mechanism of genome remodeling in higher eukaryotes. The specific aim of this proposal is to continue to examine the mechanism of amplification of the CAD gene in eukaryotic tissue culture cells. This gene encodes the multifunctional CAD protein which catalyzes the first three steps of uridine biosynthesis. Cells with amplified CAD genes have been obtained after selection with the transition state analog N- (phosphonacetyl) -L-aspartata (PALA). We will extend our previous studies of how the chromosomal location of a gene affects the size, structure and stability of the units in which it is amplified. These studies will be performed by transferring to different genomic locations a functional CAD gene we have cloned, followed by amplification of these genes in their new positions. Molecular, genetic and cytogenetic analyses will then be used to characterize the effects of changing gene position on gene amplification. In addition, we have recently developed the methodology to link other genes to the purified CAD gene using recombination in vivo. This enables us to create novel gene arrangements which we can use to study the early events in CAD gene amplification and to analyze how amplified sequences can be lost. In the context of recombinations we will study some of the factors which may be important for homologous recombination and we will attempt to target genes into specific genomic locations using homologous recombination. We will also pursue our studies on the consequences of CAD gene amplification in its normal chromosomal location. We will expand upon our previous observation that ribosomal sequences are co-amplified with CAD sequences in order to determine whether such sequences mediate CAD gene amplification and we will explore further the mechanism by which altered CAD transcripts are produced from CAD genes in some PALA-resistant cell lines.