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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027754-08
Application #
3275008
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1980-04-01
Project End
1988-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
8
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Salk Institute for Biological Studies
Department
Type
DUNS #
005436803
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Nonet, G H; Wahl, G M (1993) Introduction of YACs containing a putative mammalian replication origin into mammalian cells can generate structures that replicate autonomously. Somat Cell Mol Genet 19:171-92
Carroll, S M; DeRose, M L; Kolman, J L et al. (1993) Localization of a bidirectional DNA replication origin in the native locus and in episomally amplified murine adenosine deaminase loci. Mol Cell Biol 13:2971-81
Kimmel, M; Axelrod, D E; Wahl, G M (1992) A branching process model of gene amplification following chromosome breakage. Mutat Res 276:225-39
Von Hoff, D D; McGill, J R; Forseth, B J et al. (1992) Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity. Proc Natl Acad Sci U S A 89:8165-9
Yin, Y; Tainsky, M A; Bischoff, F Z et al. (1992) Wild-type p53 restores cell cycle control and inhibits gene amplification in cells with mutant p53 alleles. Cell 70:937-48
Carroll, S M; Trotter, J; Wahl, G M (1991) Replication timing control can be maintained in extrachromosomally amplified genes. Mol Cell Biol 11:4779-85
Ruiz, J C; Wahl, G M (1990) Chromosomal destabilization during gene amplification. Mol Cell Biol 10:3056-66
Wahl, G M (1989) The importance of circular DNA in mammalian gene amplification. Cancer Res 49:1333-40
Ruiz, J C; Choi, K H; von Hoff, D D et al. (1989) Autonomously replicating episomes contain mdr1 genes in a multidrug-resistant human cell line. Mol Cell Biol 9:109-15
Ruiz, J C; Wahl, G M (1988) Formation of an inverted duplication can be an initial step in gene amplification. Mol Cell Biol 8:4302-13

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