The broad, long-term objectives of this project are to study the structure, organization and replication mechanism of submicroscopic extrachromosomal elements (termed amplisomes) containing amplified dihydrofolate reductase (DHFR) genes in a human methotrexate (MTX) resistant cell line. These amplisomes are unique since they exist as both linear and circular forms. The small size (about 650 kilobase pairs) of these amplisomes provides us with an excellent system to study in detail the structure and origin of replication of extrachromosomal elements.
The specific aims of this project are 1) to clone the amplified DNA sequences present on the amplisomes, 2) to determine the organization of DNA sequences on the amplisomes, 3) to determine the expression of amplisomal DNA sequences other than the DHFR gene. Transcribed DNA sequences will be isolated and sequenced, and 4) to determine the replication mechanism of the amplisomes. We plan to achieve these goals by using the following methods: 1) cosmid and yeast artificial chromosome (YAC) vectors and polymerase chain reaction for the cloning of amplisomes DNAs, 2) restriction enzyme mapping of the cosmid and YAC clones and pulsed field gel electrophoresis to determine the structure and organization of the amplisomes, 3) Northern blot hybridizations and RNA protection assay to determine transcribed DNA sequences on the amplisomes, and 4) pulsed labelling, nuclease digestion and 2-D gel experiments to determine the replication mechanism. Analysis of these amplisomes might provide a better understanding on the general process of gene amplification. In addition, information obtained from studies of these structures will provide major advances in the understanding of the process of cancer generation, tumor progression or both, and of how drug-resistance occurs in tumors.
| Wang, M; Chen, X N; Shouse, S et al. (1994) Construction and characterization of a human chromosome 2-specific BAC library. Genomics 24:527-34 |
| Esnault, C; Lee, H; Lai, E (1994) Structure and organization of a stable extrachromosomal element in human cells. Gene 144:205-11 |
