GENE AMPLIFICATION AND ELIMINATION IN TETRAHYMENA

这里提出的研究是为了调查改变的各种过程

Studies are proposed here to investigate the various processes which alter the structure and dosage of genes during development. The simple ciliate Tetrahymena is chosen as a model system for these studies. Tetrahymena can be easily cultivated and manipulated in the laboratory for genetic, biochemical and developmental studies. Previous studies from this and other laboratories have shown that tRNA gene amplification and specific DNA elimination occur during the development of the somatic macronucleus of this organism. The amplification process is coupled with the fragmentation of the chromosome at specific sites, the addition of telomeric sequences and the formation of reverse repeats. The elimination process is coupled with the deletion (which involves breakage and rejoining) of DNA at specific sites. In these studies we will try to determine the molecular mechanisms and the developmental regulation of these processes. DNA at sites of chromosomal breakage and DNA deletion will be isolated by cloning and analyzed by hybridization and sequencing. Comparisons of these structures at several different sites should reveal the features important for site specific DNA breakage, telomeric DNA addition, DNA elimination and site specific DNA deletion. DNA from several different strains which share an identical genetic origin (caryonidal clones) will be examined to determine whether the processes are precise at the sequence level. We will analyze these processes directly in synchronously developing macronuclei to determine the sequence events and possible intermediates involved. Transcriptional activities of DNA at or near deletion sites will also be examined to determine the interrelationships between DNA deletion and transcription. To fully understand the developmental regulation of these events we will study the chromatin structure of the DNAs at breakage and deletion junctions in nuclei at various stages of development. Finally, we will explore the possibility of establishing an in vitro system for analyzing these processes using cloned DNAs and cellular extracts. It is hoped that these studies could further elucidate the nature of gene structure and cell differentiation, and help us understand how normal development is regulated and how abnormal cell growth might occur.