Programmed DNA rearrangements play major roles in cell differentiation in a wide variety of organisms including human, and are relevant to understanding human diseases such as cancer. One unusual class of DNA rearrangements occur globally in the genome to alter overall structure of chromosomes. Although known for more than a century, their molecular mechanisms and functional significances are poorly understood. This proposal will examine this class of DNA rearrangements in the model eukaryote Tetrahymena thermophila. Past studies by this and other laboratories have revealed two major processes: site specific chromosome breakage and DNA deletion. Chromosome breakage occurs at approximately 200 specific sites marked by a 15 bp sequence signal. It cleaves DNA, eliminate approximately 55 bp of DNA around the site, and adds new telomeric sequences to the free ends. These minichromosomes are maintained indefinitely in the somatic nucleus, which divides by amitosis during vegetative growth. This study will use a specially constructed transgenic strains to search for genetic mutants and identify genes that control this process. In addition, it will also characterize the freshly broken DNA ends generated by the cleavage reaction. This information will be used to help establish a cell-free system for studying the DNA cleavage and telomere addition reaction to further understand its biochemical details. DNA deletion is a complex process that occurs at several thousand genomic locations to excise DNA fragments of diverse sizes and sequences, which together comprise about 15 percent of the genome. Two such excised deletion elements have been studied in detail. Each was found to be controlled by a pair of flanking regulatory sequences and a set of internal promoting sequences. The internal promoting sequences have several interesting properties that suggest an un-conventional mode of action. Although not coding for proteins, they are transcribed during specific stages of nuclear differentiation and produce RNA of heterogeneous sizes. This proposal will investigate the molecular details of these transcripts, including their specificity and subcellualr locations, in hope to understand their roles in DNA rearrangements. The internal promoting sequences will inhibit DNA deletion if they are misplaced in the old somatic nucleus. This inhibition perpetuates itself in the following generations and becomes an unusual epigenetic trait. This proposal will study the sequence components responsible for this inhibition, their effects on transcription, and the potential role of RNA transcripts in this inhibition. Regulation of DNA deletion may involves changes in chromatin structure, which will be directly examined. Special transgenic strains will also be constructed to identify genes that affect DNA deletion. Finally, a new approach for cloning genes based on the location of their protein products has been developed, and will be used to isolate genes participating in nuclear differentiation including those involved in DNA deletion and chromosome breakage. By combining these approaches, the study hope to reveal the underlying molecular mechanisms of these processes, and uncover their relationships to other cellular processes such as chromosome condensation and mitosis.
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