Research
Gene Amplification, DNA Rearrangements and Chromosome Instability in Eukaryotes
The primary focus of this laboratory is on molecular processes that affect genome stability, including chromosome breakages, DNA deletions and gene amplification. We have been using the ciliated protozoan Tetrahymena thermophila as a model eukaryote for this analysis. Tetrahymena cells carry out extensive DNA rearrangements during somatic nuclear differentiation, including chromosome breakage at hundred of specific sites, deletion of thousands of specific DNA segments and amplification of the ribosomal RNA gene. We have identified critical cis-acting sequences that regulate these processes, including a 15 bp sequence for chromosome breakage sites, two sets of sequences for DNA deletion boundaries, and a pair of inverted repeats for large DNA palindrome formation during gene amplification. We have also begun to identify proteins involved in their regulations. We found a chromodomain protein, pdd1p, expressed specifically during nuclear differentiation that is closely associated with deleted DNA based on cytological and biochemical criteria. Reverse genetics studies showed that pdd1p plays an essential role in nuclear differentiation and DNA deletion. We are investigating the possible roles of other chromosomal proteins as well, and have established genetic and cytological approaches to screen for new genes involved in these processes. Most recently, we found that the deletion process can remove a bacterial sequence inserted into the germline genome during nuclear differentiation. Furthermore, we found that injection of double stranded RNA into developing cells triggers deletion of the targeted DNA. It is likely that DNA deletion is evolved from, and shares significant mechanistic details with, RNA-interference, and serves to defend the genome against invading genetic agents. We will determine how Tetrahymena recognizes foreign sequences and uses double stranded RNA to guide DNA deletion.
The mechanism of gene amplification described in Tetrahymena also occurs in other organisms. We found that large DNA palindromes can be produced in budding yeast, just like in Tetrahymena, when a double stranded DNA break is made at a site adjacent to a pair of short inverted repeats. This finding suggests a general mechanism for gene amplification in eukaryotes. We have now successfully tested this idea in Chinese hamster ovary cells, and developed a genome-wide defection method to examine its role in human cancer progression, which is often accompanied by gene amplification.
