Ribosomal Gene Repeats and Site-Specific Retrotransposons

University Of Rochester, Rochester NY

Investigators

Abstract

Transposable elements have played a major role in determining the size, structure and expression of eukaryotic genomes. R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. The specificity of these elements has enabled them to serve as a convenient model system, such that R1 and R2 are now among the best characterized transposable elements. Taking advantage of the diverse array of 5' truncations that frequently result from non-LTR retrotransposition, preliminary experiments have shown that individual R1 and R2 insertions and deletions can be scored among members of the same population as well as among 19 Harwich mutation collection lines of Drosophila melanogaster. Therefore new copies are continually being inserted into and old elements eliminated from the rDNA loci. In this project, experiments are described to address a) the recombinational forces that bring about the concerted evolution of this locus, b) how R1 and R2 elements are affected by these recombinations, and c) how often and when R1 and R2 retrotranspose. Attempts to answer these questions will focus on the following specific aims. The rDNA loci from one Harwich line will be cloned as a series of overlapping bacterial artificial chromosomes (BACs). While the cloning of rDNA loci are avoided in genome projects, it should be achievable in this project because nearly 80 uniquely marked R1 and R2 insertions have been identified to serve as reference points for the assembly of overlapping clones. Based on this physical map, as well as the 18 examples of how that locus has changed over a period of 350 generations, it can be determined where R1 and R2 insert into the loci and whether they are uniformly removed. Second, by scoring changes in the variable intergenic spacer region between the rRNA genes, a detailed view can be obtained of the recombinational processes responsible for the concerted evolution of the rDNA locus. Third, a study will be conducted of the differential replication of rDNA units in larval tissues. This latter experiment is of interest because Drosophila larvae grow normally even when large fractions of their rDNA units are inserted by under-replicating in polyploid tissues those units that have R1 and R2 insertions. Finally, to determine the tempo and developmental timing of retrotransposition, R1 and R2 activity will be monitored from one generation to the next. R1 retrotransposition is sufficiently high in the Harwich lines that new events can be monitored per generation. A search will be made for a lab strain that will allow similar studies with the R2 elements. These studies should provide an unprecedented understanding of the life cycle of two retrotransposable elements. Because the target site for R1 and R2, the rDNA locus and the nucleolus that forms from it, plays a key role in all cellular metabolism, these studies will also provide insights into the evolution, replication and function of this critical cellular component.