Telomeres are specialized structures at the ends of eukaryotic chromosomes essential for the stable maintenance and complete replication of chromosome ends. Broken chromosomes lacking telomeres are quite unstable, and a broken chromatid end may fuse after replication to its sister chromatid broken end. However, under some conditions, broken chromosomes can be "healed" and recover as terminal deficiencies. We now have the opportunity to study the dynamic behavior of terminal chromosome deficiencies and the mechanisms of healing events at the molecular level in Droposhila, because terminal chromosome deficiencies at the X-chromosome have been recovered at high frequency in females carrying a homozygous mutation of the mu-2 locus. We have observed that these terminal deficiencies recede by losing DNA sequences at an apparently constant rate of 75 bp per fly generation, but that some terminal deficiencies acquire new DNA sequences at their distal ends. This novel DNA addition apparently results in a phenotypic "stabilization" because the stock no longer changes phenotype over time from y2 to the more extreme y1 due to loss of sequences from the first exon of the yellow gene. We have cloned and analyzed twelve different terminal fragments from one of the deficiencies (RT94). None of the DNA fragments contains only typical telomeric sequences. Three of the clones contain newly added DNA sequences at their distal end that are not present at the original break, while the other nine do not. We propose to further investigate the properties of terminal deficiencies at the molecular level. We will study the extent of heterogeneity of terminal DNA fragment lengths, and determine the origin, genomic distribution, and sequence of DNA fragments that occasionally become attached to the broken chromosome ends. Moreover, we will investigate why and when during development DNA sequences are lost from the terminal deficiencies which have apparently not been "capped" by a natural telomere. Since these terminal deficiencies do not behave like broken chromosome ends, yet apparently do not contain DNA sequences that are normally present in natural telomeres, they must be protected by some other components, possibly by proteins. We will therefore search for such proteins that might be associated with the chromosome ends of terminal deficiencies. These studies will provide insights into the molecular mechanisms that are responsible for the behavior of such broken chromosome ends. They will also identify reactions that might occur at natural telomeres and that are causing the well known dynamic behavior of telomeres in many eukaryotes.
