| Description |
DNA molecules encoding two proteins, HipHop and MSK81, were cloned into bacteria so as to make large amounts of these proteins that bind to telomeres of Drosophila (fruit fly) chromosomes. The goal was to determine whether known DNA binding motifs are present in HipHop and MSK81. Understanding how these proteins interact with telomeres is relevant due to the important role telomeres play in maintaining genome stability. Drosophila telomeres differ from most other eukaryotic telomeres because the telomere DNA in fruit flies is made by retrotransposition and not by telomerase. This retrotransposition-based method of making telomere DNA is especially interesting because certain types of cancer cells employ similar methods to elongate their telomeres without the use of telomerase. We hypothesized that the proteins HipHop and MSK81 bind DNA at telomeres and tested this in a reductionist approach involving purification of the individual proteins. If HipHop and MSK81 bind telomere DNA in fruit flies, then the purified proteins should also bind to molecules that mimic fruit fly telomeres. Cloning the DNA encoding HipHop and MSK81 involved PCR amplification and insertion into a plasmid DNA molecule capable of being incorporated into bacterial cells. This plasmid DNA was placed into bacterial cells and protein expression was tested under different induction times and temperatures. Polyacrylamide gel electrophoresis with Coommassie staining was employed to confirm protein expression. Analysis via size exclusion chromatography showed the proteins to exist in several differently sized molecular complexes. pectrophotometric absorbance ratios of A260/A280 measured higher than expected suggesting, in the absence of fruit fly telomere DNA, the proteins bind non-specifically to nucleic acids (RNA or DNA) present In bacteria. These puzzling results lead to a model that posits DNA or RNA molecules present in bacteria serve as a landing platform to complex with MSK81 and HipHop. |
