Genetic Analysis of Mechanisms of Gene Silencing in a First-Year Scientific Research Learning Community

Our broad goal is to use Drosophila melanogaster to identify new genes required for a form of gene regulation known as gene silencing. As participants in iFocus, an interdisciplinary first-year orientation camp for undergraduate sciences that introduces students to scientific research, we learned how classical genetics is used to identify novel genes essential for a particular biological process such as gene silencing. Specifically, we learned how to determine which chromosomes are present in a fruit fly by observing that fly’s physical characteristics, or phenotypes. We further learned how mutations in genes are mapped to a particular location on a chromosome using Drosophila. Previous work in our lab indicated that the Regena/NOT2 gene is essential for microRNA-mediated gene silencing. We set out to locate nucleotide change that is responsible for Regena/NOT2 mutant phenotype. To do this, we started a genomics learning community and taught 8 additional first-year Linfield students how to analyze DNA sequence data. Through our collective efforts, we determined that the existing DNA sequence data only covered a portion of the Regena gene, and additional DNA needed to be sequenced. We performed fly crosses, selected relevant progeny, and prepared genomic DNA from these flies to amplify the Regena gene by polymerase chain reaction (PCR). Amplified DNA has been sent for DNA sequencing, and sequence analysis is ongoing

Validation of a Requirement for Regena/NOT-2 in miRNA Mediated Gene Silencing

miRNAs are small non-coding RNAs that silence gene expression. A forward genetic screen led to the discovery of a mutant with defective gene silencing that contains a mutation in the gene Regena/NOT2. We hypothesize that the defect in silencing is a result of the mutation in Regena/NOT2 rather than another mutation inadvertently generated in the genetic screen. To formally test this hypothesis, we must either perform a genetic rescue, by adding a functional copy of the Regena/NOT2 gene into the mutant fly and observing restoration of silencing or recreating the mutant phenotype by generating an independent mutation in Regena/NOT2 and observing the silencing defect as seen in the original mutant flies. We opted to generate an independent mutation in Regena/NOT2 and assay silencing with a GFP-based reporter of silencing. To do this, we generated flies that contained the new mutation in Regena/NOT2, the reporter of silencing, and the ability to compare silencing in adjacent cells with or without the new mutation in Regena/NOT2. We hypothesize that the independent line of flies containing the new mutation in Regena/NOT2 will show the same defect in silencing as our original mutants