Morgan's legacy: fruit flies and the functional annotation of conserved genes

Abstract

In 1915, "The Mechanism of Mendelian Heredity" was published by four prominent Drosophila geneticists. They discovered that genes form linkage groups on chromosomes inherited in a Mendelian fashion and laid the genetic foundation that promoted Drosophila as a model organism. Flies continue to offer great opportunities, including studies in the field of functional genomics.

Figure 1. Functional Annotation of Conserved Genes using Drosophila

Rapid functional annotation of conserved genes is possible in Drosophila by combining a number of technologies and resources. First, the potential fly ortholog of a human gene of interest is identified. An insertion of an artificial exon that functions as a gene trap and allows expression of GAL4 (Trojan exon cassette (Diao et al., 2015)) can be introduced in an intron between two coding exons via Recombination Mediated Cassette Exchange (RMCE) of available MiMIC (Minos Mediated Integration Cassette) insertions (Venken et al., 2011). Alternatively, this can be achieved via Homology Directed Repair (HDR) using CRISPR. This Trojan exon consist of splice acceptor (SA) followed by a ribosomal skipping peptide (2A), the GAL4 gene, and a polyadenylation (polyA) sequence, allowing the expression of GAL4 in the pattern of the gene of interest in loss-of-function (LOF) mutants. By crossing these lines with flies that carry a transgene of the human cDNA under the control of UAS (DNA sequence recognized by GAL4), it can be determined if a human cDNA is able to rescue the fly mutant phenotype. If rescue is achieved with the wild-type (reference sequence) protein, one can further assess the function of variants found in human patients. UAS-human cDNA lines can also be used to assess dominant phenotypes (antimorphic, hypermorphic, or neomorphic) by overexpressing the human gene in a wild-type fly. MiMIC or Trojan gene-traps can be converted into protein-traps via RMCE, allowing intronic tagging of the gene of interest. GFP-tagged genes/proteins can be further knocked down using strategies to degrade the transcript (iGFPi) or protein (deGradFP) in a conditional and tissue specific manner (Nagarkar-Jaiswal et al., 2015), providing stage and tissue specific gene function information.