Electronic Thesis and Dissertation Repository

Thesis Format

Monograph

Degree

Master of Science

Program

Biology

Supervisor

Moehring, Amanda J.

Abstract

The fruitless gene is highly conserved across many insect species, and its role in sex determination and sexual behaviour in Drosophila melanogaster males has been well characterized. The fruitless gene is alternatively spliced to produce at least 15 transcripts, but little is known about the alternative transcripts not involved in sexual traits. Transcripts beginning with the P3 and P4 first exons have previously been shown to be more heavily expressed during developmental stages of the D. melanogaster life cycle and have been implicated in developmental and fitness traits. Yet, these transcripts have never been assessed for their involvement in developmental traits. To study the role of these transcripts individually, the expression of fruitless P3 and P4 transcripts were reduced through RNA interference (RNAi) in combination with the Gal4/UAS binary system. Subsequent verification through quantitative PCR (qPCR) found that P3 transcript was successfully knocked down but P4 transcript was not. Assays scoring the influence of fruitless P3 on development found that knockdown of P3 transcript expression causes lethality at the pupal stage of development, where larvae form pupae but do not progress past this stage of development to form adult flies. This demonstrates that fruitless P3 plays a critical role in development, and that knockdown of P3 alone is sufficient to induce lethality.

Summary for Lay Audience

What makes a genome more complex than another has been debated extensively. Interestingly, eukaryotes have processes that lead to increased genome complexity without heavily altering the size of their genome or the number of protein coding genes in their genome. One of these processes is called alternative splicing, where parts of a gene can be arranged and come together in different ways to make similar, but differing proteins. A gene that produces alternatively spliced transcripts and has high complexity is the fruitless gene in fruit flies. While we know a lot about the fruitless gene’s role in the development of sexual characteristics and male behaviour in fruit fly species, there are components of the gene that haven’t been explored. The fruitless gene produces multiple different transcripts, RNA intermediates of transcription/translation, that aren't related to these traits. Two of these lesser-known transcripts are the fruitless P3 and P4 transcripts, which are active during key development periods and may be heavily tied to the survival of these flies. Determining the function of fru P3 and P4 in development and survival could help shed light on the complexity of this alternatively spliced gene.

In this project, I introduced a tool that significantly reduces the amount of P3 and P4 transcripts throughout the whole fly. Unfortunately, after quantifying the expression of P4 transcript, the results demonstrated that the P4 transcript was not being reduced, and therefore could not be tested further. I tested the flies expressing reduced fru P3 to determine if the transcript had an effect on development, whether its reduction influenced survival and developmental time. With reduced fru P3, flies couldn't progress past the developmental stage to become adult flies and progressed slower through the developmental phase compared to controls. This finding highlights the crucial role of fruitless P3 transcripts in development and its effect on viability. It also demonstrates the importance of fruitless’ role outside of the development of sexual characteristics and male sexual behaviour.

Download

Share

COinS