Epsilon Archive for Student Projects

Abstract

Promoters are genetic elements that facilitate the transcription of a gene and they have been found in front of non-coding RNA (ncRNA) genes in different organ-isms, e.g. the model plant Arabidopsis thaliana. A similar element, DUSE, has been found in front of ncRNAs in the social amoeba Dictyostelium discoideum and a part of this project has been to analyze the function of this putative promoter element through cloning and expression studies. A construct to analyze the func-tion of DUSE was successfully designed and introduced into D. discoideum but full expression studies were not finished because of shortage of time. However, the first preliminary tests with northern blot showed a distinct loss in the expres-sion of a model gene when the putative promoter was rendered non-functional by site-directed mutagenesis, indicating that DUSE truly functions as a promoter.

The main part of this project has been to set up an infection system for Legio-nella pneumophila utilizing the social amoeba Dictyostelium discoideum as a host organism. A big focus of the infection studies has been to study the RNA interfe-rence response to infection by utilizing two D. discoideum knock out strains and SOLiD sequencing. Deep sequencing using the SOLiD technique has been carried out on infected- and uninfected cells to compare the difference in the small RNA population and to analyze the response to infection.

The infection system was successfully set up with good confirmation of intra-cellular bacteria by two different screening methods. The infection efficiency showed to differ substantially between the tested strains AX4 and AX2, 10,0% to 3,4% respectively. The protein Dicer B has shown to play a big role in the bioge-nesis of miRNA and a D. discoideum strain with this gene knocked out showed to be more resistant to infection, having an infection efficiency of only 1.2%. At the same time, the growth of this knock out strain was inhibited when grown in prox-imity to L. pneumophila. This could indicate that there are miRNA(s) that are in-volved in the amoeba’s response to bacterial infection.

SOLiD sequencing showed that the non-coding Class I RNA gene, DdR-31,was the gene most affected in infected cells compared to uninfected cells also indicat-ing the role for a non-coding RNA in infection.