Study of group II introns in Euglena chloroplast genomes: Structure, processing and evolution

Abstract

The chloroplast genome of Euglena gracilis contains at least 155 introns, accounting for 39.2% of the genome. Among them are 88 group II introns of size 277-671 nucleotide (nt). Questions about the processing of these introns and their origin and spread in Euglena genera is the subject of this thesis. My working hypothesis is that the Euglena chloroplast genome evolved from an intron less ancestral genome by the invasion of mobile genetic elements relying in part on internally encoded enzyme activities for mobility. These internal enzymes may also be group II maturases. My research target was the largest intron in Euglena gracilis chloroplast, psbC intron 2 (4144 nt). I characterized psbC intron 2 as a cis-spliced group II intron encoding a 758 codon maturase-like protein (mat 2) that is interrupted by three additional group II introns. I also identified a putative RNA-binding domain (X), that relates to fungal mitochondrial group II intron maturases, and a reverse transcriptase (RT) domain, that has been found in other group II intron maturases and has been shown to have intron-translocation activity (Lazowska, 1994, Moran, 1995). In order to examine the distribution of mat 2, I characterized psbC intron 2 homologues from several species. I found that the mat 2 locus well conserved in most of the Euglena species tested, indicative of a deep root and important function of mat 2. Interestingly, the entire intron is absent in E. myxocylindracea. In order to investigate the effect of mat 2 loss, I analyzed group II intron content in E. myxocylindracea chloroplast genome. No group II introns have been identified in the genome. These data also provided evidence that mat 2 might be responsible for the acquisition and the processing of most of the group II introns in Euglena chloroplast. The activity of the mat 2 protein has been difficult to examine since the protein is hard to obtain. The development of protocols to overexpress mat 2 in E. coli and yeast are reported in the thesis.