Gene structure and expression of the Euglena gracilis chloroplast psbB and petB operons.

Abstract

Euglena gracilis has been used as a model for studying the chloroplast genome and gene expression during chloroplast biogenesis. My graduate research has been focused on the following two areas: (1) DNA sequencing of part of the Euglena gracilis chloroplast genome, (2) Euglena chloroplast RNA metabolism, such as intercistronic RNA processing and intron splicing. The region that I characterized is between the psbB and rbcL loci on the Euglena gracilis chloroplast genome. Three photosystem II genes (psbT, psbN and psbH), one cytochrome b6 gene (petB) and two ATPase subunit genes (atpB and atpE) have been identified. Based on northern hybridization analysis, the Euglena gracilis chloroplast petB, atpB and atpE genes are cotranscribed as a tricistronic operon. Through cDNA analysis of petB-atpB-atpE pre-mRNA, eight introns have been identified. Two independent intercistronic RNA processing events and 11 splicing reactions lead to the accumulation of the mature petB, atpB and atpE monocistronic mRNAs. The mRNAs from psbB, psbT, psbH and psbN genes have been analyzed by northern hybridization, S1 nuclease protection and primer extension RNA sequencing. psbB and psbT are cotranscribed, while psbH and psbN are cotranscribed on the opposite strand. The 5' end of the psbN-psbH transcript and the intercistronic cleavage sites between psbB-psbT and psbN-psbH were determined. Through a combination of cDNA cloning and sequencing, northern hybridization and S1 nuclease protection analysis of the partially spliced psbT pre-mRNAs, the 1352 nt psbT intron has been characterized as a complex twintron with overlapping internal introns and alternative splicing pathways. In the predominant pathway, two internal group II introns are not orderly spliced. In an alternative pathway, splicing of a group III intron occurs. This group III intron is recruited from sequences of two group II introns. The generation of this group III intron is the first evidence that a group III intron can be derived from portions of existing group II introns. The mechanism of group III intron formation may also be relevant to the evolution of nuclear introns from putative group II intron ancestors.