The origin and characterisation of new nuclear genes originating from a cytoplasmic organellar genome.

Abstract

Endosymbiotic transfer of DNA and functional genes from the cytoplasmic organelles (mitochondria and chloroplasts) to the nucleus has been a major factor driving the origin of new nuclear genes, a process central to eukaryote evolution. Recent developments have allowed the experimental reconstruction of DNA transfer and functional gene transfer, enabling investigation of the molecular mechanisms involved in these important evolutionary processes. To simulate the process of functional endosymbiotic gene transfer, a chloroplast reporter gene aadA, which had been transferred from the chloroplast to the nucleus, was monitored for nuclear activation. In total 16 plant lines were screened, each line representing an independent nuclear insertion of the aadA gene. For each line ~50 million cells were screened resulting in three plants being recovered in which aadA showed strong nuclear activation. Activation occurred by acquisition of the CaMV 35S nuclear promoter or by nuclear activation of the native chloroplast promoter. Two fortuitous sites resident within the 3' UTR of aadA mRNA both promoted polyadenylation without any sequence change. In addition, cryptic nuclear activity of the chloroplast promoter was revealed which became conspicuous when present in multiple nuclear copies. To determine the method of chloroplast DNA insertion into the nucleus the insertion site was sequenced in line kr2.2. Complete characterisation of the nuclear sequence before and after gene transfer demonstrated simultaneous insertion of multiple chloroplast DNA fragments via synthesis dependent non-homologous end joining, probably at a site of double strand break (DSB) repair. To further investigate the role of DSB repair in the nuclear insertion of organelle DNA, DSBs were induced at a specific nuclear location using the rare-cutting endonuclease I-SceI and the resulting repair events were observed. Analysis of ~300 DSB repair events indicated that most involved the loss of nucleotides from one or both ends being joined. Insertions were observed in five repair events. None of the inserted sequences were of organelle origin. Notably, the amount of nuclear sequence deleted was significantly larger in repair events involving insertion than in those without insertion, indicating that the two types of repair may be mediated by distinct pathways.