California Sea Grant College Program

Ciliates are single cell eukaryotic organisms with two nuclei and unusual genome biology. The purpose of this project is to study the molecular evolution of macronuclear (MAC) and mitochondrial (Mt) genomes of ciliate protist Tetrahymena (T.) to decipher and interpret their evolutionary processes. Tetrahymena species contain rapidly evolving mitochondrial (Mt) genomes, which have apparently diverged significantly from the ancestral pattern leaving half of their 44 genes without an assigned function. Thus I sequenced three Mt genomes of Tetrahymena paravorax, Tetrahymena pigmentosa, and Tetrahymena malaccensis via shotgun sequencing. I found unique features in these linear 47kb long genomes including long telomeric repeats, long noncoding sequences, a pseudo-tRNA gene, unusual DNA secondary structure for T. paravorax, and the transcription initiation site, a GC box. More analyses were carried out using programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (oo) to discover mutation hotspots and relaxed selective constraint in Ymf genes. Hence I concluded that the presence of mutation hot spots in Tetrahymena Mt genomes is a major reason and obstacle in identifying their function.

To study the MAC evolution I annotated a Tetrahymena thermophila chromosome, 1Mb in size, based on the translation of its possible open reading frames (ORFs) and their homologues in the GenBank. An objective of this study was to examine the TIGR automated annotation programs capability in identifying all of the biologically meaningful ORFs. Through searching the databases using BLAST I found proteins and ORFs with homologous domains that were not present in the TIGR automated annotation. Thus, I suggest phylogenetic footprinting to complement automated annotations. I also sought to reveal the extent of chromosome rearrangements between T. thermophila and the largest chromosome (LC) of Paramecium (P.) tetraurelia. Mapping all 463 putative proteins from the LC of P. tetraurelia to the chromosomes in T. thermophila genome revealed no orthologous chromosome. Interestingly, gene order between the Mt genome of these two ciliates was almost perfectly conserved, which suggests that the most plausible explanation for such an extensive gene order loss and rearrangement could be the developmentally programmed elimination of germ line sequences and randomization of MAC gene order.