Horizontal gene transfer greatly accelerates genome evolution and Innovation. We are comparing the genomes of eight prokaryotes and related horizontal gene transfer (HGT) to environmental and genomic properties of the organisms and their habitats. Extensive statistical analysis of HGT among the ortholog trees for eight taxa has revealed that HGT is strongly influenced by genomic and environmental factors, so that organisms living in similar environments preferentially exchange genes with other organisms that have similar environmental parameters. As a result, we infer that HGT has accelerated prokaryotic genome innovation and evolution by a factor of about 104. In practical terms, the number of unique prokaryotic genes that would be "invented" in a ten thousand year period if HGT were absent, can in fact be "invented" in a single year.

This year also saw publication of the transorientation hypothesis. This hypothesis presents a mechanistic, structural model for decoding and proofreading during protein synthesis. The model invokes a 5'-stacked transfer ribonucleic acid (tRNA) bound to a decoding site, the D-site, which is distinct from the well-known A-site. Upon hydrolysis of GTP by EF-Tu, the D-site tRNA switches from the 5'-stacked conformation to the 3'-stacked geometry. This conformational change causes the tRNA to rotate (transorient) about the relatively fixed codon-anticodon pair, from the D-site into, or close to, the A-site. The mechanism described by the transorientation hypothesis is dominated by tRNA conformational changes as well as tRNA-mRNA interactions, and points to those molecules being the progenitors of protein synthesis in a pre-existing RNA world.

Figure 1. a) 5'-stack tRNA (yellow ribbon) is shown docked in the D-site, on the 30S ribosome (blue) (PDB ID: 1FJF). The 5'-stacked anticodon (green) base-pairs with the message RNA (red). b) The tRNA is shown docked in the A-site of the 30S tRNA (adapted from Simonson and Lake, 2002).

Progress was made on our work on the developmental genetics relevant to the rapid evolution of morphology at the base of the Cambrian Period. In terms of sensory structures, our work on the POU gene family indicates the presence of a suite of genes involved in sensory structure development and sensory cell differentiation in cnidarians, ctenophores and sponges. We are examining the expression of these genes in the jellyfish Aurelia. Rapid progress has been made with another family of genes, the sine oculis /Six genes, as well. We have obtained sequence of these genes throughout the basal Metazoa. The sine oculis work is the subject of a recently completed Master's thesis (Ilona Bebenek). We have accumulated data relating to the posterior addition as a basal aspect of the developmental genetic program of Bilateria. We have some sequence data on the gene caudal accumulated by a Ph.D. student (Chris Winchell). We intend to integrate these data with morphologic evidence of the mode of development available in the fossil record. Our previous work on the engrailed gene has suggested the possibility that one aspect of the ancestral function of this gene is in bounding the ectodermal skeletons of bilaterian invertebrates. Artem Kouchinsky, a Russian Post Doc, is joining the lab to further pursue this evolution from the molecular side of the question. Simple analysis for the issue of rates, dates and the timing of the metazoan radiation establish that the early dates of the protostome/deuterostome divergence are based on biased data. The demonstration of this bias is useful as it allows one to understand the disparity in dates recovered in molecular clock analyses.

The long term goals in collaboration with our cross-team partner Dr. Andrew Roger of Dalhousie University, is to determine the origin and evolution of eukaryotic energy-generating organelles derived from eubacterial sources (e.g. plastids, mitochondria, hydrogenosomes) to test hypotheses for the origin of eukaryotic cells. The mitochondrion arose by an endosymbiotic event involving a proteobacteria that was either subsequent to or concurrent with eukaryogenesis. It is clear that aerobic metabolic capacity came into eukaryotes with the mitochondrion. There are however, diverse anaerobic eukaryote lineages that lack mitochondria, for which it is not clear how they acquired their energy producing pathways. Some of these possess an alternative energy-generating organelle, the hydrogenosome. Phylogenetic evidence suggests that this organelle is derived from the same endosymbiont that gave rise to the mitochondrion; however, it is not clear whether its constituent energy producing enzymes are of the same origin. To address this issue, two genes encoding proteins associated with hydrogenosomal energy production have been analyzed using phylogenetic assays, in two amitochondriate protists, the free living anaerobic flagellate, Trimastix and the anaerobic commensal gut, Retortamonas. The complete sequence of pyruvate:ferredoxin oxidoreductase (PFO) from Trimastix and Retortamonas and gene sequences for two paralogs of a second key enzyme in anaerobic metabolism, hydrogenase, from Retortamonas, have been obtained. Comprehensive phylogenetic analyses reveal that PFO acquisition occurred very early and only once in eukaryotic history. The data, however, do not allow the gene to be traced back to its prokaryotic progenitor. Unlike PFO, hydrogenase has come into eukaryotes multiple times. Interestingly, our phylogenetic trees of hydrogenase have uncovered the best-supported evidence for horizontal gene transfer (HGT) between two distantly related eukaryotic lineages (Retortamonas and Entamoeba). HGT and extensive prokaryotic gene duplications renders these data unsuitable for tracing the ancestries of hydrogenase to any particular bacterial group. Overall, these data do not support the notion that key enzymes in anaerobic metabolism share a common ancestry with the progenitor of the mitochondrion, the so-called 'hydrogen hypothesis' for the origin of eukaryotes. Nevertheless, current data on phylogenies are insufficient to refute this hypothesis, hence relevant gene sequences from additional divergent anaerobic eukaryotes and eubacteria will be necessary to clarify the origin and evolution of energy metabolism in eukaryote.

Sorel Fitz-Gibbon continued her collaboration with Christopher H. House (Penn State University Astrobiology) refining, applying and interpreting phylogenetic trees based on whole-genome sequence data. Their results suggest that the last common ancestor of the Archaea was a sulfur reducer and that methanogenesis arose only once, later, after the divergence of Crenarchaea and Euryarchaea.

Study on Protein-Protein Interactions and HGT in collaboration with Dr. Monica Riley, Lake et al. has attempted to define sets of genes that are subject to more frequent horizontal gene transfer (HGT) and genes that are less frequently horizontally transferred. This analysis has been based on protein interactions in metabolic pathways in E. coli. In parallel, this sort of analysis has been extended to yeast, using the Database of Interacting Proteins in collaborations with Dr. Ionnis Xenario and Dr. David Eisenberg. While our results are preliminary, we have found that there are several discriminating factors that influence the frequency of HGT.

-Sorel Fitz-Gibbon (UCLA) and Christopher House (PSU) collaborate on whole-genome based phylogenetic methods and applications, resulting in a publication.