Appendix 1B The Evolutionary History of Proteins Involved in Pre-replication Complex Assembly

Abstract

The analysis presented here provides a glimpse into the genomic era where the availability of several completely sequenced genomes from poorly studied eukaryotes challenges existing notions and models of replication derived from model organisms. At the same time, these genomes provide a deeper insight into the evolutionary histories of the proteins involved in the process.

METHODOLOGY
The analysis of the phyletic distribution and evolutionary histories of proteins was done using sequence-based searches for homologs among a diverse sampling of complete or nearly complete genomes. Homologous genes can be divided, on the basis of their evolutionary origins, into orthologs and paralogs. Orthologs are genes in different species that evolved from a common ancestral gene through speciation. Paralogs are genes of a given species that have been derived by duplication from a common ancestor. The process of assigning orthology or paralogy to genes was done using the cluster of orthologous groups (COG) approach (Tatusov et al. 1997). Briefly, homologs of the pre-replication complex (pre-RC) proteins from different eukaryotes were identified by PSI-BLAST searches performed against the non-redundant (nr) database using the human pre-RC proteins as query. Each of these was further used as a query in BLAST searches against the genomes of the 13 other eukaryotes in the list. An orthologous group consists of proteins that gave the maximum number (>3) of symmetrical best hits to each other. In cases that were difficult to resolve by this method, we used phylogenetic trees derived from multiple alignments to distinguish between orthologs and paralogs.