2014 Annual Science Report

University of Illinois at Urbana-Champaign Reporting  |  SEP 2013 – DEC 2014

Project 13: Experimental Determination of the Existence of the Darwinian Transition

Project Summary

Life on our planet can be divided into three domains: Archaea, Bacteria and Eukarya. While some genes may be shared among the domains of life, others especially those involved in information processing namely DNA replication, transcription and translation are often unique to a particular domain. It has, therefore, been proposed that the molecular machineries that carry out these processes (replication, transcription and translation) have crossed a so-called Darwinian threshold where the molecular machineries have become gelled and therefore intolerant of new components. This project is examining the Darwinian threshold hypothesis by testing the interchangeability of the components of the DNA replication machinery across the domains of life. Further experiments will examine the capacity of biomolecules involved in translation and transcription to substitute for their counterparts across the domains of life.

4 Institutions
3 Teams
0 Publications
0 Field Sites
Field Sites

Project Progress

Over the past year we have developed a random transposon insertion library with the goal of identifying conserved hypothetical genes, whose cellular functions are essential. To begin with we developed a genetic marker with strong positive selection for agmatine prototrophy. Using selection for this marker we constructed a transposon mutagenesis system and tested for random insertions by selecting and sequencing insertions. In order to saturate the genome and identify essential genes we estimate that we need to select 20,000 individual random insertions in the 2.59 Mbp genomic background of M.16.4. Great progress has been made toward this goal. We currently have nearly 10,000 clones isolated and estimate that our complete ordered random library will be constructed and sequenced by February 2015.

Figure 1.
Figure 1. Interaction of clamp loader and clamp from different domains of life. The first three panels represent gel filtration profiles of individual proteins, i.e., M. acetivorans clamp loader (MacRFC), M. acetivorans sliding clamp (MacPCNA) and the E. coli sliding clamp (beta-subunit). The last two panels show a mixture of MacRFC/MacPCNA and MacRFC/beta-subunit)

Figure 2.
Figure 2. The M. acetivorans Cdc6-1, a protein with very high similarity to one of the eukaryotic origin recognition complex is required for proper cell division in the archaeon. Unlike wild type cells, cells with expression of Cdc6-1 repressed (Cdc6-; Tetracycline minus) expand and fail to divide. It can be concluded that the before the archaea and eukarya diverged their ancestor had already evolved a protein that marked where in the genome DNA replication will start.