Research

BSGC Role

Clyde A. Hutchison III, will oversee Component Project VII, correlation to cellular function. He comes to the Center with experience in mycoplasma genomic analysis, with a focus on identifying the minimal essential gene set for mycoplamas. He will serve as a liaison between the Center and the mycoplasma and genomics communities. He will also assemble the current state of knowledge of function of mycoplasma genes selected as targets for structural studies at the BSGC.

Component Project Information

		Scanning electron microscope image of Mycoplasma genitalium. Photo: K. Frantz, A. Albay and K. Bott, The University of North Carolina at Chapel Hill.

The Hutchison lab will provide a liaison between the structural work going on in the Center, the mycoplasma research community, and the functional genomics community. In particular we will provide an interface between the structural work in the Center and ongoing functional genomic studies of mycoplasmas at The Institute for Genomic Research (TIGR). We will also undertake experimental approaches to defining the cellular function of genes selected as candidates for structural studies. The work under this subproject will be directed toward the following goals:

1. Collection of existing information concerning the functions of target genes.

We propose to collect existing information from the literature and from the mycoplasma research community for each gene selected for structural studies. If homologs exist in other species we will collect information on these homologs if the function has not been studied directly in mycoplasma. The information will be made available in the form of a spreadsheet over the web for all the participants in the project.

2. We will extend our analysis of gene disruptions using transposons to test whether target genes are essential for growth in the laboratory.

We will use gene-specific PCR to detect transposon insertion events in target genes. This could allow the identification of non-essential genes that have not so far been identified in our published work. We also propose a strategy using DNA microarrarys to detect gene-specific transposon disruption events.

3. Detection of mRNA as a test for expression of the gene.

RT PCR will be used to make a semiquantitative assessment of the level of expression of each target gene. As an alternative approach, in collaboration with TIGR, the expression will be studied using DNA microarray technology.

4. Targeted gene knockouts as a tool for assessing cellular function.

Since homologous recombination has been used to specifically disrupt a gene in M. genitalium (MG218), we expect that it can be developed into a tool that can be used to aid in the determination of unknown cellular functions.

5. Complementation of E. coli or yeast mutants.

We will attempt to complement E. coli or yeast mutants with mycoplasma genes as a way to identify gene functions.

6. We will attempt to establish a system for regulated expression of a gene introduced into mycoplasma.

If this can be done then the chromosomal copy of the gene could be knocked out and the phenotypic effect studied. This would permit study of genes of unknown function that are essential. Establishment of such a system can use existing methods to introduce a gene using a transposon. We will attempt to develop a repressor-regulated system based on the E. coli lac system.

7. Localization of the gene product to a cellular compartment.

Cells expressing an epitope tagged mycoplasma gene product will be fractionated and the distribution of the gene product within the cell will be studied to obtain clues concerning gene function.