In the first phase of this project, we propose to carry out three-fold (3X) random DNA sequencing of the genome of the methanogenic archaeon Methanococcus maripaludis. 3X sequencing will cover 95 percent of the nucleotides and provide partial or complete sequence of 95 percent of the genes. 3X sequencing is relatively inexpensive and is cost-effective. As the sequence information accumulates, we will organize the list of genes into functional categories according to apparent homologies with genes in available databases. Data analysis will be carried out automatically using existing software. The results will be made available on the World Wide Web the same day that they are generated. The result of this effort will be the availability of a genome sequence for a species of Archaea that is easily manipulated by genetic methodology. Therefore, the genome sequence will form the basis for studies by a large number of laboratories around the world that are interested in various aspects of the biology of the Archaea. The genome sequence will also be useful to study the basis for therophily vs. mesophily, because M. maripaludis is mesophilic, and genome sequence is already available for other Archaea that are thermophilic. The second phase of this project is to use the genome sequence information, and already developed genetic methodology, to learn about mechanisms of regulation in Archaea. These studies will begin as soon as sufficient sequence information is generated. Contig lengths in areas of particular interest will be extended by targeted sequencing. Potential regulatory genes will be identified by homology to known regulatory genes, and to known DNA binding motifs. The functions of these genes will be tested by introducing null mutations into M. maripaludis and determining their effects of the expression of potential target genes. This effort will be carried out with genes that are likely to regulate certain systems of interest to our lab, by virtue of their particular sequence homologies and their proximity to predicted target genes. At the same time that we are carrying out mutagenesis studies, we will use array technology to expand our knowledge of regulons. Genes will be identified that either vary in expression with changing conditions, or change in expression after mutagenesis of regulatory genes.
