We aim to use metagenomic methods to access portions of Antarctic low temperature crenarchaeotal genomes. Large insert metagenomic fosmid clones identified by colony hybridization with crenarchaeotal 16S rRNA gene probes will be sequenced and ORFs identif ied. The design of probes to terminal regions of the positive clones can provide access to proximal sequences, allowing us to genome walk along the crenarchaeotal chromosome. Detailed bioinformatic analysis (particularly using COG databases) will allow us to infer the function of a proportion of the identified ORFs, from which we aim to derive information on physiological function. The long term aim of this project would be to use physiological information to guide the development of isolation strategies in order to access pure cultures of these recalcitrant organisms. Dr. Cowan?s laboratory has a wide variety of source material from the Dry Valleys of Eastern Antarctica. These samples have been collected as part of an on-going research program on the di versity and function of microorganisms in the desert soils of the Antarctic Dry Valleys. Based on the metagenomic clones, we will also investigate the mechanisms of cold-adaptation of enzymes, using isocitrate dehydrogenase (IDH) as a model. This will she d light on how the organisms have adapted to the arctic environment. Furthermore, we intend to study the Cdc6 and MCM proteins from a variety of archaeal species identified in the libraries. This will broaden our knowledge about the mechanism of initiati on of archaeal DNA replication. A comparative functional analysis as well as protein-protein and protein-DNA- interactions of purified Cdc6 and MCM proteins encoded on identical cloned fragments will be carried out. This will include studies on how Cdc6 s timulates or inhibits the helicase activity of MCM, and how they physically interact with each other. Finally, functional analyses using homologous replication origins will be pursued.