The utility of 5-bromo-2-deoxyuridin (BrdU) for identification of active micro-fungi from Antarctic soil

L. Connell1, A. Barrett1, R. Redman2, R. Rodreguez3
1School of Marine Sciences, University of Maine, Orono, ME, 04469, USA
2Department of Biology, University of Washington, Seattle, WA, 98195, USA
3U.S. Geological Survey, WFRC, Seattle, WA, 98115, USA

The thymidine analog, 5-bromo-2-deoxyuridin (BrdU), has been used primarily for the study of DNA replication in tissues and more recently for determination of microbial populations that are active under specific environmental conditions. BrdU is incorporated into the newly synthesized DNA during the S phase.  DNA extracted from cells that have incorporated BrdU can be isolated using an anti-BrdU antibody for use in the generation clone libraries.  Therefore, the use of BrdU directly in environmental samples could be a powerful tool in establishing which microbes are most actively dividing under specific conditions.  However, it has been shown that some organisms, most notably laboratory strains of the bakers yeast Saccharomyces cerevisiae, do not incorporate BrdU.  This study determined the BrdU incorporation of 20 microfungal species isolated from Antarctic mineral soils.  Cultured cells were grown with BrdU and extracted DNA was used in subsequent PCR reactions using internal transcribes spacer (ITS) ribosomal gene primers ITS4 and ITS5.  Additionally, Antarctic soil samples were inoculated with BrdU and extracted DNA was cloned for comparison to both cell culture and pre-BrdU inoculation data.  The use of BrdU for determination of micro-fungal activity from Antarctic soils should provide a powerful new tool.