Isolation of Extremely AT-Rich Genomic DNA and Analysis of Genes Encoding Carbohydrate-Degrading Enzymes from Orpinomyces sp. Strain PC-2 |
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| Authors: | Huizhong Chen Sherryll L. Hopper Xin-Liang Li Lars G. Ljungdahl Carl E. Cerniglia |
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| Affiliation: | (1) Division of Microbiology, National Center for Toxicological Research, U.S. FDA, 3900 NCTR Rd.,, Jefferson, AR 72079, USA;(2) Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, USDA/ARS, 1815 N. University Street, Peoria, IL 61604, USA;(3) Department of Biochemistry and Molecular Biology and Center for Biological Resource Recovery, The University of Georgia, Athens, GA 30602, USA |
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| Abstract: | An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 β-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA). |
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