Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2.
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 beta-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).
Chen
Huizhong
H
Division of Microbiology, National Center for Toxicological Research, U.S. FDA, Jefferson, AR, 72079, USA. huizhong.chen@fda.hhs.gov
Hopper
Sherryll L
SL
Li
Xin-Liang
XL
Ljungdahl
Lars G
LG
Cerniglia
Carl E
CE
D020856:AT Rich Sequence; D044602:Cellulases / Q000235:genetics*; D004271:DNA, Fungal / Q000302:isolation & purification*; D043364:Endo-1,4-beta Xylanases / Q000235:genetics*; D016681:Genome, Fungal; D020085:Neocallimastigales / Q000235:genetics* / Q000378:metabolism; D001617:beta-Glucosidase / Q000235:genetics*
D004271:DNA, Fungal; D044602:Cellulases; D001617:beta-Glucosidase; D043364:Endo-1,4-beta Xylanases