|Chen, Huizhong - JEFFERSON, AR|
|Hopper, Sherryll - JEFFERSON, AR|
|Li, Xin Liang|
|Ljungdahl, Lars - UNIV. OF GEORGIA|
|Cerniglia, Carl - JEFFERSON, AR|
Submitted to: Current Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 30, 2006
Publication Date: June 1, 2006
Citation: Chen, H., Hopper, S.L., Li, X., Ljungdahl, L.G., Cerniglia, C.E. 2006. Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2. Current Microbiology. 53:396-400. Interpretive Summary: Anaerobic fungi serve as an excellent source of genes coding for high active hydrolytic enzymes with promising utility in biomass conversion. One characteristic of anaerobic fungi is that they possess genes and genomes with unusually high AT-nucleic acid (DNA) contents. Extraction of high quality DNA from organisms with AT-rich genomes has been very challenging. It is even more challenging from fungi with high AT-content genomes because fungal cell walls are co-extracted with nucleic acids. To overcome these challenges, an efficient method to purify genomic DNA from Orpinomyces PC-2, an anaerobic fungus, has been developed based on repeated extraction and washing with a detergent solution (hexadecyltrimethylammonium bromide). The purification of sufficient amounts of highly purified DNA is the key to successfully construct genomic libraries and conduct Southern blot analysis, PCR, and genetic studies. High quality genomic libraries are required for the cloning of genes of interest from the organism and to sequence the whole genomes. This work will greatly accelerate the progress on studies of anaerobic fungi and biomass conversion enzymes.
Technical Abstract: An effective method for extraction of intact genomic DNA from extremely AT-rich anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogenlike storage polysaccharides using repeated hexadecyltrimethylammonium bromide (CTAB) and high salt wash. The DNA was digestible with various restriction enzymes and suitable for using as PCR temperate, Southern blot, and genomic library construction. Genomic DNA analysis of four representative genes encoding (hemi-) cellulolytic enzymes of the fungus revealed that there are multiple copies of family 5 endocellulase gene (celE), family 6 cellobiohydrolase II-like gene (celA), and family 1 beta-glucosidase gene (bgl1), but there is only one copy of family 11 xylanase gene (xynA) in the genome.