Isolation of fungal DNA from plant tissues and removal of DNA amplification inhibitors

Specific primers and the polymerase chain reaction (PCR) are increasingly used for detection of fungi in plants. Detection depends on isolation of DNA that is free of compounds that inhibit amplification. We report on the significance of inhibition in soybean stems with a focus on studies of the vascular fungal pathogen Phialophora gregata. A simple DNA extraction procedure based on a FastDNA® kit is described that allows consistent detection of fungi in soybean stems using PCR. The addition of polyvinylpyrrolidone and supplemental DNA purification steps overcame inhibition in over 90% of samples. These methods should also facilitate studies with other plant and fungal species.     

Isolation and characterization of glycogen from mycelium ofAscobolus furfuraceus

A polysaccharide with the properties of glycogen has been isolated by water or phenol-water extraction from homogenates ofAscobolus furfuraceus. After 4 days of growth a maximum glycogen content of 28% of the cell dry weight was found by a specific enzymic method or by the iodine method. Taking into account the cell growth, the highest total amount of glycogen (2.04 mg/ml of broth) occurred on the 10th day. The average chain length determined by periodate oxidation was about 11. This result shows that the reserve polysaccharide fromA. furfuraceus has a highly branched structure similar to liver glycogen.     

Peroxidase from cultured peanut cells and fungal mycelium as auxin receptors

The electrophoretic patterns of peroxidase isozymes from cultured peanut cells and from the mycelium of Trametes versicolor showed minor difference     

Isolation of DNA from fungal mycelia and sclerotia without use of density gradient ultracentrifugation

A rapid method for extracting total DNA from Aspergillus flavus and Aspergillus parasiticus has been developed. The procedure can be completed in 2 h and yields 200 to 350 micrograms of DNA from 0.5 to 1.0 g wet wt of mycelia and 150 micrograms from 0.5 g of sclerotia. DNA samples had an OD260/OD280 of 1.6 to 1.8. Most of the DNA was at least 50 kb pairs in size and showed little degradation. DNA prepared by this method was used for restriction endonuclease digestion and Southern blotting. A DNA fragment containing the repeat unit of the ribosomal RNA genes of A. flavus has been identified.     

Isolation and properties of chitin synthetase from Agaricus bisporus mycelium

Using the standard procedure for the isolation of chitosomes (S. Bartnicki-Garcia, C. E. Bracker, E. Reyes, and J. Ruiz-Herrera, 1978, Exp. Mycol. 2, 173) a 50-fold purification has been achieved of chitin synthetase (ChS) from Agaricus bisporus hyphae grown in stirred, pellet-free liquid culture. Some properties of the enzyme were determined and compared with those of chitosomal ChS of other organisms. Effects of some antifungal compounds upon enzyme activity were also investigated. Inclusion of digitonin into the extraction medium afforded a second species of chitosomes, which have a higher sedimentation coefficient than the “standard” chitosomes. As regards ultrastructure, behavior in sucrose density gradients, level of activities, zymogenicity, cofactor requirements, kinetics, apparent K_m values for UDPGlcNAc and GlcNAc, inhibitory constants for nikkomycin X, nikkomycin Z, and amphotericin B methyl ester (AME), as well as modulation by digitonin, the mushroom chitosomal ChS displayed basically the same features as the corresponding enzyme from Mucor rouxii yeast cells. Some differences were, however, observed: Rennilase was almost ineffectual in overcoming latency of the enzyme, and trypsin performed satisfactorily only in a narrow concentration range, whereas pronase was very good. Furthermore, stimulation of ChS by digitonin in low concentrations was considerably higher (attaining more than 300%) with the mushroom than with the Mucor enzyme. The strong stimulatory effect of digitonin seems to be specific for the spirostanol glycoside since it could be produced by neither the closely related saponin α-tomatine, nor by AME—both compounds, like digitonin, sterol-complexing agents. The suggestion is made that stimulation of ChS by digitonin may be causally related to its antimycotic activity. Several possibilities are discussed as to its mode of action in stimulating ChS.     

Isolation and properties of macrotetrolide synthase from the mycelium of Actinomycetes

The formation of cyclic polyester antibiotics (macrotetrolides) from nactinic acids in a cell-free system in the presence of a mycelium homogenate of Streptomyces chryzomallus var. macrotetrolidi, a producer of a complex of homologous macrotetrolide antibiotics, was demonstrated. An enzyme catalyzing the formation of an ester macrotetrolide ring and possessing a specific activity of 360 mumol/min/mg of protein has been isolated for the first time from the mycelium homogenate and purified 176-fold with a 18% yield. Macrotetrolide synthase represents a macromolecular complex with a molecular mass of 360 kDa formed by several heterogeneous polypeptides. The effects of physico-chemical environmental factors on the stability and activity of the enzyme were demonstrated. The optimal conditions for the manifestation of the synthase activity (10 mM Tris-HCl, pH 8.0, 10% ethanol, 2% glycerol, 200 micrograms/ml of nactinic acids) were selected. The kinetic parameters of the enzyme-catalyzed macrotetrolide synthesis reaction (Km = 2.9.10(-4) M, V = 22.3 microM/min) were determined.     

Isolation and characterization of a mannoglucan from edible Cordyceps sinensis mycelium

Cordyceps sinensis is a well known tonic food or invigorant with broad-spectrum medicinal properties that is widely used in the People's Republic of China. A neutral mannoglucan 1 with a molar mass of approximately 7.7x10(3) Da was obtained from the 0.05 M acetate buffer extract of C. sinensis mycelium. It had [alpha](D)(20)+126 (c 0.2, H(2)O) and consisted of Man and Glc units in the molar ratio of 1:9. A combination of chemical analysis and NMR and IR spectroscopy together with digestion with alpha-D-amylase showed 1 to have a alpha-D-glucan backbone with (1-->4)- and (1-->3)-linkages, and the side chains of alpha-D-(1-->6)-Manp were attached to the backbone via O-6 of alpha-(1-->3)-Glcp residues. Compound 1 showed weak cytotoxicity activity against SPC-I (IC(50)=63 microg/mL) cancer line, and no obvious cytotoxicity activities against BCAP37 (IC(50)>100 microg/mL) and SW480 (IC(50)>100 microg/mL) cancer lines.     

Isolation and characterization of new highly polymorphic DNA markers from the Huntington disease region.

The defect causing Huntington disease (HD) has been mapped to 4p16.3, distal to the DNA marker D4S10. Subsequently, additional polymorphic markers closer to the HD gene have been isolated, which has led to the establishment of predictive testing programs for individuals at risk for HD. Approximately 17% of persons presenting to the Canadian collaborative study for predictive testing for HD have not received any modification of risk, in part because of limited informativeness of currently available DNA markers. Therefore, more highly polymorphic DNA markers are needed, which will further increase the accuracy and availability of predictive testing, specifically for families with complex or incomplete pedigree structures. In addition, new markers are urgently needed in order to refine the breakpoints in the few known recombinant HD chromosomes, which could allow a more accurate localization of the HD gene within 4p16.3 and, therefore, accelerate the cloning of the disease gene. In this study we present the identification and characterization of nine new polymorphic DNA markers, including three markers which detect highly informative multiallelic VNTR-like polymorphisms with PIC values of up to .84. These markers have been isolated from a cloned region of DNA which has been previously mapped approximately 1,000 kb from the 4p telomere.     

Isolation of a DNA fraction highly enriched in transfer RNA genes from Xenopus laevis.

A DNA fraction highly enriched in tRNA genes can be isolated from the Xenopus laevis genome by the use of Ag+/Cs2SO4 density gradients. Ag+ shows a low affinity for some tRNA cistrons, allowing their separation from bulk DNA upon equilibrium centrifugation in a Cs2SO4 density gradient. Contaminating DNA in the resulting tDNA fraction is further removed by two additional CsCl density gradient centrifugations. The final DNA fraction is 60-fold enriched in tRNA genes, compared to the starting DNA material.     

Isolation of DNA from yeasts

Methods are described that allow DNA to be prepared from widely different yeasts (Candida utilis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe). The methods are reliably reproducible, and the DNA obtained is of appropriate quality for the construction of gene libraries (upper limit of size range consistently 50-150 kbp). In method A, yeast cells are converted into spheroplasts by treatment with a highly purified mixture of enzymes from Trichoderma harzianum, the spheroplasts are lysed in a lauroylsarcosinate/EDTA buffer, and the lysate is incubated with proteinase K and then directly centrifuged through a cesium trifluoroacetate gradient. DNA is recovered from the appropriate fractions by ethanol precipitation, and the redissolved precipitate is incubated with ribonuclease. For the rest of the isolation, two protocols are given, one avoiding and one including phenol/chloroform extraction. In this way, DNA up to about 150 kbp in size can be obtained. In method B, spheroplasts are not made. Yeast cells are broken by grinding under liquid nitrogen and are then worked up in a manner similar to method A, protocol 2. Subsequent steps depend on the purpose for which the DNA is required. Traditional methods of sucrose or salt density gradient centrifugation or agarose gel electrophoresis are applicable for size selection. A sodium iodide/silica matrix technique allows fast and effective DNA recovery from agarose gels.     

Beringian paleoecology inferred from permafrost-preserved fungal DNA

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.     

Isolation of Chloroplast DNA from Pinus

Chloroplast DNA isolated from Japanese black pine [Pinus thunbergii)was digested with three restriction endonucleases. The sizeof this DNA was estimated to be 120 kbp. Chloroplast DNA fromJapanese red pine (P. densiflora) could be distinguished fromthat of P. thunbergii by BamHI restriction patterns. ³Present address: Kanto Forest Tree Breeding Institute, Kasahara,Mito, Ibaraki 310, Japan. (Received December 10, 1985; Accepted March 8, 1986)     

Isolation of plasmid DNA from Bifidobacterium

Conditions for accumulation of the biomass and a procedure for isolation of plasmid DNA from bifidobacteria in microquantities were developed. It was shown that all the strains tested had 1 to 3 plasmids of different molecular weights electrophoretically detected. Relation between the detected plasmid DNA and bifidobacteria resistance to tetracycline and fusidin as well as utilization of some carbohydrates is discussed.     

Isolation of DNA from blood.

A rapid, economical method of DNA isolation from blood was developed that yields DNA suitable for Southern analysis and polymerase chain reactions without organic solvent extractions. Bovine DNA was prepared from peripheral leukocytes and nuclei using pronase E digestion and ethanol precipitation. This isolation method readily adapts to multiple samples. The DNA is characterized by high yield, solubility, lack of protein contamination, and ease of restriction endonuclease digestion.     

Isolation of polysaccharide-free DNA from plants

A quick procedure for the isolation of polysaccharide-free DNA from different plant species and cell suspension or callus cultures is described. The originality of the method lies in the use of a mixture of glycoside hydrolases that leads, after phenol and chloroform extraction, to the isolation of pure DNA without any polysaccharide contamination. The highly purified DNA can be used for nucleotide analysis by HPLC, RFLP analysis and PCR amplification.     

Image analysis of space-filling by networks: Application to a fungal mycelium

Summary Image analysis methods are described which calculate and display the structure of a fungal mycelium grown on Cellophane. The basic network is found by identifying nodes and the sections joining them. Lengths, widths and volumes of hyphae are displayed in graphical form to show variation over the mycelium. The variation of fractal dimension shows the relative importance of feeding/foraging. These approaches can be applied to the analysis of networks other than mycelial.