A method for extracting high-molecular-weight deoxyribonucleic acid from fungi

Specht, Charles A.; DiRusso, Concetta C.; Novotny, Charles P.; Ullrich, Robert C.

doi:10.1016/0003-2697(82)90680-7

Cited by 190 publications

(88 citation statements)

References 13 publications

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“…In preliminary studies, the two sources of DNA stimulated similar quantities of cytokines (data not shown). A. fumigatus was grown on minimal liquid media consisting of yeast extract supplemented with 2.5% glucose and 3.4 g/liter yeast nitrogen base supplemented with ammonium sulfate and amino acids at 37°C for 40 to 48 h. Fungi were harvested through a nylon mesh filter and washed with Tris-sodium-EDTA (0.05 M Tris-HCl, pH 8.0, 0.150 M NaCl, 0.1 M EDTA, pH 8) (28). Subsequently, hyphae were freeze-dried and ground to a powder, and the DNA was extracted with chloroform/isopropanol.…”

Section: Methodsmentioning

confidence: 99%

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA

et al. 2008

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Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.

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Section: Methodsmentioning

confidence: 99%

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA

et al. 2008

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“…DNA was extracted according to Specht et al 19 The primers ITS5 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) were used for amplification of the ITS region. 20 The PCR products were purified and sequenced by the Sanger method.…”

Section: Fungal Strains and Inoculation Of Ex Vitro-grown And Previoumentioning

confidence: 99%

Development and validation of an HPLC-DAD analytical method to quantify 5-methoxyflavones in methanolic extracts of Vochysia divergens Pohl cultured under stress conditions

Pimenta¹,

Kellner²,

Parpinelli³

et al. 2017

Quim. Nova

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Vochysia divergens Pohl, known as "Cambara" in Brazil, is an invasive species that is expanding throughout Pantanal in Brazil, to form mono-dominant communities. This expansion is affecting the agricultural areas that support the typical seasonal flood and drought conditions of this biome. This article describes the development and validation of an HPLC-DAD analytical method to quantify 5-methoxyflavones in methanolic extracts of greenhouse-grown V. divergens associated with one of two endophytic fungal species Zopfiella tetraspora (Zt) or Melanconiella elegans (Me) and later subjected to water stress. The developed method gave good validation parameters and was successfully applied to quantify the flavones 3′,5-dimethoxy luteolin-7-O-β-glucopyranoside (1), 5-methoxy luteolin (2), and 3′,5-dimethoxy luteolin (3) in the target extracts. Inoculation of the plant with Zt decreased the concentration of flavone 1 in the extract by 2.69-fold as compared to the control. Inoculation of the plant with Zt or Me did not significantly alter the contents of flavones 2 and 3 in the extracts as compared to the control. Therefore, the aerial parts of germinated V. divergens plants inoculated with either Zt or Me responded differently in terms of the production of flavones. These results can cast light on the symbiosis between fungal microorganisms and V. divergens, which most likely influences the response of V. divergens to changes in the availability of water in Pantanal.

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“…The extraction of total DNA was done according to the method of Speacht et al (1982). The PCR was done in a reaction mixture of 25 µL, containing 50 ng of total DNA in 1x thermophilic DNA poly buffer (Promega), 0.5 µM of each primer, 100 µM of dNTP, 50 mM of KCl, 2.5 mM of MgCl 2 (Promega) and 1 U of Taq DNA polymerase (Promega).…”

Section: Total Dna Extraction and Pcrmentioning

confidence: 99%

The distribution of a transposase sequence in Moniliophthora perniciosa confirms the occurrence of two genotypes in Bahia, Brazil

Ignacchiti

Santana

Araújo

et al. 2011

Trop. plant pathol.

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Transposase sequence analysis is an important technique used to detect the presence of transposable elements in a genome. Putative transposase sequence was analyzed in the genome of the phytopathogenic fungus Moniliophthora perniciosa, the causal agent of witches' broom disease of cocoa. Sequence comparisons of the predicted transposase peptide indicate a close relationship with the transposases from the elements of the Tc1-Mariner superfamily. The analysis of the distribution of transposase sequence was done by means of PCR and Southern blot techniques in different isolates of the fungus belonging to C-, L-, and S-biotypes and collected from various geographical areas. The distribution profile of the putative transposase sequence suggests the presence of polymorphic copies among the isolates from C-biotypes. The total DNA hybridization profile of each isolate was used to calculate genetic distance and group by the UPGMA method. C-biotype isolates colleted from of the Bahia showed two hybridization profiles for the transposase sequence. Thus the two different fingerprinting profiles for transposase sequence reported here by Southern analysis could also be correlated to the presence of two different genotypes in Bahia, Brazil.

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A method for extracting high-molecular-weight deoxyribonucleic acid from fungi

Cited by 190 publications

References 13 publications

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA

Toll-Like Receptor 9-Dependent Immune Activation by Unmethylated CpG Motifs in Aspergillus fumigatus DNA

Development and validation of an HPLC-DAD analytical method to quantify 5-methoxyflavones in methanolic extracts of Vochysia divergens Pohl cultured under stress conditions

The distribution of a transposase sequence in Moniliophthora perniciosa confirms the occurrence of two genotypes in Bahia, Brazil

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