Release of high molecular weight DNA from Neurospora crassa using enzymic digestions.
Methods are described that allow extraction of high molecular weight DNA from germinated conidia of Neurospora crassa. By labelling DNA with ribonucleosides, early conidia were shown to be active in DNA synthesis. These cells when treated with the enzyme Zymolyase became fragile and could be readily lysed with ionic detergents to release high molecular weight DNA. The DNA extracted from Zymolyase treated cells on to alkaline sucrose gradients sedimented as a heterogeneous species of up to 150 x 10(6) molecular weight. A minor DNA species (presumably mitochondrial) of 20 x 10(6) molecular weight comprised 2-7% of the total. The identity of the DNA was confirmed by sensitivity to DNAase, the diphenylamine assay and TLC. Sedimentation patterns were unaffected by protease digestions and no anomalous high speed rotor effects were evident. Isopycnic gradients suggested that the DNA released was uncomplexed with either protein or carbohydrates. Sepharose chromatography of extracted, RNAase-treated Zymolyase lysate resulted in clearly separate high molecular weight DNA and RNA-protein elution profiles. UV light preferentially inhibited nuclear DNA synthesis and drastically reduced the size and amount of nascent DNA being synthesized in the excision defective uvs-2 mutant. Sites in parental DNA sensitive to Micrococcus luteus UV endonuclease were measured in cells made permeable with Triton X-100.
D002499:Centrifugation, Density Gradient; D002847:Chromatography, Agarose; D004260:DNA Repair; D004271:DNA, Fungal / Q000032:analysis / Q000096:biosynthesis*; D004720:Endonucleases; D004798:Enzymes / Q000494:pharmacology*; D006867:Hydrolases; D008970:Molecular Weight; D009491:Neurospora / Q000378:metabolism*; D009492:Neurospora crassa / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D014466:Ultraviolet Rays
D004271:DNA, Fungal; D004798:Enzymes; C037833:zymolyase; D006867:Hydrolases; D004720:Endonucleases