Electrophoretic characterization of chromosomal DNA from two microsporidia.
Electrophoretic characterization of chromosomal DNA from two microsporidia.
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DOI：
10.1016/0022-2011(90)90107-h
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发表时间：
1990-09
影响因子：
3.4
通讯作者：
U. Munderloh;T. Kurtti;S. Ross
中科院分区：
文献类型：
--
作者：
U. Munderloh;T. Kurtti;S. Ross
Spores of two microsporidia,Nosema pyrausta(from the European corn borer,Ostrinia nubilalis) andN. furnacalis(from the Asian corn borer,O. furnacalis) were harvested from laboratory-rearedO. nubilaliscaterpillars and purified by centrifugation through Percoll. Conditions permitting in vitro germination were defined for both species and found to be different.N. pyraustaspores were incubated in 0.1nKOH for 30 min, recovered by centrifugation, and resuspended in 1 ml of an equal mixture of 1% low melting point (LMP) agarose and L-15B medium at 37°C to induce germination.N. furnacalisspores were first washed in 10 mmNa2EDTA in 1 mmTris base,pH 7.5, exposed to 0.01nKOH in 0.17mKCl for 30 min, centrifuged, and germinated in 1 ml of an equal mixture of 1% LMP agarose and 0.17mKCl in 10 mmNa2EDTA (pH 8), at 37°C. Eighty to 90% of the spores of each species germinated. Germinated spores were pipetted into a casting mold. Before electrophoresis, agarose blocks were incubated 48 hr at 50°C in 10 mmTris base/100 mmNa2EDTA,pH 7.8, with 1 mg/ml proteinase K and 1%N-laurylsarcosine to release the chromosomal DNA from sporoplasms. After pulsed-field electrophoresis, ethidium bromide staining revealed 13 chromosomal bands ranging in size from 1390- to 440-kb pairs and 1360- to 440-kb pairs inN. pyraustaandN. furnacalis, respectively. The difference in size estimates of corresponding chromosomes in the two species was not more than 60-kb pairs.
