Yao C, Frederiksen R A, Magill C W
Department of Plant Pathology and Microbiology, Texas A and M University, College Station 77843.
Curr Genet. 1992 Nov;22(5):415-20. doi: 10.1007/BF00352443.
The polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and two of P. zeae. In contrast to the situation found in most-fungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.
聚合酶链反应（PCR）使用与保守侧翼序列互补的引物，来扩增5株霜霉属分离株核糖体DNA重复单元的内部转录间隔区2（ITS 2），其中包括高粱霜霉1株、玉米霜霉1株、甘蔗霜霉1株以及玉米霜霉2株。与大多数已检测真菌的情况不同，每个样本中均明显存在长度异质性。使用从高粱霜霉扩增得到的ITS 2以及粗糙脉孢菌的克隆核糖体DNA作为探针，通过Southern杂交证实了扩增条带的核糖体DNA组成。使用相同探针在基因组DNA消化产物中也检测到了长度异质性。除了每个分离株有一个优势片段外，还有几个频率较低的不同大小的片段，并且每个物种的分离株具有独特的ITS 2条带模式。通过用MspI和HpaII消化基因组DNA后用核糖体DNA探针产生相同的条带模式，以及用CfoI完全消化，证明了这4种霜霉属物种核糖体基因的CCGG和GCGC序列中不存在5 - 甲基胞嘧啶残基。
