Rapid detection of functional expression of C-5-DNA methyltransferases in yeast.
Rapid detection of functional expression of C-5-DNA methyltransferases in yeast.
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DOI：
10.1093/nar/26.5.1354
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发表时间：
1998-03
影响因子：
14.9
通讯作者：
M. Kladde;R. Simpson
中科院分区：
文献类型：
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作者：
M. Kladde;R. Simpson
我们之前使用了胞嘧啶-5- dna甲基转移酶（MTase）， m.s ssi作为完整细胞中染色质结构的探针。尽管M. Sss I是目前可用的MTase中分辨率最高的，但难以建立稳定的甲基化阳性菌株，这对其作为染色质探针的普遍应用构成了障碍。我们描述了一种简单的筛选表达M. sssi的菌株，它消除了从亚硫酸根处理的DNA中扩增的PCR产物的纯化，使用放射性同位素，聚丙烯酰胺测序凝胶电泳和放射自显影。该方法的高通量使得将M. Sss I引入到各种野生型和突变遗传背景中成为可能。
We have previously employed the cytosine-5-DNA methyltransferase (MTase), M. Sss I, as a probe for chromatin architecture in intact cells. Although M. Sss I offers the highest resolution of any currently available MTase, the difficulty in establishing stable, methylation-positive strains poses a barrier to its general utility as a chromatin probe. We describe a simple screen for M. Sss I-expressing strains that eliminates the purification of PCR products amplified from bisulfite-treated DNA, use of radioisotopes, polyacrylamide sequencing gel electrophoresis, and autoradiography. The high throughput of the method now makes it feasible to introduce M. Sss I into a variety of wild-type and mutant genetic backgrounds.