Mizuno S, Cox R F
Biochemistry. 1979 May 15;18(10):2049-56. doi: 10.1021/bi00577a032.
We have analyzed the effect of estrogen on the kinds of unique DNA sequences which are transcriptionally expressed in chick oviduct with an "expressed" DNA probe. Steady-state nRNA in estrogen-stimulated chick oviduct represents about 25% of the complexity of total chick unique DNA. To purify this expressed DNA fraction, chick unique DNA was isolated, nick-translated, and hybridized to chemically mercurated oviduct nRNA (Hg-nRNA); the resulting hybrids were bound to sulfhydryl-Sepharose, and DNA was selectively recovered by thermal elution in formamide buffer. To compare the sequence homology between nRNAs isolated from oviduct before or up to 6 days after estrogen withdrawal, trace amounts of expressed DNA derived from estrogen-stimulated oviduct were hybridized in RNA-excess reactions. All nRNAs hybridized with equal efficiency. Furthermore, hybridization of expressed DNA to nRNA mixtures showed that nRNA from nonwithdrawn and withdrawn oviduct contained a similar set of unique sequences. The data indicate that, at most, only a small percentage (0--5%) of transcriptionally active unique DNA sequences are shut down when estrogen is removed from the circulation.
我们用一种“表达的”DNA探针分析了雌激素对在鸡输卵管中转录表达的独特DNA序列种类的影响。雌激素刺激的鸡输卵管中的稳态mRNA约占鸡总独特DNA复杂度的25%。为了纯化这个表达的DNA组分,分离鸡的独特DNA,进行缺口平移,然后与化学汞化的输卵管mRNA(Hg-mRNA)杂交;所得杂交体与巯基琼脂糖结合,DNA通过在甲酰胺缓冲液中热洗脱进行选择性回收。为了比较从雌激素撤药前或撤药后长达6天的输卵管中分离的mRNA之间的序列同源性,将微量来自雌激素刺激的输卵管的表达DNA在RNA过量反应中进行杂交。所有mRNA的杂交效率相同。此外,表达DNA与mRNA混合物的杂交表明,来自未撤药和撤药输卵管的mRNA含有一组相似的独特序列。数据表明,当雌激素从循环中去除时,转录活性独特DNA序列中最多只有一小部分(0 - 5%)被关闭。