Ganguly R, Banerjee M R
Nucleic Acids Res. 1978 Nov;5(11):4463-77. doi: 10.1093/nar/5.11.4463.
Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA.
在25摄氏度的高离子强度条件下，泌乳小鼠乳腺的分离细胞核能够在未修饰的和汞化的CTP（Hg-CTP）存在的情况下支持体外DNA依赖性RNA合成。在未修饰的CTP存在下，[3H]UMP掺入RNA在180分钟内呈线性增加。当CTP被Hg-CTP取代时，反应的动力学模式和RNA合成速率基本相似。在未修饰的和含Hg-CTP的反应中，70-80%的RNA合成被α-鹅膏蕈碱抑制。通过寡聚（dT）纤维素层析可检测到体外合成的类似信使RNA的一小部分中存在poly（A）。在甲酰胺-蔗糖变性梯度中，poly（A）+和poly（A）-RNA都在15S区域附近以清晰的峰沉降。通过SH-琼脂糖亲和柱层析从内源性核RNA中分离出的Hg-RNA在甲酰胺-蔗糖梯度中也在15S区域附近沉降。现在，体外在分离的乳腺细胞核中合成的Hg-RNA应该可以通过Hg-RNA与酪蛋白mRNA的cDNA进行分子杂交来监测乳腺细胞中特定基因（酪蛋白）转录的激素调节。
