Lau A S, Baliga B S, Roy R K, Sarkar S, Munro H N
Placenta. 1980 Apr-Jun;1(2):169-81. doi: 10.1016/s0143-4004(80)80025-7.
Conditions for isolating intact and active nuclei from human term pacenta and for studying their transcription products are described. The isolated nuclei can synthesize cell-free RNA for a prolonged period at 29 degree C in a medium containing 100 mM KCl and 5 mM MgCl2. Actinomycin D inhibited 92 per cent of RNA synthesis, whereas approximately 60 per cent of RNA synthesis was sensitive to alpha-amanitin. When nuclei were incubated at 29 degrees C for 1 h, about 27 per cent of the newly synthesized RNA was released into the medium outside the nucleus. Analysis of this released material by affinity chromatography on an oligo(dT)-cellulose column revealed that 2.4 per cent of the total released RNA was adsorbed at high salt concentration. Most of this fraction was eluted with a low-salt buffer at 45 degrees C and the remainder by 50 per cent formamide, conditions that are necessary for elution of poly(A)-containing mRNP particles from oligo(dT)-cellulose. These results show that placental nuclei incubated in vitro synthesize poly(A)-containing RNA, which is released as a protein-bound complex. This procedure allows exploration of changes in mRNA release during placental development.
本文描述了从足月人胎盘中分离完整且有活性的细胞核以及研究其转录产物的条件。分离出的细胞核能在含100 mM KCl和5 mM MgCl2的培养基中于29℃长时间合成无细胞RNA。放线菌素D抑制了92%的RNA合成，而约60%的RNA合成对α-鹅膏蕈碱敏感。当细胞核在29℃孵育1小时时，约27%新合成的RNA释放到细胞核外的培养基中。通过在寡聚（dT）-纤维素柱上进行亲和层析分析这种释放的物质，发现在高盐浓度下2.4%的总释放RNA被吸附。该部分的大部分在45℃用低盐缓冲液洗脱，其余部分用50%甲酰胺洗脱，这些条件是从寡聚（dT）-纤维素上洗脱含多聚（A）的mRNP颗粒所必需的。这些结果表明，体外孵育的胎盘细胞核合成含多聚（A）的RNA，并以蛋白质结合复合物的形式释放。该方法可用于探索胎盘发育过程中mRNA释放的变化。
