Sagher D, Grosfeld H, Edelman M
Proc Natl Acad Sci U S A. 1976 Mar;73(3):722-6. doi: 10.1073/pnas.73.3.722.
RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT)-cellulose at 4 degrees. The poly(A)-containing and poly(A)-lacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1.1.39; 3-phospho-D-glycerate carboxy-lyase (dimerizing)]. The large subunit polypeptide (59,000 molecular weight) was identified in the in vitro reaction by two-dimensional electrophoresis involving isoelectric focusing and size filtration on polyacrylamide gels. Template activity for the large subunit was detected in wild-type cells but not in aplastidic mutant strains; it was highly enriched in the isolated chloroplasts. This messenger was present only in the poly(A)-lacking RNA fraction, where it constituted the most prominent template species of chloroplast RNA. The large subunit message was freed of considerable non-messenger RNA contamination and localized to the 10-20S fraction by sucrose gradient centrifugation.
将来自野生型眼虫、无质体突变细胞和纯化叶绿体的RNA在4℃下于寡聚（dT）-纤维素上进行层析。然后，将来自每种样品的含聚腺苷酸（poly(A)）和不含聚腺苷酸的组分在小麦胚芽的无细胞蛋白质合成系统中进行测试，以检测它们特异性刺激大亚基核酮糖二磷酸羧化酶[EC 4.1.1.39；3-磷酸-D-甘油酸羧基裂解酶（二聚化）]合成的能力。通过二维电泳（包括等电聚焦和在聚丙烯酰胺凝胶上的尺寸过滤）在体外反应中鉴定大亚基多肽（分子量59,000）。在野生型细胞中检测到了大亚基的模板活性，但在无质体突变菌株中未检测到；它在分离的叶绿体中高度富集。这种信使仅存在于不含聚腺苷酸的RNA组分中，在那里它构成了叶绿体RNA中最突出的模板种类。通过蔗糖梯度离心，大亚基信使摆脱了相当多的非信使RNA污染，并定位于10 - 20S组分。
