大豆幼苗下胚軸核仁染色質與核仁先質核糖體的分離及其生化組成的研究

Abstract

由2,4-D處理過之大豆幼苗下胚軸分離核仁，再利用30mM dithiothreitol使核仁瓦解而分離核仁染色質及先質核糖體。核仁內的RNA聚台?Ⅰ的活性佔總活性的90%以上，當把核仁瓦解分出核仁染色質及先質核糖體後，約有90%以上的RNA聚合?Ⅰ出觀在核仁染色質上，而先質核糖體部份只佔10%左右。但先質核糖體卻含有較高的RNA聚合?Ⅱ的活性。 核仁染色質的RNA與蛋白質的比例約為1:3；若以核仁的結DNA當 100，則核仁染色質的DNA約佔90%，而只有10％在先質核糖體部份。若把分離核仁染色質後的上清液用200,000×g將先質核糖體離心下來，則其RNA 與蛋白質的比值為1:1.13。將此先質核糖體經15-60%連續蔗糖濃度梯度離心後，發現其沉降係數約在80 S附近，與細胞質單核糖體類似。 試以2.4露膠體電泳分析核仁、核仁染色質與先質核糖體的RNA類型，並以細胞質80 S單核糖體之RNA作為對照標準。由結果知先質核糖體的rRNA類型與細胞質單核糖體類似，即主要為25 S及18 S二種rRNA所組成；但核仁染色質則除了有25 S及18 S RNA外，尚有大量的小分子RNA，這些少分子RNA所佔的比率比核仁大，由此可推知核仁染色質是這些小分子RNA存在的主要場所。 細胞質80 S單核糖體的鹼性蛋白質，在二度膠體電泳分析後，發現共有86個蛋白質點；而先質核糖體的鹼性蛋白質同樣做二度膠體電泳分析，共有77點，其中71點與核糖體蛋白質一樣。核仁染色質的鹼性蛋白質則共有54個，其中最主要的有9點；這9點經鑑定後是屬於組蛋白，而其約佔全部核仁染色質鹼性蛋白質80％以上。

Nucleoli isclated from auxin-treated soybean hpocotyl were fractionated into nucleclar chromatin and nucleolar preribosoral particles by suspending the nucleolar preparation in 30 mM DTT. Majority of RNA polymerase I activity in nucleoli was recevered in nucleolar chromatin fraction. Nucleolar chrocmatin also contained most of nucleolar DNA and RNA to protein ratio was 1:3. Only less than l0% of RNA polymerase I activity in nucleoli was found in nucleolar preribosonal fraction. Nucleolar preribosomal particles contained RNA to protein ratio of 1:1. When the nucleolar preribosomal particles were layered on 15-60% 1inear sucrcse gradient for centrifugation. they sedinented lighter than run-off 80 S single ribosorres. RNA species extracted from preribosomal particles were analyzed with 2.4% polvacrylamide gel using rRNAs from 80 S run-off side ribosomes as reference. The results showed that preribosomal particles mainly consisted of 25 S and 18 S rENA in a ratio of 1:1. However, RNA species isolated from nucleolar chrorratin consisted of low molecular weight RNAs besides 25 S and 18 S rNAS. Acid-soluble proteins associated with preribosomal particles were resolved into 77 protein spots on 2-D gel electrophoresis and 71 protein spots were presented in 80 S run-off ribosomes. They contributed core thar. 80% of total acid-soluble proteinss in nueleolar preribosoral particles. Acid-soiub1e proteins associated with nucleclar chromatin. were resolved into 54 protein spots and 9 of which were identified as histones. They constituted core than 80% of acid-soluble proteins in nucleolar chromatin.