大豆白化幼苗下胚軸核仁染色質之核小體的分離與其生化性質的研究

Abstract

由2,4-D處理過的大豆白化幼苗下胚軸分離的核仁，以不連續percoll梯度離心部分純化，然後將它解離成核仁染色質，分別用Micrococcal nuclease, DNase I及DNase II(三者都屬內切去氧核糖核酸?)局部水解後，萃取DNA用1.6％洋菜凝膠電泳分析，得到相似的水解圖型，顯示大豆核仁染色質具某種特殊的結構。 用Micrococcal nuclease局部水解大豆核仁染色質，其水解產物以5?20％蔗糖梯度離心(包含 ethidium bromide)分離後，在波長360nm下可覜祭到至少五個吸收帶，由離心管頂至底依序為：單一核小體、核小體二借體、核小體三倍體、核小體四倍禮和核小體五倍體；在離心管最上層另有許多吸收紫外線的物質，可能為遊離的核歸甘酸。分別萃取這五個吸收帶處的DNA,用2.0％洋菜凝膠分析，並根據分子量標準曲線圖換算各DNA片段的分子量，得呈倍數比的相關性，且最小單位的DNA約含一百八十個鹽基對，顯示大豆核仁染色質與其他高等生物細胞核染色質相同，亦具重覆的構造單位，而盤繞此構造單位上的DNA約含一百八十個鹽基對。 經2,4-D處埋的大豆核仁染色質校未經2,4-D處理者，對Ｍicrococcal nuclease 較具敏感性，由此可推測核仁染色質基因被２，４-D活化從,染色質結構鬆散以利遺傅訊息(基因)的裸露及表現。

Nucleoli isolated from 2, 4-D treated soybean hypocotyl were fractionated into nucleolar chromatin by suspending the nucleolar preparation in 30mM DTT. Nucleolar chromatin were digested by Micrococcal nuclease, DNase I or DNase II. DNA fragments generated by the nuclease were analyzed by the technique of agarose gel electrophoresis. We found that the DNA fragments cleaved from nucleolar chromatin by these nucleases formed a regular pattern of bands in the gel; the pattern showed the fragments were like multiples of the smallest unit size. Such a result was in striking contrast to the uniform smear pattern across a gel formed by nuclease-cleaved fragments of naked DNA. Partial Micrococcal nuclease digested nucleolar chromatin were fractionated by a 5% to 20% linear sucrose density gradient (containing ethidium bromide), five 0.D. 254 absorbance peaks were observed when the gradient was scanned with Isco gradient fractionator. DNA. extracted from each peak was subjected to 2.0% agarose gel electrophoresis on a separate track of a gel and molecular size in base pairs calibrated by comparison with unfractionated nuclease digest。The DNA from each fraction formed a single band corresponding either to the size of a 180-base-pair unit or to a dimer, a trimer, a tetrameer or a pentarner of that unit. The nucleolar chromatins from 2, 4-D treated were cleaved faster by Micrococcal nuclease than those from the untreated. These results suggest that nucleolar chromatins were made up of nucleosome which corresponding to the size of a 180 base pairs unit as nuclear chromatin shown in the other system. 2, 4-D treatment caused the nucleolar chromatin more susceptible to Micrococcal nuclease digestion suggest that these nucleolar chromatin was more active for rjbosomal RNA synthesis.