Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption.
复制标题
通过多核糖体免疫吸附从大鼠肝脏中纯化低丰度信使 RNA。
DOI：
10.1073/pnas.79.13.4015
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发表时间：
1982
影响因子：
11.1
通讯作者：
Rosenberg,LE
中科院分区：
文献类型：
--
作者：
Kraus,JP;Rosenberg,LE
我们已经纯化了三个低丰度肝mRNA的多核糖体免疫吸附接近同质。编码鸟氨酸转氨甲酰酶前体[氨甲酰磷酸：L-鸟氨酸氨甲酰转移酶，EC 2.1.3.3]、丙酰辅酶A羧化酶β亚基前体[丙酰辅酶A：二氧化碳连接酶（ADP形成），EC 6.4.1.3]和胱硫醚β合酶[L-丝氨酸水解酶（添加同型半胱氨酸），EC 4.2.1.22]的mRNA分别约占总肝脏mRNA的0.20、0.02和0.015%，纯化450- 6，300倍。我们使用了以下步骤：大鼠肝多核糖体与针对每种多肽产生的单特异性抗血清的IgG部分的相互作用;多核糖体-抗体复合物在蛋白A-琼脂糖柱上的固定化;通过大量洗涤去除大部分多核糖体;核糖体亚基的解离和用EDTA洗脱特异性mRNA;以及通过在寡聚（dT）-纤维素柱上的色谱分离洗脱的mRNA。这一过程似乎将允许分离其他低丰度mRNA并随后克隆它们各自的cDNA。
We have purified three low-abundance hepatic mRNAs to near homogeneity by polysome immunoadsorption. The mRNAs coding for the precursor of ornithine transcarbamoylase [carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3], the precursor of the beta-subunit of propionyl-CoA carboxylase [propionyl-CoA:carbon dioxide ligase (ADP-forming), EC 6.4.1.3], and cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22], representing approximately 0.20, 0.02, and 0.015% of total hepatic mRNA, respectively, were purified 450- to 6,300-fold. We used the following steps: interaction of rat liver polysomes with an IgG fraction of monospecific antisera raised against each polypeptide; immobilization of polysome-antibody complexes on a protein A-Sepharose column; removal of the bulk of polysomes by extensive washing; dissociation of ribosomal subunits and elution of specific mRNA with EDTA; and isolation of the eluted mRNA by chromatography on an oligo(dT)-cellulose column. It seems likely that this procedure will permit isolation of other low-abundance mRNAs and subsequent cloning of their respective cDNAs.