Kraus J P, Rosenberg L E
Proc Natl Acad Sci U S A. 1982 Jul;79(13):4015-9. doi: 10.1073/pnas.79.13.4015.
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.
我们通过多核糖体免疫吸附将三种低丰度肝脏mRNA纯化至接近均一状态。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（生成ADP），EC 6.4.1.3]以及胱硫醚β-合酶[L-丝氨酸水解酶（添加高半胱氨酸），EC 4.2.1.22]的mRNA，分别约占肝脏总mRNA的0.20%、0.02%和0.015%，被纯化了450至6300倍。我们采用了以下步骤：大鼠肝脏多核糖体与针对每种多肽产生的单特异性抗血清的IgG组分相互作用；将多核糖体-抗体复合物固定在蛋白A-琼脂糖柱上；通过大量洗涤去除大部分多核糖体；解离核糖体亚基并用EDTA洗脱特异性mRNA；以及通过在寡聚（dT）-纤维素柱上进行层析分离洗脱的mRNA。看来该方法将有可能分离其他低丰度mRNA并随后克隆其各自的cDNA。
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