Isolation of Rat α1-Fetoprotein Messenger RNA from Morris Hepatoma 7777
Isolation of Rat α1-Fetoprotein Messenger RNA from Morris Hepatoma 7777
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从莫里斯肝癌 7777 中分离大鼠 α1-胎蛋白信使 RNA
DOI：
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发表时间：
1979
期刊：
影响因子：
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通讯作者：
J. Chiu
中科院分区：
文献类型：
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作者：
L. Bélanger;P. Commer;J. Chiu
建立了一种从Morris肝癌7777细胞中分离合成甲胎蛋白(α)多聚体的双抗体方法。含多聚腺苷酸的RNA经蔗糖梯度沉淀和寡脱氧胸腺酸-纤维素层析纯化后，在聚丙烯酰胺凝胶电泳中以单一的21S组分迁移；在无细胞翻译系统中，它产生的多肽产物可被抗大鼠AFP抗血清免疫沉淀，但不能被抗大鼠白蛋白免疫沉淀，其迁移速度略快于血清AFP在十二烷基硫酸钠-尿素-聚丙烯酰胺凝胶上的迁移。该信使RNA组分被用于合成放射性互补DNA。在杂交实验中，互补DNA与其纯化的模板以1.5×10-2[RNA浓度(每升核苷酸摩尔)×半衰期(秒)的乘积]的Crt1/2重新结合。参照该值，可杂交序列分别占Morris肝癌7777、10日龄大鼠肝脏和成年大鼠肝脏含多聚腺苷酸的多聚体RNA总量的3、2和0.01%。多聚体免疫沉淀系统的高度特异性，分离的信使RNA组分的电泳均一性，其选择性地翻译成AFP，以及杂交探针的特异性，表明所述的方法可获得高纯度的大鼠AFP信使RNA。
A double-antibody procedure has been developed for the isolation of α1-fetoprotein (AFP)-synthesizing polysomes from Morris hepatoma 7777. The polyadenylic acid-containing RNA, subsequently purified by differential sedimentation on sucrose gradient and oligodeoxythymidylic acid-cellulose chromatography, migrates as a single 21S component in polyacrylamide gel electrophoresis; in a cell-free translation system, it yields a peptide product immunoprecipitable by anti-rat AFP antiserum, but not by anti-rat albumin, and which migrates slightly faster than serum AFP on sodium dodecyl sulfate-urea-polyacrylamide gels. This messenger RNA fraction was used for the synthesis of a radioactive complementary DNA. In hybridization assays, the complementary DNA reassociated with its purified template at a Cr t 1/2 [product of RNA concentration (mol of nucleotides per liter) × half-time (sec)] of 1.5 × 10-2. By reference to this value, hybridizable sequences were found to constitute 3, 2, and <0.01% of total polyadenylic acid-containing polysomal RNA of Morris hepatoma 7777, 10-day-old-rat liver, and adult rat liver, respectively. The high specificity of the polysome immunoprecipitation system, the electrophoretic homogeneity of the isolated messenger RNA fraction, its selective translation into AFP, and the specificity of the hybridization probe indicate that the procedure described yields a highly purified rat AFP messenger RNA.