Nuclear ribonucleoprotein particles probed in living cells.
Nuclear ribonucleoprotein particles probed in living cells.
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在活细胞中探测核糖核蛋白颗粒。
DOI：
10.1073/pnas.78.4.2208
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发表时间：
1981
影响因子：
11.1
通讯作者：
Pederson,T
中科院分区：
文献类型：
--
作者：
Mayrand,S;Pederson,T
在完整的HeLa或Friend红白血病细胞中，通过剂量为10(1)至10(5)ergs/mm2（1至10(4)microJ/mm2）的254 nm光照射，异质核RNA （hnRNA）与核核糖核蛋白颗粒中的蛋白质之间的接触进行了光化学交联。分离得到的交联颗粒并与常规hnRNA进行比较。蛋白（hnRNP）的制备。通过核分离行为、沉降系数、核酸酶消化谱和Cs2SO4密度梯度带测RNA-to-protein比值的标准，体内交联的hnRNP颗粒与未辐照的颗粒完全相同。凝胶印迹杂交来自Friend细胞hnRNP交联的RNA显示，辐照后β -珠蛋白RNA序列保持完整和可杂交。交联的hnRNA-蛋白键在8 M尿素/0.5%十二烷基硫酸钠溶液中稳定，并且经得起初始密度为1.50 g/cm3的Cs2SO4梯度离心。这些结果表明，hnRNA在体内与核蛋白紧密络合，并且通过核分离分离的hnRNP颗粒代表天然结构。
Contacts between heterogeneous nuclear RNA (hnRNA) and protein in nuclear ribonucleoprotein particles have been photochemically crosslinked in intact HeLa or Friend erythroleukemia cell by irradiation with 254-nm light at doses of 10(1) to 10(5) ergs/mm2 (1 to 10(4) microJ/mm2). The resulting crosslinked particles were isolated and compared with conventional hnRNA . protein (hnRNP) preparations. By the criteria of nuclear fractionation behavior, sedimentation coefficients, nuclease digestion profiles, and RNA-to-protein ratio measured by banding in Cs2SO4 density gradients, the hnRNP particles crosslinked in vivo are identical to nonirradiated particles. Gel blot hybridization of RNA from Friend cell hnRNP crosslinked in vivo reveals that beta-globin RNA sequences remain both intact and hybridizable after the irradiation procedure. The crosslinked hnRNA--protein bonds are stable in 8 M urea/0.5% sodium dodecyl sulfate and withstand centrifugation in Cs2SO4 gradients of initial density 1.50 g/cm3. These results establish that hnRNA is tightly complexed with nuclear proteins in vivo and that hnRNP particles isolated by nuclear fractionation represent native structures.