PRESENCE OF N-FORMYL1-METHIONY1-TRNA IN HELA CELL MITROCHONDRIA
PRESENCE OF N-FORMYL1-METHIONY1-TRNA IN HELA CELL MITROCHONDRIA
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DOI：
10.1016/0006-291x(69)90633-0
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发表时间：
1969-01-01
影响因子：
3.1
通讯作者：
DARNELL, JE
中科院分区：
文献类型：
--
作者：
GALPER, JB;DARNELL, JE
方法：HeLa细胞在添加有7%马血清的Eagle培养基（Eagle，1959）中悬浮培养。在所有实验中，在37分钟收集细胞，重悬并在相同体积的含有5%透析马血清的无甲硫氨酸培养基中孵育。5分钟后，再次离心细胞并以4 × 106个细胞/ml重悬2分钟，然后加入均匀标记的甲硫氨酸（1.5-3 × 105 μ m）。为了使细胞在氨基蝶呤存在下生长，在Hakala和Taylor（1959）的条件之后，向培养基中补充胸苷、腺嘌呤、甘氨酸和丝氨酸。已经描述了通过冷酚提取分离RNA（Pene，Knight和Darnell，1968）。所有的RNA制备物或者对O. OlM乙酸钠-EDTA，pH 5.1，或在RNA酶处理之前用乙醇沉淀三次。RNA酶消化和通过RNA酶消化产物的电泳鉴定涉及Marker和桑格（1964）的程序。将电泳图切成1 cm的条带，用1 ml浓NH 401 H处理，然后用20 ml Bray溶液处理，并在液体闪烁光谱仪中计数。E.大肠杆菌提取物和充电5.根据--Marker（1965）进行大肠杆菌f-met-tRNA。
METHODS-m HeLa cells were grown iin suspension culture, in Eagle’s medium (Eagle, 1959) supplemented with 7% horse serum. In all experiments cells were co1 lected at 37', resuspended and incubated in the same volume of methionineless medium containing 5% dialyzed horse serum. After 5 minutes the cells were again centrifuged and resuspended at 4 x lo6 cells/ml for 2’before the addition of uniformly labeled methionine(1.5-3 &ml; 205 &pm01 e). For the growth of cells in the presence of aminopterin, the medium was supplemented with thymidine, adenine, glycine and serine after the conditions of Hakala and Taylor(1959). Isolation of RNA by cold phenol extraction has been described(Pene, Knight & Darnell, 1968). All RNA preparations were either dialyzed overnight against O. OlM Na acetate-EDTA, pH 5.1, or precipitated with ethanol three times before RNAse treatment. RNAse digestion and identification by electrophoresis of the products of RNAse digestion involved the procedures of Marker and Sanger(1964). The electropherograms were cut into 1 cm strips, treated with 1 ml of concentrated NH401H followe’d by 20 ml of Bray’s solution and counted in a liquid scintillation spectrometer. Preparation of E. coli extracts and charging of 5. coli f-met-tRNA was performed according to--Marker(1965).