STUDIES ON POLYNUCLEOTIDES .88. ENZYMATIC JOINING OF CHEMICALLY SYNTHESIZED SEGMENTS CORRESPONDING TO GENE FOR ALANINE-TRNA
STUDIES ON POLYNUCLEOTIDES .88. ENZYMATIC JOINING OF CHEMICALLY SYNTHESIZED SEGMENTS CORRESPONDING TO GENE FOR ALANINE-TRNA
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DOI：
10.1073/pnas.60.4.1338
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发表时间：
1968-01-01
影响因子：
11.1
通讯作者：
KHORANA, HG
中科院分区：
文献类型：
--
作者：
GUPTA, NK;OHTSUKA, E;KHORANA, HG
材料和方法-脱氧核多核苷酸：图1所示的所有脱氧核糖核酸、寡核苷酸和多核苷酸都是化学合成的产物。除了四核苷酸，dTCTC，/这些多核苷酸的合成仍有待发表。3在DNA连接酶的孵育混合物中，只有短链寡核苷酸组分dTCTC、dTCTCC含有5‘-磷酸末端基团，并用p32标记，如图1所示。5’-P32标记产物是用y-P32标记的三磷酸腺苷(ATP)和多核苷酸激酶(PK)将5‘-OH基团磷酸化而成。5我们感谢张世昌博士制备了y标记的三磷酸腺苷和多核苷酸激酶。连接反应测定及产物分离：标准孵育混合物(0.015~0.06ml)每毫升含8 mM三(羟甲基)氨基甲烷-氯缓冲液，pH 7.6，5 mM氯化镁，，三磷酸腺苷，8 mM二硫苏糖醇，ICOSA-I和ICOSA-II各0.4ODX0，等量5‘-P32标记的短寡核苷酸。在T4-连接酶的实验中，酶的浓度为1100个单位/毫升，并在一些实验中添加了可比的水平。大肠杆菌连接酶以200单位/毫升的速度使用。(用前面描述的方法同时测试两种酶的比活力。5)孵化温度为5~20℃。取不同间隔取出的等量(1-5，Au)，用0.1M Tris-Cl缓冲液，pH 8.0，取0.08ml，1000℃煮沸2min后，在700℃与25/Ag细菌碱性磷酸酶孵育30mnin。然后将混合物应用于O-(二乙氨基乙基)纤维素纸条进行层析，并如前所述对纸条进行放射性计数。5保留在原产地的接合产物用1M三乙基铵碳酸氢钠-1338洗脱
Materials and Methods.-Deoxyribopolynucleotides: All of the deoxyribo-, oligo-, and polynucleotides shown in Figure 1 are chemically synthesized products. Except for the tetranucleotide, dTCTC,/syntheses of these polynucleotides remain to be published. 3 In the incubation mixtures for theDNA-joining enzymes, only the short oligonucleotidic components, eg, dTCTC, dTCTCC, contained 5'-phosphate end groups and these were labeled with p32 as shown in Figure 1. The 5'-P32-labeled products were prepared by phosphorylation of the 5'-OH groups using y-P32-labeled adenosine 5'-triphosphate (ATP) and polynucleotide kinase as described previously. 5 We are grateful to Dr. S. Chang for preparation of the y-labeled ATP and of the polynucleotide kinase. Assay of the joining reactions and isolation of the products: The standard incubation mixture (0.015-0.06 ml) contained per ml: 8 mM tris (hydroxymethyl) aminomethane (Tris)-Cl buffer, pH 7.6, 5 mM MgCl2, 64, uM ATP, 8 mM dithiothreitol, 0.4 ODX0 each of Icosa-I and Icosa-II, and equivalent amounts of 5'-P32-labeled short oligonucleotides. In the experiments with T4-joining enzyme, the concentration of the enzyme was 1100 units/ml and in some experiments subsequent additions of comparable level were made. The E. coli joining enzyme was used at 200 units/ml.(Both enzymes were tested for specific activity side by side by using the assay described previously. 5) Incubations were at 5-20'. Aliquots (1-5, Au) removed at different intervals were takell to 0.08 ml with 0.1 M Tris-Cl buffer, pH 8.0, and after the solutiols had been boiled at 1000 for 2 min, they were incubated with 25/Ag of bacterial alkaline phosphatase at 700 for 30mnin. The mixtures were then applied to O-(diethylaminoethyl) cellulose paper strips for chromatography and the strips were counted for radioactivity as described before. 5 The joined product which remained at the origin was eluted with 1 M triethylammonium bicarbo-1338