大肠杆菌酪氨酸抑制性转移核糖核酸前体结构基因的全合成。11. 酶促连接形成完整的DNA双链体。

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

作者信息

Kleppe R, Sekiya T, Loewen P C, Kleppe K, Agarwal K L, Büchi H, Besmer P, Caruthers M H, Cashion P J, Fridkin M, Jay E, Kumar A, Miller R C, Minamoto K, Panet A, RajBhandary U L, Ramamoorthy B, Sidorova N, Takeya T, van de Sande J H, Khorana H G

出版信息

J Biol Chem. 1976 Feb 10;251(3):667-75.

PMID:175058

Abstract

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.

摘要

已合成了与大肠杆菌酪氨酸转运RNA前体全长（126个核苷酸）相对应的DNA双链体。双链体[I]（关谷彻、贝斯默、竹谷隆和霍拉纳（1976年）《生物化学杂志》251卷，634 - 641页），对应核苷酸序列1 - 26，含有单链末端并带有一个适当标记的5'-磷酸基团，与双链体[II]（洛文、米勒、帕内、关谷彻和霍拉纳（1976年）《生物化学杂志》251卷，642 - 650页）（核苷酸序列23 - 66或23 - 60）相连，双链体[II]在5'-羟基末端用[γ-33P]ATP进行了磷酸化。双链体[III]（帕内、克莱佩、克莱佩和霍拉纳（1976年）《生物化学杂志》251卷，651 - 657页）（核苷酸序列57 - 94（图2））也在5'-末端用[γ-33P]ATP进行了磷酸化，并与双链体[IV]（卡拉瑟斯、克莱佩、克莱佩和霍拉纳（1976年）《生物化学杂志》251卷，658 - 666页）（核苷酸序列90 - 126）相连，双链体[IV]在核苷酸90上带有一个33P标记的磷酸基团。连接后的产物双链体[III + IV]（核苷酸序列57 - 126）得到了表征。后一个双链体与双链体[I + II]相连得到完整的双链体。后者含有单链末端（核苷酸1至6和121至126），这些末端既可以“补齐”以产生完全碱基配对的双链体，也可用于在适当末端添加启动子和终止子区域。