Tompson J G, Agris P F
Nucleic Acids Res. 1979 Oct 10;7(3):765-79. doi: 10.1093/nar/7.3.765.
Escherichia coli C6 rel met cys was cultured in a stringently defined minimal medium containing 13C-enriched metabolites in order to (1) achieve maximal 13C isotopic enrichment of tRNA; and (2) produce site specific but natural, non-perturbing NMR probes of tRNA structure and function. Growth conditions were manipulated to achieve optimal culture growth concomitant with maximal in vivo incorporation of various 13C-enriched nucleic acid precursors, including L-[methyl-13C] methionine, [2-(13)C] adenine, and [2-(13)C] uracil. Effective blockage of purine biosynthesis de novo was accomplished with the addition of the antimetabolite 6-mercaptopurine to the growth medium. Transfer RNAs specifically 13C-enriched in all methyl groups (57 atom %), C2 of adenine (60 atom %), and C2 of uracil (82 atom %) and C2 of cytosine (73 atom %) have been produced.
大肠杆菌C6 rel met cys在含有13C富集代谢物的严格定义的基本培养基中培养,目的是:(1)实现tRNA的最大13C同位素富集;(2)产生tRNA结构和功能的位点特异性但天然的、无干扰的NMR探针。通过控制生长条件,实现最佳的培养生长,同时使各种13C富集的核酸前体在体内最大程度地掺入,包括L-[甲基-13C]甲硫氨酸、[2-(13)C]腺嘌呤和[2-(13)C]尿嘧啶。通过向生长培养基中添加抗代谢物6-巯基嘌呤,有效地阻断了嘌呤的从头生物合成。已经产生了在所有甲基(57原子%)、腺嘌呤的C2(60原子%)、尿嘧啶的C2(82原子%)和胞嘧啶的C2(73原子%)中特异性13C富集的转移RNA。