Chemical synthesis and sequence studies of deoxyribooligonucleotides which constitute the duplex sequence of the lactose operator of Escherichia coli

• Published in: The Journal of biological chemistry 1975-06, Vol.250 (12), p.4592-4600
• Main Authors: Itakura, K, Katagiri, N, Narang, S A, Bahl, C P, Marians, K J, Wu, R
• Format: Article
• Language: English
• Subjects: Base Sequence; Chromatography, Thin Layer; Deoxyribonucleotides; Escherichia coli - metabolism; Kinetics; Lactose - biosynthesis; Oligonucleotides - chemical synthesis; Oligonucleotides - metabolism; Operon; Phosphoric Diester Hydrolases; Snake Venoms
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)41343-4

We have synthesized the deoxyribooligonucleotide fragments, constituting the sequence of the lac operator of Escherichia coli. Two of these fragments, d(pApApTpTpGpTpTpApT) (nonamer) and d(pApApTpTpGpTpGpApG) (nonamer), corresponding to the 5' termini of lac operator have been synthesized by the phosphodiester method. The remaining four fragments, d(ApCpApApTpT) (hexamer), d(ApTpApApCpApApTpT) (nonamer), d(ApApTpTpGpTpGpApGpCpGpG) (dodecamer), and d(ApApTpTpGpTpTpApTpCpCpGpCpTpC) (pentadecamer), have been synthesized by an improved phosphotriester method. All of the compounds were first characterized by venom and spleen phosphodiesterase digestion to obtain their base composition. The sequence of these oligonucleotides was fully confirmed by the characteristic mobility shifts of their partial venom phosphodiesterase digestion products on two-dimensional homochromatography. A comparative study of the two methods for the synthesis of oligonucleotides has revealed that the phosphotriester method is more convenient than the phosphodiester method because of higher yields and ease of handling large scale preparations.