MOLECULAR-CLONING OF THE GENE FOR PHOSPHOFRUCTOKINASE-2 OF ESCHERICHIA-COLI AND THE NATURE OF A MUTATION, PFKB1, CAUSING A HIGH-LEVEL OF THE ENZYME
MOLECULAR-CLONING OF THE GENE FOR PHOSPHOFRUCTOKINASE-2 OF ESCHERICHIA-COLI AND THE NATURE OF A MUTATION, PFKB1, CAUSING A HIGH-LEVEL OF THE ENZYME
复制标题
DOI：
10.1016/s0022-2836(83)80019-9
复制
发表时间：
1983-01-01
影响因子：
5.6
通讯作者：
DALDAL, F
中科院分区：
文献类型：
--
作者：
DALDAL, F
已知大肠杆菌的 pfkB 基因在野生型菌株中指定一种次要的磷酸果糖激酶 Pfk-2； pfkB1 突变导致 PFk-2 的量增加 25 倍，从而足以替代主要磷酸果糖激酶 Pfk-1（由 pfkA 指定）的突变损失；另一个密切相关的突变 pfkB10 影响 Pfk-2 的结构。本文是关于 pfkB1 突变的。将pfkB+、pfkB1和pfkB1 pfkB10克隆并亚克隆到质粒pBR322上：在所有3个例子中，它们的功能均由2.1×携带。 103个碱基对片段具有相似或相同的限制模式。 maxicells 实验证实，克隆片段包含结构基因及其表达水平的决定因素。酶的 N 端测序结果与 DNA 序列相匹配，确定了基因的位置和方向。 408个碱基对的HindIII-SmaI片段，其中包括非编码5''区的294个碱基对和蛋白质编码序列的114个碱基对，与启动子克隆载体pK01上的galK融合； pfkB1 融合引起 galK 高水平表达。 pfkB+和pfkB1的体外转录允许确定两种情况下的+1位置，位于起始甲硫氨酸密码子之前约19个碱基对； pfkB1 的转录水平比 pfkB+ 的转录水平高得多。 pfkB+ 和 pfkB1 的 408 个碱基对片段的 DNA 序列仅在一个残基上不同，即 pfkB1 突变，证明是转录起始后约 -12 位处的 C 到 T 变化。
The pfkB gene of E. coli is known to specify a minor phosphofructokinase, Pfk-2, in the wild-type strain; the pfkB1 mutation causes a 25-fold increase in the amount of PFk-2 so that it adequately substitutes for mutational loss of the major phosphofructokinase, Pfk-1 (specifed by pfkA); and another closely linked mutation, pfkB10, affects the structure of Pfk-2. This paper is about the pfkB1 mutation. pfkB+, pfkB1 and pfkB1 pfkB10 were cloned and subcloned on plasmid pBR322: their functions were carried, in all 3 cases, by a 2.1 .times. 103 base-pair fragment with a similar or identical restriction pattern. Experiments with maxicells confirmed that the cloned fragments included the structural gene as well as the determinants of its level of expression. Results of N-terminal sequencing of the enzyme matched with the DNA sequence and established the position and direction of the gene. A HindIII-SmaI fragment of 408 base-pairs, which included 294 base-pairs of the non-coding 5'' region and 114 base-pairs of the protein coding sequence, was fused to galK on the promoter cloning vector pK01; in the fusion pfkB1 caused a high level expression of galK. Transcription in vitro from pfkB+ and pfkB1 allowed the determination of the +1 position in both cases, at about 19 base-pairs before the initiating methionine codon; the level of transcription was much higher from pfkB1 than from pfkB+. The DNA sequence of the 408 base-pair fragments from pfkB+ and pfkB1 differ only in a single residue, the pfkB1 mutation, proving to be a C to T change at position about -12 from the initiation of transcription.