Fructose bisphosphatase of Saccharomyces cerevisiae. Cloning, disruption and regulation of the FBP1 structural gene.
Fructose bisphosphatase of Saccharomyces cerevisiae. Cloning, disruption and regulation of the FBP1 structural gene.
复制标题
酿酒酵母的果糖双磷酸酶。
DOI：
10.1016/0022-2836(85)90107-x
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发表时间：
1985
影响因子：
5.6
通讯作者：
Fraenkel,DG
中科院分区：
文献类型：
--
作者：
Sedivy,JM;Fraenkel,DG
果糖双磷酸酶催化糖异生的关键反应。我们通过筛选基因组文库来补充大肠杆菌 fbp 缺失突变，从酿酒酵母中克隆了果糖双磷酸酶 (FBP1) 结构基因。克隆的 DNA 在 E 中表达。大肠杆菌果糖双磷酸酶活性可通过酵母酶特异性抗体沉淀，并且对果糖 2,6-二磷酸的抑制作用敏感。有证据表明整个基因，包括全顺式作用调节序列，已被克隆。通过置换将破坏FBP1的取代突变整合到酵母基因组中，构建果糖双磷酸酶无效突变。 FBP1突变菌株是己糖营养缺陷型，否则生长正常。 Southern印迹杂交分析证实了置换结构并证明FBP1在单倍体基因组中以单拷贝存在。 Northern印迹杂交分析显示大约1350个核苷酸的mRNA，其存在可被培养基中的葡萄糖抑制。当FBP1基因存在于酵母中的多拷贝载体上时，果糖双磷酸酶活性并没有大大过量产生。
Fructose bisphosphatase catalyzes a key reaction of gluconeogenesis. We have cloned the fructose bisphosphatase (FBP1) structural gene fromSaccharomyces cerevisiaeby screening a genomic library for complementation of anEscherichia coli fbpdeletion mutation. The cloned DNA expresses inE. colia fructose bisphosphatase activity which is precipitable with antibodies specific for the yeast enzyme and is sensitive to inhibition by fructose 2,6-bisphosphate. Evidence is presented demonstrating that the entire gene, including allcis-acting regulatory sequences, has been cloned. A substitution mutation that disruptsFBP1 was incorporated into the yeast genome by transplacement to construct a fructose bisphosphatase null mutation. TheFBP1 mutant strain is a hexose auxotroph, otherwise growing normally. Southern blot hybridization analysis confirmed the structure of the transplacement and demonstrated thatFBP1 is present in single copy in the haploid genome. Northern blot hybridization analysis revealed an mRNA of about 1350 nucleotides, whose presence was repressible by glucose in the medium. Fructose bisphosphatase activity was not greatly overproduced when theFBP1 gene was present on a multicopy vector in yeast.