NUCLEOTIDE SEQUENCE AND CONTROL OF THE GDH OPERON

在导致孢子产生的发育过程中， 枯草芽孢杆菌，合成许多蛋白质。 我们有 一直在研究这样一种蛋白质，葡萄糖脱氢酶， 仅在孢子形成期间和前孢子中产生 车厢 编码葡萄糖脱氢酶（GDH）的基因是 双顺反子单元的一部分。 来自S1核酸酶图谱 实验中，转录起始位点的位置是 确定为距离核糖体结合位点12个碱基对 在操纵子的第一个基因之前。 该区域包含 确定GDH表达所需的核苷酸序列。 DNA片段与启动子的Bal 31核酸酶双链反应 区域与无启动子的lacZ基因融合， 将融合物插入到B中。枯草染色体 还通过 寡核苷酸定点突变，碱基改变 在-35和-22区域进行测序。 变化- 35区域导致lacZ表达的缺失，而没有影响 在lacZ表达的变化，注意到在- 22区。 目前正在对-10区域进行改造 化验。 同样的技术也被用来修改 在B。 枯草杆菌gdh基因位于His 148和Trp 154区域附近 这对巨大芽孢杆菌中的亚基结合很重要 葡萄糖脱氢酶 插入两个氨基酸（瓦尔， Asn）进入B。枯草杆菌gdh基因产生的提取物中只有5%的 活性与未改变的葡萄糖脱氢酶相比。

During the developmental process leading to spore production in Bacillus subtilis, numerous proteins are synthesized. We have been studying one such protein, glucose dehydrogenase, which is made exclusively during sporulation and only in the forespore compartment. The gene encoding glucose dehydrogenase (gdh) is part of a bicistronic unit. From S1 nuclease mapping experiments, the location of the transcriptional initiation site was determined to be 12 base pairs from the ribosome binding site preceding the first gene of the operon. The region containing the nucleotide sequences needed for gdh expression was determined. Bal31 nuclease digestions of DNA fragments with the promoter region were fused to a promoterless lacZ gene and the gene fusions were inserted into the B. subtilis chromosome. Also, by oligonucleotide site directed mutagenesis, alterations in the base sequence at the -35 and -22 region were made. Changes in the - 35 region resulted in the loss of lacZ expression whereas no effect on lacZ expression was noted by change of base sequences in the - 22 region. Alterations in the -10 region are currently being assayed. The same technique was employed to make alterations in the B. subtilis gdh gene near the His 148 and Trp 154 region which is important for subunit binding in the Bacillus megaterium glucose dehydrogenase. An insertion of two amino acids (Val, Asn) into the B. subtilis gdh gene yielded extracts with only 5% of activity as compared to unaltered glucose dehydrogenase.