NUCLEOTIDE SEQUENCE AND CONTROL OF THE GDH OPERON

在导致产孢子的发育过程中 枯草芽孢杆菌，合成了大量的蛋白质。我们有 我一直在研究一种这样的蛋白质，葡萄糖脱氢酶，它是 仅在孢子形成期间和仅在前孢子中产生 车厢。编码葡萄糖脱氢酶(GDH)的基因是 双声道单元的一部分。从S1核酸酶图谱 实验中，转录起始点的位置是 确定为核糖体结合部位的12个碱基对 在操纵子的第一个基因之前。该区域包含 确定了GDH表达所需的核苷酸序列。 启动子对DNA片段的Bal31核酸酶消化 区域与无启动子的LacZ基因和该基因融合 融合基因被插入到枯草杆菌染色体中。此外，由 寡核苷酸位点定向突变，碱基改变 测定了-35区和-22区的序列。变化-- 35区导致LacZ表达缺失，但无影响 在LacZ的表达上，通过碱基序列的变化来记录- 22个地区。-10区域的变化目前正在进行中 化验过了。同样的技术也被用来进行修改 枯草杆菌His 148区和Trp 154区附近的GdH基因 这对巨大芽孢杆菌中的亚基结合是重要的 葡萄糖脱氢酶。插入两个氨基酸(Val， Asn)转化枯草杆菌GdH基因，得到的提取物仅有5% 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.