CLONING AND CHARACTERIZING B. LICHENIFORMIS APASE GENES

我们的初步结果已经确定，至少有两个结构 地衣芽孢杆菌中的APase基因，APase I和APase II，以及这2个基因 是由不同的RNA聚合酶全酶转录的。我们将决定 如果这是之前仅有的两个与APase物种有关的基因 通过使用克隆的基因来定位和分离这种有机体 构建地衣芽孢杆菌APase I-APase II双突变株。如果 双突变体不是APase阴性，我们将克隆另一个APase基因(S) 并将这个克隆的基因(S)包括在下面所有拟议的实验中。 这些实验旨在检验这样一种假设，即唯一的 碱性磷酸酶在地衣芽孢杆菌中的分布(1) 膜相关(完整--外膜蛋白；外周--外膜--和 内源蛋白)和(2)分泌--主要在静止生长中)是 相似的多个结构基因在不同调控下的结果 控制力。我们建议： 1.确定B.中是否存在2个以上的APase结构基因。 并构建了地衣芽孢杆菌MC14突变体，该突变体 仅保留1个功能性APase基因。 2.分析各克隆APase基因的转录调控。 3.测定各APase基因的DNA序列，比较 由衍生的APase蛋白确定的功能结构域 氨基酸序列。 4.在APase突变体中确定APase的最终目的地 只保留1个APase基因。

Our preliminary results have established there are at least 2 structural APase genes in B. licheniformis, APase I and APase II, and that the 2 genes are transcribed by different RNA polymerase holoenzymes. We will determine if these are the only 2 genes responsible for the APase species previously localized and isolated from this organism, by using the cloned genes to construct a APase I-APase II double mutant of B. licheniformis. If the double mutant is not APase negative, we will clone the other APase gene(s) and include this cloned gene(s) in all the proposed experiments below. These experiments are designed to examine the hypothesis that the unique distribution of alkaline phosphatase in B. licheniformis ((1) membrane-associated (integral--ecto-protein; peripheral--ecto- and endo-protein) and (2) secreted - primarily in stationary growth) is the result of similar multiple structural genes under different regulatory control. We propose: 1. To determine if there are more than 2 APase structural genes in the B. licheniformis genome and construct B. licheniformis MC14 mutants which retain only 1 functional APase gene. 2. To analyze the transcriptional regulation of each clone APase gene. 3. To determine the DNA sequence of each APase gene and compare the functional domains of the APase proteins as determined from the derived amino acid sequence. 4. To determine the final destination of the APase in APase mutants which retain only 1 APase gene.