MICROBIAL DEGRADATION OF AGENT ORANGE

该提案的主要目的是了解 基因的进化、组织和调控 降解2，4-二氯苯氧乙酸（2，4-D）， 2，4，5-三氯苯氧乙酸（2，4，5-T）- 除草剂橙子。 橙子的出现，特别是一个 它的成分，2，4，5-T，在大浓度的各个部分， 世界已经造成了公共卫生问题，只有这样才能最大限度地减少， 这些污染物是通过微生物从污染场地中清除的， 降解 已知质粒如pAC 27编码降解的 而质粒如pJP 2和pJP 4编码一个氯儿茶酚途径， 完整的2，4-D降解途径，包括氯儿茶酚。 由于这些 三种质粒在大小和不相容性特征上不同， 证明了氯儿茶酚基因的广泛聚类和同源性， 将尝试克隆单独的氯儿茶酚和 来自质粒pJP 2和pJP 4的苯氧乙酸降解基因，测定 它们的完整核苷酸序列，并比较开放阅读框， 以及pAC 27的上游序列，以观察 氯儿茶酚基因是相同的。 每个基因的产物 将在tac启动子的转录控制下过量产生 并对其酶活性进行了研究。 使用2，4，5-T降解菌株 洋葱假单胞菌AC 1100，其中Tn 5诱变允许分离 在大量的2，4，5-T阴性突变体中，将尝试 将Tn 5定位在质粒上或质粒上， 染色体DNA，从而证明2，4，5-T基因的位置。 在一种情况下，转座子诱变后与Tn 5杂交， DNA已经证明了2，4，5-T基因的染色体定位， 似乎在这个基因上或附近有一个重复的序列。 这种反复 在洋葱假单胞菌、恶臭假单胞菌、P. 铜绿假单胞菌、门多萨假单胞菌等。 序列这个重复的序列，以确定其在自然界中的出现 除假单胞菌外的微生物，以确定是否有其他类型的 重复序列与其他2，4，5-T降解基因有关， 确定这种重复序列的可转位性。 此类研究 将提供关于进化模式的重要线索， 苯氧乙酸除草剂降解基因的组织 假单胞菌

The major goal of this proposal is to obtain an understanding of the evolution, organization and regulation of genes involved in the total degradation of 2,4,-Di-chlorophenoxyacetic acid (2,4-D) and 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) - the two components of the herbicide Agent Orange. The occurrence of Agent Orange, particularly one of its components, 2,4,5-T, in large concentrations in various parts of the world has created public health problems, that can only be minimized if such pollutants are removed from the contaminated sites through microbial degradation. Plasmids such as pAC27 are known to encode the degradative pathway for chlorocatechols, while plasmids such as pJP2 and pJP4 encode a complete 2,4-D degradative pathway including chlorocatechols. Since these three plasmids differ in sizes and incompatibility characteristics, yet demonstrate extensive clustering and homology for the chlorocatechol genes, attempts would be made to clone the individual chlorocatechol and the phenoxyacetate degradative genes from plasmids pJP2 and pJP4, determine their complete nucleotide sequences, and compare the open reading frames as well as the upstream sequences to those of pAC27 to see if the chlorocatechol genes are identical. The products for each individual genes will be overproduced under the transcriptional control of the tac promoter and their enzymatic activities studied. Using the 2,4,5-T degrading strain of Pseudomonas cepacia AC1100, where Tn5 mutagenesis has allowed isolation of a large number of 2,4,5-T negative mutants, attempts would be made to localize the presence of the Tn5 either on the plasmid or on the chromosomal DNA, thereby demonstrating the location of the 2,4,5-T genes. In one instance, transposon mutagenesis followed by hybridization with Tn5 DNA have demonstrated the chromosomal location of a 2,4,5-T gene which appears to harbor a repeated sequence on or near this gene. This repeated sequence is absent in the laboratory strains of P. cepacia, P. putida, P. aeruginosa, P. mendocina, etc. Attempts would be made to characterize and sequence this repeated sequence, to determine its occurrence in natural microorganisms other than Pseudomonas, to determine if other types of repeated sequences are involved with other 2,4,5-T degradative genes and to determine the translocatibility of such repeated sequences. Such studies will provide important clues regarding the mode of evolution and organization of the phenoxyacetate herbicide degradative genes in Pseudomonas.