REGULATION OF CARAB IN P AERUGINOSA

第一个目标是阐明间隔区的功能 铜绿假单胞菌CARA内序列分析。两个版本的比较 含氨基端氨基酸的CARA衍生氨基酸序列 氨甲酰磷酸合成酶小亚基序列公布 由四个密码子指定的氨基酸残基不存在于 多肽。这种共线性的缺乏可以在以下基础上解释 核糖体在翻译或多肽剪接过程中的跳跃。在……里面 体内和体外Chase标记实验及诱变 研究将区分这两种模式。关于基因表达的研究 在异源系统中的CARA将评估对 交易因素。将采用定点突变技术来 研究间隔区序列的功能，并确定 它在表达调控中起着重要作用。诱变也将是 用来鉴定决定核糖体的序列特征 跳跃或多肽剪接。结果应该是一般性的 对我们理解间隔区序列功能的意义 在结构基因中。 第二个目标是阐明调控的机制。 CarAB表达式。定量引物的延伸与诱变 实验将被用来检验假设的倍数 CARA上游启动的转录本受不同的 嘧啶、精氨酸和另一种西格玛因子。研究对象： 融合和突变将检验这样一种假设，即对 CarAB涉及一种衰减机制。编码调节性蛋白的基因 参与控制某些基因表达的因子 精氨酸的生物合成和有氧分解代谢将通过 插入突变。这个基因将被克隆、鉴定和 其产品经鉴定。一个长期目标是隔离监管机构 并研究其与控制区的相互作用。这个 鉴于信息有限，建议的研究具有重要意义。 在这个机会主义者中对染色体基因的调节 病原体。 还将进行关于监管的比较但有限的研究 在P.Stutzeri中确定非共线性的缺失是否与 一种不同的监管模式。

The first objective is elucidation of the function of the spacer sequence within carA from Pseudomonas aeruginosa. Comparison of the derived amino acid sequence of carA with the amino terminal amino acid sequence of the small subunit of carbamoylphosphate synthetase revealed that the amino acid residues specified by four codons are not present in the polypeptide. This lack of colinearity can be explained on the basis of ribosomal hopping during translation or polypeptide splicing. In vivo and in vitro chase-labeling experiments as well as mutagenesis studies will distinguish between the two models. Studies on expression of carA in heterologous systems will assess the requirement of a transacting factor. Site-directed mutagenesis will be employed to investigate the function of the spacer sequence and to determine whether it plays a role in regulation of expression. Mutagenesis will be also employed to identify the sequence features that determine ribosomal hopping or polypeptide splicing. The results should be of general significance to our understanding of the function of spacer sequences within structural genes. The second objective is elucidation of the mechanism of regulation of carAB expression. Quantitative primer extension and mutagenesis experiments will be employed to test the hypothesis that the multiple transcripts initiated upstream of carA are differentially regulated by pyrimidines, arginine, and an alternative sigma factor. Studies with fusions and mutagenesis will test the hypothesis that regulation of carAB involves an attenuation mechanism. The gene encoding a regulatory factor involved in control of expression of certain genes of biosynthesis and aerobic catabolism of arginine will be identified by insertional mutagenesis. This gene will be cloned, characterized, and its product identified. A long term goal is to isolate the regulatory product and to study its interactions with the control region. The proposed studies are of significance in view of the limited information available on regulation of chromosomal genes in this opportunistic pathogen. Comparative but limited studies on regulation will be carried out also in P. stutzeri to determine if the absence of non-colinearity relates to a different regulatory pattern.