Identification of new catalytic and regulative components of purine nucleotide catabolism of Arabidopsis thaliana using a genetic screen.

植物能够完全分解嘌呤核苷酸，以重新调动嘌呤环系统中所含的氮。近年来，在嘌呤核苷酸降解酶的遗传鉴定和生化表征方面取得了重大进展。尽管如此，这种代谢途径的几个组成部分仍然未知。此外，到目前为止，对嘌呤核苷酸分解代谢的调节成分一无所知。这种催化或调节成分将通过基因筛选确定。用甲基磺酸乙酯（EMS）诱变拟南芥（Arabidopsis thaliana）的一个脲酸氧化酶T-DNA插入突变体。有尿酸氧化酶缺陷的植物的种子不能完全形成幼苗，因为它们含有在胚胎发育过程中积累的有毒水平的尿酸。这种表型在嘌呤核苷酸降解途径中缺乏尿酸氧化酶上游酶的双突变体中受到抑制。这种突变体不会积累尿酸。在遗传筛选中，我们将确定新的抑制因子，以防止尿酸氧化酶背景中的尿酸积累。这可能是尿酸氧化酶上游代谢途径的酶基因或对嘌呤核苷酸分解代谢表达重要的调控基因。或者，可以发现抑制因子，在幼苗建立期间赋予对高水平尿酸的耐受性增加。这组基因的鉴定可能会揭示尿酸如何对幼苗的建立产生负面影响。除了植物，这可能会导致对尿酸对人体代谢的毒性作用的更深层次的理解。抑制基因的鉴定将随后进行相应蛋白质的全面生化表征。突变体和双突变体核苷酸代谢的代谢组学分析将阐明这些抑制因子在体内的功能。

Plants are able to catabolize purine nucleotides completely to remobilize the nitrogen that is contained within the purine ring system. Substantial advances have been made recently in the genetic identification and biochemical characterization of enzymes that are involved in the degradation of purine nucleotides. Nonetheless, there are still several components of this metabolic route which remain unknown. In addition, nothing is known so far about the regulatory components of purine nucleotide catabolism.Such catalytic or regulative components will be identified through a genetic screen. A urate oxidase T-DNA insertion mutant of Arabidopsis thaliana will be mutagenized with ethyl methanesulfonate (EMS). Seeds from plants with a defect in urate oxidase do not complete the establishment of the seedling because they contain toxic levels of uric acid which accumulates during embryo development. This phenotype is suppressed in double mutants that additionally lack an enzyme upstream of urate oxidase in the degradation route of the purine nucleotides. Such mutants do not accumulate uric acid. In a genetic screen we will identify new suppressors which prevent the accumulation of uric acid in the urate oxidase background. This could be genes for enzymes of this metabolic route upstream of urate oxidase or regulative genes which are important for the expression of purine nucleotide catabolism. Alternatively, suppressors may be found which confer an increased tolerance to high levels of uric acid during seedling establishment. This group comprises genes whose identification may shed light on how uric acid exerts its negative effect on seedling establishment. Beyond plants, this may result in a deeper understanding of the toxic effects of uric acid on human metabolism as well.The identification of the suppressor genes will be followed by a comprehensive biochemical characterization of the corresponding proteins. Metabolomic analyses of nucleotide metabolism in mutants and double mutants will elucidate the function of the suppressors in vivo.