Polyamine Biosynthesis And Physiological Functions

多年来，我们一直在研究这些多胺是如何合成的，它们的生物合成和降解是如何被调节的，它们的生理功能，它们在体内是如何起作用的。为此，我们在大肠杆菌和酿酒酵母的每个生物合成步骤中构建了零突变体，并制备了生物合成酶的过表达系统。我们的总体研究旨在利用这些突变体来阐明多胺的生理功能。在之前的报告中，我们描述了使用微阵列和蛋白质组学技术以及我们的多胺需求突变体的酿酒酵母，以发现在多胺缺乏培养物中补充亚精胺会上调或下调哪些基因和蛋白质。我们还报道了一株大肠杆菌的构建，该菌株含有所有参与多胺生物合成的基因缺失；即speA（精氨酸脱羧酶）、speB（谷丙氨酸脲水解酶）、speC（鸟氨酸脱羧酶）、spe D（腺苷蛋氨酸脱羧酶）、speE（亚精胺合成酶）、speF（诱导型鸟氨酸脱羧酶）、cadA（赖氨酸脱羧酶）和ldcC（赖氨酸脱羧酶）。尽管完全没有所有的多胺，菌株在无胺的空气培养基中无限生长；尽管增长率略有下降（约40-50%）。最近，我们也在用我们的大肠杆菌突变体进行微阵列研究，这些突变体缺乏参与多胺生物合成的所有酶。为此，我们开发了一种化学调节剂，我们还发现，在多胺剥夺过程中，使用空气和氮气的混合物来避免细胞因氧化应激而死亡是很重要的。与其他实验室报告的一些研究相反，我们认为在添加亚精胺后，不让结果因生长速率的变化而复杂化是非常重要的。赖氨酸和精氨酸营养不良细胞也在多胺突变背景下构建，以便使用SILAC技术进行蛋白质组学研究，比较多胺加培养和缺培养之间的蛋白质阵列。在一项初步研究中，与添加多胺的培养相比，在不添加胺的情况下，多胺突变体也显示出更高的氟尿嘧啶耐药突变体频率。

For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared over-expression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. In previous reports we have described the use of microarray and proteomic techniques together with our polyamine-requiring mutants of Saccharomyces cerevisiae to find which gene and proteins are upregulated or downregulated by spermidine supplementation to polyamine -deficient cultures. We have also reported the construction of a strain of Escherichia coli that contained deletions in all of the genes involved in polyamine biosynthesis; namely, speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase). Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free media; albeit at a slightly (ca 40-50%) reduced growth rate. Recently, we are also carrying out microarray studies with our Escherichia coli mutants that lack all of the enzymes involved in polyamine-biosynthesis. For this purpose we have developed the use of a chemostat and also we have found it is important to use a mixture of air and nitrogen to avoid cell death due to oxidative stress during polyamine deprivation. As opposed to a number of studies reported from other laboratories, we feel that it is very important not to have the results complicated by changes in the growth rates after spermidine addition. Lysine and arginine auxotrophs have also being constructed in the polyamine mutant background to permit the use of the SILAC technique for proteomic studies to compare the protein arrays between polyamine plus and deprived culture. In a preliminary study, polyamine mutants also show higher frequency of fluorouracil resistant mutants in the absence of added amines as compared to polyamine supplemented culture.