Polyamine Biosynthesis And Physiological Functions
多胺生物合成和生理功能
基本信息
- 批准号:8349661
- 负责人:
- 金额:$ 32.7万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:
- 资助国家:美国
- 起止时间:至
- 项目状态:未结题
- 来源:
- 关键词:Adenosylmethionine DecarboxylaseAgmatinaseAirAminesAnabolismArginine decarboxylaseCell DeathCellsDifferentiation and GrowthEnzymesEscherichia coliFluorouracilFrequenciesGene ProteinsGenesGrowthLaboratoriesLifeLysine decarboxylaseNitrogenOrnithine DecarboxylaseOxidative StressPhysiologicalPolyaminesProtein ArrayProteomicsPutrescineReportingResistanceSaccharomyces cerevisiaeSpermidineSpermidine SynthaseSpermineSupplementationSystemTechniquesarginyllysinedeprivationin vivomutant
项目摘要
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.
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调节的,它们的生理功能,它们如何在体内发挥作用。为了这个目的,我们已经构建了空突变体在大肠杆菌和酿酒酵母的生物合成步骤中的每一个,并已准备过表达系统的生物合成酶。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。在以前的报告中,我们已经描述了使用微阵列和蛋白质组学技术与我们的多胺需要的酿酒酵母突变体一起,以发现哪些基因和蛋白质被亚精胺补充剂上调或下调多胺缺乏的文化。我们还报道了一种大肠杆菌菌株的构建,该菌株包含参与多胺生物合成的所有基因的缺失;即speA(精氨酸脱羧酶)、speB(胍丁胺脲水解酶)、speC(鸟氨酸脱羧酶)、spe D(腺苷甲硫氨酸脱羧酶)、speE(亚精胺合酶)、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)。尽管完全不存在所有多胺,但菌株在空气中在无胺培养基中无限期生长;尽管生长速率略微降低(约40-50%)。最近,我们也正在进行微阵列研究与我们的大肠杆菌突变体,缺乏所有的酶参与多胺的生物合成。为此目的,我们已经开发了恒化器的使用,并且我们还发现使用空气和氮气的混合物以避免由于多胺剥夺期间的氧化应激导致的细胞死亡是重要的。与其他实验室报告的一些研究相反,我们认为,加入亚精胺后,不要使结果因生长速率的变化而复杂化,这一点非常重要。 赖氨酸和精氨酸营养缺陷型也被构建在多胺突变体的背景下,允许使用的SILAC技术的蛋白质组学研究比较多胺加和剥夺文化之间的蛋白质阵列。在初步研究中,与多胺补充培养物相比,多胺突变体在不存在添加的胺的情况下也显示出更高频率的氟尿嘧啶抗性突变体。
项目成果
期刊论文数量(0)
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Herbert Tabor其他文献
Herbert Tabor的其他文献
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{{ truncateString('Herbert Tabor', 18)}}的其他基金
Biophysical studies on the interaction of antizyme and ornithine decarboxylase
抗酶与鸟氨酸脱羧酶相互作用的生物物理学研究
- 批准号:
7593451 - 财政年份:
- 资助金额:
$ 32.7万 - 项目类别:
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