Department of Microbiology and School of Ocean and Earth Sciences and Technology, Department of Oceanography, University of Hawaii, Honolulu, Hawaii 96822.
Appl Environ Microbiol. 1990 May;56(5):1245-54. doi: 10.1128/aem.56.5.1245-1254.1990.
The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which >/=95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [H]thymidine experiments and compared the degree of nonspecific labeling by [H]adenine with that derived from [H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.
研究了五个微生物生态系统中氚标记胸腺嘧啶的掺入以及氚在 DNA、RNA 和蛋白质中的分布。通过差异酸碱水解法测定，所有测试的微生物组合都表现出 RNA 和蛋白质的显著标记（即非特异性标记）。非特异性标记在沉积物样品中最大，其中 >/=95%的氚与 RNA 和蛋白质部分一起回收。在 DNA 部分回收的氚百分比范围为从总标记大分子中回收的 15%到 38%。非特异性标记与非常宽的孵育时间和胸苷浓度无关。四种不同的 RNA 水解试剂（KOH、NaOH、哌啶和酶）从冷三氯乙酸沉淀中溶解氚。哌啶水解产物的高压液相色谱分离，然后测量分离的单磷酸酯，证实了 RNA 的标记，并表明氚主要回收在 CMP 和 AMP 残基中。我们还评估了 [2-H]腺嘌呤与 [H]胸苷实验平行掺入 RNA 和 DNA 中腺苷酸残基的特异性，并比较了 [H]腺嘌呤和 [H]胸苷衍生的非特异性标记程度。通过确定氚标记胸腺嘧啶从孵育介质中的消失以及通过无细胞介质的高压液相色谱分离来检测降解产物来评估氚标记胸腺嘧啶的快速代谢。降解产物的形成，包括挥发性和非挥发性化合物，远远大于氚掺入稳定大分子的速度。与利用克雷布斯循环中间体合成氨基酸、嘌呤和嘧啶相结合的胸苷标准降解途径很容易解释环境样品中观察到的非特异性标记。
