Development of a Non-Radioisotopic Method to Measure Bacterial Production in the Sea

通过3h -胸腺嘧啶掺入的Azam细菌产量是生物海洋学和海洋生物地球化学的“核心”参数。目前还没有可用的非放射性同位素方法，但出于各种后勤、安全和科学原因，一种方法是非常可取的。该项目将开发一种基于胸腺嘧啶类似物5-溴脱氧尿嘧啶（BrdU）的非放射性同位素方法，用于测量海水样品中细菌DNA合成速率。BrdU表现为胸腺嘧啶类似物，并取代胸腺嘧啶并入新合成的DNA中。通过免疫化学方法定量其与DNA的结合。BrdU掺入后，细胞被渗透，并用一种与DNA中的BrdU结合的抗体处理。第二抗体与荧光染料或染料偶联，然后用于定量BrdU结合。已经采用了许多不同的方法来定量BrdU并入DNA。目前的方案将根据需要进行测试和修改，以提供最简单可靠的水生微生物组合方法。这种方法的另一个好处是可以估计单个细菌在原位杂交样品中的生长速率。单个细菌的荧光信号可以用荧光显微镜或流式细胞术检测。原则上，brdu标记甚至可以与rRNA基因探测相结合，使用流式细胞术，可以估计复杂组合中任何特定细菌组的生长速率。这种能力将彻底改变微生物生态学的研究，以目前的放射性标签程序无法提供的详细程度提供关于微生物种群生长速度的信息。

9708153 Azam Bacterial production, via 3H-thymidine incorporation, is a "core" parameter in biological oceanography and marine biogeochemistry. No non-radio-isotopic method is currently available but one would be highly desirable for a variety of logistical, safety and scientific reasons. This project will develop a non-radioisotopic method based on the use of the thymidine analog, 5-bromo-deoxyuridine (BrdU), for measuring the rate of bacterial DNA synthesis in seawater samples. BrdU behaves as a thymidine analog and becomes incorporated into newly synthesized DNA in place of thymidine. Its incorporation into DNA is quantified by an immunochemical method. After BrdU incorporation, the cells are permeabilized and treated with an antibody which binds to BrdU in the DNA. A second antibody, conjugated with a fluorochrome or a dye, is then used to quantify BrdU incorporation. Many different procedures for quantifying BrdU incorporation into DNA have been employed. Current protocols will be tested and modified as necessary to provide the simplest reliable method for aquatic microbial assemblages. An additional benefit of this approach is the possibility to estimate growth rates of individual bacteria in a sample in situ hybridization. The fluorescent signal of individual bacteria could be determined by epifluorescence microscopy or by flow cytometry. In principle, the BrdU-labeling could even be combined with rRNA gene probing and, using flow cytometry, growth rates could then be estimated for any specific group of bacteria in a complex assemblage. Such a capacity would revolutionize the study of microbial ecology, providing information on growth rates in microbial populations at a level of detail not possible with the current radiolabeling procedures.