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

9708153通过~3H-胸腺嘧啶核苷掺入的阿扎姆细菌产量是生物海洋学和海洋生物地球化学中的一个“核心”参数。目前还没有非放射性同位素方法，但出于各种后勤、安全和科学原因，人们非常希望有一种方法。该项目将开发一种基于胸苷类似物5-溴脱氧尿苷(BrdU)的非放射性同位素方法，用于测量海水样本中细菌DNA合成的速度。BrdU的行为类似于胸苷类似物，并取代胸苷被结合到新合成的DNA中。它与DNA的结合是通过免疫化学方法进行定量的。在BrdU掺入后，细胞被渗透，并用与DNA中BrdU结合的抗体处理。第二种抗体与荧光或染料结合，然后用来定量BrdU的掺入。已经采用了许多不同的方法来量化BrdU在DNA中的掺入。目前的方案将在必要时进行测试和修改，以便为水生微生物组合提供最简单可靠的方法。这种方法的另一个好处是有可能估计样本原位杂交中单个细菌的生长速度。单个细菌的荧光信号可以用荧光显微镜或流式细胞仪来测定。原则上，BrdU标记甚至可以与rRNA基因探测相结合，然后使用流式细胞术，可以估计复杂组合中任何特定细菌组的生长速度。这种能力将彻底改变微生物生态学的研究，提供有关微生物种群生长率的详细信息，这是目前的放射性标记程序不可能做到的。

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.