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

9708153 Azam细菌通过3 H-胸腺嘧啶核苷掺入产生的产物是生物海洋学和海洋生物地球化学中的“核心”参数。 目前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，非常需要一种方法。 该项目将开发一种基于使用胸苷类似物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合成的速率。 BrdU作为胸苷类似物，取代胸苷掺入新合成的DNA中。 通过免疫化学方法定量其掺入DNA中。 BrdU掺入后，将细胞透化并用结合DNA中BrdU的抗体处理。 然后使用与荧光染料或染料缀合的第二抗体来量化BrdU掺入。 已经采用了许多不同的方法来定量BrdU掺入DNA中。 目前的协议将进行测试和必要的修改，以提供最简单的可靠的方法，水生微生物组合。 这种方法的另一个好处是可以估计样品中单个细菌的原位杂交生长速率。 单个细菌的荧光信号可以通过落射荧光显微镜或流式细胞术来确定。 原则上，BrdU标记甚至可以与rRNA基因探测相结合，并且使用流式细胞术，然后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，提供关于微生物种群增长率的详细信息，而目前的放射性标记程序是不可能做到的。

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.