Collaborative Resarch: Development of Phytoplankton PCNA Antibodies for Use in Growth Studies: Emiliania huxleyi and Karlodinium micrum as Model Systems

增殖细胞核抗原（PCNA）是一种细胞周期标志物，对评估浮游植物生长状况和估计原位生长速率具有潜在的应用价值。然而，由于缺乏有效的浮游植物PCNA抗体，其应用受到阻碍。 主要种类的浮游植物的PCNA编码基因已被克隆和测序最近，允许生产和有效的抗体的特性。该项目将产生一套10种浮游植物PCNA抗体。三种通用的藻类PCNA抗血清和三种甲藻特异性PCNA抗血清将使用合成肽与抗体生产公司合作生产。 这种安排将降低生产成本，并将提供可供其他研究人员使用的抗体供应。 将根据在细菌中过表达的纯化的PCNA，从各自的PCNA基因中产生四种抗血清，分别用于球石藻属物种赫氏艾美尼亚藻（2）和鱼毒性甲藻微小卡甲藻（2）。该项目将包括使用这些抗体的免疫荧光的优化方案的开发，基于落射荧光显微镜和流式细胞术，与E。huxleyi和K. micrum作为模型系统。PCNA在E. huxleyi和K.将使用蛋白质印迹法对微球进行表征。PCNA与细胞分裂周期的关联将通过PCNA的免疫荧光标记，结合流式细胞术，基于DNA的细胞周期分析来表征。将这些种属基于PCNA的生长速率与培养物中的实际生长速率（使用细胞计数）进行比较，可评估新方法的准确度和精密度。 总共有10种多克隆抗体将提供给世界各地感兴趣的研究人员。这些PCNA抗血清将被证明是非常有用的浮游植物种群的生长状况进行评估，并估计在原位总生长率（GGR）使用各种检测格式atr的自由裁量权的研究人员。 实例包括蛋白质印迹、免疫荧光或潜在的酶联免疫吸附测定（ELISA）。PCNA抗体产品的可用性将促进未来的应用，包括两个模式物种和其他浮游植物的细胞周期和生长特性的研究，以及现场应用的PCNA方法原位生长速率估计。该项目包括对研究生和本科生进行分子技术和流式细胞术分析方面的培训。两个参与机构之间的旅行将允许PI和他们的学生扩展他们的专业知识。研究结果将纳入PI教授的课程。该项目还将为PI提供一个极好的机会，继续履行其鼓励少数民族学生和女学生参与科学研究的承诺。

Proliferating cell nuclear antigen (PCNA) is a cell cycle marker that is potentially useful for assessing phytoplankton growth status and estimating in situ growth rate. However, its application has been hindered by the lack of effective antibodies for phytoplankton PCNA. PCNA-encoding genes for major classes of phytoplankton have been cloned and sequenced recently, permitting production and characterization of effective antibodies. This project will produce a suite of ten phytoplankton PCNA antibodies. Three universal algal PCNA antisera and three dinoflagellate-specific PCNA antisera will be produced using synthetic peptides, in collaboration with an antibody producing company. This arrangement will reduce production costs and will provide a supply of antibody that can be made available to other researchers. Four antisera will be produced for the coccolithophorid species Eminiliania huxleyi (2) and the ichthyotoxic dinoflagellate Karlodinium micrum (2) based on purified PCNA overexpressed in bacteria from respective PCNA genes. This project will include the development of an optimized protocol for immunofluorescence using these antibodies, based on both epifluorescence microscopy and flow cytometry, with E. huxleyi and K. micrum as model systems. Expression of PCNA in E. huxleyi and K. micrum will be characterized using Western Blots. Association of PCNA with the cell division cycle will be characterized by means of immunofluorescence labeling of PCNA, coupled with flow cytometric, DNA-based analysis of the cell cycle. Comparisons of PCNA-based growth rates of these species to actual growth rates in culture (using cell counts) will allow assessment of the accuracy and precision of the new method. A total of 10 polyclonal antibodies will be available to interested researchers worldwide. These PCNA antisera will prove extremely useful for assessing the growth status of phytoplankton populations and for estimating in situ gross growth rates (GGR) using various detection formats atr the discretion of the researcher. Examples include Western Blotting, immunofluorescence, or potentially, enzyme linked immunoabsorbent assays (ELISA). The availability of PCNA antibody products will facilitate future applications including studies of the cell cycle and growth characteristics of the two model species and other phytoplankton, as well as field applications of the PCNA approach to in situ growth rate estimation. This project includes training for graduate and undergraduate students in molecular techniques and flow cytometric analyses. Travel between the two participating institutions will allow both PIs and their students to expand their expertise. Results of the research will be integrated in courses taught by the PIs. This project will also provide an excellent opportunity for the PIs to continue with their commitment to encourage minority and female students to participate in scientific research.