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.
