CORE--TOXIN PROBES

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Description (provided by applicant): From its inception in 1992, the Center has housed a Toxin Probes Facility Core. The Core provided purified toxins and toxin antibodies to Center Investigators. This Core was temporarily discontinued in 1998 with the departure of the Core Leader and Center Director, Daniel Baden. The Core will be re-established at Florida International University and will be led by Dr. Kathleen Rein, beginning with the April 1, 2001 budget year. The new Core will provide similar services as the old Core. However, the new Core is envisioned to be a significant expansion of the previous Core at the University of Miami MFBS Center with an emphasis on DNA technology. In addition to Center support, funding is being sought from NIEHS via the ARCH (Advanced Research Cooperation in Environmental Health Sciences) mechanism that pairs a minority institution (FIU) with a research institution (UM). In addition, this facility will provide the basis for ongoing collaborations between institutions. Cultures will be grown in 4L carboys to maximum density. In addition to dinoflagellates the investigators will grow toxic prymesiophytes and cyanobacteria. The cyanobacteria have been isolated from the Florida Everglades by Dr. Miro Gantar and Evelyn Gaiser at FIU and will be made available to Center members. The volume of each individual strain at any given time will be dictated by the needs of the Center scientists. Cells will be harvested either by centrifugation prior to toxin purification or DNA/RNA isolation. Alternatively, depending on the specific circumstance, cultures will be extracted directly for toxin. Toxins will be purified by a combination of solvent extraction, column chromatography, preparative TLC and HPLC. Purified material will be analyzed by 400 MHz NMR and stored in sealed ampoules in a locked freezer. DNA and RNA will be isolated by standard protocols, quantitated spectrophotometrically, analyzed by gel electrophoresis for quality and stored at 80 C. This core will interact with the analytical chemistry and electron microscopy core for quality control of purified toxins.