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Project Progress

Work on the isolation of symbiosis-specific genes from algal-sarcodine photosymbiotic relationships has begun with the utilization of the cultures of free-living symbionts that we have in the laboratory. During the past year we have optimized several kits and methods from Clontech for the generation of cDNA from mRNA, and suppression-subtractive hybridization. We have used RNA isolated from two different dinoflagellate symbionts in their free-living states to test the methods. cDNA libraries were generated for both, and we have successfully performed suppression-subtractive hybridization utilizing them. Our next step is the collection of intact symbioses, and we are currently planning two field sessions for the collection of the sarcodines in June and in August (see below). RNA isolation methods that we have been using allow us to extract nucleic acids from a small number of cells (about 10,000), and we have decided to utilize intact symbioses rather than continue to try to refine the inducible system that we originally proposed. Symbionts will be microdissected from their hosts immediately upon collection, and the symbionts from at least 20 of the same host will be pooled for extraction. This should provide us with at least 20,000 symbiont cells for RNA collection. We will then use the cDNAs from the free-living symbionts to perform the suppression-subtraction hybridization and begin our identification and analysis of the differentially expressed genes.

Although this project has been slow in developing, we have made a significant step forward in our progress this year with the free-living symbionts. We are poised to make the next step during 2000-2001. Our work will focus largely upon recovering and identifying genes expressed in the symbiotic state of the algal symbionts that we are studying. The initial analyses will utilize the natural symbionts of the host organism, Thalassicolla nucleata. Once this has been accomplished, we will begin to work with some of the foraminiferan hosts and their symbiont.