nai

Project Progress

We are studying the genetic communication involved in photosymbiotic associations to determine what makes an alga an acceptable symbiont. We are working with symbiotic and free-living dinoflagellate symbionts from the radiolarian Thalassicolla nucleata. We have used suppression subtractive hybridization to prospective differentially expressed clones. Sequence analysis identifies three distinct gene families, and Blast analyses showed that the F7 clone is a series of 30 basepair repeats with strong homology to repeats found in the Cyplasin S protein. The other clones are not significantly similar to any nucleotide or protein sequences in the databases.

We have confirmed differential expression of these clones through dot blot hybridization analyses, and believe that all of the genes we have reported here represent ones that are up-regulated in the symbiotic state of the algae. There are also clones that represent genes that are down-regulated. We used RACE to recover 5’ sequence information from our B8 clone, and will now begin working with the remaining clones. There is still no significant homology with the extended B8 clone to anything in the GenBank nucleotide database, but the translated sequences show some structural homology with several protein sequences. Whether this represents functional homology remains to be determined.

Our next steps in this project are directed towards obtaining 5’ RACE sequences for the remaining 4 clones and to begin the construction of traditional cDNA libraries for the free-living and symbiotic states of the dinoflagellate symbiont. We plan to use these libraries in the generation of microarrays that will be screened for the recovery of more symbiotically regulated genes. This method was not a viable option when we began our work, but the very recent acquisition of a microarray facility at the MBL has made it feasible. We feel that the development and application of these microarrays will allow us to recover more genes up and down regulated in the association.