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Project Progress

Genes that Regulate Photosymbiotic Interactions (dm)

We are interested in understanding how photosymbiotic relationships are controlled through the genetic communication of two cells, and perhaps how this may relate to the evolution of organelles. The presence of an algal symbiont represents the acquisition of novel functions by the host cell. There is an increasing body of evidence for the eukaryotic origin of chloroplasts in cryptophytes, chlorarachniophytes, euglenophytes, heterokonts, haptophytes and dinoflagellates. In these different hosts, the endosymbiotic algae are in various states of genetic and physical reduction. In all cases it is impossible to remove the alga from its host and culture it independently.

Planktonic foraminifera, radiolaria and acantharia are ameboid protists that occur in the pelagic environment of the world’s oceans. Many of the species in this group harbor algal symbionts, particularly dinoflagellates. These symbiotic relationships have not evolved to the point where the symbiont is dependent upon the host, so it is possible to separate the two and study the changes in algal gene expression that occur when the free-living organism becomes a symbiont. The sarcodine symbioses will also allow the comparison of symbiotically regulated genes in taxonomically distinct algae, which can lead to the identification of the common genetic elements involved in the associations, as well as what makes each one specific.

In order to reach our overall goal of determining the genetic mechanisms behind algal symbioses and, potentially, organelle evolution, we have begun comparing the mRNA expressed in symbiotic and free-living dinoflagellate symbionts from the radiolarian Thalassicolla nucleata. We have cultures of the dinoflagellate (Scrippsiella velellae) in our laboratory, which serve as a source of free-living RNA. To obtain RNA from the symbiotic state of S. velellae, we collected intact symbioses and dissected the symbionts away from the host. RNA was isolated from cultures and dissected symbionts using Ambion’s RNAqueous Kit. The two samples of RNA were processed further by suppression subtractive hybridization. Messenger RNA is converted to cDNA, and hybridization of cDNA tester (symbiotic cDNA) with cDNA driver (free-living cDNA) results in a population of cDNAs enriched for those of the tester (symbiotic). The differentially expressed cDNAs are amplified, and the PCR products are cloned and screened for confirmation of differential expression. This process enriches/selects for genes that are differentially regulated and is available in a kit format from Clontech.

We have completed a subtractive selection for S. velellae, and have recovered over 20 differentially expressed clones. Sequence analysis of these clones has indicated that 11 are essentially identical (C4). Although their Blast searches recover unidentified human chromosomal DNA as the most similar sequences, there is actually not a significant match to anything in the database, potentially indicating a novel gene sequence. Another clone (F7) was analyzed and found to be similar to the gene for Cyplasin S. The matching region is actually a repeat unit, potentially indicating a conserved function or structure.