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Project Progress

Isolation of Differentially Expressed cDNA by rapid amplification of cDNA ends (RACE).

We are interested in identifying genes that may impart symbiotic competence to the dinoflagellate symbionts of planktonic protists (foraminifera and radiolaria). Our work has focused on the symbiotic and free-living states of the dinoflagellate symbiont (Scrippsiella nutricula) from the radiolarian Thalassicolla nucleata. Clones of gene fragments expressed in one messenger ribonucleic acid (mRNA) population (symbiotic state) but not in another population (free-living state) were identified using a method called cDNA Suppression Subtractive Hybridization. Sequence analysis and comparison of these clones suggested that most, if not all, of these genes were previously unknown.

Over the past year, we have attempted to isolate the complete gene sequence of several of these target clones using 5’- and 3’-rapid amplification of cDNA ends (RACE). Clone-specific primers have been designed for 3 different clones (E7, B8, C4) originating from 2 separate experiments. Typical RACE amplifications result in fragments ranging from approximately 200-400 nucleotides in length with a short overlapping region of close similarity to the target clone. To date, at least 1 additional fragment has been amplified from each of the 3 clones mentioned above. We intend to continue designing clone-specific primers as needed for RACE analysis until the longest possible ORF is identified for each target gene.

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Symbiont Surface Protein Analysis.

It is unknown what traits impart symbiotic competence to certain organisms and not others. Presumably, a potential symbiont must have certain identifying features by which a prospective host may distinguish it from other similar non-symbiosis forming organisms. It is possible that proteins expressed on the surface of symbiotic cells play an important role in identifying and retaining likely symbionts.

Using cell surface protein labeling reagents, we investigated surface protein expression in 3 dinoflagellates. Two of these, Scrippsiella nutricula, and Gymnodinium beii, are known to form symbiotic associations with radiolaria and foraminifera respectively. The third, Gymnodinium simplex, is a strictly free-living organism. We hypothesized that proteins might be expressed by both symbiosis-capable dinoflagellates, but not G. simplex, might be important for the successful recognition/initiation of the symbiotic state.

The surface proteins of live cells were biotinylated with either amine-reactive or sulfhydryl-reactive reagents. Total protein was then separated by SDS-PAGE. Labeled proteins were visualized on Western Blots by chemiluminescent detection and the protein patterns of all 3 dinoflagellates were compared.

Although many surface proteins from each organism were successfully labeled, we were unable to identify any that were specifically shared by the symbiotically competent algae. In fact, even the two symbiosis competent dinoflagellates showed few, if any, proteins in common. It is possible that not all of the surface exposed proteins were labeled in our experiments. We chose labeling reagents that differentiate between amine containing and sulfhydryl containing protein residues. It is possible that continued experimentation with additional reagents such as glycoprotein or tyrosine targeting reagents would yield different results.

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Gymnodinium beii / Gymnodinium simplex Subtraction Results.

A second suppression subtraction experiment was accomplished to examine the differences between free-living states of a symbiotically competent alga and one that is never symbiotic (but genetically very similar). Analysis of the SSH products from a known foraminifera symbiont Gymnodinium beii, and the closely related but strictly free-living dinoflagellate Gymnodinium simplex is under way. Our motivation for this work is similar to that of the surface protein analysis. We hypothesize that genes expressed by G. beii, but not G. simplex, may play an important role in recognition of the symbiont by the host.

Nine clones (A2, A7, A12, B12, C3, C7, D2, F12, and G8) have been identified as potential differentially expressed gene fragments that are present in G. beii, but not G. simplex. Expression levels of these clones are currently being confirmed by Dot Blot analysis of the cDNA libraries. DNA sequencing has been completed on some of the clones, and results of BLAST searches on these clone fragments indicate that they are likely new genes. Clones C7 and A2 do have short regions of high similarity to Symbiodinium photosystem 11 and alpha tubulin genes respectively. Following sequencing, we will attempt to retrieve full-length gene sequences by RACE as was done previously.

Scrippsiella nutricula cDNA Library Construction.

Probes targeting the differentially expressed clones isolated from our suppression subtraction experiments will be made and used to screen a symbiont cDNA library. Recently, Scrippsiella symbionts were collected from Velella velella, an oceanic chondrophore. Gast and Caron (1996) previously showed that these dinoflagellate symbionts are the same as those from the radiolarian (Thalassicolla) that were used in the initial SSH. Full-length cDNAs were generated from Scrippsiella mRNA by Long-Distance polymerase chain reaction (PCR), and these cDNAs were then inserted into a donor vector that will enable us to perform functional analysis of individual genes at a later date.

Gast, R. J., and Caron, D. A. 1996. Molecular Phylogeny of Symbiotic Dinoflagellates from Planktonic Foraminifers and Radiolaria. Mol. Biol. Evol 13(9):1192-1197.