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Project Progress

This project focused on recovering and identifying genes expressed in photosymbiotic relationships of planktonic sarcodines. Genes involved with symbiont identification, acquisition, and symbiosis maintenance were of particular interest. Initial experiments concentrated on comparing cDNA populations from the symbiotic and free-living states of a single dinoflagellate symbiont Scrippsiella nutricula but were expanded to include comparisons of a known foraminifera symbiont ( Gymnodinium beii ) with a closely related but strictly free-living dinoflagellate, Gymnodinium simplex . In all cases, a technique called cDNA Suppression Subtractive Hybridization followed by cloning and DNA sequencing was the method used.

As reported previously, we were successful in isolating and cloning a number of partial gene fragments whose expression was up regulated in the symbiotic state. All of these gene fragments appeared to originate from previously unknown genes. Clone-specific primers were designed for several different clones (E7, B8, and C4 to name a few) and Rapid Amplification of cDNA ends (RACE) used to isolate the full-length genes. Unfortunately, typical RACE amplifications resulted in fragments of only 200-400 nucleotides in length with a short overlapping region of close similarity to the target clone. Because of the large number of separate amplifications needed to generate full-length sequences and extremely limited starting material we were unable to recover full-length genes.

Thanks to additional funding through the Astrobiology supplement we were able to collect a significant number of symbionts from the west coast in April of 2003. The RNA extracted from these samples was used directly in continued RACE experiments and also in an attempt to establish traditional cDNA libraries. We hoped that a cDNA library would facilitate full-length gene recovery and permit better evaluation of their potential identity and function.

1. Scripsiella symbiont vs. free-living state comparison results:
   
   Three clones were identified as potential RACE targets based on their apparent increased expression in the symbiont state. One of these clones, B8, harbored a long poly-A tail and permitted RACE amplifications in only 1 direction. Amplifications of the other clones were attempted in both directions and were repeated until the longest open reading frame (ORF) was found. Single RACE amplifications of clones B8 and C4 resulted in ORFs of 295 bp and 189 bp respectively. BLAST results showed no strong homology to anything in the GenBank database. Clone E7 was successfully extended by 2 rounds of RACE in the 5’ direction (see Figure 1). No stop codons were detected throughout the entire fragment length. The last 225-bp fragment amplified was largely comprised of a repeat sequence. This repeat showed high identity to a fragment of the Cyplasin S gene from the gastropod Aplysia punctata . Because of the presence of this repeat, we were limited in our ability to design suitable primers for continued RACE amplifications and were unsuccessful in further extending this fragment.

2. Gymnodinium simplex/Gymnodinium beii comparison results:
   
   Nine clones were identified as potential differentially expressed gene fragments that were up regulated in G. beii . Based on apparent expression levels, clone-specific primers targeting clones G8 and F12 were initially designed. A short ~100 bp fragment was recovered from G8 RACE experiments. This fragment included a long poly-A repeat and resulted in a 1020bp ORF. BLAST results of the G8 clone showed high homology to the psaA_psaB photosystem 1 psaA/psaB protein. The clone F12 RACE experiments were all unsuccessful.
   

3. cDNA library construction results:
   
   Our goal was to generate a cDNA library of full-length cDNAs. This library was then to be used to screen for differentially expressed genes thus eliminating the need for multiple RACE experiments. A small library of cDNAs from the Scrippsiella symbiont of the oceanic chondrophore Velella velella was established. These symbionts are known to be identical to the radiolarian symbiont used to generate our clones. Isolation of full-length cDNAs proved difficult, and the titer on the library was low (a common problem for dinoflagellate cDNA libraries). The size range of our library was 250bp-1kb with the average size being roughly 500bp. The number of clones in our library is estimated at 5000. Despite repeated attempts, we were unable to generate clones containing inserts greater than 1kb.

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