CLONING AND EXPRESSION ANALYSIS OF THE GLUTAMINE SYNTHETASE GENE OF THE SEA ANEMONE AIPTASIA PALLIDA

Abstract

The association between the sea anemone Aiptasia pallida and its zooxanthellae algae enables the symbiosis to flourish in tropical nutrient-poor seas. The presence of symbiotic algae can alter the biochemical dynamics of cnidarian host. Previous studies have found that anemones containing algae (zooxanthellate anemones) not fed for two weeks, exhibit elevated glutamine synthetase (GS) activity relative to anemones without algae (aposymbiotic anemones) not fed for two weeks. It has been suggested that the activity of anemone GS plays a key role in controlling the growth of endosymbionts by limiting the amount of nitrogen available to the algae. Further studies were performed to determine if increased GS activity is correlated with increased GS mRNA steady-state levels. To examine this, the GS cDNA was cloned and the sequencing information was used to develop a semi-quantitative RT-PCR to measure GS mRNA steady-state levels between starved zooxanthellate anemones and fed aposymbiotic anemones. Sequence analysis revealed that the cloned GS cDNA is about 1600 bp in length and the protein consists of 369 amino acids with a predicted molecular weight of 41.7 kDa. A northern blot specific for anemone GS mRNA detected the presence of a 1.6-kb GS transcript, which agrees with the size from the sequenced cDNA. A phylogenetic analysis of the amino acid sequence showed greater homology to animal GS relative to protist GS protein. The results of the semi-quantitative RT-PCR assay, normalized to 18S ribosomal RNA, showed that increased GS activity in the starved zooxanthellate anemones was not correlated with increased GS mRNA steady-state levels. Furthermore, the aposymbiotic anemones had higher GS mRNA steady-state levels than the zooxanthellate anemones. This indicates that increased GS enzyme activity is not associated with increased GS mRNA steady-state levels and that the presence of symbiotic algae may play a role in reducing GS mRNA steady-state levels in the host. Thus, the results suggest that GS enzyme activity is regulated by events that may occur at the level of protein synthesis, protein stability, or protein modifications.