<p> This study investigates the eIF4E family members in Dinoflagellates. Dinoflagellates are eukaryotic algae with large genomes and a minimal role for transcriptional regulation. All mRNA in dinoflagellates is <i>trans </i>-spliced with a 22-nucleotide 5'-spliced-leader sequence bearing a multi-methylated cap. Like other eukaryotes, dinoflagellates encode multiple eIF4E family members that are anticipated to fulfill a range of functions. Three distinct and novel clades of eIF4E have been recognized in dinoflagellates that are separate from the three metazoan classes of eIF4E. The dinoflagellate <i> Amphidinium carterae</i> encodes eight eIF4E family members while <i> Karlodinium veneficum</i> encodes fifteen eIF4E family members. I assayed six of these family members from <i>A. carterae</i> for expression levels, m<sup>7</sup>GTP binding, yeast knockout complementation and affinity for three mRNA cap analogs using surface plasmon resonance (SPR). Transcripts of each are expressed through a diel cycle, but only eIF4E-1 family members and eIF4E-2a are expressed at the level of protein. Recombinant eIF4E-1 family members and eIF4E-3a, but not eIF4E-2a, are able to bind to m<sup> 7</sup>GTP-agarose beads. Of the clade 1 eIF4Es, only eIF4E-1a and -1d1 complement a S. cerevisiae strain conditionally deficient in functional eIF4E, consistent with their function as translation initiation factors. However, only eIF4E-1a can be recovered from <i>A. carterae</i> extracts by m<sup>7</sup>GTP affinity binding. Using SPR analysis, the affinity of <i>A. carterae</i> eIF4E-1a for m<sup>7</sup>GTP is lower than that of murine eIF4E-1A. By the same analysis, <i>A. carterae</i> eIF4E-1a has a higher affinity for m<sup>7</sup>GpppG than m<sup>7</sup>GTP. In addition, <i>K. veneficum</i> eIF4E-1a1 displays many of the same characteristics as <i>A. carterae</i> eIF4E-1a. Four eIF4E-1 and one eIF4E-2 family members from <i>K. veneficum</i> were characterized for m<sup>7</sup>GTP binding capacity, only the eIF4E-1 family members can be pulled down with m<sup>7</sup>GTP. Three eIF4E family members were tested for their ability to interact with a putative eIF4E-interacting protein, although none interacted. Overall, the eIF4E-1a sub-clade emerges with characteristics consistent with the role of a prototypical translation initiation factor. These initial analyses will allow for a better understanding of specific translational control of gene expression through mRNA recruitment in the unique dinoflagellate lineage.</p>
| Identifer | oai:union.ndltd.org:PROQUEST/oai:pqdtoai.proquest.com:10118645 |
| Date | 09 June 2016 |
| Creators | Jones, Grant D. |
| Publisher | University of Maryland, Baltimore |
| Source Sets | ProQuest.com |
| Language | English |
| Detected Language | English |
| Type | thesis |
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