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UBC Theses and Dissertations

Characterization of genes of the elongation factor 2 (EF-2) family of caenorhabditis elegans Ofulue, Esther Ngozi


A gene eft- 1 encoding a protein synthesis elongation factor 2-like protein was isolated from chromosome III of C. elegans, and was mapped approximately 50 kb downstream from ubq-l. Five overlapping cosmids spanning 150 kb in this region were mapped by restriction endonuclease digestion, and hybridized to cDNA probes made from embryo polyA+RNA. One positive fragment B3255 was analyzed by sequencing, and was also used as a specific probe to isolate a eDNA clone pEF1.35 encoding eft-1 mRNA. The entire eft-l gene of 3.8 kb predicted a protein (EFT-l) of 849 amino acid residues which shared 38% overall identity with mammalian and Drosophila elongation factor 2 (EF-2) sequences. Sequence segments implicated in GTP-binding and GTPase activity in EF-2 were found in the N-terminal region, while segments characteristic of EF-2 and its prokaryotic counterpart EF-G were found at the coffesponding C-terminal portion of EFT-1; the latter region shared 40 - 50% similarity with, the hamster EF-2. However, the histidyl residue target for ADP-ribosylation and inactivation of EF-2 by diphtheria toxin, which is thought to be of functional importance in EF-2, was replaced by a tyrosyl residue in EFT- 1. By rapid amplification of C elegans DNA sequences using primers specific for highly conserved regions of mammalian and Drosophila EF-2 using the polymerase chain reaction, cDNA and genomic clones containing a C. elegans gene (eft-2) were isolated and characterized. One cDNA clone, pCef6A, encoding the entire eft-2 mRNA predicted a polypeptide of 852 amino acid residues which shared greater than 80% identity with the hamster and Drosophila EF-2 sequences. The GTP-binding domains, ADP-modifiable histidyl residue, and high homology regions shared between EF-2 and EF-G were 80 - 100% conserved in the C elegans protein. These results suggested that eft-2 and not eft-1 encoded the C elegans homolog of EF-2. The conservation of functional domains of EF-2 in EFT- 1 implied that the two genes were derived at least in part from a common ancestor. The copy numbers of eft- 1 and eft-2 were examined and their expression was monitored throughout nematode development. The results revealed that both genes are unique and each encodes a 3 kilobase mRNA species which does not appear to be under nutritional or developmental regulation.

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