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Title: The isolation and functional analysis of the eukaryotic translation initiation factor eIF4E from tobacco
Author: Combe, Jonathan P.
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1998
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An investigation into the regulatory characteristics of plant eIF4E was initiated. Two cDNAs, NeIF4E1 and NeIF4E2 encoding putative tobacco eIF4E homologues were isolated from a pollen cDNA library. NeIF4E2 contained an incomplete open reading frame (ORF) truncated at the 5' terminus, whereas NeIF4E2 consisted of a complete ORF, encoding a predicted 222 amino acid polypeptide. The predicted polypeptide sequences of NeIF4E1 and NeIF4E2 were 95% identical. The complete NeIF4E1 translation product was 67, 69 and 64% identical to wheat, rice and the Arabidopsis eIF4E, respectively. The expression pattern of NeIF4E in tobacco was investigated at the mRNA and protein level. The latter involved the raising of rabbit polyclonal antibodies against bacterially expressed NeIF4E1 protein. Both NeIF4E1 mRNA and protein were detected in all tissues and developmental stages investigated. To investigate whether NeIF4E was essential for tobacco translation, NeIF4E1 antisense constructs, incorporating either the constitutive CaMV 35S and pollen-specific lat52 promoter, were introduced into tobacco by stable genetic transformation. No visible phenotype was obtained with either construct, although NeIF4E protein was reduced by more than 50% of wild type levels with the CaMV35S-NeIF4E1-antisense transgene. The effect of overexpressing NeIF4E1 on plant gene expression was also investigated. Sense NeIF4E1 constructs containing the CaMV35S and lat52 promoters were transiently expressed, with a luciferase test plasmid, in leaves and pollen respectively. Luciferase gene expression was consistently inhibited by NeIF4E1 overexpression. Further experiments with mutant NeIF4E derivatives indicated the inhibition was exerted by the NeIF4E polypeptide not the transcript.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available