Yeast 'knockout-and-rescue' system for identification of eIF4E-family members possessing eIF4E-activity

摘要

Evidence from several laboratories and sequencing projects has revealed that many eukaryotes contain multiple proteins related in sequence to the human mRNA-cap binding translation initiation factor 4E (eIF4E-1). Although some have been shown to bind cap-analogues, whether all eIF4E-family members function as translation initiation factors is unclear. Furthermore, the existence of proteins related to eIF4E complicates the identification of the translation factor by sequence-based approaches. Methods to assess the functionality of eIF4E are limited. The most informative, single assay to identify proteins with eIF4E-activity is that of rescue of the lethal disruption of the single Saccharomyces cerevisiae eIF4E gene. We have developed a simplified yeast eIF4E "knockout-and-rescue" system, the characteristics of which are (i) a haploid system that obviates the need for a "plasmid shuffle", (ii) a simple G418-based selection for yeast lacking a chromosomal eIF4E gene, and (iii) a glucose-based selection to deplete the strain of a human eIF4E-1 substitute and to assess the eIF4E-activity of an untested eIF4E family member. In this,form, the yeast eIF4E knockout-and-rescue system becomes a tool available to any laboratory experienced in the selection of microbial strains with antibiotics and standard media for the identification and isolation of cDNAs encoding proteins with eIF4E-activity.