Yeast "knockout-and-rescue" system for identification of eIF4E-family members possessing eIF4E-activity.
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 needfor 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 elF4E-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.
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore 21202, USA. joshi@umbi.umd.edu
Bhavesh
B
Joshi
Javier
J
Robalino
Eric J
EJ
Schott
Rosemary
R
Jagus
D000595:Amino Acid Sequence; D000818:Animals; D002238:Carbohydrate Epimerases; D002460:Cell Line; D039561:Eukaryotic Initiation Factor-4E / Q000032:analysis / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D005786:Gene Expression Regulation; D006238:Haploidy; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity; D019076:Transgenes; D015003:Yeasts / Q000235:genetics; D015027:Zebrafish / Q000235:genetics* / Q000378:metabolism
D039561:Eukaryotic Initiation Factor-4E; D002238:Carbohydrate Epimerases; C019646:aldose 1-epimerase