Identification of eukaryotic internal ribosome entry site (IRES) elements
A method of search for and identification of a eukaryotic IRES element active in cap-independent translation of RNA in eukaryotic cells is provided, comprising the following steps: i) screening eukaryotic mRNA sequences or corresponding DNA sequences for a potential IRES element having a block of nucleotides having: a) a length of at least 30 nucleotides; b) an adenine nucleotide content of at least 40 mol-%; and c) a pyrimidine nucleotide content of less than 40 mol-%; ii) inserting said potential IRES element into a linear dicistronic construct between an upstream gene and a downstream GUS reporter gene, whereby said potential IRES element is positioned for IRES-dependent translation of said downstream GUS gene and whereby said upstream gene is preceded by a stable hairpin structure to prevent IRES-independent translation of said genes; and iii) testing said potential IRES element for IRES-dependent translation of said GUS gene in a rabbit reticulocyte lysate or in a wheat germ extract in vitro translation assay, whereby GUS gene expression is quantitated preferably relative to a construct having a reference IRES element or a non-IRES element between said upstream gene and said GUS gene.
1. A process of expressing a nucleotide sequence of interest in eukaryotic cell(s), wherein the process comprises introducing into said cell(s) a vector comprising said nucleotide sequence of interest operably linked to an upstream IRES element of eukaryotic origin, wherein the IRES element comprises a nucleotide sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, and SEQ ID NO:20, whereby said nucleotide sequence of interest is translated cap-independently by way of said IRES element.
2. The process of claim 1 , wherein said nucleotide sequence of interest is expressed as part of a bicistronic or a polycistronic mRNA.
3. The process of claim 1 , wherein the IRES element is of mammalian origin.
4. The process of claim 1 , wherein said eukaryotic cell is a plant cell.
5. The process of claim 1 , wherein said eukaryotic cell is an animal cell.
6. The process of claim 1 , wherein said eukaryotic cell is a yeast cell.
7. A process of expressing a nucleotide sequence of interest in eukaryotic cell(s), wherein the process comprises introducing into said cell(s) a vector comprising said nucleotide sequence of interest operably linked to an upstream IRES element of plant origin, wherein the IRES element comprises SEQ ID NO:20, whereby said nucleotide sequence of interest is translated cap-independently by way of said IRES element.
8. A process of expressing a nucleotide sequence of interest in eukaryotic cell(s), wherein the process comprises introducing into said cell(s) a vector comprising said nucleotide sequence of interest operably linked to an upstream IRES element, wherein the IRES element comprises SEQ ID NO:20, whereby said nucleotide sequence of interest is translated cap-independently by way of said IRES element.