Abstract
Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo) on a construct bearing dual reporter genes (gfp and neo) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes.
Highlights
Eukaryotic genomes are rich in repetitive sequences, transposable elements (TEs) that are capable of jumping from one genomic locus to another
These results suggest that gene cassettes carried in T7 are excised efficiently
Even though a cargo gene cassette carried by the original miniature inverted transposable elements (MITEs) Ost35 did not result in efficient transposition activity, the internal sequence of this MITE is critical to the high transposition efficiencies for the hybrid Stowaway T7 based vectors
Summary
Eukaryotic genomes are rich in repetitive sequences, transposable elements (TEs) that are capable of jumping from one genomic locus to another. One type of nonautonomous element is wrecked autonomous element, bearing mutations in the transposase coding sequences. The entire transposase coding sequences are deleted [1,2]. Another type of nonautonomous elements does not bear protein coding regions, often they share very limited (if any) sequence similarities to transposase coding elements in the genome. Despite the apparent absence of cognate autonomous elements for these orphan elements, many of them appear to be highly efficient in transposition, and exist in high copy numbers in a genome. The human SINE element Alu is mobilized by the transposase produced by L1 elements that share very little sequence similarity with Alu [3]. The most abundant orphan type of non-autonomous class II elements are miniature inverted transposable elements (MITEs) [7]
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