Site-specific integration of heterologous genes in yeast via Ty3 retrotransposition

Abstract

The Ty3 retrotransposon of Saccharomyces cerevisiae was employed for the site‐specific integration of heterologous genes into the yeast genome. A GAL‐regulated promoter allowed induction of the retrotransposition process, and a bacterial neor gene inserted in the Ty3 element was used as a selectable model heterologous gene. The frequency of transposition of this neor‐marked element was found to be comparable to that of an unmarked element. Three amplification systems were constructed; the systems varied with respect to the location and number of the GAL‐regulated helper and neor‐marked Ty3 elements. For all three systems, neor integrations were readily selected with a maximum of two insertions obtained per round of amplification. A sequential amplification strategy was effective for further increasing the number of integrated cloned genes, and families of strains varying by only one neor insertion were easily obtained. Resistance to the antibiotic G418 correlated well with the number of integrated neor genes, and Northern blots verified the relationship between cloned gene number (up to four) and neor expression. Structural stability of the integrated genes was also demonstrated. By controlling the number of rounds of amplification and the level of G418 selection, precise numbers of integrated heterologous genes could be obtained. Because the amplification process can be repeated using different cloned genes inserted in the Ty3 element, these results demonstrate the potential of retrotransposition for the regulated integration of a series of different genes at nondeleterious chromosomal locations.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

“…tRNA genes have been associated with reduced expression of associated RNA polymerase II promoters (Hull et al , 1994; Ji et al , 1993; Kinsey and Sandmeyer, 1991). Nonetheless, expression of marker genes associated with Ty1 and Ty3 elements can be readily detected at levels expected for single gene copies (Chalker and Sandmeyer, 1990; Lee and Da Silva, 1996; Wang and Da Silva, 1996). Cumulatively, Ty1 RNAs have been shown to account for approximately 10% of the total poly(A) RNA in the yeast cells and the individual expression of 31 Ty1 elements has been characterized using lacZ reporter fusions (Lesage and Todeschini, 2005).…”