SITE-SPECIFIC INTEGRATION OF HETEROLOGOUS GENES IN YEAST VIA TY3 RETROTRANSPOSITION

The Ty3 retrotransposon of Saccharomyces cerevisiae was employed for t he site-specific integration of heterologous genes into the yeast geno me. A GAL-regulated promoter allowed induction of the retrotranspositi on process, and a bacterial neo(r) gene inserted in the Ty3 element wa s used as a selectable model heterologous gene. The frequency of trans position of this neo(r)-marked element was found to be comparable to t hat of an unmarked element. Th ree amplification systems were construc ted; the systems varied with respect to the location and number of the GAL-regulated helper and neo(r)-marked Ty3 elements. For all three sy stems, neo(r) integrations were readily selected with a maximum of two insertions obtained per round of amplification. A sequential amplific ation strategy was effective for further increasing the number of inte grated cloned genes, and families of strains varying by only one neo(r ) insertion were easily obtained. Resistance to the antibiotic G418 co rrelated well with the number of integrated neo(r) genes, and Northern blots verified the relationship between cloned gene number (up to fou r) and neo(r) expression. Structural stability of the integrated genes was also demonstrated. By controlling the number of rounds of amplifi cation and the level of G418 selection, precise numbers of integrated heterologous genes could be obtained. Because the amplification proces s can be repeated using different cloned genes inserted in the Ty3 ele ment, these results demonstrate the potential of retrotransposition fo r the regulated integration of a series of different genes at nondelet erious chromosomal locations. (C) 1996 John Wiley & Sons, Inc.