Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae.

Abstract

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GAL] promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin strujcture.In general, the genome of eucaryotic cells is larger and more structurally complex than that of procaryotic cells. Moreover, the eucaryotic genome is composed of multiple linear chromosomes, whereas bacteria tend to have a single circular chromosome. A distinctive eucaryotic feature is that the genomic DNA is organized into nucleosomes by intimate association with histones and other proteins to form chromatin. Treatment of chromatin with micrococcal nuclease results in a characteristic nucleosome repeat structure (16). Moreover, chromatin can exist in both transcriptionally active and inactive forms. This difference is most likely due to differences in accessibility of the DNA, as measured by susceptibility to nuclease digestion (34).The ability of an enzyme to access DNA in chromatin may not be a property intrinsic to its eucaryotic source. Certainly eucaryotic DNA polymerases, RNA polymerases, and topoisomerases can act on naked DNA in vitro. Conversely, procaryotic proteins such as the lexA repressor and the EcoRI endonuclease can act in vivo at specific DNA sequences on the chromosomes of the yeast Saccharomyces cerevisiae (3, 5). These observations suggest that procaryotic proteins may also be able to conduct more sophisticated transactions on the DNA of a eucaryotic chromosome. For instance, can DNA recombination events in eucaryotes be promoted by a bacterial protein? Such events demand not only recognition of DNA sequences but also synapsis, DNA cleavage, strand exchange, and religation. In particular, can a site-specific recombination system of Escherichia coli function in a eucaryotic cell? The cre-lox site-specific recombination system of coliphage P1 is well suited to answer such questions.Phage P1 encodes an efficient site-specific recombination system consisting of a short asymmetric DNA sequence called loxP and a 38-kilodalton protein called Cre (1,12,29). The loxP site is a 34-base-pair sequence composed of two 13-base-pair inverted repeats separated by an asymmetric 8-base-pair core sequence. Recombination between loxP sites (i) can occur either inter-or intramolecularly, (ii) can occur when the sites are present on either supercoiled or linear DNA, and (iii) is independent of the re...

DNA Topoisomerase II Is a Determinant of the Tensile Properties of Yeast Centromeric Chromatin and the Tension Checkpoint

“…Standard genetic crosses were used to transfer loxCENlox to other strains. To induce CEN excision, loxP(CEN1:kanMX4)loxP strains were transformed with pBS49 (pGAL-Cre CEN/ARS/URA3; Sauer, 1987) and processed into galactose media.…”

Polar Transmembrane Domains Target Proteins to the Interior of the Yeast Vacuole

“…A ⌬pho13 ⌬pho8 mutant was obtained by disrupting PHO13 with HIS5 flanked by loxP sites and subsequently disrupting PHO8 with TRP1 also flanked by coliphage loxP sites. The selectable markers were removed by excisive recombination at the loxP sites with the transient expression of Cre recombinase (Sauer, 1987).…”

Cre Mutants with Altered DNA Binding Properties

“…In eukaryotes it was first demonstrated by Cremediated deletion of a LEU2 gene flanked by two lox sites (16). Afterward, by Cre-mediated recombination, the LEU2 gene was deleted.…”