Recombinational instability of a chimeric plasmid in Saccharomyces cerevisiae.

Abstract

Wild-type strains of Saccharomyces cerev'isiae exhibit mitotic recombination between the chimeric plasmid TLC-1 and the endogenous 2,u circle that involves sequence homologies between the two plasmids that are not acted on by the 2,u circle site-specific recombination system. This generalized recombination can be detected because it separates the LEU2 and CAN! markers of TLC-1 from each other through the formation of a plasmid containing only the S. cerev'isiae LEU2 region and the 2,u circle. This derivative plasmid is maintained more stably during vegetative growth than TLC-1, and strains which carry it frequently lose the endogenous 2,u circle. Therefore, TLC-1 can provide a convenient selection for [cir°] cells. Formation of this new plasmid is greatly reduced, but not eliminated, in strains containing the rad52-1 mutation. This indicates that generalized mitotic recombination between plasmid sequences utilizes functions required for chromosomal recombination in S. cerevisiae.Most laboratory strains of Saccharomyces cerevisiae contain an endogenous plasmid commonly referred to as the 2,u circle (4). Although this plasmid has not yet been shown to carry out an essential function in yeast cells, its properties have made it suitable for studies on yeast DNA replication (15,16,29) and site-specific recombination (5). The origin of replication of this plasmid has also been introduced into a number of yeast cloning vectors (6,24,25) to allow their autonomous, high-copy-number replication.One of the cloning vectors that contains the 2,u circle origin of replication is YEp13 (6). This plasmid also includes the yeast P-isopropyl malate dehydrogenase (LEU2) gene. A derivative of YEp13 has been constructed which contains the yeast arginine permease (CAN!) gene (6). This plasmid (TLC-1) is unstable, because S. cereVisiae strains which contain it can form derivatives which have lost the CANI gene while retaining the LEU2 gene. Such derivatives can be identified because the CANI gene confers dominant sensitivity to the arginine analog canavanine (28), so that loss of the gene from the plasmid results in a canavanine-resistant phenotype in a canl host. This separation of plasmid markers is due to recombination between TLC-1 and the endogenous 2,u circle and involves sequences that are not part of the site-specific recombination system that operates on the inverted repeat sequences. We have used this marker separation to investigate the role of the RAD52 gene product on generalized mitotic recombination between autonomously replicating plasmids in S. cerei'isiae. MATERIALS AND METHODSStrains. S. cerevisiae strains MSW28-1OC(x (leu2-3,2-112 his3-11,3-15 canl-100 iura3-1 trp5-2) and MSW152-lAa (leu2-3,2-112 hisl-7 can!-100 lura3-1 rad52-1) were constructed for this study from strain GRF18a obtained from G. R. Fink.The rad52-1 strain E053-6Doa (rad52-1 arg4-17 ade2-1 lysl-l hisl-7) was supplied by S. Plasmids and probes. Plasmid TLC-1 (6) was obtained from J. Hicks, and pBR322 (3) was obtained from J. Calvo. The LEU2-specifi...

Behavior of a Drosophila melanogaster Transposable Element in Saccharomyces cerevisiae

“…lc), had only 1% (1 of 105) of the plasmids deleted by recombination within the direct repeats after transformation into DBY747 (Table 1). The 412 results were surprising since intermolecular (15) and intramolecular (7) recombination of plasmids in yeast rarely occurs unless stimulated by plasmid breakage. A notable exception is the Flp recombination system of 2,um (3).…”