Identification of functional lox sites in the plastid genome

Abstract

SummaryOur objective was to test whether or not cyclization recombination (CRE), the P1 phage site-speci®c recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3 H rps12 gene leader (lox-rps12 ) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CREactive plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are speci®c to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.

Plastid marker gene excision by the phiC31 phage site-specific recombinase

“…Testing Cre-mediated marker gene excision revealed fortuitous ptDNA sequences or pseudo lox sites in the plastid genome that are recognized by the Cre. As the consequence, when a loxP site is present, Cre mediates deletions between loxP and lox-rps12 or loxP and lox-psbA sequences (Corneille et al 2003). In this study no deletions were found on DNA blots that would be indicative of recombination between attP or attB sequences and fortuitous ptDNA sequences that function as pseudo-attP or pseudo-attB sites.…”

“…When excision of the marker gene is required, a plastid-targeted cre gene is expressed in the nucleus and the Cre protein is imported into chloroplasts where it excises the plastid marker gene (Corneille et al 2001;Hajdukiewicz et al 2001;Lutz et al 2006a). Problems encountered with Cre-mediated marker excision were recombination of loxP sequences with fortuitous pseudo lox sites in the plastid genome (Corneille et al 2003), and enhanced homologous recombination adjacent to loxP sites that resulted in the loss of ptDNA segments between directly repeated sequences (Corneille et al 2001;Tungsuchat et al 2006).…”

Study of Plastid Genome Stability in Tobacco Reveals That the Loss of Marker Genes Is More Likely by Gene Conversion Than by Recombination between 34-bp loxP Repeats

“…INT-mediated excision was confirmed by DNA gel-blot analyses. The functionality of loxP sites flanking the bar au gene in Nt-pSS42 plants was shown by excision of the bar au gene by the Cre recombinase after cocultivation with Agrobacterium carrying plasmid pKO31 and confirmed by DNA gel-blot analyses (Corneille et al, 2003).…”

Scientific Opinion on an application (EFSA-GMO-NL-2009-70) for the placing on the market of genetically modified drought tolerant maize MON 87460 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto

“…Site-specific Cre-mediated recombination has been shown to occur between sites of varying degrees of sequence similarity to loxP with a high frequency in vitro (Hoess et al, 1982;Sauer, 1992Sauer, , 1996Thyagarajan et al, 2000;Corneille et al, 2003). Cryptic sites containing as few as 14 out of 34 bases in common with loxP have been shown to be effective (Sauer, 1996;Siegel et al, 2001;Chatterjee et al, 2010).…”