Single-site manipulation of tomato chromosomes in vitro and in vivo using Cre-lox site-specific recombination

Abstract

With the aim of developing new techniques for physical and functional genome analysis, we have introduced the Cre-lox site-specific recombination system into the cultivated tomato (Lycopersicon esculentum). Local transposition of a Ds(lox) transposable element from a T-DNA(lox) on the long arm of chromosome 6 was used to position pairs of lox sites on different closely linked loci. In vitro Cre-lox recombination between chromosomal lox sites and synthetic lox oligonucleotides cleaved the 750 Mb tomato genome with 34 bp specificity to release unique 65 kb and 130 kb fragments of chromosome 6. Parallel in vitro experiments on Saccharomyces cerevisiae chromosomes show the efficiency of cleavage to be 50% per chromosomal lox site at maximum. By expressing the Cre recombinase in tomato under control of a constitutive CaMV 35S promoter, efficient and specific somatic and germinal in planta inversion of the 130 kb fragment is demonstrated. The combined use of in vitro and in vivo recombination on genetically mapped lox sites will provide new possibilities for long range restriction mapping and in vivo manipulation of selected tomato genome segments.

Biochemical, Genetic, and Zoosporicidal Properties of Cyclic Lipopeptide Surfactants Produced by Pseudomonas fluorescens

“…DNA flanking the Tn5lacZ-Km insertions in different mutants of P. fluorescens isolate SS101 was obtained by anchored PCR (44). Anchored PCR was performed in three steps.…”

Size Does Matter: Cre-mediated Somatic Deletion Efficiency Depends on the Distance Between the Target lox-Sites

“…The location of the D3-5 integration was determined on a molecular linkage map, by RFLP mapping of closely linked Ds flanking sequences as described before (Stuurman et al, 1996), using two independent mapping populations. As shown in Figure 1B, D3-5 is located on the long arm of chromosome 6, between markers TG 25 and TG 253.…”

“…To map D3-3, the plant DNA flanking the T-DNA was isolated by means of plasmid rescue (Rommens et al, 1992) and mapped on chromosome 7, using a population of 38 L. esculentum · L. pennellii F2 plants as described before (Stuurman et al, 1996). To confirm the mapping position of D3-3 on chromosome 7, the Lycopersicon pennellii introgression lines IL 7-1 to -4 (Eshed and Zamir, 1995) were used.…”

Identification and Ds‐tagged isolation of a new gene at the Cf‐4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5

“…The flanking sequences of the Ds element from plant 65 were isolated using anchored PCR (Stuurman et al, 1996) with the following modifications. Plant DNA was digested with BglII or BclI instead of BstY1 and ligation was done in the absence of the restriction enzyme (this was removed by a phenol/chloroform extraction) because the ligated products were recleavable.…”