T-DNA recombination and replication in maize cells.
T-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.
Zhao
Xiaoxia
X
Transformation Research, Pioneer Hi-Bred International Inc., Johnston, IA 50131, USA.
Coats
Isabelle
I
Fu
Ping
P
Gordon-Kamm
Bill
B
Lyznik
L Alexander
LA
D016960:Agrobacterium tumefaciens / Q000235:genetics; D002478:Cells, Cultured; D004261:DNA Replication / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005966:Glucuronidase / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D014170:Transformation, Genetic; D003313:Zea mays / Q000235:genetics*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; C036483:T-DNA; D005966:Glucuronidase