Efficient transfer of base changes from a vector to the rice genome by homologous recombination: involvement of heteroduplex formation and mismatch correction.
Gene targeting refers to the alteration of a specific DNA sequence in an endogenous gene at its original locus in the genome by homologous recombination. Through a gene-targeting procedure with positive-negative selection, we previously reported the generation of fertile transgenic rice plants with a positive marker inserted into the Adh2 gene by using an Agrobacterium-mediated transformation vector containing the positive marker flanked by two 6-kb homologous segments for recombination. We describe here that base changes within the homologous segments in the vector could be efficiently transferred into the corresponding genomic sequences of rice recombinants. Interestingly, a few sequences from the host genome were flanked by the changed sequences derived from the vector in most of the recombinants. Because a single-stranded T-DNA molecule in Agrobacterium-mediated transformation is imported into the plant nucleus and becomes double-stranded, both single-stranded and double-stranded T-DNA intermediates can serve in gene-targeting processes. Several alternative models, including the occurrence of the mismatch correction of heteroduplex molecules formed between the genomic DNA and either a single-stranded or double-stranded T-DNA intermediate, are compared to explain the observation, and implications for the modification of endogenous genes for functional genomic analysis by gene targeting are discussed.
Johzuka-Hisatomi
Yasuyo
Y
National Institute for Basic Biology, Myodaiji, Okazaki 444-8585, Japan. yasuyo@nibb.ac.jp
Terada
Rie
R
Iida
Shigeru
S
D053903:DNA Breaks, Double-Stranded; D053843:DNA Mismatch Repair; D004269:DNA, Bacterial / Q000378:metabolism; D018390:Gene Targeting; D005822:Genetic Vectors / Q000737:chemistry; D018745:Genome, Plant; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D012275:Oryza / Q000235:genetics*; D017354:Point Mutation; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; C036483:T-DNA