Illegitimate recombination in plants : a model for T-DNA integration

Agrobacterium tumefaciens is a soil bacterium capable of transferring DNA (the T-DNA) to the genome of higher plants, where it is then stably integrated. Six T-DNA inserts and their corresponding preinsertion sites were cloned from Arabidopsis thaliana and analyzed. Two T-DNA integration events from Nicotiana tabacum were included in the analysis. Nucleotide sequence comparison of plant target sites before and after T-DNA integration showed that the T-DNA usually causes only a small (13-28 bp) deletion in the plant DNA but larger target rearrangements can occur. Short homologies between the T-DNA ends and the target sites, as well as the presence of filler sequences at the junctions, indicate that T-DNA integration is mediated by illegitimate recombination and that these processes in plants are very analogous to events in mammalian cells. We propose a model for T-DNA integration on the basis of limited base-pairing for initial synapsis, followed by DNA repair at the junctions. Variations of the model can explain the formation of filler DNA at the junctions by polymerase slipping and template switching during DNA repair synthesis and the presence of larger plant target DNA rearrangements

Gene expression associated with water-stress adaptation of rice cells and identification of two genes as hsp 70 and ubiquitin

We have isolated polyethylene glycol (PEG)-adapted rice cells that can proliferate in a medium with 20% PEG as well as in a medium with 1% NaCl. The adapted-cells overproduce a set of proteins whose roles may be associated with water-stress adaptation. To isolate genes encoding these proteins, we differentially screened a cDNA library and obtained 5 cDNA clones which showed preferential hybridization to mRNA of adapted cells. The present paper describes the pattern of expression and the sequence analysis of these 5 genes. Sequence analysis of partial cDNAs indicates that two genes encode the 70 kDa heat shock protein and the ubiquitin but the identities of the other 3 are not known. The expression of all 5 genes fluctuates slightly during the growth cycle of the PEG-adapted cells grown in the control or in the PEG-containing medium. In contrast, gene expression in the parental cells fluctuates to a much greater extent and always begins with an enhanced expression during the first day after subculture. Sub-lethal concentrations of PEG or NaCl have no immediate effect on gene expression in parental cells but NaCl may have a long term effect in enhancing the expression of two genes after 5 days. Abscisic acid (ABA) has no effect on the expression of 4 genes but suppresses the expression of one gene. The roles of these genes in water-stress adaptation of plant cells are discussed

The use of selectable markers for the isolation of plant-DNA/T-DNA junction fragments in a cosmid vector

DNA of the crown gall tumor line W38T37::Tn7-1 was partially digested with Sau3A to an average molecular weight of 25 Md and ligated either directly or after size fractionation to BamHI cut cosmid pJC81 DNA. After in vitro packaging in phage λ particles and transduction to E. coli HB101, recombinants that expressed the Tn7 coded resistances to spectinomycin and trimethoprim were selected. The recombinant plasmids thus isolated contained part or the whole of Tn7 together with adjacent T-DNA. Four independent, large clones are described, three containing the left border of the T-DNA, one containing the right border and an intact copy of the Tn7 transposon. In this case all the Tn7 encoded genes were shown to have remained fully functional since the reisolated Tn7 was found to be capable of normal transposition in E. coli. The T-DNA in the W38T37::Tn7 tumor line is flanked both to the left and to the right by highly AT rich repetitive plant sequences. These results further demonstrate that foreign genes can be transferred, integrated and stably maintained in chromosomes of plant cells without undergoing any observable rearrangements. This method of cosmid cloning combined with direct selection for the desired recombinant colonies is of general application for the genomic cloning of transformed eukaryotic cells

Complete nucleotide sequence of the T-DNA region of the plant tumour-inducing Agrobacterium tumefaciens Ti plasmid pTiC58

The complete nucleotide sequence has been determined of the T-DNA region from the plant tumour-inducing Agrobacterium tumefaciens nopaline Ti plasmid pTiC58. The T-DNA itself consists of 24 782 bp flanked by two direct 25 bp repeats, the border sequences. In addition, 3622 bp located at the left and 1070 bp at the right of the T-DNA borders were sequenced. Twenty-two open reading frames that code for proteins larger than 125 amino acids have been identified

CLONING AND SEQUENCE-ANALYSIS OF THE GENE ENCODING ISOCITRATE LYASE FROM RHODOCOCCUS FASCIANS.

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