PubMed · 9304210
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E Pennisi. 1997-09-05. Laboratory workhorse decoded.. https://doi.org/10.1126/science.277.5331.1432
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Agrobacterium transfers DNA into plant cells, leading to tumors, hairy roots (HR), and natural genetically modified organisms (nGMOs). Transferred DNAs (T-DNAs) from agrobacteria and T-DNA-derived cellular T-DNAs (cT-DNAs) from nGMOs vary considerably and may carry up to 15 different genes. Among these, opine synthase (ops) genes encode the synthesis of opines used as nutrients by the agrobacteria. Earlier studies predicted large numbers of naturally transformed plant species, but only few have been identified and studied so far. We therefore developed a general method to detect cT-DNAs in all publicly available whole genome sequences (WGS) and Sequence Read Archive (SRA) data from land plants. To avoid false positives, we only retained DNA sequences coding for T-DNA proteins. A total of 2614 nGMO species were identified, most are eudicots. However, cT-DNAs were also found in 82 mosses and 75 ferns, showing that Agrobacterium can also generate natural transformants among the early land plants. Analysis of 149 cT-DNA maps revealed different types of T-DNAs. Most notably, these included small T-DNAs (mini T-DNAs) with a single opine synthase gene. Mini T-DNAs are not expected to induce tumors or HRs. The predominance of mini cT-DNAs in mosses and ferns, and the presence of more complex cT-DNAs in spermatophytes, indicate that mini T-DNAs represent the earliest types of T-DNA. Our study also detected unusual T-DNA integration patterns, with multiple copies spread out over several hundreds of kilobases.
Repetitive sequences in DNA molecules, some of which are palindromic, tend to form stable cruciforms. These are frequently located in promoter regions of a specific operon and origin of replication. Temperature gradient gel electrophoresis can be used to distinguish among various supercoiled DNA topoisomers and to ascertain whether or not the cruciform motif has been extruded. In the current study, this technique is implemented for the first time to address the role of temperature in cruciform extrusion from plasmids.
We describe a general methodology for the direct detection of DNA by the design of a split-protein system that reassembles to form an active complex only in the presence of a targeted DNA sequence. This approach, called SEquence Enabled Reassembly (SEER) of proteins, combines the ability to rationally dissect proteins to construct oligomerization-dependent protein reassembly systems and the availability of DNA binding Cys2-His2 zinc-finger motifs for the recognition of specific DNA sequences. We demonstrate the feasibility of the SEER approach utilizing the split green fluorescent protein appended to appropriate zinc fingers, such that chromophore formation is only catalyzed in the presence of DNA sequences that incorporate binding sites for both zinc fingers.