PubMed · 7527224
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A Yule. 1994. Amplification-based diagnostics target TB.. https://doi.org/10.1038/nbt1294-1335
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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.
We have used two new dye sets for automated dye-labeled terminator DNA sequencing. One set consists of four, 4,7-dichlororhodamine dyes (d-rhodamines). The second set consists of energy-transfer dyes that use the 5-carboxy-d-rhodamine dyes as acceptor dyes and the 5- or 6-carboxy isomers of 4'-aminomethylfluorescein as the donor dye. Both dye sets utilize a new linker between the dye and the nucleotide, and both provide more even peak heights in terminator sequencing than the dye-terminators consisting of unsubstituted rhodamine dyes. The unsubstituted rhodamine terminators produced electropherograms in which weak G peaks are observed after A peaks and occasionally C peaks. The number of weak G peaks has been reduced or eliminated with the new dye terminators. The general improvement in peak evenness improves accuracy for the automated base-calling software. The improved signal-to-noise ratio of the energy-transfer dye-labeled terminators combined with more even peak heights results in successful sequencing of high molecular weight DNA templates such as bacterial artificial chromosome DNA.
The osmotic stress technique was used to measure the changes in macromolecular hydration that accompany binding of the Escherichia coli CAP protein to its transcription-regulatory site (C1) in the lactose promoter and that accompany the transfer of CAP from site C1 to nonspecific genomic DNA. Formation of the C1 complex is accompanied by the net release of 79 +/- 11 water molecules. If all water molecules were released from macromolecular surfaces, this result would be consistent with a net reduction of solvent-accessible surface area of 711 +/- 189 A2. This area is only slightly smaller than the solvent-inaccessible macromolecular interface in crystalline CAP-DNA complexes. The transfer of CAP from site C1 to nonspecific sites is accompanied by the net uptake of 56 +/- 10 water molecules. Taken with the water stoichiometry of sequence-specific binding, this value implies that formation of a nonspecific complex is accompanied by the net release of 2-44 water molecules. The enhanced stabilities of CAP-DNA complexes with increased osmolality (decreased water activity) may contribute to the ability of E.coli cells to tolerate dehydration and/or high external salt concentrations.