PubMed · 7979081
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M Spigelman. 1994. Ancient DNA.. https://pubmed.ncbi.nlm.nih.gov/7979081/
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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.
BACKGROUND: The causes of sarcoidosis are not known. The DNA of Mycobacterium tuberculosis has been detected in some sarcoid lesions. In Japan, Propionibacterium acnes has been isolated from such lesions, but whether this indigenous bacterium is related to the disease is unclear. We used PCR to estimate the number of genomes of these bacteria in sarcoid lesions, to identify any link between sarcoidosis and these two bacterial species. METHODS: We examined formalin-fixed and paraffin-embedded sections of biopsy and surgical samples from lymph nodes of 15 patients with sarcoidosis, 15 patients with tuberculosis, and 15 patients with gastric cancer (controls). Quantitative PCR was done to amplify segments of 16 S ribosomal RNA of P. acnes and P. granulosum and of insertion sequence 6110 of M. tuberculosis. PCR products were identified and the quantities of the products were estimated in terms of the fluorescence of oligonucleotide reporter probes. The numbers of bacterial genomes in samples were estimated from standard curves of serially diluted bacterial DNA. FINDINGS: Genomes of M. tuberculosis were found in samples from all 15 patients with tuberculosis, from three patients with sarcoidosis, and in one control sample. Genomes of P. acnes were found in 12 of the 15 patients with sarcoidosis, in two tuberculosis patients, and three controls. The difference in the estimated number of P. acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M. tuberculosis between people with and without tuberculosis. There were 5x10(5) P. acnes genomes in sarcoidosis and 3x10(6) M. tuberculosis genomes in tuberculosis, respectively, on average per microg of total DNA. The three patients with sarcoidosis but without P. acnes all had P. granulosum DNA in their biopsy samples; the number of genomes of the bacterium was 5x10(5). INTERPRETATION: These findings suggest that propionibacteria had resided or proliferated ectopically in the sarcoid lesions, whether there was a connection with the disease or not. Propionibacteria are a more likely cause than mycobacteria of sarcoidosis.
We use a synthetic "primed bubble-duplex" model elongation complex developed previously to examine certain structural and thermodynamic features of the transcription elongation complex of Escherichia coli. The nucleic acid framework of this model complex consists of a linear base-paired DNA molecule with a central "bubble" of non-complementary nucleotide residues, together with a single-stranded RNA molecule that is complementary (at its 3'-end) to three to 12 nucleotide residues of one of the DNA strands within the bubble. RNA polymerase is added to this framework in trans, and on addition of rNTPs the resulting complex can elongate the 3'-end of the RNA primer in a template-dependent manner with functional properties that are indistinguishable from those of a "natural" promoter-initiated transcription elongation complex operating under the same conditions. In this study we use this model system to show that the formation of a stable elongation complex at any particular template position can be treated as an equilibrium process, and that semi-quantitative dissociation constants can be estimated for the complex by using a gel band-shift assay to monitor the binding of the RNA oligomer to the complex. We then show that the formation of a stable complex depends on the presence of a complementary RNA-DNA hybrid that is at least 9 bp in length, and in addition that several nucleotide residues of non-complementary RNA located upstream of the RNA-DNA hybrid bind strongly to the putative single-stranded RNA binding site of the polymerase and significantly enhance the stability of the resulting elongation complex. Finally, we demonstrate that the measured stabilities of the model constructs in which the length of the RNA-DNA hybrid is varied correlate well with the transcriptional processivity of the functioning complex that results when rNTPs are added. These findings are discussed in the context of related studies of both model systems and natural elongation complexes. The general concepts that emerge are used to define some central structural and functional features of the transcription complex.