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- Germann, Markus W., et al.
(author)
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Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition : physical (NMR and UV) and enzymatic (EcoRI) studies
- 1990
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 18:6, s. 1489-1498
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Journal article (peer-reviewed)abstract
- We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.
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| 3. |
- Lundberg, Peter, et al.
(author)
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Nuclear magnetic resonance studies of cellular metabolism
- 1990
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In: Analytical Biochemistry. - 0003-2697 .- 1096-0309. ; 191:2, s. 193-222
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Journal article (peer-reviewed)abstract
- Nuclear magnetic resonance (NMR) spectroscopy was described in 1946 (1,2), initially as a method that had appeal only for nuclear physicists who used it to accurately determine nuclear magnetic moments. Thissituation changed rapidly, however, when it was demonstrated that the NMR frequency for the same nucleus in different chemical compounds was different (3). For example, two separate signals are observed in a 14N NMR spectrum of a solution of NH,NO,, representing the NH: and NO; ions, respectively (4). Since individual atoms within one molecule also give rise to resolved signals (5) it became clear that the NMR technique held great analytical potential, in particular since the spectra can be recorded in such a way that the area under a signal is directly proportional to its concentration. Such phenomena and various theoretical aspects of NMR are currently quite well understood (6,7). Because of these features NMR has become the foremost spectroscopic method for the analysis of all sorts of chemical compounds.
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| 4. |
- Vogel, Hans J, et al.
(author)
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Uptake, metabolism, and storage of phosphate and nitrogen in plant cells; an NMR perspective
- 1990
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In: NMR applications in biopolymers. - Boston, MA : Plenum Press, New York, USA. - 9781468458701 - 9781468458688 ; , s. 329-348
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Book chapter (peer-reviewed)abstract
- A number of Nuclear Magnetic Resonance (NMR) Spectroscopy techniques can be used to study pH regulation and various aspects of nutrient metabolism in plant material. In this study phosphorus-31 NMR has been used to determine the energy state (ATP) and the intracellular cytoplasmic and vacuolar pH of cultured plant cells and algae. For the algae it was found that the chemical shift of the terminal polyphosphate resonance provided a good monitor of the vacuolar pH which was estimated at pH 5.5. A cytoplasmic pH of 7.2 was determined from the chemical shifts of the Pi and glucose-6-phosphate resonances. Phosphate uptake could also be followed by 31P NMR and these studies showed that Pi was stored as polyphosphates in algae, but as vacuolar Pi in certain higher plants such as Catharanthus roseus and Nicotiana tabacum.
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