Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Enhancing disease resistance in poplar through modification of its natural defense pathway

Plant Molecular Biology - Modification of the poplar defense pathway through pathogen-induced expression of an amphibian host defense peptide modulates plant innate immunity and confers robust and...     

Supramolecular non-amyloid intermediates in the early stages of α-synuclein aggregation

The aggregation of α-synuclein is associated with progression of Parkinson's disease. We have identified submicrometer supramolecular structures that mediate the early stages of the overall mechanism. The sequence of structural transformations between metastable intermediates were captured and characterized by atomic force microscopy guided by a fluorescent probe sensitive to preamyloid species. A novel ~0.3-0.6 μm molecular assembly, denoted the acuna, nucleates, expands, and liberates fibers with distinctive segmentation and a filamentous fuzzy fringe. These fuzzy fibers serve as precursors of mature amyloid fibrils. Cryo-electron tomography resolved the acuna inner structure as a scaffold of highly condensed colloidal masses interlinked by thin beaded threads, which were perceived as fuzziness by atomic force microscopy. On the basis of the combined data, we propose a sequential mechanism comprising molecular, colloidal, and fibrillar stages linked by reactions with disparate temperature dependencies and distinct supramolecular forms. We anticipate novel diagnostic and therapeutic approaches to Parkinson's and related neurodegenerative diseases based on these new insights into the aggregation mechanism of α-synuclein and intermediates, some of which may act to cause and/or reinforce neurotoxicity.     

Substrate binding and activation in the active site of cytochrome <Emphasis Type=Italic>c</Emphasis> nitrite reductase: a density functional study

Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His₂₇₇, whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg₁₁₄. The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr₂₁₈, which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.     

Can DNA-binding proteins of replisome tautomerize nucleotide bases? Ab initio model study

Ab initio quantum-chemical study of specific point contacts of replisome proteins with DNA modeled by acetic acid with canonical and mutagenic tautomers of DNA bases methylated at the glycosidic nitrogen atoms was performed in vacuo and continuum with a low dielectric constant (??～?4) corresponding to a hydrophobic interface of protein-nucleic acid interaction. All tautomerized complexes were found to be dynamically unstable, because the electronic energies of their back-reaction barriers do not exceed zero-point vibrational energies associated with the vibrational modes whose harmonic vibrational frequencies become imaginary in the transition states of the tautomerization reaction. Additionally, based on the physicochemical arguments, it was demonstrated that the effects of biomolecular environment cannot ensure dynamic stabilization. This result allows suggesting that hypothetically generated by DNA-binding proteins of replisome rare tautomers will have no impact on the total spontaneous mutation due to the low reverse barrier allowing a quick return to the canonical form.