Multiple physical forms of excised group II intron RNAs in wheat mitochondria

Plant mitochondrial group II introns do not all possess hallmark ribozymic features such as the bulged adenosine involved in lariat formation. To gain insight into their splicing pathways, we have examined the physical form of excised introns in germinating wheat embryos. Using RT–PCR and cRT–PCR, we observed conventional lariats consistent with a two-step transesterification pathway for introns such as nad2 intron 4, but this was not the case for the cox2 intron or nad1 intron 2. For cox2, we detected full-length linear introns, which possess non-encoded 3′terminaladenosines, as well as heterogeneous circular introns, which lack 3′ nucleotide stretches. These observations are consistent with hydrolytic splicing followed by polyadenylation as well as an in vivo circularization pathway, respectively. The presence of both linear and circular species in vivo is supported by RNase H analysis. Furthermore, the nad1 intron 2, which lacks a bulged nucleotide at the branchpoint position, comprised a mixed population of precisely full-length molecules and circular ones which also include a short, discrete block of non-encoded nucleotides. The presence of these various linear and circular forms of excised intron molecules in plant mitochondria points to multiple novel group II splicing mechanisms in vivo

Group II intron in Bacillus cereus has an unusual 3′ extension and splices 56 nucleotides downstream of the predicted site

All group II introns known to date fold into six functional domains. However, we recently identified an intron in Bacillus cereus ATCC 10987, B.c.I4, that splices 56 nt downstream of the expected 3′ splice site in vivo (Tourasse et al. 2005, J. Bacteriol., 187, 5437–5451). In this study, we confirmed by ribonuclease protection assay that the 56-bp segment is part of the intron RNA molecule, and computational prediction suggests that it might form a stable stem-loop structure downstream of domain VI. The splicing of B.c.I4 was further investigated both in vivo and in vitro. Lariat formation proceeded primarily by branching at the ordinary bulged adenosine in domain VI without affecting the fidelity of splicing. In addition, the splicing efficiency of the wild-type intron was better than that of a mutant construct deleted of the 56-bp 3′ extension. These results indicate that the intron has apparently adapted to the extra segment, possibly through conformational adjustments. The extraordinary group II intron B.c.I4 harboring an unprecedented extra 3′ segment constitutes a dramatic example of the flexibility and adaptability of group II introns

Extensive mis-splicing of a bi-partite plant mitochondrial group II intron

Expression of the seed plant mitochondrial nad5 gene involves two trans-splicing events that remove fragmented group II introns and join the small, central exon c to exons b and d. We show that in both monocot and eudicot plants, extensive mis-splicing of the bi-partite intron 2 takes place, resulting in the formation of aberrantly spliced products in which exon c is joined to various sites within exon b. These mis-spliced products accumulate to levels comparable to or greater than that of the correctly spliced mRNA. We suggest that mis-splicing may result from folding constraints imposed on intron 2 by base-pairing between exon a and a portion of the bi-partite intron 3 downstream of exon c. Consistent with this hypothesis, we find that mis-splicing does not occur in Oenothera mitochondria, where intron 3 is further fragmented such that the predicted base-pairing region is not covalently linked to exon c. Our findings suggest that intron fragmentation may lead to mis-splicing, which may be corrected by further intron fragmentation

Trans-splicing of the Ll.LtrB group II intron in Lactococcus lactis

The Ll.LtrB intron from the Gram-positive bacterium Lactococcus lactis is one of the most studied bacterial group II introns. Ll.LtrB interrupts the relaxase gene of three L. lactis conjugative elements. The relaxase enzyme recognizes the origin of transfer (oriT ) and initiates the intercellular transfer of its conjugative element. The splicing efficiency of Ll.LtrB from the relaxase transcript thus controls the conjugation level of its host element. Here, we used the level of sex factor conjugation as a read-out for Ll.LtrB splicing efficiency. Using this highly sensitive splicing/conjugation assay (107-fold detection range), we demonstrate that Ll.LtrB can trans-splice in L. lactis when fragmented at various positions such as: three different locations within domain IV, within domain I and within domain III. We also demonstrate that the intron-encoded protein, LtrA, is absolutely required for Ll.LtrB trans-splicing. Characteristic Y-branched trans-spliced introns and ligated exons are detected by RT-PCR from total RNA extracts of cells harbouring fragmented Ll.LtrB. The splicing/conjugation assay we developed constitutes the first model system to study group II intron trans-splicing in vivo. Although only previously observed in bacterial-derived organelles, we demonstrate that assembly and trans-splicing of a fragmented group II intron can take place efficiently in bacterial cells

Lateral transfer of introns in the cryptophyte plastid genome

Cryptophytes are unicellular eukaryotic algae that acquired photosynthesis secondarily through the uptake and retention of a red-algal endosymbiont. The plastid genome of the cryptophyte Rhodomonas salina CCMP1319 was recently sequenced and found to contain a genetic element similar to a group II intron. Here, we explore the distribution, structure and function of group II introns in the plastid genomes of distantly and closely related cryptophytes. The predicted secondary structures of six introns contained in three different genes were examined and found to be generally similar to group II introns but unusually large in size (including the largest known noncoding intron). Phylogenetic analysis suggests that the cryptophyte group II introns were acquired via lateral gene transfer (LGT) from a euglenid-like species. Unexpectedly, the six introns occupy five distinct genomic locations, suggesting multiple LGT events or recent transposition (or both). Combined with structural considerations, RT–PCR experiments suggest that the transferred introns are degenerate ‘twintrons’ (i.e. nested group II/group III introns) in which the internal intron has lost its splicing capability, resulting in an amalgamation with the outer intron

Laser Interactions for the Synthesis and In Situ Diagnostics of Nanomaterials

Laser interactions have traditionall been at thec center of nanomaterials science, providing highly nonequilibrium growth conditions to enable the syn- thesis of novel new nanoparticles, nanotubes, and nanowires with metastable phases. Simultaneously, lasers provide unique opportunities for the remote char- acterization of nanomaterial size, structure, and composition through tunable laser spectroscopy, scattering, and imaging. Pulsed lasers offer the opportunity, there- fore, to supply the required energy and excitation to both control and understand the growth processes of nanomaterials, providing valuable views of the typically nonequilibrium growth kinetics and intermediates involved. Here we illustrate the key challenges and progress in laser interactions for the synthesis and in situ diagnostics of nanomaterials through recent examples involving primarily carbon nanomaterials, including the pulsed growth of carbon nanotubes and graphene

Observation of the metallic-type selective etching of single walled carbon nanotubes by real-time in situ two-laser Raman spectroscopy

Real time, in situ Raman spectroscopy with two simultaneously incident laser excitation wavelengths is used to investigate the dynamics of single-walled carbon nanotube etching. For the source material, nanotubes of diameter 1.4 nm, a 532 nm laser is resonant with semiconducting nanotubes and a 633 nm laser is resonant with metallic nanotubes. Changes in metallic and semiconducting population are tracked separately and simultaneously. In oxygen, metals consistently etch faster than semiconductors, and all etch rates increase with the process temperature and the defect density in the source material. A similar evolution is observed in carbon dioxide. Simultaneous two-color Raman spectroscopy provides information beyond standard Raman spectroscopy and can be effective as an instantaneous measure of metallicity for nanotubes.Peer reviewed: YesNRC publication: Ye

Type and species selective air etching of single-walled carbon nanotubes tracked with in situ Raman spectroscopy

The thermal oxidation of carbon nanotubes in air is investigated by in situ Raman spectroscopy. Etching rates are directly seen to be diameter, chirality, and type dependent. We directly track the evolution of bundled nanotube networks that undergo air etching from 300 to 600 \ub0C. Some species are more robust than others. Changes to radial breathing mode (RBM) and G\u2013 peak structures suggest that metallic species etch away more rapidly, with smaller diameter semiconducting species etching more slowly and large diameter nanotubes, including semiconductors, etching last. The decay in integrated G and D band intensities is tracked and fit reasonably well with biexponential decay. The RBM evolution is better represented by a single exponential. All bands are fit to activation plots with RBMs showing significantly different rates.Peer reviewed: YesNRC publication: Ye

The Dynamics of the Nucleation, Growth and Termination of Single-Walled Carbon Nanotubes from in situ Raman Spectroscopy During Chemical Vapor Deposition

The dynamics of the chemical vapor deposition (CVD) of single-walled carbon nanotubes (SWNTs) is extracted experimentally using in situ Raman spectroscopy. Nanotubes are grown using a thin fi lm cobalt catalyst and an ethanol precursor in a miniature hot walled reactor with optical access. Raman spectra at room temperature and at the growth temperature are compared for two growth temperatures. The evolution of the G-band, D-band, and radial breathing mode (RBM) is tracked at the growth temperature with time resolution of a few seconds. There are three identifiable phases in the evolution of the Raman signal intensity: an initial exponential increasing phase, a linear growth phase, and a saturation phase. In situ optical spectroscopy thus enables the study of nucleation, steady growth, and deactivation processes to be investigated separately in real time. The evolution curves for all bands (G, D, and RBM), when scaled, collapse onto the same curve, to within experimental uncertainty.Peer reviewed: YesNRC publication: Ye

RT–PCR and cRT–PCR detection of lariat (and/or circularized) versus linear wheat mitochondrial intron molecules

<p><b>Copyright information:</b></p><p>Taken from "Multiple physical forms of excised group II intron RNAs in wheat mitochondria"</p><p>Nucleic Acids Research 2006;34(9):2782-2790.</p><p>Published online 22 May 2006</p><p>PMCID:PMC1464410.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> () RT–PCR across the excised intron junction using 24 h wheat mitochondrial RNA as template, either pre-treated with ligase (+L; lanes 2, 5, 7 and 9) or untreated (−L; lanes 1, 4, 6 and 8) generated products of sizes 450, 520, 630 and 400 bp (denoted by arrowheads) for mRNA, intron 4, intron and intron 2, respectively. Size markers are shown in lane 3. () Southern blot of gels (shown in A, lanes 4–9) using intron-specific oligomer probes for intron 4, intron and intron 2. () Schematic showing positions of primers (arrows 1 and 2) used for RT–PCR and cRT–PCR in panels (A) and (B). Branchpoint is shown by A. Dotted lines indicate regions of amplification. () Direct sequencing of RT–PCR products for (D) intron (+L), (E) intron 2 (−L) and (F) intron 2 (+L) using oligomers 6, 11 and 16, respectively (Supplementary data). Note that an inverted orientation of the sequencing gel is shown for (E). Arrows show positions of the 5′ terminal nucleotide of the introns, stars highlight short non-encoded A-rich stretches and the discrete non-encoded insert sequence is boxed in (E)