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Abstract: Phylogenetic relationships within the Grimmiaceae/Ptychomitriaceae were studied using a plastid tRNA cluster, including four tRNAs (trnS, trnT, trnL, trnF), a fast evolving gene (rps4), four spacers separating the coding regions, as well as one group I intron. Secondary structure analyses of the spacers as well as the trnL intron P8 domain identified several homoplastic inversions. Tracing the structural evolution of P8 we were able to identify lineage specific modifications that are mainly explained by inversions often in combination with large indel events. Phylogenetic analyses using maximum parsimony, maximum likelihood, and Bayesian methods indicate that Jaffueliobryum and Indusiella are closely related to Ptychomitrium and form the Ptychomitriaceae s. str. As Campylostelium is neither resolved within Ptychomitriaceae s. str. nor Grimmiaceae s. str., we prefer to treat it in its own family, Campylosteliaceae De Not. The systematic position of Glyphomitrium, as also found by other authors, should be considered in a broader analysis of haplolepidous mosses as our analyses indicate that it is not part of Campylosteliaceae, Grimmiaceae, or Ptychomitriaceae. Within Grimmiaceae s. str., Racomitrium is recognized as a monophyletic group sister to a clade including Dryptodon, Grimmia, and Schistidium. Coscinodon species appear disperse in Grimmia s. str. next to species sharing the same gametophyte morphology, and thus the genus is synonymized with Grimmia. Finally, Schistidium is resolved monophyletic with high statistical support, and seems to represent a rapidly evolving group of species. Our results are not fully congruent with recently published treatments splitting Grimmiaceae in a fairly high number of genera, neither with a comprehensive Grimmia including Dryptodon and Grimmia s. str. [Copyright &y& Elsevier]

Abstract: Olpidiopsis porphyrae sp. nov., a marine oomycete endoparasite that infects the commercially cultivated red alga Porphyra yezoensis, is described and its phylogenetic position based on molecular data and ultrastructural morphology is discussed. O. porphyrae infects the host Porphyra by means of encysted zoospores. Spherical-shaped holocarpic thalli develop within the cytoplasm of its algal host, which produce monoplanetic, subapically biflagellate zoospores. The characteristic features of this isolate are the ellipsoidal, unicellular thallus and simple holocarpic zoosporangial development, which show morphological similarity with the genus Olpidiopsis. Laboratory infection experiments with a wide range of green, brown, and red algae revealed that O. porphyrae infects several stages of the bangialean red algae (the genera Bangia and Porphyra). Molecular phylogenetic analyses inferred from both SSU rRNA and cox2 genes showed O. porphyrae branched before the main saprolegnian and peronosporalean lineages within the monophyletic oomycete clade, indicating its phylogenetic separation from them. A single or double K-body-like organelle, which contains tubular inclusions, is found located to one side of the zoospore nucleus and shows similarities to homologous organelles previously described in O. saprolegniae. The ultrastructural morphology of O. porphyrae with zoospore initials containing K-bodies and tubular mitochondrial cristae is characteristic of oomycetes. Group I intron-like multiple insertions were found in the SSU rRNA gene of O. porphyrae. This is the first report of SSU group I introns in the class Oomycetes. [Copyright &y& Elsevier]

Although the examination of large subunit ribosomal RNA genes (LSU rDNA) is advanced in phylogenetic studies, no corresponding sequence data from trebouxiophytes have been published, with the exception of ‘ Chlorella’ ellipsoidea Gerneck. We determined the LSU rDNA sequence of Chlorella vulgaris Beijerinck and of the symbiotic alga of green paramecium, Chlorella sp. NC64A. A total of 59 nucleotide substitutions were found in the LSU rDNA of the two species, which are disproportionately distributed. Primarily, 65% of the substitutions were encountered in the first 800 bp of the alignment. This segment apparently has evolved eight times faster than the complete SSU rDNA sequence, making it a good candidate for a phylogenetic marker and giving a resolution level intermediate between small subunit (SSU) rDNA and internal transcribed spacers. Green algae are known as a group I intron-rich group along with rhodophytes and fungi. NC64A is particularly rich in the introns; five introns were newly identified from the LSU rDNA sequence, which we named Cnc.L200, Cnc.L1688, Cnc.L1926, Cnc.L2184 and Cnc.L2437, following the insertion positions. In the present study we analyzed these introns with three others (Cnc.S943, Cnc.S1367 and Cnc.S1512) that had already been found in NC64A SSU rDNA. Secondary structure modeling placed these introns in the group I intron family, with four introns belonging to subgroup C1 and the other four introns belonging to subgroup E. Five of the intron insertion positions are unique to the paramecian symbiont, which may indicate relatively recent events of intron infections that includes transpositions. Intron phylogeny showed unprecedented relationships; four Cnc. IC1 introns made a clade with some green algal introns with insertions at nine different positions, whereas four Cnc. IE introns made a clade with the S651 intron ( Chlorella sp. AN 1–3), which lay as a sister to the S516 insertion position subfamily. [ABSTRACT FROM AUTHOR]

Twin-ribozyme introns contain a branching ribozyme (GIR1) followed by a homing endonuclease (HE) encoding sequence embedded in a peripheral domain of a group I splicing ribozyme (GIR2). GIR1 catalyses the formation of a lariat with 3 nt in the loop, which caps the HE mRNA. GIR1 is structurally related to group I ribozymes raising the question about how two closely related ribozymes can carry out very different reactions. Modelling of GIR1 based on new biochemical and mutational data shows an extended substrate domain containing a GoU pair distinct from the nucleophilic residue that dock onto a catalytic core showing a different topology from that of group I ribozymes. The differences include a core J8/7 region that has been reduced and is complemented by residues from the pre-lariat fold. These findings provide the basis for an evolutionary mechanism that accounts for the change from group I splicing ribozyme to the branching GIR1 architecture. Such an evolutionary mechanism can be applied to other large RNAs such as the ribonuclease P. [ABSTRACT FROM AUTHOR]

Abstract: Metal ions play key roles in the folding and function for many structured RNAs, including group I introns. We determined the X-ray crystal structure of the Azoarcus bacterial group I intron in complex with its 5′ and 3′ exons. In addition to 222 nucleotides of RNA, the model includes 18 Mg2+ and K+ ions. Five of the metals bind within 12 Å of the scissile phosphate and coordinate the majority of the oxygen atoms biochemically implicated in conserved metal–RNA interactions. The metals are buried deep within the structure and form a multiple metal ion core that is critical to group I intron structure and function. Eight metal ions bind in other conserved regions of the intron structure, and the remaining five interact with peripheral structural elements. Each of the 18 metals mediates tertiary interactions, facilitates local bends in the sugar-phosphate backbone or binds in the major groove of helices. The group I intron has a rich history of biochemical efforts aimed to identify RNA–metal ion interactions. The structural data are correlated to the biochemical results to further understand the role of metal ions in group I intron structure and function. [Copyright &y& Elsevier]

Ribosomal DNA (rDNA) is a specialised chromosomal location that is dedicated to high-level transcription of ribosomal RNA genes. Interestingly, rDNAs are frequently interrupted by parasitic elements, some of which carry protein genes. These are non-LTR retrotransposons and group II introns that encode reverse transcriptase-like genes, and group I introns and archaeal introns that encode homing endonuclease genes (HEGs). Although rDNA-embedded protein genes are widespread in nuclei, organelles and bacteria, there is surprisingly little information available on how these genes are expressed. Exceptions include a handful of HEGs from group I introns. Recent studies have revealed unusual and essential roles of group I and group I-like ribozymes in the endogenous expression of HEGs. Here we discuss general aspects of rDNA-embedded protein genes and focus on HEG expression from group I introns in the nucleolus. [ABSTRACT FROM AUTHOR]

Abstract: Although hepatocellular carcinoma (HCC) is one of the world-wide common malignancies, development of more specific and controlled therapeutic methods should be warranted. In this study, we describe a novel approach to HCC therapy that is based on trans-splicing ribozyme-mediated replacement of HCC-associated specific RNAs. We have developed a specific ribozyme that can target and replace human alpha-fetoprotein (AFP) RNA, which is highly expressed in HCC, with new transcript exerting therapeutic activity selectively in AFP-expressing liver cancer cells. The RNA replacement was employed via a high-fidelity trans-splicing reaction with the targeted residue in the AFP-expressing cells. Noticeably, the ribozyme could selectively deliver activity of suicide gene, herpes simplex virus thymidine kinase gene, into the liver cancer cells expressing the AFP RNA and thereby specifically and effectively retarded the survival of these cells with ganciclovir treatment. Simultaneously with the specific induction of therapeutic gene activity, the ribozyme reduced expression level of the targeted AFP RNA in the cells. These results suggest that the AFP RNA-targeting trans-splicing ribozyme could be a useful genetic agent for HCC-targeted efficient gene therapy. [Copyright &y& Elsevier]

The homing endonuclease I-Ssp6803I causes the insertion of a group I intron into a bacterial tRNA gene—the only example of an invasive mobile intron within a bacterial genome. Using a computational fold prediction, mutagenic screen and crystal structure determination, we demonstrate that this protein is a tetrameric PD-(D/E)-XK endonuclease—a fold normally used to protect a bacterial genome from invading DNA through the action of restriction endonucleases. I-Ssp6803I uses its tetrameric assembly to promote recognition of a single long target site, whereas restriction endonuclease tetramers facilitate cooperative binding and cleavage of two short sites. The limited use of the PD-(D/E)-XK nucleases by mobile introns stands in contrast to their frequent use of LAGLIDADG and HNH endonucleases—which in turn, are rarely incorporated into restriction/modification systems. [ABSTRACT FROM AUTHOR]

Analyses of mitochondrial sequences revealed the existence of a group I intron in the cytochrome oxidase subunit 1 ( cox1) gene in 13 of 41 genera (20 out of 73 species) of corals conventionally assigned to the suborder Faviina. With one exception, phylogenies of the coral cox1 gene and its intron were concordant, suggesting at most two insertions and many subsequent losses. The coral introns were inferred to encode a putative homing endonuclease with a LAGLI-DADG motif as reported for the cox1 group I intron in the sea anemone Metridium senile. However, the coral and sea anemone cox1 group I introns differed in several aspects, such as the intron insertion site and sequence length. The coral cox1 introns most closely resemble the mitochondrial cox1 group I introns of a sponge species, which also has the same insertion site. The coral introns are also more similar to the introns of several fungal species than to that of the sea anemone (although the insertion site differs in the fungi). This suggests either a horizontal transfer between a sponge and a coral or independent transfers from a similar fungal donor (perhaps one with an identical insertion site that has not yet been discovered). The common occurrence of this intron in corals strengthens the evidence for an elevated abundance of group I introns in the mitochondria of anthozoans. [ABSTRACT FROM AUTHOR]

Abstract: Myxomycetes (plasmodial slime molds) belonging to the order Physarales contain obligatory group I introns at positions 1949 and 2449 in their large subunit ribosomal RNA gene. Here, we report 36 group I introns from the Didymiaceae family (order Physarales) from 18 isolates representing three genera and seven species, and have reconstructed both host and intron phylogenies. The introns, named L1949 and L2449, were found in all isolates analyzed, consistent with an obligatory distribution in Didymiaceae. The introns fold at the RNA-level into typical group I ribozyme core structures that are relatively conserved, but contain large and highly variable extension sequences in peripheral domains without any detectable protein coding capacities. Furthermore, the L1949 and L2449 introns have probably become dependent on host factors for folding or activity. This assumption is based on that all introns tested for self-splicing in vitro failed to ligate the flanking exon regions. Phylogenies based on LSU rDNA and intron sequences are consistent with that the L1949 and L2449 introns follow a strict vertical inheritance within Didymiaceae. We suggest that the Didymiaceae L1949 and L2449 introns are well suited as high-resolution markers in genetic assessments at various taxonomic levels, from closely related strains of a single species to separating genera. [Copyright &y& Elsevier]

Abstract: Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the β-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS–RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex. [Copyright &y& Elsevier]

Abstract: Carcinoembryonic antigen (CEA) has been shown to be involved in a variety of neoplasia process, such as tumor cell adhesion, metastasis, blocking of cellular immune mechanisms, and anti-apoptosis function. Therefore, CEA has been a potential target for anti-cancer therapy. In this study, we developed a specific ribozyme that can target CEA RNA and then reprogram the RNA with new transcripts, resulting in triggering of transgene activity selectively in cancer cells that express the RNA. The ribozyme-mediated induction of the transgene expression was caused via a highly accurate and specific RNA replacement through trans-splicing reaction with the targeted residue in the CEA-expressing cells. Simultaneously with the specific RNA replacement, the ribozyme efficiently reduced expression level of the targeted CEA RNA in the cells. Importantly, the ribozyme could selectively deliver activity of suicide gene, herpes simplex virus thymidine kinase gene, into cancer cells expressing the CEA RNA and thereby specifically retarded the survival of these cells with ganciclovir treatment. These suggest that the CEA RNA-targeting trans-splicing ribozyme could be a powerful genetic agent for specific cancer gene therapy. [Copyright &y& Elsevier]

Abstract: Like most cellular RNA enzymes, the bI5 group I intron requires binding by a protein cofactor to fold correctly. Here, we use single-molecule approaches to monitor the structural dynamics of the bI5 RNA in real time as it assembles with its CBP2 protein cofactor. These experiments show that CBP2 binds to the target RNA in two distinct modes with apparently opposite effects: a “non-specific” mode that forms rapidly and induces large conformational fluctuations in the RNA, and a “specific” mode that forms slowly and stabilizes the native RNA structure. The bI5 RNA folds though multiple pathways toward the native state, typically traversing dynamic intermediate states induced by non-specific binding of CBP2. These results suggest that the protein cofactor-assisted RNA folding involves sequential non-specific and specific protein–RNA interactions. The non-specific interaction potentially increases the local concentration of CBP2 and the number of conformational states accessible to the RNA, which may promote the formation of specific RNA–protein interactions. [Copyright &y& Elsevier]

Abstract: Aminoglycoside antibiotics inhibit several types of ribozymes, including group I introns, by displacing critical Mg2+ ions. However, they stimulate activity of the small hairpin ribozyme. We show here that aminoglycosides promote self-splicing of the Cr.psbA2 group I intron at subthreshold Mg2+ concentrations. Neomycin is the most effective of the aminoglycosides tested; it stimulates splicing of Cr.psbA2 at micromolar concentrations, and, in this respect, is >100-fold more effective than spermidine. At optimal Mg2+ for Cr.psbA2 splicing, these drugs, especially kanamycin B and tobramycin, promote GTP attack at the 3′ splice-site. Kinetic analysis suggests that this is due to an alternatively folded state of the ribozyme that is induced, or stabilized, by aminoglycosides. A similar effect is observed at high Mg2+ concentrations. Comparing the effects of structurally related aminoglycosides indicates that splicing promotion is more sensitive to drug structure than misfolding and occurs at lower drug concentrations. These data show that aminoglycosides can promote biochemical activities of a large ribozyme by acting as a Mg2+ mimic. The results also underscore the functional diversity of group I introns in nature. [Copyright &y& Elsevier]

The self-assembly of RNA structure depends on the interactions of counterions with the RNA and with each other. Comparison of various polyamines showed that the tertiary structure of the Tetrahymena ribozyme is more stable when the counterions are small and highly charged. By monitoring the folding kinetics of the ribozyme as a function of polyamine concentration, we now find that the charge density of the counterions determines the positions of the folding transition states. The transition state ensemble (TSE) between U and N moves away from the native state as the counterion valence and charge density increase, as predicted by the Hammond postulate. The TSE is broader and less structured when the RNA is refolded in polyamines rather than Mg2+. That the charge density of the counterions determines the plasticity of the TSE demonstrates the importance of interactions among condensed counterions for the self-assembly of RNA structures. We propose that the major barrier to RNA folding is dominated by entropy changes when counterion charge density is low and enthalpy differences when it is high. [Copyright &y& Elsevier]

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g. three of the five group I introns in Chlamydomonas reinhardtii. We have investigated one of these ribozymes, an intron from the 23S LSU rRNA gene of Chlamydomonas reinhardtii ( Cr.LSU), by determining if the inhibition by Mn2+ involves catalysis, folding, or both. Kinetic analysis of guanosine-dependent cleavage by a Cr.LSU ribozyme, 23S.5ΔGb, that lacks the 3′ exon and intron-terminal G shows that Mn2+ does not affect guanosine binding or catalysis, but instead promotes misfolding of the ribozyme. Surprisingly, ribozyme misfolding induced by Mn2+ is highly cooperative, with a Hill coefficient larger than that of native folding induced by Mg2+. At lower Mn2+ concentrations, metal inhibition is largely alleviated by the guanosine cosubstrate (GMP). The concentration dependence of guanosine cosubstrate-induced folding suggests that it functions by interacting with the G binding site, perhaps by displacing an inhibitory Mn2+. Because of these and other properties of Cr.LSU, the tertiary structure of the intron from 23S.5ΔGb was examined using Fe2+-EDTA cleavage. The ground-state structure shows evidence of an unusually open ribozyme core: the catalytic P3–P7 domain and the nucleotides that connect it to the P4–P5–P6 domain are exposed to solvent. The implications of this structure for the in vitro and in vivo properties of this intron ribozyme are discussed. [ABSTRACT FROM AUTHOR]

Trans-splicing group I ribozymes have been introduced in order to mediate RNA reprogramming (including RNA repair) of therapeutically relevant RNA transcripts. Efficient RNA reprogramming depends on the appropriate efficiency of the reaction, and several attempts, including optimization of target recognition and ribozyme catalysis, have been performed. In most studies, the Tetrahymena group IC1 ribozyme has been applied. Here we investigate the potential of group IC1 and group IE intron ribozymes, derived from the myxomycetes Didymium and Fuligo, in addition to the Tetrahymena ribozyme, for RNA reprogramming of a mutated α-mannosidase mRNA sequence. Randomized internal guide sequences were introduced for all four ribozymes and used to select accessible sites within isolated mutant α-mannosidase mRNA from mammalian COS-7 cells. Two accessible sites common to all the group I ribozymes were identified and further investigated in RNA reprogramming by trans-splicing analyses. All the myxomycete ribozymes performed the trans-splicing reaction with high fidelity, resulting in the conversion of mutated α-mannosidase RNA into wild-type sequence. RNA protection analysis revealed that the myxomycete ribozymes perform trans-splicing at approximately similar efficiencies as the Tetrahymena ribozyme. Interestingly, the relative efficiency among the ribozymes tested correlates with structural features of the P4–P6-folding domain, consistent with the fact that efficient folding is essential for group I intron trans-splicing. [ABSTRACT FROM AUTHOR]

Specimens of Rhizoplaca chrysoleuca from Mount Wuling can be divided into two distinct groups based on obvious differences in morphological characters. Here we investigated 26 specimens of R. chrysoleuca from Mount Wuling, 10 specimens of this species from other areas and seven specimens of other Rhizoplaca species by analyzing morphology, chemistry and genetics. Nine chemotypes were detected among the specimens of R. chrysoleuca from Mount Wuling, and five of them were reported for the first time. Based on the ITS phylogenetic analysis, the chemotypes and the insertion distribution patterns in SSU rDNA, the samples of R. chrysoleuca from Mount Wuling were grouped in two distinct clades corresponding to two phenotypic groups and no gene flow was detected between these two groups. Our results establish all individuals of Rhizoplaca chrysoleuca are conspecific although some populations have been isolated on Mount Wuling, indicating that they are in the process of speciation. Our study also reveals that the relationships between genotypes and chemotypes are complicated and should be avoided, and we instead recommend using single individuals or few individuals from the same site to represent the population or whole species in systematics study. The results also indicate that Rhizoplaca chrysoleuca might provide a good model for studying the speciation of saxicolous lichenized fungi. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we describe a novel approach to human cancer therapy that is based upon trans-splicing ribozyme-mediated replacement of cancer-specific RNAs with new transcripts that exert therapeutic activities. We have developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce transgene activity selectively in cancer cells that express the RNA. The ribozyme-mediated triggering of the transgene expression was accomplished via a high-fidelity trans-splicing reaction with the targeted residue in the hTERT-expressing cells. The ribozyme also induced cytotoxic activity in various hTERT-expressing cancer cells, hence selectively retarding the growth of those cells. Efficient and specific cell regression was also detected with ganciclovir (GCV) treatment only in hTERT-positive cancer cells, which were established to express stably the specific ribozyme that contains the herpes simplex virus thymidine kinase (HSV-tk) gene. Tissue-specific expression of the ribozyme could further augment the target specificity of the ribozyme. Importantly, we observed efficient regression of tumors with GCV treatment in mice that had been inoculated subcutaneously with hTERT-positive cancer cells that stably expressed the specific ribozyme that contains HSV-tk. These results suggest that the hTERT RNA-targeting trans-splicing ribozyme could be a powerful agent for tumor-targeted specific gene therapy. [Copyright &y& Elsevier]

Intron origin and evolution are of high interest, yet the rates of insertion and loss are unclear. To investigate their spread, we studied ribosomal (r)DNA introns from the closely related lichens Physcia aipolia and P. stellaris. Both taxa are replete with rDNA spliceosomal introns and autocatalytic group I introns, many of which show presence/absence polymorphism when screened with the PCR approach. This initially suggested that Physcia could be a model for studying intron retention and loss. However, during the course of a population-level analysis, we discovered widespread intron presence/absence heterogeneity within lichen thalli. To address this result, we sequenced multiple clones encoding nuclear rDNA and the single-copy elongation factor-1α ( EF-1α) from individual thalli. These data showed extensive rDNA heterogeneity within individuals, rather than the presence of multiple fungi within a thallus. Our results suggest that considerable care must be taken when interpreting intron presence/absence in lichen rDNA, an observation that has general implications for the study of rDNA intron evolution. [ABSTRACT FROM AUTHOR]

Group I introns are thought to be self-propagating mobile elements, and are distributed over a wide range of organisms through horizontal transmission. Intron invasion is initiated through cleavage of a target DNA by a homing endonuclease encoded in an open reading frame (ORF) found within the intron. The intron is likely of no benefit to the host cell and is not maintained over time, leading to the accumulation of mutations after intron invasion. Therefore, regular invasional transmission of the intron to a new species at least once before its degeneration is likely essential for its evolutionary long-term existence. In many cases, the target is in a protein-coding region which is well conserved among organisms, but contains ambiguity at the third nucleotide position of the codon. Consequently, the homing endonuclease might be adapted to overcome sequence polymorphisms at the target site. To address whether codon degeneracy affects horizontal transmission, we investigated the recognition properties of a homing enzyme, I-CsmI, that is encoded in the intronic ORF of a group I intron located in the mitochondrial COB gene of the unicellular green algaChlamydomonas smithii. We successfully expressed and purified three types of N-terminally truncated I-CsmI polypeptides, and assayed the efficiency of cleavage for 81 substrates containing single nucleotide substitutions. We found a slight but significant tendency that I-CsmI cleaves substrates containing a silent or tolerated amino acid change more efficiently than nonsilent or nontolerated ones. The published recognition properties of I-SpomI, I-ScaI, and I-SceII were reconsidered from this point of view, and we detected proficient adaptation of I-SpomI, I-ScaI, and I-SceII for target site sequence degeneracy. Based on the results described above, we propose that intronic homing enzymes are adapted to cleave sequences that might appear at the target region in various species, however, such adaptation becomes less prominent in proportion to the time elapsed after intron invasion into a new host. [ABSTRACT FROM AUTHOR]

RNA reprogramming represents a new concept in correcting genetic defects at the RNA level. However, for the technique to be useful for therapy, the level of reprogramming must be appropriate. To improve the efficiency of group I ribozyme-mediated RNA reprogramming, when using theTetrahymenaribozyme, regions complementary to the target RNA have previously been extended in length and accessible sites in the target RNAs have been identified. As an alternative to theTetrahymenamodel ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomyceteDidymium iridis, represents a new and attractive tool in RNA reprogramming. We reported recently that the deletion of a structural element within the P9 domain of DiGIR2 turns off hydrolysis at the 3′ splice site (side reaction) without affecting self-splicing[Haugen, P., Andreassen, M., Birgisdottir,Å.B.&Johansen, S.D. (2004)Eur. J. Biochem.271, 1015–1024]. Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3′ splice site, for application in group I ribozyme-mediatedtrans-splicing of RNA. The improved ribozyme catalyses bothcis-splicing andtrans-splicingin vitroof a humanglycosylasparaginasemRNA sequence with the same efficiency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis. [ABSTRACT FROM AUTHOR]

The genome of bacteriophage Tuc2009 consists of 38347 base pairs on which 57 open reading frames (ORFs) were identified, divided in two oppositely transcribed regions. The leftward-transcribed region harbors three ORFs, two of which are involved in the establishment of lysogeny. The rightward-transcribed region contains 54 ORFs, which are assumed to be required for the lytic life cycle. An exception to the above organization is ORF 10, of unknown function, located within the rightward-transcribed region that has an orientation opposite to the ORFs surrounding it. Transcriptional analysis of the Tuc2009 genome following infection of a sensitive host revealed that most ORFs are transcribed in a sequential manner. ORFs that are presumed to form (part of) the genetic switch along with the superinfection exclusion-encoding gene are transcribed immediately after infection, followed by transcription of the presumed replication region. Subsequent to this, several small transcripts could be identified followed by a single 24-kb transcript. This latter transcript was shown to specify most of the identified structural proteins as well as two proteins required for host lysis. Interestingly, the 24-kb mRNA was shown to undergo splicing through the activity of a type I intron whose removal from the mRNA resulted in the formation of an ORF specifying a major structural protein. Primer extension analysis was employed to identify the 5′ ends of mRNA transcripts and the genome and transcriptional data are discussed in relation to other lactococcal bacteriophages. [Copyright &y& Elsevier]

The fungi Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are mainly encountered in (sub)tropical areas as plant pathogens and agents of human dermatomycosis. Because the classification and differentiation of these two species is unclear, we studied 22 S. dimidiatum and 15 S. hyalinum isolates in order to identify potential species-specific insertions and polymorphisms in the 18S subunit ribosomal gene. The presence of an IE intron in S. dimidiatum, together with a single polymorphism (A in S. dimidiatum, G in S. hyalinum) in the coding region, allowed us to differentiate these two species in most cases. Moreover, in one S. dimidiatum isolate we found a group IC1 intron containing a putative truncated His-Cys endonuclease gene. This enzyme shows strong similarity to the intronic homing endonuclease of Physarum polycephalum. Based on these results and our previous findings, we propose an evolutionary pathway for 18S rDNA S. dimidiatum insertions, implying independent events. [Copyright &y& Elsevier]

A phylogenetic investigation of the autosporic chlorophycean alga speciesSelenastrum capricornutumusing the small subunit (SSU) rRNA gene revealed the unusual presence of six group IC1 introns. Previous studies showed that numerous green algal taxa contain group IC1 introns. However, whereas some algal species harbor multiple introns in a single ribosomal gene, none have contained as many asS. capricornutum. Three of theS. capricornutumintrons are located at conserved algal intron sites and the remaining three are located at novel eukaryotic positions. The SSU rRNA genes and their introns have been sequenced and putative secondary structures are proposed for the introns. Also, their similarity to other group IC1 introns from algal, fungal, and viral sources is investigated. Results suggest the initial presence of introns at conserved locations, followed by duplication and insertion to novel positions within the SSU rRNA gene. [ABSTRACT FROM AUTHOR]

The protein encoded by the second intron (bi2) of the mitochondrial cyt b gene from Saccharomyces cerevisiae functions as a maturase promoting intron splicing. This protein belongs to a large family characterized by the presence of two conserved motifsauthor="alan" time="20040513T082332+0000":author="alan" time="20040513T082334+0000" data=";" LAGLIDADG (or P1 and P2). We have isolated and characterized spontaneous revertants from two mis-sense mutations, G85D and H92Pauthor="alan" time="20040513T082434+0000" data=","author="alan" time="20040513T082437+0000" (localized in the P1 motif of the bi2author="alan" time="20040513T083211+0000"-author="alan" time="20040513T082359+0000" data="-"maturaseauthor="alan" time="20040513T082439+0000"), that have a detrimental effect on intron splicing. All analyzed revertants are intragenic and resulted from monosubstitutions in the mutated codons. Only true backauthor="alan" time="20040513T082508+0000"-author="alan" time="20040513T082508+0000" data=" "mutations author="alan" time="20040513T082526+0000"that restorauthor="alan" time="20040513T082528+0000"eauthor="alan" time="20040513T082529+0000" data="ing" the initial glycineauthor="alan" time="20040513T082515+0000" author="alan" time="20040513T082512+0000" data=" "85 and a pseudoreversion that replaces the deleterious aspartic acidauthor="alan" time="20040513T082531+0000" author="alan" time="20040513T082530+0000" data=" "85 by alanine were found in revertants of the mutant G85D. In contrast, all possible monosubstitutions in the mutated codon H92P were identified among the revertants of this mutant. The maturase activity of all novel forms of the protein is similar to the wild-type protein. [ABSTRACT FROM AUTHOR]

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3′ end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype. [Copyright &y& Elsevier]

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis. [ABSTRACT FROM AUTHOR]

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 Å resolution. The structure demonstrates the remarkable structural conservation of the β-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage. [ABSTRACT FROM AUTHOR]

To assess phylogenetic relationships and speciation modes in Closterium , we sequenced two noncoding regions of the nuclear ribosomal cistron, the 1506 group I intron in small subunit and the internal transcribed spacer 2, for a total of 58 strains of the Closterium moniliferum-ehrenbergii species complex. These include both homothallic and heterothallic C. moniliferum Erenberg ex Ralfs v. moniliferum , heterothallic C. moniliferum v. submoniliferum (Woronichin) Krieger, and heterothallic C. ehrenbergii Meneghini ex Ralfs that can be divided into several mating groups. We found no or very little sequence divergence within single mating groups of C. ehrenbergii and among all heterothallic strains of C. moniliferum v. moniliferum or C. moniliferum v. submoniliferum. Nevertheless, sequence divergence was much greater between those mating groups of C. ehrenbergii and also among the three traditional taxa. Maximum parsimony and maximum likelihood analyses showed that the taxon C. ehrenbergii was not monophyletic. The two varieties of C. moniliferum appeared as a sister clade to certain mating groups of C. ehrenbergii . Among the clades that were recovered in different trees by maximum parsimony and maximum likelihood analyses, we consistently found two large conspicuous clades: clade I consisted of mating groups A, B, C, H, K, and L of C. ehrenbergii whose zygospores have smooth-walls, and clade II contained the mating groups D, E, I, J, and S whose zygospores are scrobiculate. Phylogenetic incongruences observed are discussed from the viewpoints of the different molecular nature of the group I intron and internal transcribed spacer 2, as well as putative rapid diversification of the mating groups and probable ancient ancestral hybridization. [ABSTRACT FROM AUTHOR]

Abstract Recent contributions from DNA sequences have revolutionized our concept of systematic relationships in angiosperms. However, parts of the angiosperm tree remain unclear. Previous studies have been based on coding or rDNA regions of relatively conserved genes. A phylogeny for basal angiosperms based on noncoding, fast-evolving sequences of the chloroplast genome region trnT-trnF is presented. The recognition of simple direct repeats allowed a robust alignment. Mutational hot spots appear to be confined to certain sectors, as in two stem-loop regions of the trnL intron secondary structure. Our highly resolved and well-supported phylogeny depicts the New Caledonian Amborella as the sister to all other angiosperms, followed by Nymphaeaceae and an Austrobaileya –Illicium –Schisandra clade. Ceratophyllum is substantiated as a close relative of monocots, as is a monophyletic eumagnoliid clade consisting of Piperales plus Winterales sister to Laurales plus Magnoliales. Possible reasons for the striking congruence between the trnT-trnF based phylogeny and phylogenies generated from combined multi-gene, multi-genome data are discussed. [ABSTRACT FROM AUTHOR]

The P5 or P7 extensions in the group I intron ribozyme serve as “modular activator units” by stabilizing the conserved core of the ribozyme. The P5 extension of a group IC1 intron was introduced to a barely active group IA2 intron lacking its original P7 extension. The inserted P5 extension significantly activated the chimeric construct. Because the CYT-18 protein factor is also known to activate mutant group IA2 and IC1 introns lacking their P7 and P5 extensions, respectively, the RNA and protein activator units function in an analogous manner. [ABSTRACT FROM PUBLISHER]

The folding pathway of the Tetrahymena ribozyme correlates inversely with the sequence distance between native interactions, or contact order. The rapidly folding P4–P6 domain has a low contact order, while the slowly folding P3–P7 region has a high contact order. To examine the role of topology and contact order in RNA folding, we screened for circular permutants of the ribozyme that retain catalytic activity. Permutants beginning in the P4–P6 domain fold 5 to 20 times more slowly than the wild-type ribozyme. By contrast, 50% of a permuted RNA that disjoins a non-native interaction in P3 folds tenfold faster than the wild-type ribozyme. Hence, the probability of rapidly folding to the native state depends on the topology of tertiary domains. [Copyright &y& Elsevier]

Summary: The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1–P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group. [Copyright &y& Elsevier]

The catalytic core of the Tetrahyemena group I ribozyme consists of two functionally different domains, P4–P6 and P3–P7, that are conjugated via multiple tertiary interactions. The sequence encoding the P3–P7 domain is divided into two fragments in its primary sequence although the two domains are physically separable in the three dimensional (3D-) structure of the ribozyme: The sequence encoding the P4–P6 domain is inserted into that of the P3–P7 domain. An artificial rearrangement was designed and attempted for the primary sequence of the P3–P7 domain on the basis of a 3D-structural model and the biochemical data on the ribozyme. The domain in the primary structure was relocated to form a contiguous region while retaining the 3D-structure of the ribozyme required for self-splicing. The topologically rearranged ribozyme exhibited self-splicing activity. [ABSTRACT FROM PUBLISHER]

Effects of the coenzyme thiamine pyrophosphate and its analogs on the inhibition of self-splicing of primary transcripts of the phage T4 thymidylate synthase gene (td) were investigated. Of all compounds tested, the coenzyme thiamine pyrophosphate was the most potent inhibitor and the order of inhibitory efficiency for compounds tested was as follows: thiamine pyrophosphate>thiamine monophosphate>thiamine>thiochrome. Increasing guanosine concentration overcame the suppression of self-splicing by thiamine pyrophosphate close to the level of normal splicing. Kinetic analysis demonstrated that thiamine pyrophosphate acts as a competitive inhibitor for the td intron RNA with a Ki of 2.2 mM. The splicing specificity inhibition by thiamine pyrophosphate is predominantly due to changes in Km. [Copyright &y& Elsevier]

During starvation induced encystment, cells of the myxomycete Didymium iridis accumulate a 7.5-kb RNA that is the result of alternative processing of pre-rRNA. The 5′ end corresponds to an internal processing site cleaved by the group I-like ribozyme DiGIR1, located within the twin-ribozyme intron Dir.S956-1. The RNA retains the majority of Dir.S956-1 including the homing endonuclease gene and a small spliceosomal intron, the internal transcribed spacers ITS1 and ITS2, and the large subunit rRNA lacking its two group I introns. The formation of this RNA implies cleavage by DiGIR1 in a new RNA context, and presents a new example of the cost to the host of intron load. This is because the formation of the 7.5-kb RNA is incompatible with the formation of functional ribosomal RNA from the same transcript. In the formation of the 7.5-kb RNA, DiGIR1 catalysed cleavage takes place without prior splicing performed by DiGIR2. This contrasts with the processing order leading to mature rRNA and I-Dir I mRNA in growing cells, suggesting an interplay between the two ribozymes of a twin-ribozyme intron. [ABSTRACT FROM AUTHOR]

The group I-like ribozyme GIR1 is a unique example of a naturally occurring ribozyme with an evolved biological function. GIR1 generates the 5′-end of a nucleolar encoded messenger RNA involved in intron mobility. GIR1 is found as a cis-cleaving ribozyme within two very different rDNA group I introns (twin-ribozyme introns) in distantly related organisms. The Didymium GIR1 (DiGIR1) and Naegleria GIR1 (NaGIR1) share fundamental features in structural organization and reactivity, and display significant differences when compared to the related group I splicing ribozymes. GIR1 lacks the characteristic P1 segment present in all group I splicing ribozymes, it has a novel core organization, and it catalyses two site-specific hydrolytic cleavages rather than splicing. DiGIR1 and NaGIR1 appear to have originated from eubacterial group I introns in order to fulfil a common biological challenge: the expression of a protein encoding gene in a nucleolar context. [Copyright &y& Elsevier]

The first group I intron in the cox1 gene (cox1I1b ) of the mitochondrial genome of the fission yeast Schizosaccharomyces pombe is a mobile DNA element. The mobility is dependent on an endonuclease protein that is encoded by an intronic open reading frame (ORF). The intron-encoded endonuclease is a typical member of the LAGLIDADG protein family of endonucleases with two consensus motifs. In addition to this, analysis of several intron mutants revealed that this protein is required for intron splicing. However, this protein is one of the few group I intron-encoded proteins that functions in RNA splicing simultaneously with its DNA endonuclease activity. We report here on the biochemical characterization of the endonuclease activity of this protein artificially expressed in Escherichia coli . Although the intronic ORF is expressed as a fusion protein with the upstream exon in vivo , the experiments showed that a truncated translation product consisting of the C-terminal 304 codons of the cox1I1b ORF restricted to loop 8 of the intron RNA secondary structure is sufficient for the specific endonuclease activity in vitro . Based on the results, we speculate on the evolution of site-specific homing endonucleases encoded by group I introns in eukaryotes. [ABSTRACT FROM AUTHOR]

Summary • The diversity and phylogenetic affinities of symbiotic root-associated ascomycetes of the Helotiales are reported here based on ITS1–5.8S-ITS2 (internal transcribed spacer, ITS) nrDNA sequences. • Mycobionts were obtained from roots of ericoid plants and grasses and from Piceirhiza bicolorata ectomycorrhizas (pb ECM) on conifers and hardwoods, predominantly in burnt and metal-polluted habitats. The mycobionts were sequenced through the ITS and compared with sequences of known helotialean taxa. • We recognized 132 fungal ITS-sequences with affinity to the Helotiales, of which 75% (54 different ITS-genotypes) grouped within the Hymenoscyphus ericae aggregate including Phialophora finlandia . This aggregate showed stronger affinity to members of the Hyaloscyphaceae and Dermateaceae than to Hymenoscyphus fructigenus (genus-type species; Helotiaceae). Most of the pb ECM mycobionts grouped with P. finlandia , although some grouped with H. ericae . Two genotypes co-occurred in ericoid and ectomycorrhizal roots. • The H. ericae aggregate may be referable to a generic unit, and includes a diverse group of closely related, more or less darkly pigmented, root-associated ascomycetes where the borders between intra- and interspecific ITS-sequence variation, as well as different mycorrhizal and nonmycorrhizal root-symbioses, remains unclear. [ABSTRACT FROM AUTHOR]

One family within the Euascomycetes (Ascomycota), the lichen-forming Physciaceae, is particularly rich in nuclear ribosomal [r]DNA group I introns. We used phylogenetic analyses of group I introns and lichen-fungal host cells to address four questions about group I intron evolution in lichens, and generally in all eukaryotes: 1) Is intron spread in the lichens associated with the intimate association of the fungal and photosynthetic cells that make up the lichen thallus? 2) Are the multiple group I introns in the lichen-fungi of independent origins, or have existing introns spread into novel sites in the rDNA? 3) If introns have moved to novel sites, then does the exon context of these sites provide insights into the mechanism of intron spread? and 4) What is the pattern of intron loss in the small subunit rDNA gene of lichen-fungi? Our analyses show that group I introns in the lichen-fungi and in the lichen-algae (and lichenized cyanobacteria) do not share a close evolutionary relationship, suggesting that these introns do not move between the symbionts. Many group I introns appear to have originated in the common ancestor of the Lecanorales, whereas others have spread within this lineage (particularly in the Physciaceae) putatively through reverse-splicing into novel rRNA sites. We suggest that the evolutionary history of most lichen-fungal group I introns is characterized by rare gains followed by extensive losses in descendants, resulting in a sporadic intron distribution. Detailed phylogenetic analyses of the introns and host cells are required, therefore, to distinguish this scenario from the alternative hypothesis of widespread and independent intron gains in the different lichen-fungal lineages. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of the mitochondrial genome of the coral Acropora tenuis has been determined. The 18,338 bp A. tenuis mitochondrial genome contains the standard metazoan complement of 13 protein-coding and two rRNA genes, but only the same two tRNA genes (trnM and trnW) as are present in the mtDNA of the sea anemone, Metridium senile. The A. tenuis nad5 gene is interrupted by a large group I intron which contains ten protein-coding genes and rns; M. senile has an intron at the same position but this contains only two protein-coding genes. Despite the large distance (about 11.5 kb) between the 5?-exon and 3?-exon boundaries, the A. tenuis nad5 gene is functional, as we were able to RT-PCR across the predicted intron splice site using total RNA from A. tenuis. As in M. senile, all of the genes in the A. tenuis mt genome have the same orientation, but their organization is completely different in these two zoantharians: The only common gene boundaries are those at each end of the group I intron and between trnM and rnl. Finally, we provide evidence that the rns-cox3 intergenic region in A. tenuis may correspond to the mitochondrial control region of higher animals. This region contains repetitive elements, and has the potential to form secondary structures of the type characteristic of vertebrate D-loops. Comparisons between a wide range of Acropora species showed that a long hairpin predicted in rns-cox3 is phylogenetically conserved, and allowed the tentative identification of conserved sequence blocks. [ABSTRACT FROM AUTHOR]

An optional group I intron was characterized at a single insertion point in nuclear small subunit rRNA (nuSSU rRNA) genes of the imperfect entomopathogenic fungi, Beauveria bassiana and B. brongniartii. Insertion points were conserved among nuSSU rRNA genes from 35 Beauveria isolates. PCR-RFLP and DNA sequencing identified 12 group I intron variants and were applied to the identification of strains isolated from insect hosts. Alignment of 383–404-nt subgroup IB3 group I introns indicated that four insertion/deletion (indel) mutations were the main basis of fragment length variation. Phylogeny reconstruction using parsimony and neighbor-joining methods suggested six lineages may be present among nuSSU rRNA group I intron sequences from Beauveria and related ascomycete fungi. Terminal node placement of Beauveria introns conflicted with previously published phylogenies constructed from gene sequences, suggesting horizontal transfer of group I introns. PCR-RFLP among introns provided a means for the differentiation of Beauveria isolates. [ABSTRACT FROM AUTHOR]

The highly conserved P3–P7 domain of the Group I intron ribozymes is known to contain essential elements, such as the binding site for the cofactor guanosine, required for conducting the splicing reaction. We investigated the domain of the Tetrahymena intron ribozyme and its variants in order to clarify the relationship between its stability and function. We found that the destabilization of the P3–P7 domain facilitates the active structure formation at high magnesium ion concentrations where the formation is retarded for the wild type. The destabilized domain also increases KGTPm although this can be compensated by increasing the concentration of Mg2+, indicating that the stable domain is required for establishing a tight guanosine binding site. The results suggest that the stability of the domain affects the rate-limiting step in the RNA folding pathway and also regulates the efficiency of the splicing reaction. [Copyright &y& Elsevier]

The molds Scytalidium dimidiatum (Nattrassia mangiferae synanamorph) and Scytalidium hyalinum are responsible for dermatomycosis in humans. We sequenced their 18S subunit ribosomal RNA gene to identify these species with molecular biology-based methods. The coding sequences differed by a single polymorphism (A in S. dimidiatum, G in S. hyalinum). Moreover, we found an insert at position 1199 in the 18S rRNA gene sequence of S. dimidiatum. Its potential secondary structure was characteristic of a group IE intron. Bioinformatic and phylogenic group IE intron analyses generated four main homogeneous clusters. The S. dimidiatum intron is original and not related with other known IE group introns. [Copyright &y& Elsevier]

The Tetrahymena group I intron ribozyme folds into a complex three dimensional structure for performing the self-splicing reaction. Catalysis depends on its core structure comprising two helical domains, P4-P6 and P3-P7. The two domains are joined by three sets of conserved base-triple(s) and other tertiary interactions. We found that the disruption of J8/7 X P4, one such conserved base-triple, causes the catalytic ability to deteriorate without altering the folding rate. This suggests that the base-triple stabilizes the active structure of the ribozyme but plays no significant role in RNA folding. By combining the present and previous results, it can be concluded that three sets of conserved base-triples play distinct roles in the Tetrahymena ribozyme. [ABSTRACT FROM AUTHOR]

Sequence data are presented for approximately 85% of the nuclear large subunit (LSU) rDNA gene for one member of the Bangiophyceae and 47 members of the Florideophyceae, the latter representing all but one of the currently recognized florideophyte orders. Distance, parsimony, and maximum likelihood analyses of these data were used to generate phylogenetic trees, and bootstrap resampling was implemented to infer robustness for distance and parsimony results. LSU phylogenies were congruent with published nuclear small subunit (SSU) rDNA results in that four higher level florideophyte lineages were resolved: lineage 1, containing the order Hildenbrandiales; lineage 2, recovered only under distance analysis, composed of the orders Acrochaetiales, Balliales, Batrachospermales, Corallinales, Nemaliales, Palmariales, and Rhodogorgonales; lineage 3, containing the Ahnfeltiales; and lineage 4, composed of the orders Bonnemaisoniales, Ceramiales, Gelidiales, Gigartinales, Gracilariales, Halymeniales, Plocamiales, and Rhodymeniales. Analyses were also performed on a combined LSU–SSU data set and an SSU-only data set to account for differences in taxon sampling relative to published studies using this latter gene. Combined LSU–SSU analyses resulted in phylogenetic trees of similar topology and support to those obtained from LSU-only analyses. Phylogenetic trees produced from SSU-only analyses differed somewhat in particulars of branching within lineages 2 and 4 but overall were congruent with the LSU-only and combined LSU–SSU results. We close with a discussion of the phylogenetic potential that the LSU has displayed thus far for resolving relationships within the Florideophyceae. [ABSTRACT FROM AUTHOR]

Ribonucteoproteins (RNPs) consisting of derivatives of a ribozyme and an RNA-binding protein were designed and constructed based upon high-resolution structures of the corresponding prototype molecules, the Tetrahyinena group I self-splicing intron RNA and two proteins (bacteriophage λN and HIV Rev proteins) containing RNA-binding motifs. The splicing reaction proceeds efficiently only when the designed RNA associates with the designed protein either in vivo or in vitro. In vivo mutagenic protein selection was effective for improving the capability of the protein. Kinetic analyses indicate that the protein promotes RNA folding to establish an active conformation. The fact that the conversion of a ribozyme to an RNP can be accomplished by simple molecular design supports the RNA world hypothesis and suggests that a natural active RNP might have evolved readily from a ribozyme. [ABSTRACT FROM AUTHOR]

Four strains of ballistoconidium-forming yeast-like fungi (K-95, K-125, K-132 and K139), isolated from plants collected in Bangkok, Thailand, were assigned to the genus Tilletiopsis based on morphological and chemotaxonomical characteristics. On the basis of sequence data of 18S rDNA and the D1/D2 region of 26S rDNA, strains K-95, K-125 and K-132 were close to T. flava and T. fulvescens, and strain K-139 each formed related to T. minor. DNA-DNA reassociation experiments with related species revealed that strains K-125, K-132 and K-139 each formed a new and distinct species whereas strain K-95 was identified as T. flava. Tilletiopsis derxii Takashima et Nakase sp. nov. (JCM 10217T; K-125), Tilletiopsis oryzicola Takashima et Nakase sp. nov. (JCM 10218T; K-132), and Tilletiopsis penniseti Takashima et Nakase sp. nov. (JCM 10216T; K-139) are the names proposed for the new taxa. [ABSTRACT FROM AUTHOR]

How group I introns originate in nuclear ribosomal (r)RNA genes is an important question in evolutionary biology. Central to this issue is the multitude of group I introns present in evolutionarily distantly related plant, fungal, and protist lineages, together with an understanding of their origin and lateral transfer from one exon to another, between cell organelles, and between cells. These introns vary considerably in primary and secondary structure; and their provenance from a few or perhaps many mobile elements that have spread in rRNAs is unknown. Here we show that a novel lineage of group IC1 introns inserted at position 516 (Escherichia coli gene numbering) in the small subunit rRNA in bangiophyte red algae and a brown alga (Aureoumbra lagunensis) are specifically related, although their host cells are not. These bangiophyte and Aureoumbra introns are the only known cases that have a helical insertion in the P5b helix. The highly conserved primary and secondary structure of the extra P5b helix suggests that it is important, although its specific function is unknown. Our study attempts to understand the origin and movement of these IC1 introns. [ABSTRACT FROM AUTHOR]