Explosive invasion of plant mitochondria by a group I intron

Group I introns are mobile, self-splicing genetic elements found principally in organellar genomes and nuclear rRNA genes. The only group I intron known from mitochondrial genomes of vascular plants is located in the cox1 gene of Peperomia, where it is thought to have been recently acquired by lateral transfer from a fungal donor. Southern-blot surveys of 335 diverse genera of land plants now show that this intron is in fact widespread among angiosperm cox1 genes, but with an exceptionally patchy phylogenetic distribution. Four lines of evidence-the intron's highly disjunct distribution, many incongruencies between intron and organismal phylogenies, and two sources of evidence from exonic coconversion tracts-lead us to conclude that the 48 angiosperm genera found to contain this cox1 intron acquired it by 32 separate horizontal transfer events. Extrapolating to the over 13,500 genera of angiosperms, we estimate that this intron has invaded cox1 genes by cross-species horizontal transfer over 1,000 times during angiosperm evolution. This massive wave of lateral transfers is of entirely recent occurrence, perhaps triggered by some key shift in the intron's invasiveness within angiosperms.     

Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron

Recently, the 2.8 A crystal structure of one domain of the self-splicing Tetrahymena group I intron was reported. Although it revealed much about RNA tertiary interactions, it contained only half of the active site. We have now designed a series of larger molecules that contain about 70% of the intron and all of the catalytic core. These RNAs were efficient in cleavage of a substrate RNA, consisting of the approximately 100 nucleotides from the 5' end of the intron, at a site corresponding to the 5' splice site. A sparse matrix was designed specifically for large RNAs and used to screen for preliminary crystallization conditions. Of the six RNAs initially tested, five were crystallized in this initial trial. Two of these crystals were further examined. The first diffracted X-rays to only approximately 16 A resolution, even when the crystal were very large. The second diffracted as high as 3.5 A, but the crystals were twinned and therefore unusable for structural studies. Site-specific mutagenesis was performed on the latter RNA to disrupt interactions that might have been responsible for the twinning. One of these mutant RNAs produced large, single, diffraction-quality crystals. The crystals belong to the tetragonal space group P42212 and have large unit cell dimensions, a=b=178 A and c=199 A. Thus, by variation of both sequence elements and crystallization conditions, crystals of a 247 nucleotide catalytic RNA were obtained.     

The Peperomia mitochondrial coxI group I intron: timing of horizontal transfer and subsequent evolution of the intron

PURPOSE: To review the results of recent studies on radiation-induced germline instability at mammalian minisatellite loci. RESULTS: Evidence has been obtained recently that germline mutation at minisatellites is remarkably sensitive to ionizing radiation, in both mice and humans. In mice, an elevated mutation rate was found after acute irradiation of pre-meiotic spermatogonia, with a doubling dose of 0.33 Gy, a value close to those obtained in mice after acute spermatogonia irradiation using other systems for mutation detection. In humans, analysis of germline mutation rate at minisatellites among children born in areas of the Mogilev district of Belarus, which was heavily polluted after the Chernobyl accident, has shown a twofold higher mutation rate in exposed families compared with non-irradiated families from the United Kingdom. Within the Belarus cohort, the mutation rate was significantly greater in families exposed to a higher parental radiation dose, consistent with radiation induction of germline mutation. The data in this study also demonstrate the indirect nature of radiation-induced germline mutation at mammalian minisatellite loci suggesting a strong similarity with the phenomenon of genomic instability in somatic cells. CONCLUSIONS: Minisatellite loci provide a powerful system for the efficient monitoring of germline mutation in humans and are capable of detecting induced mutations in relatively small population samples.     

Intron open reading frames as mobile elements and evolution of a group I intron

Group I introns are proposed to have become mobile following the acquisition of open reading frames (ORFs) that encode highly specific DNA endonucleases. This proposal implies that intron ORFs could behave as autonomously mobile entities. This was supported by abundant circumstantial evidence but no experiment of ORF transfer from an ORF-containing intron to its ORF-less counterpart has been described. In this paper we present such experiments, which demonstrate the efficient mobility of the mitochondrial nad1-i4-orf1 between two Podospora strains. The homing of this mobile ORF was accompanied by a bidirectional co-conversion that did not systematically involve the whole intron sequence. Orf1 acquisition would be the most recent step in the evolution of the nad1-i4 intron, which has resulted in many strains of Podospora having an intron with two ORFs (biorfic) and four splicing pathways. We show that two of the splicing events that operate in this biorfic intron, as evidenced by PCR experiments, are generated by a 5'-alternative splice site, which is most probably a remnant of the monoorfic ancestral form of the intron. We propose a sequential evolution model that is consistent with the four organizations of the corresponding nad1 locus that we found among various species of the Pyrenomycete family; these organizations consist of no intron, an intron alone, a monoorfic intron, and a biorfic intron.     

A minor groove RNA triple helix within the catalytic core of a group I intron

Close packing of several double helical and single stranded RNA elements is required for the Tetrahymena group I ribozyme to achieve catalysis. The chemical basis of these packing interactions is largely unknown. Using nucleotide analog interference suppression (NAIS), we demonstrate that the P1 substrate helix and J8/7 single stranded segment form an extended minor groove triple helix within the catalytic core of the ribozyme. Because each triple in the complex is mediated by at least one 2'-OH group, this substrate recognition triplex is unique to RNA and is fundamentally different from major groove homopurine-homopyrimidine triplexes. We have incorporated these biochemical data into a structural model of the ribozyme core that explains how the J8/7 strand organizes several helices within this complex RNA tertiary structure.     

Stimulation of gene expression by introns: conversion of an inhibitory intron to a stimulatory intron by alteration of the splice donor sequence

Efficient expression of many mammalian genes depends on the presence of at least one intron. We previously showed that addition of almost any of the introns from the mouse thymidylate synthase (TS) gene to an intronless TS minigene led to a large increase in expression. However, addition of intron 4 led to a reduction in minigene expression. The goal of the present study was to determine why TS intron 4 was unable to stimulate expression. Insertion of intron 4 into an intron-dependent derivative of the ribosomal protein L32 gene did not lead to a significant increase in expression, suggesting that its inability to stimulate expression was due to sequences within the intron. Deleting most of the interior of intron 4, improving the putative branch point, removing purines from the pyrimidine stretch at the 3' end of the intron, or removing possible alternative splice acceptor or donor sites within the intron each had little effect on the level of expression. However, when the splice donor sequence of intron 4 was modified so that it was perfectly complementary to U1 snRNA, the modified intron 4 stimulated expression approximately 6-fold. When the splice donor site of TS intron 1 (a stimulatory intron) was changed to that of TS intron 4, the modified intron 1 was spliced very inefficiently and lost the ability to stimulate mRNA production. Our observations support the idea that introns can stimulate gene expression by a process that depends directly on the splicing reaction.     

In vitro protein folding by ribosomes from Escherichia coli, wheat germ and rat liver: the role of the 50S particle and its 23S rRNA

Ribosomes from a number of prokaryotic and eukaryotic sources (e.g. Escherichia coli, wheat germ and rat liver) can refold a number of enzymes which are denatured with guanidine/HC1 prior to incubation with ribosomes. In this report, we present our observations on the refolding of denatured lactate dehydrogenase from rabbit muscle and glucose-6-phosphate dehydrogenase from baker's yeast by ribosomes from E. coli, wheat germ and rat liver. The protein-folding activity of E. coli ribosomes was found to be present in 50S particles and in 23S rRNA. The 30S particle or 16S rRNA did not show any protein-folding activity. The protein-folding activity of 23S rRNA may depend on its tertiary conformation. Loss of tertiary structure, by incubation with low concentrations of EDTA, inhibited the protein-folding activity of 23S rRNA. This low concentration of EDTA had no effect on folding of the denatured enzymes by themselves.     

Genetic survey of a group of children with clefting: implications for genetic counseling

A cleft lip, cleft palate, or both are associated with a high frequency of other anomalies. This study gives an inventory of associated anomalies in a consecutive group of children (n = 36) with clefts, referred to a local multidisciplinary cleft team in the Netherlands. In 47.2% of cleft patients associated anomalies were found, allowing diagnosis of provisionally unique syndromes or known entities. In 17 patients family history was positive for clefting; in five patients (13.9%) this influenced the occurrence risks for siblings. Both findings had an effect on genetic counseling of the parents of these children. Additional evidence is provided that all children with clefting should be carefully evaluated by a trained clinician for additional anomalies, including dysmorphic features.     

Probing the interplay between the two steps of group I intron splicing: competition of exogenous guanosine with omega G

One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.     

Sporadic distribution of tRNA(Arg)CCU introns among alpha-purple bacteria: evidence for horizontal transmission and transposition of a group I intron

A group I intron interrupts the tRNA(Arg)CCU gene of the alpha-purple bacterium Agrobacterium tumefaciens (B. Reinhold-Hurek and D. A. Shub, Nature [London] 357:173-176, 1992). In this study, we assess the distribution of the corresponding intron among 12 additional species of alpha-purple bacteria. Of 10 newly identified tRNA(Arg)CCU genes, we found only two that contained an intron homologous to that of the Agrobacterium intron. This restricted and scattered distribution of the tRNA(Arg)CCUg intron among alpha-purple bacteria is consistent with a recent origin and horizontal transmission. Primary and secondary structural similarities between tRNA(Leu)UAA introns found in strains of the cyanobacterium Microcystis aeruginosa (K. Rudi and K. S. Jacobsen, FEMS Microbiol. Lett. 156:293-298, 1997) and alpha-purple tRNA(Arg)CCU introns suggest that these introns share a more recent common ancestor than either does with other known cyanobacterial tRNA(Leu)UAA introns.     

A 30 S precursor of 30 S ribosomes in a mutant of Escherichia coli

Escherichia coli 15-28, a mutant with a defect in ribosome metabolism, accumulates a ribonucleoprotein particle that is indistinguishable from 30S subunits by sedimentation but contains the precursor form of 16S RNA. This particle is probably a precursor of 30 S ribosomes.     

Association of elongation factor 2 with ribosomes during growth of a murine ascitic tumor

The amount of elongation factor 2 (EF-2) associated with different ribosomal fractions (mono- and polyribosomes) isolated from a methylcholanthrene-induced sarcoma is estimated during tumor growth (exponential and plateau phase of growth). Direct EF-2 quantification is obtained by a modification of the method of the diphtheria toxin-catalyzed transfer of (14C)ADP-ribose from (14C)NAD+ to the enzyme. Data reported show that the amount of EF-2 associated with the monoribosomal fraction changes during tumor growth, and particularly, that this amount increases when the tumor cells enter into the plateau phase. In contrast, the EF-2 content of the polyribosomal fraction does not change during the different phases of tumor growth. Data also show that the amount of EF-2 bound to the monoribosomal fraction isolated from tumor cells is significantly and constantly lower than that of the corresponding fraction isolated from reticulocytes or hepatocytes. Moreover the tumor monoribosomes generated by the polyribosome breakdown induced by the "starvation" procedure did not show significant changes in their EF-2 content with respect to monoribosomes isolated from tumor cells maintained in physiological conditions. Besides, tumor monoribosomes generated by the polyribosome breakdown induced by puromycin or by running-off treatment exhibit a relevant increase of the EF-2 content. In these conditions the amount of EF-2 associated with the monoribosomes is similar to that associated with the monoribosomes of control cells (hepatocytes and reticulocytes). Results are discussed in view of a possible regulative role of the EF-2 enzyme in the ribosomal cycle of eukaryotic cells.     

Affinity modification of 80S ribosomes from human placenta with mRNA analogs--derivatives of oligouridylates with and alkylating group at the 5'-end

Affinity labelling of 80S ribosomes from human placenta with 4-(N-methylamino-N-2-chloroethyl)benzylmethylphosphoramide derivatives of oligouridylates pUn (n = 3, 4, 6, 12) bearing 5'-32P-label was studied. Complexes of these derivatives with 80S ribosomes where codon-anticodon interaction took place either in P-site (in the case of pU3-and pU4-derivatives), or in P- and A-site simultaneously (in the case of pU6- and pU12-derivatives) were obtained in the presence of Phe-tRNA(Phe). All the reagents modified only the 40S subunit. The extent of 18S rRNA modification by pU3-, pU4-, pU6- and pU12-derivatives as a fraction of the total modification extent of 18S rRNA and proteins in the 40S subunit equaled 96, 93, 24 and 4%, respectively. The pU4-derivative was covalently attached at positions 976-1061 and 1058-1164 and pU12-derivative was covalently attached within regions 976-1061, 1058-1164, 593-673 and 1748-1869 of the 18S rRNA. By means of the primer extension technique, modified bases in 18S rRNA were determined to be: A-1023, C-1026, A-1027, A-1058, G-1059 for pU3- and pU4-derivatives and A-1058 for pU6-derivative.     

Cardiovascular toxicity of nicotine: implications for nicotine replacement therapy

This review discusses the known cardiovascular effects of smoking and the effects of nicotine without tobacco smoke and interprets the available data on cardiovascular risk during nicotine replacement therapy (NRT). Nicotine gum and patches are now approved for over the counter sale in the United States. Smokers with cardiovascular disease are advised to seek physician counseling before using nicotine products, but information regarding the safety of these products in such patients is not readily available to most physicians. Nicotine may contribute to cardiovascular disease, presumably by hemodynamic consequences of sympathetic neural stimulation and systemic catecholamine release. However, there are many potential cardiovascular toxins in cigarette smoke other than nicotine. The doses of nicotine obtained by regular cigarette smoking generally exceed those delivered by NRTs, and the cardiovascular effects of nicotine are, in general, more intense when delivered rapidly by cigarette smoking than the slower delivery by transdermal nicotine or nicotine gum. Because the dose-cardiovascular response relation for nicotine is flat, the effects of cigarette smoking in conjunction with NRT are similar to those of cigarette smoking alone. Cigarette smoking increases blood coagulability, a major risk factor for acute cardiovascular events, whereas transdermal nicotine does not appear to do so. Clinical trials of NRT in patients with underlying, stable coronary disease suggest that nicotine does not increase cardiovascular risk. At worst, the risks of NRT are no more than those of cigarette smoking. The risks of NRT for smokers, even for those with underlying cardiovascular disease, are small and are substantially outweighed by the potential benefits of smoking cessation.     

Morphometric approaches for evaluating pulmonary toxicity in mammals: implications for risk assessment

Recent advances in quantitative morphology provide all the tools necessary to obtain structural information in the lung that can be quantified and interpreted in the three-dimensional world of toxicology. Structural hierarchies of conducting airways and parenchyma of the lung provide: (1) numbers of cells per airway, lobe, or lung; (2) surface areas of cells, airways, and alveoli; (3) length of airways and vessels; and (4) volumes of cells, alveoli, airways, vessels, and individual lobes or the entire lung. Unbiased sampling of these subcompartments of the lung requires fractionation of lobes or individual airways. Individual airways of proximal and distal generations are obtained by airway microdissection along one axial pathway and comparisons made between airway generations. Vertical sections of selected airways are used to sample epithelium and interstitium. Using this unbiased approach of quantitative morphology, we have shown that inhalation of low ambient concentrations of ozone ([O3]0.15 ppm) near or at the United States National Ambient Air Quality Standard (NAAQS) (0.12 ppm O3) induces significant alterations in bronchiolar epithelium and interstitium in nonhuman primates but not rats. The alterations do not appear to be concentration- or time-dependent, thereby bringing into question the current NAAQS that may be at or above the threshold for distal airway injury in primates. Unbiased morphometric methods are critical in a quantitative evaluation of toxicological injury of mammalian tracheobronchial airways.     

Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).     

Energetics of vinca alkaloid interactions with tubulin isotypes: implications for drug efficacy and toxicity

Capture ELISAs, for canine IgG, IgM, IgA and albumin, were developed and used to analyse immunoglobulin (Ig) concentrations in both serum and secretions. Matched samples of serum, saliva and tears were taken from 31 dogs, assigned to two groups based on age, whilst bile samples were obtained from nine adult dogs at post-mortem. Serum and tear IgA concentrations were significantly lower in dogs < or = 12 months of age compared with dogs > 12 months of age (p = 0.006 and 0.045, respectively). There was no significant correlation between serum and secretory Ig levels, with the single exception of serum and tear IgM concentrations (rp = 0.553, p = 0.004). IgG and IgM concentrations were significantly correlated in matched tear and saliva samples (IgG: rp = 0.470, p = 0.023; IgM: rp = 0.651, p < 0.0001). Albumin concentrations were significantly correlated with IgG, but not IgM or IgA, in both saliva and tears (saliva, rp = 0.581, p = 0.004; tears, rp = 0.843, p < 0.0001) whilst IgA and IgM concentrations were significantly correlated with each other in both secretions (saliva, rp = 0.644, p = 0.001; tears, rp = 0.555, p = 0.009). Significantly, more Ig of all classes was secreted into saliva than tears as calculated by a secretory index. A large diurnal and day-to-day variation was observed in Ig concentrations in serial saliva and tear samples taken from a further four dogs. Serum Ig concentrations are therefore, poor indicators of mucosal secretion in this species and significant intra-individual variation exists in secretory Ig levels. Both findings should be taken into account in future studies of canine mucosal immunoglobulins.     

A Pneumocystis carinii group I intron ribozyme that does not require 2' OH groups on its 5' exon mimic for binding to the catalytic core

The recent increase in the population of immunocompromised patients has led to an insurgence of opportunistic human fungal infections. The lack of effective treatments against some of these pathogens makes it important to develop new therapeutic strategies. One such strategy is to target key RNAs with antisense compounds. We report the development of a model system for studying the potential for antisense targeting of group I self-splicing introns in fungal pathogens. The group I intron from the large ribosomal subunit RNA of mouse-derived Pneumocystis carinii has been isolated and characterized. This intron self-splices in vitro. A catalytically active ribozyme, P-8/4x, has been constructed from this intron to allow measurement of dissociation constants for potential antisense agents. At 37 degrees C, in 50 mM Hepes (25 mM Na+), 15 mM MgCl2, and 135 mM KCl at pH 7.5, the exogenous 5' exon mimic r(AUGACU) binds about 60 000 times more tightly to this ribozyme than to r(GGUCAU), a mimic of its complementary binding site on the ribozyme. This enhanced binding is due to tertiary interactions. This tertiary stabilization is increased by single deoxynucleotide substitutions in the exon mimic at every position except for the internal A, which is essentially unchanged. Thus 2' OH groups of the 5' exon mimic do not form stabilizing tertiary interactions with the P-8/4x ribozyme, in contrast to the Tetrahymena L-21 ScaI ribozyme. Furthermore, at 37 degrees C, the exogenous 5' exon mimic d(ATGACT) binds nearly 32 000 times more tightly to the P-8/4x ribozyme than to r(GGUCAU). Therefore, oligonucleotides without 2' OH groups can exploit tertiary stabilization to bind dramatically more tightly and with more specificity than possible from base pairing. These results suggest a new paradigm for antisense targeting: targeting the tertiary interactions of structural RNAs with short antisense oligonucleotides.