Presence Of Intron Research Articles

Arabidopsis DEAD-Box RNA Helicase UAP56 Interacts with Both RNA and DNA as well as with mRNA Export Factors

The DEAD-box protein UAP56 (U2AF65-associcated protein) is an RNA helicase that in yeast and metazoa is critically involved in mRNA splicing and export. In Arabidopsis, two adjacent genes code for an identical UAP56 protein, and both genes are expressed. In case one of the genes is inactivated by a T-DNA insertion, wild type transcript level is maintained by the other intact gene. In contrast to other organisms that are severely affected by elevated UAP56 levels, Arabidopsis plants that overexpress UAP56 have wild type appearance. UAP56 localises predominantly to euchromatic regions of Arabidopsis nuclei, and associates with genes transcribed by RNA polymerase II independently from the presence of introns, while it is not detected at non-transcribed loci. Biochemical characterisation revealed that in addition to ssRNA and dsRNA, UAP56 interacts with dsDNA, but not with ssDNA. Moreover, the enzyme displays ATPase activity that is stimulated by RNA and dsDNA and it has ATP-dependent RNA helicase activity unwinding dsRNA, whereas it does not unwind dsDNA. Protein interaction studies showed that UAP56 directly interacts with the mRNA export factors ALY2 and MOS11, suggesting that it is involved in mRNA export from plant cell nuclei.

Genome-wide analysis of the auxin response factor gene family in cucumber

Auxin response factors (ARFs) participate in the transcriptional regulation of a series of biological processes related to growth and development. The ARF genes comprise a large multigene family in plants. Recently, a draft of the full cucumber (Cucumis sativus) genome assembly has been released; however, none of the ARF genes have been characterized. We made a comprehensive analysis of ARF genes in this species. Fifteen ARF genes were identified and could be divided into three classes. Intron presence and position were conserved to some extent within one phylogenetic group. Based on genome distribution analysis, tandem duplication appears to have contributed to ARF gene expansion. Specific motifs were selectively distributed among specific clades. Putative cis-elements involved in auxin response, light signaling responses, phytohormones, and defense responses were identified in promoter regions of the ARF genes. Almost all of the ARF genes exhibited a constitutive expression pattern, implying that there are functional redundancies among the ARF proteins.

Evaluation of intron 22 and intron 1 inversions of the factor 8 gene using an inverse shifting PCR method in severe haemophilia A patients

Haemophilia A (HA) is an X-linked recessive bleeding disorder caused by several mutations in the F8 gene, whose inversion causes most of the mutation in 40% of severe HA patients. Methods such as long distance PCR and southern blot have been exploited to analyse intron 22 inversion in the F8 gene. For this purpose, we used an inverse shifting PCR assay, a novel genetic diagnostic method, for the detection of intron 22 inversion in severe HA patients. A screen for the presence of intron 22 and intron 1 inversions at the F8 gene in 30 Iranian severe HA patients revealed that 47% of patients showed intron 22 inversion (40% type 1 and 7% type 2), while 7% of patients had intron 1 inversion. Among the patients carrying the inversion, 12% developed inhibitors. Inverse shifting PCR is a precise method for assessment of rearrangements related to int 22h and int 1h in patients and carriers of haemophilia A. Since the previous methods of assessing inversion are time-consuming and demanding, this method can be a good replacement.

Establishment and interpretation of the genome sequence of the phytopathogenic fungus Rhizoctonia solani AG1-IB isolate 7/3/14

Anastomosis group AG1-IB isolates of the anamorphic basidiomycetous fungus Rhizoctonia solani Kühn affect various agricultural and horticultural important crops including bean, rice, soybean, figs, hortensia, cabbage and lettuce. To gain insights into the genome structure and content, the first draft genome sequence of R. solani AG1-IB isolate 7/3/14 was established. Four complete runs on the Genome Sequencer (GS) FLX platform (Roche Applied Science) yielding approx. a 25-fold coverage of the R. solani genome were accomplished. Assembly of the sequence reads by means of the gsAssembler software version 2.6 applying the heterozygotic mode resulted in numerous contigs and scaffolds and a predicted size of 87.1Mb for the diploid status of the genome. ‘Contig-length vs. read-count’ analysis revealed that the assembled contigs can be classified into five different groups. Detailed BLAST-analysis revealed that most contigs of group II feature high-scoring matches to other contigs of the same group suggesting that distinguishable allelic variants exist for many genes. Due to the supposed diploid and heterokaryotic nature of R. solani AG1-IB 7/3/14, this result has been anticipated. However, the heterokaryotic character of the isolate is not really supported by sequencing data obtained for the isolate R. solani AG1-IB 7/3/14. Coverage of group III contigs is twice as high as for group II contigs which can also be explained by the diploid status of the genome and indistinguishable alleles on homologous chromosomes. Assembly of sequence data led to the identification of the rRNA unit (group V contigs) and the mitochondrial (mt) genome (group IV contigs) which is a circular molecule of 162,751bp in size featuring a GC-content of 36.4%. The R. solani 7/3/14 mt-genome is one of the largest fungal mitochondrial genomes known to date. Its large size essentially is due to the presence of numerous non-conserved hypothetical ORFs and introns. Gene prediction for the R. solani AG1-IB 7/3/14 genome was conducted by the Augustus Gene Prediction Software for Eukaryotes (version 2.6.) applying the parameter set for the fungus Coprinopsis cinerea okayama 7#130. Gene prediction and annotation provided first insights into the R. solani AG1-IB 7/3/14 gene structure and content. In total, 12,422 genes were predicted. The average number of exons per gene is five. Exons have a mean length of 214bp, whereas introns on average are 66bp in length. Annotation of the genome revealed that 4169 of 12,422 genes could be assigned to KOG functional categories.

Analysis of phylogenetic signal in protostomial intron patterns using Mutual Information

Many deep evolutionary divergences still remain unresolved, such as those among major taxa of the Lophotrochozoa. As alternative phylogenetic markers, the intron-exon structure of eukaryotic genomes and the patterns of absence and presence of spliceosomal introns appear to be promising. However, given the potential homoplasy of intron presence, the phylogenetic analysis of this data using standard evolutionary approaches has remained a challenge. Here, we used Mutual Information (MI) to estimate the phylogeny of Protostomia using gene structure data, and we compared these results with those obtained with Dollo Parsimony. Using full genome sequences from nine Metazoa, we identified 447 groups of orthologous sequences with 21,732 introns in 4,870 unique intron positions. We determined the shared absence and presence of introns in the corresponding sequence alignments and have made this data available in "IntronBase", a web-accessible and downloadable SQLite database. Our results obtained using Dollo Parsimony are obviously misled through systematic errors that arise from multiple intron loss events, but extensive filtering of data improved the quality of the estimated phylogenies. Mutual Information, in contrast, performs better with larger datasets, but at the same time it requires a complete data set, which is difficult to obtain for orthologs from a large number of taxa. Nevertheless, Mutual Information-based distances proved to be useful in analyzing this kind of data, also because the estimation of MI-based distances is independent of evolutionary models and therefore no pre-definitions of ancestral and derived character states are necessary.

Identification of a newly isolated erythritol-producing yeast and cloning of its erythritol reductase genes

A new erythritol-producing yeast (strain BH010) was isolated in this study. Analysis of the D1/D2 domain of the 26S rDNA sequence, the ITS/5.8S rDNA sequence [corrected] and the 18S rDNA sequence allowed the taxonomic position of strain BH010 to be discussed, [corrected] and it was identified and named Moniliella sp. BH010. Physiological characteristics were described. Scanning electron micrography clearly indicated that the cells were cylindrical to elliptical with an average size of 5 × 10 μm when growing in liquid medium [corrected] and that pseudohyphae and blastoconidia were observed when cultivated in agar plates. The erythrose [corrected] reductase genes were cloned, sequenced, and analyzed. BLAST analysis and multiple sequence alignment demonstrated that erythrose [corrected] reductase genes of Moniliella sp. BH010 shared very high homology with that of Trichosporonoides megachiliensis SNG-42 except for the presence of introns. The deduced amino acid sequences showed high homology to the aldo-keto reductase superfamily.

748 Detection of Intron 22 Gene Inversion (Common Mutation) in Severe Hemophilia a in Khouzestan Province

IntroductionHemophilia A, is the most common severe coagulation disorder with an incidence of 1 in 5000 males. The most common genetic alteration is a gene inversion, which occurs in approximately...

First report of molecular diagnosis of Tunisian hemophiliacs A: Identification of 8 novel causative mutations

IntroductionHemophilia A is an X linked recessive hemorrhagic disorder caused by mutations in the F8 gene that lead to qualitative and/or quantitative deficiencies of coagulation factor VIII (FVIII). Molecular diagnosis of hemophilia A is challenging because of the high number of different causative mutations that are distributed throughout the large F8 gene. Molecular studies of these mutations are essential in order to reinforce our understanding of their pathogenic effect responsible for the disorder.AimIn this study we have performed molecular analysis of 28 Tunisian hemophilia A patients and analyzed the F8 mutation spectrum.MethodsWe screened the presence of intron 22 and intron 1 inversion in severe hemophilia A patients by southern blotting and polymerase chain reaction (PCR). Detection of point mutations was performed by dHPLC/sequencing of the coding F8 gene region. We predict the potential functional consequences of novel missense mutations with bioinformatics approaches and mapping of their spatial positions on the available FVIII 3D structure.ResultsWe identified 23 different mutations in 28 Tunisian hemophilia A patients belonging to 22 unrelated families. The identified mutations included 5 intron 22 inversions, 7 insertions, 4 deletions and 7 substitutions. In total 18 point mutations were identified, of which 9 are located in exon 14, the most mutated exonic sequence in the F8 gene. Among the 23 mutations, 8 are novel and not deposited in the HAMSTeRS database nor described in recently published articles.ConclusionThe mutation spectrum of Tunisian hemophilia A patients is heterogeneous with the presence of some characteristic features.Virtual slidesThe virtual slide(s) for this article can be found here:http://www.diagnosticpathology.diagnomx.eu/vs/1693269827490715

A primary survey on bryophyte species reveals two novel classes of nucleotide-binding site (NBS) genes.

Due to their potential roles in pathogen defense, genes encoding nucleotide-binding site (NBS) domain have been particularly surveyed in many angiosperm genomes. Two typical classes were found: one is the TIR-NBS-LRR (TNL) class and the other is the CC-NBS-LRR (CNL) class. It is seldom known, however, what kind of NBS-encoding genes are mainly present in other plant groups, especially the most ancient groups of land plants, that is, bryophytes. To fill this gap of knowledge, in this study, we mainly focused on two bryophyte species: the moss Physcomitrella patens and the liverwort Marchantia polymorpha, to survey their NBS-encoding genes. Surprisingly, two novel classes of NBS-encoding genes were discovered. The first novel class is identified from the P. patens genome and a typical member of this class has a protein kinase (PK) domain at the N-terminus and a LRR domain at the C-terminus, forming a complete structure of PK-NBS-LRR (PNL), reminiscent of TNL and CNL classes in angiosperms. The second class is found from the liverwort genome and a typical member of this class possesses an α/β-hydrolase domain at the N-terminus and also a LRR domain at the C-terminus (Hydrolase-NBS-LRR, HNL). Analysis on intron positions and phases also confirmed the novelty of HNL and PNL classes, as reflected by their specific intron locations or phase characteristics. Phylogenetic analysis covering all four classes of NBS-encoding genes revealed a closer relationship among the HNL, PNL and TNL classes, suggesting the CNL class having a more divergent status from the others. The presence of specific introns highlights the chimerical structures of HNL, PNL and TNL genes, and implies their possible origin via exon-shuffling during the quick lineage separation processes of early land plants.

SLC34A3 Intronic Deletion in a New Kindred with Hereditary Hypophosphatemic Rickets with Hypercalciuria

Objective: Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is an autosomal recessive form of hypophosphatemia with hyperphosphaturia, hypercalciuria, and hypercalcemia. In two reports on six affected kindreds with HHRH, the disease was mapped to chromosome 9q34, which contains the SLC34A3 gene that encodes the renal type 2c sodium-phosphate cotransporter. Our objective was to define the clinical course of these cases in a family with HHRH and to screen for SLC34A3 gene in order to determine whether these mutations are responsible for HHRH.Methods: After clinical and biochemical evaluations, the entire SLC34A3 gene was screened using PCR amplification followed by direct sequencing technique. In this paper, we describe a new kindred with HHRH and a case of progressive and complicated HHRH presenting at age 27 years.Results: We found 101-bp deletion in intron 9 of the SLC34A3 gene. The index patient was homozygous for this mutation which has been previously reported in a Caucasian population. This is the first report for presence of SLC34A3 intron 9 deletion in an Iranian population.Conclusions: These data showed that HHRH can be easily missed or underdiagnosed. Genetic evaluation of patients with familial hypercalciuria, hypophosphatemia and nephrolithiasis is needed for further information on the prevalence and management of this rare disorder. Conflict of interest:None declared.

Lineage-specific fragmentation and nuclear relocation of the mitochondrial cox2 gene in chlorophycean green algae (Chlorophyta)

In most eukaryotes the subunit 2 of cytochrome c oxidase (COX2) is encoded in intact mitochondrial genes. Some green algae, however, exhibit split cox2 genes (cox2a and cox2b) encoding two polypeptides (COX2A and COX2B) that form a heterodimeric COX2 subunit. Here, we analyzed the distribution of intact and split cox2 gene sequences in 39 phylogenetically diverse green algae in phylum Chlorophyta obtained from databases (28 sequences from 22 taxa) and from new cox2 data generated in this work (23 sequences from 18 taxa). Our results support previous observations based on a smaller number of taxa, indicating that algae in classes Prasinophyceae, Ulvophyceae, and Trebouxiophyceae contain orthodox, intact mitochondrial cox2 genes. In contrast, all of the algae in Chlorophyceae that we examined exhibited split cox2 genes, and could be separated into two groups: one that has a mitochondrion-localized cox2a gene and a nucleus-localized cox2b gene (“Scenedesmus-like”), and another that has both cox2a and cox2b genes in the nucleus (“Chlamydomonas-like”). The location of the split cox2a and cox2b genes was inferred using five different criteria: differences in amino acid sequences, codon usage (mitochondrial vs. nuclear), codon preference (third position frequencies), presence of nucleotide sequences encoding mitochondrial targeting sequences and presence of spliceosomal introns. Distinct green algae could be grouped according to the form of cox2 gene they contain: intact or fragmented, mitochondrion- or nucleus-localized, and intron-containing or intron-less. We present a model describing the events that led to mitochondrial cox2 gene fragmentation and the independent and sequential migration of cox2a and cox2b genes to the nucleus in chlorophycean green algae. We also suggest that the distribution of the different forms of the cox2 gene provides important insights into the phylogenetic relationships among major groups of Chlorophyceae.

Molecular Characterization of Pyraclostrobin Resistance and Structural Diversity of the Cytochrome b Gene in Botrytis cinerea from Apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.

Intron gains and losses in the evolution of Fusarium and Cryptococcus fungi.

The presence of spliceosomal introns in eukaryotic genes poses a major puzzle for the study of genome evolution. Intron densities vary enormously among distant lineages. However, the mechanisms driving intron gains are poorly understood and very few intron gains and losses have been documented over short evolutionary time spans. Fungi emerged recently as excellent models to study intron evolution and “reverse splicing” was found to be a major driver of recent intron gains in a clade of ascomycete fungi. We screened a total of 38 genomes from two fungal clades important in medicine and agriculture to identify intron gains and losses both within and between species. We detected 86 and 198 variable intron positions in the Cryptococcus and Fusarium clades, respectively. Some genes underwent extensive changes in their exon–intron structure, with up to six variable intron positions per gene. We identified a very recently gained intron in a group of tomato-infecting strains belonging to the F. oxysporum species complex. In the human pathogen C. gattii, we found recent intron losses in subtypes of the species. The two studied fungal clades provided evidence for extensive changes in their exon–intron structure within and among closely related species. We show that both intronization of previously coding DNA and insertion of exogenous DNA are the major drivers of intron gains.

<p><strong>Genetic factors potentially reducing fitness cost of organophosphate-insensitive acetylcholinesterase(s) in </strong><strong><em>Rhipicephalus </em></strong>(<strong><em>Boophilus</em></strong>) <strong><em>microplus </em></strong><strong>(Acari: Ixodidae)*</strong></p>

Acaricidal activity of organophosphate (OP) and carbamate acaricides is believed to result from inhibition of acetylcholinesterase (AChE). Previous studies in Rhipicephalus (Boophilus) microplus demonstrated the presence of three presumptive AChE genes (BmAChEs). Biochemical characterization of recombinant BmAChE proteins expressed in the baculovirus system demonstrated that each of the three R. (B.) microplus rBmAChEs have enzymatic properties consistent with designation as functional acetylcholinesterases. Complementary DNAs (cDNAs) for each of the three BmAChEs were cloned and sequenced from individual adult tick synganglia excised from OP-susceptible and OPresistant strains. The data revealed the presence of multiple transcript sequences within individual ticks for each of the BmAChEs, suggesting alternative mRNA splicing or expression of multiple alleles for each of the BmAChE genes. Quantitative real-time PCR provided evidence of possible gene duplication or amplification for each of the BmAChE genes, and direct sequencing of genomic DNA provided evidence of structural BmAChE gene diversity with respect to presence or absence of introns, as well as the presence or absence of sequence polymorphisms. Baculovirus expression of rBmAChE1 and rBmAChE3 proteins containing some of the predicted amino acid sequence polymorphisms resulted in production of OP-insensitive AChE, demonstrating that at least some OP-resistant individuals contain mutations that reduce OP-inhibition for at least two of the three known BmAChEs. RNA interference was utilized to silence in vivo expression of the BmAChE genes in adult ticks, resulting in tick mortality if all three BmAChEs were silenced simultaneously, strongly suggesting that the BmAChE proteins functionally complement one another in vivo. Together, the results presented provide strong evidence that OP-resistance in R. microplus is at least partially mediated by a combination of the expression of multiple genes encoding acetylcholinesterase, mutations in BmAChEs resulting in OP-insensitivity, gene duplication, and maintenance of allelic diversity, including both OP-sensitive and OPinsensitive alleles within individual ticks. The authors propose that these factors may mitigate fitness costs that might otherwise result from BmAChE mutations, and demonstrate the extreme complexity of OP-resistance in R. (B.) microplus. It is hoped that elucidation of the complex interactions among the multiple BmAChEs and their physiological roles may enable development of new opportunities for tick control.

Genome-wide analysis of retrogene polymorphisms in Drosophila melanogaster

Gene duplication via retrotransposition has been shown to be an important mechanism in evolution, affecting gene dosage and allowing for the acquisition of new gene functions. Although fixed retrotransposed genes have been found in a variety of species, very little effort has been made to identify retrogene polymorphisms. Here, we examine 37 Illumina-sequenced North American Drosophila melanogaster inbred lines and present the first ever data set and analysis of polymorphic retrogenes in Drosophila. We show that this type of polymorphism is quite common, with any two gametes in the North American population differing in the presence or absence of six retrogenes, accounting for ~13% of gene copy-number heterozygosity. These retrogenes were identified by a straightforward method that can be applied using any type of DNA sequencing data. We also use a variant of this method to conduct a genome-wide scan for intron presence/absence polymorphisms, and show that any two chromosomes in the population likely differ in the presence of multiple introns. We show that these polymorphisms are all in fact deletions rather than intron gain events present in the reference genome. Finally, by leveraging the known location of the parental genes that give rise to the retrogene polymorphisms, we provide direct evidence that natural selection is responsible for the excess of fixations of retrogenes moving off of the X chromosome in Drosophila. Further efforts to identify retrogene and intron presence/absence polymorphisms will undoubtedly improve our understanding of the evolution of gene copy number and gene structure.

Intron 4 polymorphism of the endothelial nitric oxide synthase (eNOS) gene is associated with decreased NO production in a mercury-exposed population

Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a potent vasodilator and plays a prominent role in regulating the cardiovascular system. Decreased basal NO release may predispose to cardiovascular diseases. Evidence suggests that the 27nt repeat polymorphism of the intron 4 in the eNOS gene may regulate eNOS expression. On the other hand, some recent reports strongly suggest an association between methylmercury (MeHg) exposures and altered NO synthesis. In the present study, we investigate the contribution of the 27-pb tandem repeat polymorphism on nitric oxide production, which could enhance susceptibility to cardiovascular disease in the MeHg-exposed study population. Two-hundred-two participants (98 men and 104 women), all chronically exposed to MeHg through fish consumption were examined. Mean blood Hg concentration and nitrite plasma concentration were 50.5±35.4μg/L and 251.4±106.3nM, respectively. Mean systolic and diastolic blood pressure were 120.1±19.4mm Hg and 72.0±10.6mm Hg, respectively. Mean body mass index was 24.5±4.3kg/m2 and the mean heart rate was 69.8±11.8bpm. There were no significant differences in age, arterial blood pressure, body mass index or cardiac frequency between genotype groups (all P>0.05). However, we observed different nitrite concentrations in the genotypes groups, with lower nitrite levels for the 4a4a genotype carriers. Age, gender and the presence of intron 4 polymorphism contributed to nitrite reduction as a result of blood Hg concentration. Taken together, our results show that the 27nt repeat polymorphism of the intron 4 in the eNOS gene increases susceptibility to cardiovascular diseases after MeHg exposure by modulating nitric oxide levels.

DNA barcoding in the rust genus Chrysomyxa and its implications for the phylogeny of the genus

Chrysomyxa rusts are fungal pathogens widely present in the boreal forest. Taxonomic delimitation and precise species identification are difficult within this genus because several species display similar morphological features. We applied a DNA barcode system based on the ribosomal internal transcribed spacer region (ITS), large subunit (28S) ribosomal RNA gene, mitochondrial cytochrome oxidase 1 (CO1) and mitochondrial NADH dehydrogenase subunit 6 (NAD6) in 86 strains from 16 different Chrysomyxa species, including members of the Chrysomyxa ledi species complex. The nuclear ITS and 28S loci revealed higher resolving power than the mitochondrial genes. Amplification of the full CO1 barcode region failed due to the presence of introns limiting the dataset obtained with this barcode. In most cases the ITS barcodes were in agreement with taxonomic species based on phenotypic characters. Nevertheless we observed genetically distinct (different DNA barcodes) lineages within Chrysomyxa pyrolae and Chrysomyxa rhododendri, providing some evidence for allopatric speciation within these morphologically defined species. This finding, together with the observed pattern of host specificities of the studied rust fungi, suggest that species diversification within the C. ledi species complex might be governed by a set of factors such as specialisation to certain Ericaceae species as telial hosts and to a lesser extent specialization to different spruce species as aecial hosts. Moreover allopatric speciation by geographic disruption of species also seems to take place. When our data were integrated into a broader phylogenetic framework the Chrysomyxa genus unexpectedly was not resolved as a monophyletic group. Indeed the spruce cone rusts C. pyrolae and C. monesis coalesced with the pine needle rusts belonging to the genus Coleosporium, whereas the microcyclic species Chrysomyxa weirii was embedded within a clade comprising the genus Melampsora.

The presence of multiple introns is essential for ERECTA expression in Arabidopsis

Gene expression in eukaryotes is often enhanced by the presence of introns. Depending on the specific gene, this enhancement can be minor or very large and occurs at both the transcriptional and post-transcriptional levels. The Arabidopsis ERECTA gene contains 27 exons encoding a receptor-like kinase that promotes cell proliferation and inhibits cell differentiation in above-ground plant organs. The expression of ERECTA very strongly depends on the presence of introns. The intronless ERECTA gene does not rescue the phenotype of erecta mutant plants and produces about 500-900 times less protein compared with the identical construct containing introns. This result is somewhat surprising as the region upstream of the ERECTA coding sequence effectively promotes the expression of extraneous genes. Here, we demonstrate that introns are essential for ERECTA mRNA accumulation and, to a lesser extent, for mRNA utilization in translation. Since mRNA produced by intronless ERECTA is degraded at the 3' end, we speculate that introns increase mRNA accumulation through increasing its stability at least in part. No individual intron is absolutely necessary for ERECTA expression, but rather multiple introns in specific locations increase ERECTA expression in an additive manner. The ability of introns to promote ERECTA expression might be linked to the process of splicing and not to a particular intron sequence.

Computational and molecular approaches for predicting unreported causal missense mutations in Belgian patients with haemophilia A

Haemophilia A (HA) is caused by widespread mutations in the factor VIII gene. The high spontaneous mutation rate of this gene means that roughly 40% of HA mutations are private. This study aimed to describe the approaches used to confirm private disease-causing mutations in a cohort of Belgian HA patients. We studied 148 unrelated HA families for the presence of intron 22 and intron 1 inversion by Southern blotting and polymerase chain reaction (PCR). Multiplex ligation-dependent probe amplification (MLPA) assay was used to detect large genomic rearrangements. Detection of point mutations was performed by DNA sequencing. Predicting the causal impact of new non-synonymous changes was studied by two general strategies: (i) molecular approaches such as family cosegregation, evaluation of the implicated codon based on phylogenic separated species and absence of the mutation in the general Belgian population, and (ii) bioinformatics approaches to analyse the potential functional consequences of missense mutations. Among the 148 HA patients, in addition to common intron 22 and intron 1 inversions as well as large deletions or duplications, 67 different point mutations were identified, of which 42 had been reported in the HAMSTeRS database, and 25 were novel including 10 null variants for which RNA analyses confirmed the causal effect of mutations located in a splice site consensus and 15 missense mutations whose causality was demonstrated by molecular approaches and bioinformatics. This article reports several strategies to evaluate the deleterious consequences of unreported F8 substitutions in a large cohort of HA patients.

The highly variable mitochondrial small-subunit ribosomal RNA gene of Ophiostoma minus

Mitochondrial genomes in the true fungi are highly variable both in size and organization. Most of this size variation is due to the presence of introns and intron-encoded open reading frames (ORFs). The objectives for this work were to examine the mitochondrial small-subunit ribosomal RNA ( rns) gene of strains of Ophiostoma minus for the presence of introns and to characterize such introns and their encoded ORFs. DNA sequence analysis showed that among different strains of O. minus various rns gene exon/intron configurations can be observed. Based on comparative sequence analysis and RNA secondary structure modeling group I introns with LAGLIDADG ORFs were uncovered at positions mS569 and mS1224 and group II introns were present at positions mS379 and mS952. The mS379 group II intron encoded a fragmented reverse transcriptase (RT)-like ORF and the mS952 group II intron encoded a LAGLIDADG-type ORF. Examples of intron ORF degeneration due to frameshift mutations were observed. The mS379 group II intron is the first mitochondrial group II intron to have an ORF inserted within domain II, typically RT-like ORFs are inserted in domain IV. The evolutionary dynamics of the intron-encoded ORFs have also been examined.