Intron Insertion Research Articles

Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border (RB) end of the T-DNA is largely preserved whereas the left border (LB) end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61 % of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67 % of T-DNA integrations are integrations at a single chromosomal site and 31 % of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.

Intron-derived aberrant splicing of A20 transcript in rheumatoid arthritis

Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients. Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression. In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes. The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.

I-PfoP3I: A Novel Nicking HNH Homing Endonuclease Encoded in the Group I Intron of the DNA Polymerase Gene in Phormidium foveolarum Phage Pf-WMP3

Homing endonucleases encoded in a group I self-splicing intron in a protein-coding gene in cyanophage genomes have not been reported, apart from some free-standing homing edonucleases. In this study, a nicking DNA endonuclease, I-PfoP3I, encoded in a group IA2 intron in the DNA polymerase gene of a T7-like cyanophage Pf-WMP3, which infects the freshwater cyanobacterium Phormidium foveolarum is described. The Pf-WMP3 intron splices efficiently in vivo and self-splices in vitro simultaneously during transcription. I-PfoP3I belongs to the HNH family with an unconventional C-terminal HNH motif. I-PfoP3I nicks the intron-minus Pf-WMP3 DNA polymerase gene more efficiently than the Pf-WMP4 DNA polymerase gene that lacks any intervening sequence in vitro, indicating the variable capacity of I-PfoP3I. I-PfoP3I cleaves 4 nt upstream of the intron insertion site on the coding strand of EXON 1 on both intron-minus Pf-WMP3 and Pf-WMP4 DNA polymerase genes. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a 14 bp region downstream of the cut site. I-PfoP3I requires Mg2+, Ca2+ or Mn2+ for nicking activity. Phylogenetic analysis suggests that the intron and homing endonuclease gene elements might be inserted in Pf-WMP3 genome individually after differentiation from Pf-WMP4. To our knowledge, this is the first report of the presence of a group I self-splicing intron encoding a functional homing endonuclease in a protein-coding gene in a cyanophage genome.

Conditional control of gene function by an invertible gene trap in zebrafish

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

Progesterone receptor variants associated with the PROGINS haplotype exhibit functional properties similar to those of wild-type progesterone receptor

The progesterone receptor (PR) is a ligand-activated transcription factor existing in two isoforms, A (PRA) and B (PRB), resulting from alternative promoter usage. It has long been speculated that genetic variants of PR are associated with the risk for various benign and malignant diseases, but data from clinical trials and in-vitro studies remain contradictory. The most extensively studied variant is termed PROGINS and consists of an intronic 320-bp Alu insertion and two coding (Ser344Thr, Val660Leu) and one silent single nucleotide polymorphism in complete linkage disequilibrium (allele frequency in Caucasians 9-19%). Our study aimed at elucidating the functional consequences of the PROGINS-associated single nucleotide polymorphisms of PRA and PRB (i.e. Thr344 and Leu660) as compared with wild-type PR (Ser344, Val660). The two PRA and two PRB full-length receptor variants were expressed by adenovirus in the PR-negative human breast cancer cell line T47D-Y and assayed with respect to transactivational properties, c-src activation, combined net mRNA and protein stability and hormone-binding characteristics. In all experiments the wild-type PR and the PROGINS variant were undistinguishable. Though there still might be tissue specific effects of the variants, our data indicate that these common PR variants do not functionally differ, which may provide a basis to explain the heterogeneous outcome of association studies.

Transposon insertion in a cinnamyl alcohol dehydrogenase gene is responsible for a brown midrib1 mutation in maize

Maize brown midrib1 (bm1) mutant plants have reduced lignin content and offer significant advantages when used in silage and biofuel applications. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the conversion of hydroxycinnamyl aldehydes to monolignols, a key step in lignin biosynthesis. Maize CAD2 has been implicated as the underlying gene for bm1 phenotypes since bm1 plants have reduced CAD activity and lower CAD2 transcript level. Here, we describe a Dow AgroSciences maize bm1 mutant (bm1-das1) that contains a 3,444-bp transposon insertion in the first intron of CAD2 gene. As a result of chimeric RNA splicing, cad2 mRNA from bm1-das1 contains a 409-bp insert between its 1st and 2nd exons. This insertion creates a premature stop codon and is predicted to result in a truncated protein of 48 amino acids (AA), compared to 367 AA for the wild type (WT) CAD2. We have also sequenced cad2 from the reference allele bm1-ref in 515D bm1 stock and showed that it contains a two-nucleotide (AC) insertion in the 3rd exon, which is predicted to result in a truncated protein of 147 AA. The levels of cad2 mRNA in the midribs of bm1-das1 and bm1-ref are reduced by 91 and 86 % respectively, leading to reductions in total lignin contents by 24 and 30 %. Taken together, our data show that mutations in maize CAD2 are responsible for maize bm1 phenotypes. Based on specific changes in bm1-das1 and bm1-ref, high throughput TaqMan and KBioscience's allele specific PCR assays capable of differentiating mutant and WT alleles have been developed to accelerate bm1 molecular breeding.

Activity of Ancient RTE Retroposons during the Evolution of Cows, Spiral-Horned Antelopes, and Nilgais (Bovinae)

In the genome of Artiodactyla (cow, sheep, pigs, camels, and whales), a major retroposon group originated from a presumable horizontal transfer of BovB, a retrotransposon-like element retroposon, between 52 and 70 million years ago. Since then, BovB retroposons have proliferated and today occupy a quarter of the cow's genome sequence. The BovB-related short interspersed elements (SINEs) were used for resolving the phylogeny of Bovinae (cows, spiral-horned antelopes, and nilgais) and their relatives. In silico screening of 55,000 intronic retroposon insertions in the cow genome and experimental validation of 126 introns resulted in 29 informative retroposon markers for resolving bovine evolutionary relationships. A transposition-in-transposition analysis identifies three different phases of SINE activity and show how BovB elements have expanded in the cattle genome.

Association of TP53 Polymorphisms with Primary Open-Angle Glaucoma: A Meta-Analysis

To offer a comprehensive evaluation of the potential associations of TP53 polymorphisms with primary open-angle glaucoma (POAG) through a systematic review and meta-analysis of candidate genetic association study. Medline, Embase, Science Citation Index, the Cochrane Library, and other databases (up to January 20, 2012) were searched by two investigators independently. Pooled odd ratios (ORs) and 95% confidence interval (CI) were used to assess the strength of the associations between two TP53 polymorphisms (codon 72 in exon 4 and 16 base-pair [bp] insertion in intron 3) and POAG. Statistical analysis was performed with a commercial statistical and data analysis software package. Nine independent studies on TP codon 72 (1930 cases and 1463 controls) and four articles on TP intron 3 16-bp insertion (858 cases and 683 controls) were identified. The overall results showed that there was significant association between TP53 codon 72 genotype and POAG risk in the recessive model (OR = 1.31, 95% CI 1.05-1.64, P = 0.017). Also, our analysis suggested that TP53 intron 3 16-bp insertion polymorphism was associated with decreased POAG risk in overall population when examining the contrast of Ins versus Del (OR 0.75, 95% CI = 0.57-0.97, P = 0.031). In subgroup analyses for ethnicity (Caucasian, Asian), we detected the association between codon 72 polymorphism and risk for POAG in Asian populations (recessive model: OR = 1.36, 95% CI 1.03-1.80, P = 0.026) but not in Caucasian populations. However, no significant finding was noted between P53Arg72Pro and risk for open-angle glaucoma either in high tension glaucoma or in normal tension glaucoma. Because of insufficient studies on TP53 16-bp insertion polymorphism, no subgroup analyses were conducted according to ethnicity and glaucoma subtype to detect the effect of this polymorphism on the susceptibility to POAG. This meta-analysis showed the evidence that TP53 codon 72 (CC versus CG+GG) and intron 3 16-bp insertion (Ins versus Del) polymorphisms may affect individual susceptibility to POAG. Moreover, stratified analyses that detected the effect of TP53 codon 72 polymorphism seemed to be varied by ethnicity. Given the limited sample size, further investigations are needed to validate the association.

Correlation Analysis between Single Nucleotide Polymorphism of Fatty Acid Synthase and Fatness Traits in Chickens

The F2 chicken population from China Agricultural University was selected to explore the correlation between single nucleotide polymorphism of fatty acid synthase (FAS) and fatness traits. The primers of FAS gene were designed according to the sequence of chicken fatty acid synthase gene (accession number: J02839). The PCR-SSCP was used for genotype analysis. The results showed that the insertion or deletion of the 41st intron (1418-1424 bp) in chicken FAS gene could result in AA, AB and BB genotypes. In addition, least square analysis revealed that the deletion or mutation insertion of the 41st intron (1418-1424 bp) in FAS gene could affect body weights, abdominal fat weights and abdominal fat ratios of chickens. Therefore, polymorphic loci can be used for marker-assisted selection breeding of chickens.

Identification of novel variant of EML4-ALK fusion gene in NSCLC: Potential benefits of the RT-PCR method.

e12007 Background: The discovery of the transforming fusion gene of the anaplastic lymphoma kinase (ALK) with the echinoderm microtubule-associated protein like 4 (EML4) as an oncogene in 2007 has led to its validation as a clinical target in NSCLC patients in a short period of time. The inhibition of the anaplastic lymphoma receptor tyrosine kinase has demonstrated to prolong progression-free survival compared to the standard of care chemotherapy in patients with advanced NSCLC that are ALK positive. However, the clinical implications of the 15 different variants of the EML4-ALK transforming gene described so far are currently not defined. Here we present a novel variant of the EML4-ALK fusion gene which we named variant 3c. Methods: RNA extracted from formalin fixed paraffin embedded (FFPE) specimens from patients with advanced and metastatic NSCLC was amplified, using primers and probes designed to detect specific EML4-ALK fusion gene fragments. Gel electrophoresis showed a different band for the new variant 3c compared to the known bands of positive cell lines for variant 3a and 3b. These findings were further investigated by dye-terminator Sequencing and FISH. Results: The novel variant, detected in two NSCLC specimens, is longer than v3a and shorter than v3b, representing an 18 base pair insertion of intron 19 of ALK between exon 6 of EML4 and exon 20 of ALK. All of the two samples showed exactly the same sequencing result. One of the samples was negative for FISH break apart testing and the other one showed a positive result, defined by ≥ 15% split nuclei as indicative of an ALK rearrangement. Conclusions: Compared to FISH technology, RT-PCR enables the detection of different isoforms of the EML4-ALK transforming gene, which can be validated by sequencing. Only one out of two samples that were positive for the new variant by RT-PCR could be confirmed by FISH. The clinical significance of the different variants, notably to resistance and response to ALK-Inhibitors and the concordance and sensitivity of FISH and RT-PCR should be subject to further investigations.

Mitochondrial genomes from the lichenized fungi Peltigera membranacea and Peltigera malacea: Features and phylogeny

Mitochondrial genomes from the fungal partners of two terricolous foliose lichen symbioses, Peltigera membranacea and Peltigera malacea, have been determined using metagenomic approaches, including RNA-seq. The roughly 63 kb genomes show all the major features found in other Pezizomycotina, such as unidirectional transcription, 14 conserved protein genes, genes for the two subunit rRNAs and for a set of 26 tRNAs used in translating the 62 amino acid codons. In one of the tRNAs a CAU anticodon is proposed to be modified, via the action of the nuclear-encoded enzyme, tRNA Ile lysidine synthase, so that it recognizes the codon AUA (Ile) instead of AUG (Met). The overall arrangements and sequences of the two circular genomes are similar, the major difference being the inversion and deterioration of a gene encoding a type B DNA polymerase. Both genomes encode the RNA component of RNAse P, a feature seldom found in ascomycetes. The difference in genome size from the minimal ascomycete mitochondrial genomes is largely due to 17 and 20 group I introns, respectively, most associated with homing endonucleases and all found within protein-coding genes and the gene encoding the large subunit rRNA. One new intron insertion point was found, and an unusually small exon of seven nucleotides (nt) was identified and verified by RNA sequencing. Comparative analysis of mitochondrion-encoded proteins places the Peltigera spp., representatives of the class Lecanoromycetes, close to Leotiomycetes, Dothidiomycetes, and Sordariomycetes, in contrast to phylogenies found using nuclear genes.

First draft of the genomic organization of a PIII-SVMP gene

The evolutionary pathway of highly toxic proteins expressed in snake venom glands from proteins without toxic function and expressed in non-parotid tissues remains poorly understood. Here we examine gene structure of a representative of a venom protein with an ADAMs metalloproteinase evolutionary origin. The structure of the 15,652 bp Echis ocellatus pre-pro EOC00089-like PIII-SVMP gene was assembled from PCR-amplified sequences of overlapping genomic fragments. The gene comprises 12 exons interrupted by 11 introns. In a homology model of the EOC00089-like protein, the insertion of introns interrupting coding regions lie just after or between secondary structure elements. Long interspersed nuclear retroelements (LINE) L2/CR1 and RTE/Bov-B, short interspersed nuclear retroelements SINE/Sauria, and a hobo-activator DNA (Charlie, hAT) transposon were identified within introns 1, 3, 7 and 8. Pairwise amino acid sequence comparisons between EOC00089-like PIII-SVMP and its closest orthologs, ADAM28, from a mammal, Homo sapiens, and the lizard, Anolis carolinensis, showed that the ORFs of these three proteins share 42%/59%, 49%/69%, and 48%/65% (identity/similarity), respectively. The protein-coding positions interrupted by each of the 11 introns of the Echis PIII-SVMP gene are entirely conserved in the A. carolinensis and human ADAM28 genes. However, the lizard and the human ADAM28 genes contain 5 introns not present in the E. ocellatus gene. Furthermore, Echis and Anolis introns exhibit quantitatively and qualitatively distinctions in their inserted retroelements. These findings identify introns as possible key elements in the recruitment and amplification process of SVMPs into the venom gland of extant snakes. Ongoing reptile genome sequencing projects may shed light on this intriguing aspect of the emergence and evolution of venom toxin genes. Furthermore, the organization of the PIII-SVMP reported here provides a genomic explanation for the emergence of dimeric disintegrin subunits encoded by short messengers.

TET2 is essential for survival and hematopoietic stem cell homeostasis

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2(trap)) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2(trap/trap) mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2(trap/trap) HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2(trap/trap) HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

Group II intron architecture and its implications for the development of eukaryotic splicing systems

Group II introns are self‐splicing RNA molecules that have played an important role in the evolution of terrestrial genome organization. After splicing, free group II introns are liberated into the cell, where they behave as mobile genetic elements that insert themselves into new RNA or DNA sites. As a result of this behavior, the introns have spread throughout bacteria and eukaryotic lineages, diversifying organisms through the insertion of new introns and by evolving into new types of splicing machinery. The eukaryotic spliceosome is believed to have evolved from an ancestral group II intron that was absorbed during an early endosymbiont event. The functional relationship between group II introns and the spliceosome has long been supported by similarities in splicing mechanism and conserved RNA secondary structures. Recent crystallographic studies of group II introns have provided a high resolution view of active‐site organization, resulting in additional evidence that group II‐like components lie within the catalytic heart of the eukaryotic spliceosome.

Cross-Species Analyses Identify the BNIP-2 and Cdc42GAP Homology (BCH) Domain as a Distinct Functional Subclass of the CRAL_TRIO/Sec14 Superfamily

The CRAL_TRIO protein domain, which is unique to the Sec14 protein superfamily, binds to a diverse set of small lipophilic ligands. Similar domains are found in a range of different proteins including neurofibromatosis type-1, a Ras GTPase-activating Protein (RasGAP) and Rho guanine nucleotide exchange factors (RhoGEFs). Proteins containing this structural protein domain exhibit a low sequence similarity and ligand specificity while maintaining an overall characteristic three-dimensional structure. We have previously demonstrated that the BNIP-2 and Cdc42GAP Homology (BCH) protein domain, which shares a low sequence homology with the CRAL_TRIO domain, can serve as a regulatory scaffold that binds to Rho, RhoGEFs and RhoGAPs to control various cell signalling processes. In this work, we investigate 175 BCH domain-containing proteins from a wide range of different organisms. A phylogenetic analysis with ∼100 CRAL_TRIO and similar domains from eight representative species indicates a clear distinction of BCH-containing proteins as a novel subclass within the CRAL_TRIO/Sec14 superfamily. BCH-containing proteins contain a hallmark sequence motif R(R/K)h(R/K)(R/K)NL(R/K)xhhhhHPs (‘h’ is large and hydrophobic residue and ‘s’ is small and weekly polar residue) and can be further subdivided into three unique subtypes associated with BNIP-2-N, macro- and RhoGAP-type protein domains. A previously unknown group of genes encoding ‘BCH-only’ domains is also identified in plants and arthropod species. Based on an analysis of their gene-structure and their protein domain context we hypothesize that BCH domain-containing genes evolved through gene duplication, intron insertions and domain swapping events. Furthermore, we explore the point of divergence between BCH and CRAL-TRIO proteins in relation to their ability to bind small GTPases, GAPs and GEFs and lipid ligands. Our study suggests a need for a more extensive analysis of previously uncharacterized BCH, ‘BCH-like’ and CRAL_TRIO-containing proteins and their significance in regulating signaling events involving small GTPases.

Photobiont Flexibility in <i>Paramecium bursaria</i>: Double and Triple Photobiont Co-Habitation

The green ciliate, Paramecium bursaria, has evolved a mutualistic relationship with endosymbiotic green algae (photobionts). Under culture conditions, photobionts are usually unified (to be single species) within each P. bursaria strain. In most cases, the algal partners are restricted to either Chlorella variabilis or Micractinium reisseri (Chlorellaceae, Trebouxiophyceae). Both species are characterized by particular physiology and atypical group I intron insertions, although they are morphologically indistinguishable from each other or from other Chlorella-related species. Both algae are exclusive species that are viable only within P. bursaria cells, and therefore their symbiotic relationship can be considered persistent. In a few cases, the other algal species have been reported as P. bursaria photobionts. Namely, P. bursaria have occasionally replaced their photobiont partner. This paper introduces some P. bursaria strains that maintain more than one species of algae for a long period. This situation prompts speculations about flexibility of host-photo-biont relationships, how P. bursaria replaced these photobionts, and the infection theory of the group I introns.

Quorum sensing in the probiotic bacterium Escherichia coli Nissle 1917 (Mutaflor) - evidence that furanosyl borate diester (AI-2) is influencing the cytokine expression in the DSS colitis mouse model.

Background“Quorum sensing” (QS) is the phenomenon which allows single bacterial cells to measure the concentration of bacterial signal molecules. Two principle different QS systems are known, the Autoinducer 1 system (AI-1) for the intraspecies communication using different Acyl-homoserine lactones (AHL) and AI-2 for the interspecies communication. Aim of this study was to investigate QS of Escherichia coli Nissle 1917 (Mutaflor).ResultsWhile E. coli Nissle is producing AI-2 in a density dependent manner, no AI-1 was produced. To study the effect of AI-2 in the DSS (dextran sulphate sodium) induced mouse model of acute colitis, we silenced the corresponding gene luxS by intron insertion. The mutant bacterium E. coli Nissle::luxS was equally effective in colonizing the colon and the mutation turned out to be 100% stable during the course of the experiment. Isolating RNA from the colon mucosa and performing semiquantitative RT PCR, we were able to show that the expression of the pro-inflammatory cytokine IFN-y was suppressed in mice being infected with the E. coli Nissle wild type. Mice infected with the E. coli Nissle::luxS mutant showed a suppressed expression of IL-10 compared to uninfected mice, while the expression of the pro-inflammatory cytokines IL-6 and TNF-α was higher in these mice. The expression of mBD-1 was suppressed in mice being infected with the mutant in comparison to the mice not infected or infected with the wild type. No differences were seen in the histological examination of the colon sections in the different groups of mice.ConclusionsE. coli Nissle is producing AI-2 molecules, which are influencing the expression of cytokines in the mucosa of the colon in the DSS mice. However, if QS has a direct influence on the probiotic properties of E. coli Nissle remains to be elucidated.

Biochemical Studies of a Long Noncoding RNA Involved in Breast Cancer Biology

Long non-coding RNAs, a significant fraction of the transcriptome in higher eukaryotes, are emerging as key players in many rapidly growing areas of biology, including embryonic stem cell differentiation, brain function, subcellular compartmentalization, chromatin remodeling and cancer biology. We report one of the first biochemical characterizations of a long ncRNA, which coactivates sex hormone nuclear receptors and is strongly associated with breast cancer. This was the first RNA proven to act as a regulatory non-coding RNA and to code for a protein. To date, eleven variant transcripts of the long ncRNA have been identified. We study three: 1) a noncoding isoform, 2) a coding isoform, and 3) an intron-comprising noncoding transcript, which is alternatively spliced and possesses a partial intron insertion. To assess the secondary structures of the selected RNAs and map their overall structural organization, we employ chemical probing tools at select experimental conditions.

Alternative Splicing of a Novel Inducible Exon Diversifies the CASK Guanylate Kinase Domain.

Alternative pre-mRNA splicing has a major impact on cellular functions and development with the potential to fine-tune cellular localization, posttranslational modification, interaction properties, and expression levels of cognate proteins. The plasticity of regulation sets the stage for cells to adjust the relative levels of spliced mRNA isoforms in response to stress or stimulation. As part of an exon profiling analysis of mouse cortical neurons stimulated with high KCl to induce membrane depolarization, we detected a previously unrecognized exon (E24a) of the CASK gene, which encodes for a conserved peptide insertion in the guanylate kinase interaction domain. Comparative sequence analysis shows that E24a appeared selectively in mammalian CASK genes as part of a >3,000 base pair intron insertion. We demonstrate that a combination of a naturally defective 5′ splice site and negative regulation by several splicing factors, including SC35 (SRSF2) and ASF/SF2 (SRSF1), drives E24a skipping in most cell types. However, this negative regulation is countered with an observed increase in E24a inclusion after neuronal stimulation and NMDA receptor signaling. Taken together, E24a is typically a skipped exon, which awakens during neuronal stimulation with the potential to diversify the protein interaction properties of the CASK polypeptide.

CAPN3 mRNA processing alteration caused by splicing mutation associated with novel genomic rearrangement of Alu elements

Recessive mutations of CAPN3 gene are reported to be responsible for limb girdle muscular dystrophy type 2A (LGMD2A). In all, 15-25% of intronic nucleotide changes identified in this gene were investigated by in silico analysis, but occasionally supported by experimental data or reported in some cases as a polymorphism. We report here genetic and transcriptional analyses in three Tunisian patients belonging to the same consanguineous family sharing the same mutation c.1194-9 A>G and Alu repeats insertion in intron 7 of CAPN3 gene. Reverse transcriptase-PCR experiments performed on total RNA from the patient's muscle biopsy showed retention of the eight last nucleotides of intron 9 in the CAPN3 transcript lacking the first seven exons. Our results provide evidence regarding the potential involvement of Alu elements in aberrant processing of pre-mRNA owing to the disruption of pre-existing intronic splicing regulatory elements. We also demonstrated variable mRNA alternative splicing among tissues and between LGMD2A patients. A deep intronic variation and rearrangement have been reported in the literature as causing genetic diseases in humans. However, this is the first report on a potential pathogenic CAPN3 gene mutation resulting from an Alu insertion.