Exon shuffling and the origin of the mitochondrial targeting function in plant cytochrome c1 precursor.

Since most of the examples of "exon shuffling" are between vertebrate genes, the view is often expressed that exon shuffling is limited to the evolutionarily recent lineage of vertebrates. Although exon shuffling in plants has been inferred from the analysis of intron phases of plant genes [Long, M., Rosenberg, C. & Gilbert, W. (1995) Proc. Natl. Acad. Sci. USA 92, 12495-12499] and from the comparison of two functionally unknown sunflower genes [Domon, C. & Steinmetz, A. (1994) Mol. Gen. Genet. 244, 312-317], clear cases of exon shuffling in plant genes remain to be uncovered. Here, we report an example of exon shuffling in two important nucleus-encoded plant genes: cytosolic glyceraldehyde-3-phosphate dehydrogenase (cytosolic GAPDH or GapC) and cytochrome c1 precursor. The intron-exon structures of the shuffled region indicate that the shuffling event took place at the DNA sequence level. In this case, we can establish a donor-recipient relationship for the exon shuffling. Three amino terminal exons of GapC have been donated to cytochrome c1, where, in a new protein environment, they serve as a source of the mitochondrial targeting function. This finding throws light upon an old important but unsolved question in gene evolution: the origin of presequences or transit peptides that generally exist in nucleus-encoded organelle genes.

Structure of murine and human renal type II Na+-phosphate cotransporter genes (Npt2 and NPT2).

Na+-phosphate (Pi) cotransport across the renal brush border membrane is the rate limiting step in the overall reabsorption of filtered Pi. Murine and human renal-specific cDNAs (NaPi-7 and NaPi-3, respectively) related to this cotransporter activity (type II Na+-Pi cotransporter) have been cloned. We now report the cloning and characterization of the corresponding mouse (Npt2) and human (NPT2) genes. The genes were cloned by screening mouse genomic and human chromosome 5-specific libraries, respectively. Both genes are approximately 16 kb and are comprised of 13 exons and 12 introns, the junctions of which conform to donor and acceptor site consensus sequences. Putative CAAT and TATA boxes are located, respectively, at positions -147 and -40 of the Npt2 gene and -143 and -51 of the NPT2 gene, relative to nucleotide 1 of the corresponding cDNAs. The translation initiation site is within exon 2 of both genes. The first 220 bp of the mouse and human promoter regions exhibit 72% identity. Two transcription start sites (at positions -9 and - 10 with respect to nucleotide 1 of NaPi-7 cDNA) and two polyadenylylation signals were identified in the Npt2 gene by primer extension, 5' and 3' rapid amplification of cDNA ends (RACE). A 484-bp 5' flanking region of the Npt2 gene, comprising the CAAT box, TATA box, and exon 1, was cloned upstream of a luciferase reporter gene; this construct significantly stimulated luciferase gene expression, relative to controls, when transiently transfected into OK cells, a renal cell line expressing type II Na+ -Pi cotransporter activity. The present data provide a basis for detailed analysis of cis and trans elements involved in the regulation of Npt2/NPT2 gene transcription and facilitate screening for mutations in the NPT2 gene in patients with autosomally inherited disorders of renal Pi reabsorption.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

Ancestral major histocompatibility complex DRB genes beget conserved patterns of localized polymorphisms.

Genes within the major histocompatibility complex (MHC) are characterized by extensive polymorphism within species and also by a remarkable conservation of contemporary human allelic sequences in evolutionarily distant primates. Mechanisms proposed to account for strict nucleotide conservation in the context of highly variable genes include the suggestion that intergenic exchange generates repeated sets of MHC DRB polymorphisms [Gyllensten, U. B., Sundvall, M. & Erlich, H. A. (1991) Proc. Natl. Acad. Sci. USA 88, 3686-3690; Lundberg, A. S. & McDevitt, H. 0. (1992) Proc. Natl. Acad. Sci. USA 89, 6545-6549]. We analyzed over 50 primate MHC DRB sequences, and identified nucleotide elements within macaque and baboon DRB6-like sequences with deletions corresponding to specific exon 2 hypervariable regions, which encode a discrete alpha helical segment of the MHC antigen combining site. This precisely localized deletion provides direct evidence implicating segmental exchange of MHC-encoded DRB gene fragments as one of the evolutionary mechanisms both generating and maintaining MHC diversity. Intergenic exchange at this site may be fundamental to the diversification of immune protection in populations by permitting alteration in the specificity of the MHC that determines the repertoire of antigens bound.

Banho de clorexidina para prevenção de colonização e infecção por micro-organismos multirresistentes na unidade de transplante de células tronco e hematopoiéticas

Introdução: Infecções relacionadas à assistência de saúde (IRAS) representam hoje um dos principais desafios da qualidade do cuidado do paciente, principalmente em pacientes submetido a transplante de células tronco e hematopoiéticas (TCTH) O banho diário com a clorexidina (CHG) degermante a 2% tem sido proposto principalmente em unidades de terapia intensivas (UTIs) para diminuir a colonização bacteriana do paciente e assim diminuir IRAS. O objetivo deste estudo foi avaliar o impacto do banho com CHG degermante a 2% em unidade de internação de TCTH na incidência de infecção e colonização por patógenos multirresistentes e ainda avaliar seu impacto na sensibilidade das bactérias ao antisséptico. Métodos: Foi realizado um estudo quasi-experimental, com duração de 9 anos, com início em janeiro/2005 até dezembro/2013. A intervenção foi iniciada em agosto de 2009, sendo que os períodos pré e pós-intervenção tiveram duração de 4,5 anos. As taxas de IRAS, infecção por gram-negativos multirresistentes e infecção e colonização por enterococo resistente a vancomicina (VRE) foram avaliadas através de série temporal, para estudar o impacto da intervenção. As concentrações inibitórias mínimas (CIM) das bactérias para a CHG com e sem o inibidor de bomba de efluxo (CCCP) foram avaliadas nos dois períodos. Os genes de resistência a CHG foram estudados por meio da PCR e a clonalidade dos isolados por eletroforese em campo pulsátil. Resultados: Foi observada redução significativa na incidência de infecção e colonização de VRE na unidade no período pós-intervenção (p: 0,001). Essa taxa permaneceu estável em outras UTIs clínicas do hospital. Contudo as taxas de infecção por Gram negativos multirresistentes aumentou nos últimos anos na unidade. Não ocorreu diminuição na taxa de IRAS na unidade. As CIMs testadas de CHG aumentaram nas amostras de VRE e K. pneumoniae após o período de exposição ao antisséptico, com queda importante da CIM após o uso do CCCP, revelando ser a bomba de efluxo, um importante mecanismo de resistência à CHG. As amostras de A. baumannii e P. aeruginosa não apresentaram aumento da CIM após período de exposição à clorexidina. As bombas de efluxo Ade A, B e C estiveram presentes na maioria dos A. baumannii do grupo controle (66%). A bomba cepA foi encontrada em 67% de todas as K. pneumoniae testadas e em 44,5% das P. aeruginosas do grupo pré intervenção. Observamos uma relação positiva entre a presença da CepA nas amostras de K. pneumoniae e a resposta ao CCCP: de todas as 49 amostras CepA positivas 67,3% obtiveram redução do seu MIC em 4 diluições após adição do CCCP. A avaliação de clonalidade demonstrou padrão policlonal das amostras de VRE, K. pneumoniae e A. baumannii avaliadas. Em relação às amostras de P. aeruginosa foi observado que no período pós-intervenção ocorreu predominância de um clone com > 80% semelhança em 10 das 22 amostras avaliadas pelo dendrograma. Conclusões: O banho de clorexidina teve impacto na redução da incidência de infecção e colonização por VRE na unidade de TCTH, e não teve o mesmo impacto nas bactérias gram-negativas. Os mecanismos moleculares de resistência à clorexidina estão intimamente ligados à presença de bomba de efluxo, sendo provavelmente o principal mecanismo de resistência e tolerância das bactérias ao antisséptico

RNA-Seq expression profile of genes related to neurodegenerative disorders affecting the human retina

Human neurodegenerative diseases, such as Parkinson’s disease (PD) and the neuromuscular disorders called dystroglycanopathies (DGPs), cause retinal impairments. We have used RNA-Seq technology to catalog all known genes linked to PD and DGPs expressed in the human retina and quantitate their mRNA levels in terms of FPKM. We have also characterized their expression profiles in the retina by determining their exonic, intronic and exon-intron junction expression levels, as well as the alternative splicing pattern of particular genes. We believe these data could pave the way toward understanding the molecular bases of sight deficiencies associated with neurodegenerative disorders.

Next-generation sequencing of iron-metabolism related genes in Portuguese patients with iron overload: novel pathogenic genetic variants

Objective: In Southern European countries up to one-third of the patients with hereditary hemochromatosis (HH) do not present the common HFE risk genotype. In order to investigate the molecular basis of these cases we have designed a gene panel for rapid and simultaneous analysis of 6 HH-related genes (HFE, TFR2, HJV, HAMP, SLC40A1 and FTL) by next-generation sequencing (NGS). Materials and Methods: Eighty-eight iron overload Portuguese patients, negative for the common HFE mutations, were analysed. A TruSeq Custom Amplicon kit (TSCA, by Illumina) was designed in order to generate 97 amplicons covering exons, intron/exon junctions and UTRs of the mentioned genes with a cumulative target sequence of 12115bp. Amplicons were sequenced in the MiSeq instrument (IIlumina) using 250bp paired-end reads. Sequences were aligned against human genome reference hg19 using alignment and variant caller algorithms in the MiSeq reporter software. Novel variants were validated by Sanger sequencing and their pathogenic significance were assessed by in silico studies. Results: We found a total of 55 different genetic variants. These include novel pathogenic missense and splicing variants (in HFE and TFR2), a very rare variant in IRE of FTL, a variant that originates a novel translation initiation codon in the HAMP gene, among others. Conclusion: The merging of TSCA methodology and NGS technology appears to be an appropriate tool for simultaneous and fast analysis of HH-related genes in a large number of samples. However, establishing the clinical relevance of NGS-detected variants for HH development remains a hard-working task, requiring further functional studies.

LPS regulates a set of genes in primary murine macrophages by antagonising CSF-1 action

We previously reported that bacterial products such as LPS and CpG DNA down-modulated cell surface levels of the Colony Stimulating Factor (CSF)-1 receptor (CSF-1R) on primary murine macrophages in an all-or-nothing manner. Here we show that the ability of bacterial products to down-modulate the CSF-IR rendered bone marrow-derived macrophages (BMM) unresponsive to CSF-1 as assessed by Akt and ERK 1/2 phosphorylation. Using toll-like receptor (th-)9 as a model CSF-1-repressed gene, we show that LPS induced tlr9 expression in BMM only when CSF-1 was present, suggesting that LPS relieves CSF-1-mediated inhibition to induce gene expression. Using cDNA microarrays, we identified a cluster of similarly CSF-1 repressed genes in BMM. By real time PCR we confirmed that the expression of a selection of these genes, including integral membrane protein 2B (itm2b), receptor activity-modifying protein 2 (ramp2) and macrophage-specific gene 1 (mpg-1), were repressed by CSF-1 and were induced by LPS only in the presence of CSF-1. This pattern of gene regulation was also apparent in thioglycollate-elicited peritoneal macrophages (TEPM). LPS also counteracted CSF-1 action to induce mRNA expression of a number of transcription factors including interferon consensus sequence binding protein 1 (Icsbp1), suggesting that this mechanism leads to transcriptional reprogramming in macrophages. Since the majority of in vitro studies on macrophage biology do not include CSF-1, these genes represent a set of previously uncharacterised LPS-inducible genes. This study identifies a new mechanism of macrophage activation, in which LPS (and other toll-like receptor agonists) regulate gene expression by switching off the CSF-1R signal. This finding also provides a biological relevance to the well-documented ability of macrophage activators to down-modulate surface expression of the CSF-1R. (C) 2005 Elsevier GmbH. All rights reserved.

Assessment of epidermal cell viability by near infrared multi-photon microscopy following ballistic delivery of gold micro-particles

The use of gene guns in ballistically delivering DNA vaccine coated gold micro-particles to skin can potentially damage targeted cells, therefore influencing transfection efficiencies. In this paper, we assess cell death in the viable epidermis by non-invasive near infrared two-photon microscopy following micro-particle bombardment of murine skin. We show that the ballistic delivery of micro-particles to the viable epidermis can result in localised cell death. Furthermore, experimental results show the degree of cell death is dependant on the number of micro-particles delivered per unit of tissue surface area. Micro-particles densities of 0.16 +/- 0.27 (mean +/- S.D.), 1.35 +/- 0.285 and 2.72 +/- 0.47 per 1000 mu m(2) resulted in percent deaths of 3.96 +/- 5.22, 45.91 +/- 10.89, 90.52 +/- 12.28, respectively. These results suggest that optimization of transfection by genes administered with gene guns is - among other effects - a compromise of micro-particle payload and cell death. (c) 2005 Elsevier Ltd. All rights reserved.

Engrailed genes in mammary development and tumorigenesis

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Posttranscriptional Regulation of Embryonic Neurogenesis by the Exon Junction Complex

The six-layered neuron structure in the cerebral cortex is the foundation for human mental abilities. In the developing cerebral cortex, neural stem cells undergo proliferation and differentiate into intermediate progenitors and neurons, a process known as embryonic neurogenesis. Disrupted embryonic neurogenesis is the root cause of a wide range of neurodevelopmental disorders, including microcephaly and intellectual disabilities. Multiple layers of regulatory networks have been identified and extensively studied over the past decades to understand this complex but extremely crucial process of brain development. In recent years, post-transcriptional RNA regulation through RNA binding proteins has emerged as a critical regulatory nexus in embryonic neurogenesis. The exon junction complex (EJC) is a highly conserved RNA binding complex composed of four core proteins, Magoh, Rbm8a, Eif4a3, and Casc3. The EJC plays a major role in regulating RNA splicing, nuclear export, subcellular localization, translation, and nonsense mediated RNA decay. Human genetic studies have associated individual EJC components with various developmental disorders. We showed previously that haploinsufficiency of Magoh causes microcephaly and disrupted neural stem cell differentiation in mouse. However, it is unclear if other EJC core components are also required for embryonic neurogenesis. More importantly, the molecular mechanism through which the EJC regulates embryonic neurogenesis remains largely unknown. Here, we demonstrated with genetically modified mouse models that both Rbm8a and Eif4a3 are required for proper embryonic neurogenesis and the formation of a normal brain. Using transcriptome and proteomic analysis, we showed that the EJC posttranscriptionally regulates genes involved in the p53 pathway, splicing and translation regulation, as well as ribosomal biogenesis. This is the first in vivo evidence suggesting that the etiology of EJC associated neurodevelopmental diseases can be ribosomopathies. We also showed that, different from other EJC core components, depletion of Casc3 only led to mild neurogenesis defects in the mouse model. However, our data suggested that Casc3 is required for embryo viability, development progression, and is potentially a regulator of cardiac development. Together, data presented in this thesis suggests that the EJC is crucial for embryonic neurogenesis and that the EJC and its peripheral factors may regulate development in a tissue-specific manner.

Engrailed genes in mammary development and tumorigenesis

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.