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Biomedical subjects

Georges Lutfalla

Publications and source records attributed to Georges Lutfalla.

11 recordsLinked to original sources
PubMed2006

Acetylcholinesterase genes within the Diptera: takeover and loss in true flies.

It has recently been reported that the synaptic acetylcholinesterase (AChE) in mosquitoes is encoded by the ace-1 gene, distinct and divergent from the ace-2 gene, which performs this function in Drosophila. This is an unprecedented situation within the Diptera order because both ace genes derive from an old duplication and are present in most insects and arthropods. Nevertheless, Drosophila possesses only the ace-2 gene. Thus, a secondary loss occurred during the evolution of Diptera, implying a vital function switch from one gene (ace-1) to the other (ace-2). We sampled 78 species, representing 50 families (27% of the Dipteran families) spread over all major subdivisions of the Diptera, and looked for ace-1 and ace-2 by systematic PCR screening to determine which taxonomic groups within the Diptera have this gene change. We show that this loss probably extends to all true flies (or Cyclorrhapha), a large monophyletic group of the Diptera. We also show that ace-2 plays a non-detectable role in the synaptic AChE in a lower Diptera species, suggesting that it has non-synaptic functions. A relative molecular evolution rate test showed that the intensity of purifying selection on ace-2 sequences is constant across the Diptera, irrespective of the presence or absence of ace-1, confirming the evolutionary importance of non-synaptic functions for this gene. We discuss the evolutionary scenarios for the takeover of ace-2 and the loss of ace-1, taking into account our limited knowledge of non-synaptic functions of ace genes and some specific adaptations of true flies.

Acetylcholinesterase↗
PubMed2006

Performing quantitative reverse-transcribed polymerase chain reaction experiments.

Quantitative polymerase chain reaction (PCR) is as old as PCR, but it has had to wait for the introduction of real-time PCR instruments to become widely used. These instruments allow monitoring of the PCR reaction on line; they involve the use of a fluorescent probe that allows quantification of the amplified DNA. Different fluorescent formats and different applications have been developed for quantitative PCR, but this chapter focuses on the use of the SYBR Green label for the quantification of specific cDNAs in reverse transcription mixes: RT-PCR. We propose optimal reaction conditions for the reactions to be performed on the different available instruments and discuss the important parameters for setting up experiments: specificity, efficiency, and reproducibility. We also introduce the reader to the problems of relative quantification.

Animals↗
PubMed2004

Genome duplication in the teleost fish Tetraodon nigroviridis reveals the early vertebrate proto-karyotype.

Tetraodon nigroviridis is a freshwater puffer fish with the smallest known vertebrate genome. Here, we report a draft genome sequence with long-range linkage and substantial anchoring to the 21 Tetraodon chromosomes. Genome analysis provides a greatly improved fish gene catalogue, including identifying key genes previously thought to be absent in fish. Comparison with other vertebrates and a urochordate indicates that fish proteins have diverged markedly faster than their mammalian homologues. Comparison with the human genome suggests approximately 900 previously unannotated human genes. Analysis of the Tetraodon and human genomes shows that whole-genome duplication occurred in the teleost fish lineage, subsequent to its divergence from mammals. The analysis also makes it possible to infer the basic structure of the ancestral bony vertebrate genome, which was composed of 12 chromosomes, and to reconstruct much of the evolutionary history of ancient and recent chromosome rearrangements leading to the modern human karyotype.

Animals↗
PubMed2003

A real-time polymerase chain reaction assay for quantification of allele ratios and correction of amplification bias.

Allele-specific epigenetic modifications are crucial for several important biological functions, including genomic imprinting and X-inactivation in mammals. Consequently, an ever increasing number of investigations requires accurate quantification of the relative abundance of parental alleles of a specific sequence in a DNA sample. Here, combining the use of polymorphic restriction sites with real-time polymerase chain reaction (PCR) amplification, we describe a simple and quantitative assay to measure allele ratios. The efficiency of the assay was assessed on genomic DNA for several polymorphic restriction sites located in the mouse Igf2/H19 imprinted locus. The assay was also successfully applied to quantify allele ratio in cDNA samples. In addition, we provide an experimental procedure for detection and correction of potential PCR amplification bias which significantly extends the range of application of the assay.

Data Interpretation, Statistical↗
PubMed2003

Comparative genomic analysis reveals independent expansion of a lineage-specific gene family in vertebrates: the class II cytokine receptors and their ligands in mammals and fish.

BACKGROUND: The high degree of sequence conservation between coding regions in fish and mammals can be exploited to identify genes in mammalian genomes by comparison with the sequence of similar genes in fish. Conversely, experimentally characterized mammalian genes may be used to annotate fish genomes. However, gene families that escape this principle include the rapidly diverging cytokines that regulate the immune system, and their receptors. A classic example is the class II helical cytokines (HCII) including type I, type II and lambda interferons, IL10 related cytokines (IL10, IL19, IL20, IL22, IL24 and IL26) and their receptors (HCRII). Despite the report of a near complete pufferfish (Takifugu rubripes) genome sequence, these genes remain undescribed in fish. RESULTS: We have used an original strategy based both on conserved amino acid sequence and gene structure to identify HCII and HCRII in the genome of another pufferfish, Tetraodon nigroviridis that is amenable to laboratory experiments. The 15 genes that were identified are highly divergent and include a single interferon molecule, three IL10 related cytokines and their potential receptors together with two Tissue Factor (TF). Some of these genes form tandem clusters on the Tetraodon genome. Their expression pattern was determined in different tissues. Most importantly, Tetraodon interferon was identified and we show that the recombinant protein can induce antiviral MX gene expression in Tetraodon primary kidney cells. Similar results were obtained in Zebrafish which has 7 MX genes. CONCLUSION: We propose a scheme for the evolution of HCII and their receptors during the radiation of bony vertebrates and suggest that the diversification that played an important role in the fine-tuning of the ancestral mechanism for host defense against infections probably followed different pathways in amniotes and fish.

Animals↗
PubMed2003

Down-modulation of responses to type I IFN upon T cell activation.

The immunomodulatory role of type I IFNs (IFN-alpha/beta) in shaping T cell responses has been demonstrated, but the direct effects of IFN on T cells are still poorly characterized. Particularly, because IFN exert an antiproliferative activity, it remains elusive how the clonal expansion of effector T cells can paradoxically occur in the event of an infection when large amounts of IFN are produced. To address this issue, we have studied the effects of type I IFN in an in vitro differentiation model of human primary CD4(+) T cells. We found that IFN-alpha treatment of resting naive T cells delayed their entry into the cell cycle after TCR triggering. Conversely, the ongoing expansion of effector T cells was not inhibited by the presence of IFN. Moreover, activated T cells showed a significantly reduced induction of IFN-sensitive genes, as compared with naive precursors, and this decline occurred independently of subset-specific polarization. The residual type I IFN response measured in activated T cells was found sufficient to inhibit replication of the vesicular stomatitis virus. Our data suggest that the activation of T lymphocytes includes regulatory processes that restrain the transcriptional response to IFN and allow the proliferation of effector cells in the presence of this cytokine.

Antiviral Agents↗
PubMed2003

Inventing a sex-specific gene: a conserved role of DMRT1 in teleost fishes plus a recent duplication in the medaka Oryzias latipes resulted in DMY.

DMY, the first sex-determining gene to be described in a nonmammal vertebrate was recently characterized in the medaka fish (Oryzias latipes). It is homologous to DMRT1, a conserved gene of the sex determination cascade in vertebrates. We have checked the near complete genomes of two other percomorph fishes, Tetraodon nigroviridis and Takifugu rubripes, for supplementary homologs of DMRT1 and DMY. We also compared the new gene, DMY, to its homolog DMRT1 from all available vertebrates. Finally, we found evidence for sex-specific expression and alternative splicing of the homolog from T. nigroviridis. Our results show that DMY is a recent duplicate of DMRT1 in the medaka. Its role in sex determination was not acquired through an acceleration of evolutionary rates, but by translocation to the Y chromosome and possibly changes at key positions.

Alternative Splicing↗
PubMed2003

Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2'-O-ribose methylation of a conserved rRNA position in yeast.

In yeast, guide snoRNAs have been assigned to 51 of the 55 rRNA ribose methylation sites. LSU-Um2918 is one of the four remaining positions. This residue is highly conserved and located in the peptidyl transferase center of the ribosome. The equivalent position on the E. coli 23S rRNA is methylated by FtsJ/RrmJ which has three yeast homologs: Spb1, involved in biogenesis of LSU; Trm7, a tRNA methyltransferase; and Mrm2, a mitochondrial 21S rRNA methyltransferase. We demonstrate that a point mutation in the Ado-Met binding site of Spb1p affects cell growth but does not abolish methylation of U2918. When this mutation is combined with disruption of snR52 (a snoRNA C/D), cell growth is severely impaired and U2918 is no longer methylated. In vitro, Spb1p is able to methylate U2918 on 60S subunits. Our results reveal the importance of this methylation for which two mechanisms coexist: a site-specific methyltransferase (Spb1p) and a snoRNA-dependent mechanism.

Methylation↗
PubMed2002

Selective expression of type I IFN genes in human dendritic cells infected with Mycobacterium tuberculosis.

Type I IFN regulates different aspects of the immune response, inducing a cell-mediated immunity. We have recently shown that the infection of dendritic cells (DC) with Mycobacterium tuberculosis (Mtb) induces IFN-alpha. In this work we have monitored a rapid induction of IFN-beta followed by the delayed production of the IFN-alpha1 and/or -alpha13 subtypes. The Mtb infection rapidly activates the NF-kappaB complex and stimulates the phosphorylation of IFN regulatory factor (IRF)-3, events known to induce IFN-beta expression in viral infection. In turn, the autocrine production of IFN-beta induces the IFN-stimulated genes that contain binding sites for activated STATs in their promoters. Among the IFN-stimulated genes induced in DC through STAT activation are IRF-1 and IRF-7. The expression of IRF-1 appears to be dependent on the sequential activation of NF-kappaB and STAT-1. Once expressed, IRF-1 may further stimulate the transcription of IFN-beta. Induction of IRF-7 is also regulated at the transcriptional level through the binding of phosphorylated STAT-1 and STAT-2, forming the IFN-stimulated gene factor-3 complex. In turn, the IRF-1 and IRF-7 expression appears to be required for the delayed induction of the IFN-alpha1/13 genes. Although correlative, our results strongly support the existence of a cascade of molecular events in Mtb-infected DC. Upon infection, constitutively expressed NF-kappaB and IRF-3 are activated and likely contribute to the rapid IFN-beta expression. In turn, IFN-beta-induced IRF-1 and IRF-7 may cooperate toward induction of IFN-alpha1/13 if infection persists and these factors are activated.

Cells, Cultured↗