RESUMEN
Trichinella spiralis muscle stage larvae (mL1) produce excretory-secreted products (ESPs), a complex mixture of protein, which are believed to be important for establishing or maintaining an infection niche within skeletal muscle and the intestine. Studies of both whole ESPs and individual cloned proteins have shown that some ESPs are potent immunogens capable of eliciting protective immune responses. Here we describe two novel proteins, Secreted from Muscle stage Larvae SML-4 and SML-5 which are 15 kDa and 12 kDa respectively. The genes encoding these proteins are highly conserved within the Trichinellids, are constituents of mL1 ESP and localized in the parasite stichosome. While SML-5 is only expressed in mL1 and early stages of adult nematode development, SML-4 is a tyvosylated glycoprotein also produced by adult nematodes, indicating it may have a function in the enteral phase of the infection. Vaccination with these proteins resulted in an impaired establishment of adult stages and consequently a reduction in the burden of mL1 in BALB/c mice. This suggests that both proteins may be important for establishment of parasite infection of the intestine and are prophylactic vaccine candidates.
Asunto(s)
Anticuerpos Antihelmínticos/inmunología , Antígenos Helmínticos/inmunología , Proteínas del Helminto/inmunología , Vacunas Antiprotozoos/inmunología , Trichinella spiralis/inmunología , Triquinelosis/prevención & control , Animales , Femenino , Larva/inmunología , Ratones , Ratones Endogámicos BALB C , Músculos/parasitología , Ratas , Ratas Sprague-Dawley , Células TH1/inmunología , Células Th2/inmunología , Triquinelosis/inmunología , Vacunación , Vacunas Sintéticas/inmunologíaRESUMEN
OBJECTIVE: HLA-B27 is associated with the inflammatory spondyloarthritides (SpA), although subtypes HLA-B*27:06 and HLA-B*27:09 are not. These subtypes differ from the HLA-B*27:05 disease-associated allele primarily at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerization of HLA-B27 during assembly has been implicated in disease onset. The purpose of this study was to investigate the factors that influence differences in dimerization between disease-associated and non-disease-associated HLA-B27 alleles. METHODS: HLA-B*27:05 and mutants resembling the HLA-B*27:06 and 09 subtypes were expressed in the rat C58 T cell line, the human CEM T cell line and its calnexin-deficient variant CEM.NKR. Immunoprecipitation, pulse-chase experiments, flow cytometry, and immunoblotting were performed to study the assembly kinetics, heavy-chain dimerization, and chaperone associations. RESULTS: By expressing HLA-B*27:05, 06-like, and 09 alleles on a restrictive rat transporter associated with antigen processing background, we demonstrate that a tyrosine expressed at p116, either alone or together with an aspartic acid residue at p114, inhibited HLA-B27 dimerization and increased the assembly rate. F-pocket residues altered the associations with chaperones of the early major histocompatibility complex class I folding pathway. Calnexin was demonstrated to participate in endoplasmic reticulum (ER) stress-mediated degradation of dimers, whereas the oxidoreductase ERp57 does not appear to influence dimerization. CONCLUSION: Residues within the F pocket of the peptide-binding groove, which differ between disease-associated and non-disease-associated HLA-B27 subtypes, can influence the assembly process and heavy-chain dimerization, events which have been linked to the initiation of disease pathogenesis.
Asunto(s)
Antígeno HLA-B27/clasificación , Antígeno HLA-B27/genética , Chaperonas Moleculares/fisiología , Pliegue de Proteína , Multimerización de Proteína , Animales , Línea Celular , RatasRESUMEN
Recent studies have suggested the potential of probiotic organisms to be adapted for the synthesis and delivery of oral therapeutics. The probiotic yeast Saccharomyces boulardii would be especially well suited for this purpose due to its ability, in contrast to probiotic prokaryotes, to perform eukaryotic post translational modifications. This probiotic yeast thus has the potential to express a broad array of therapeutic proteins. Currently, however, use of wild type (WT) S. boulardii relies on antibiotic resistance for the selection of transformed yeast. Here we report the creation of auxotrophic mutant strains of S. boulardii that can be selected without antibiotics and demonstrate that these yeast can express functional recombinant protein even when recovered from gastrointestinal immune tissues in mice. A UV mutagenesis approach was employed to generate three uracil auxotrophic S. boulardii mutants that show a low rate of reversion to wild type growth. These mutants can express recombinant protein and are resistant in vitro to low pH, bile acid salts, and anaerobic conditions. Critically, oral gavage experiments using C57BL/6 mice demonstrate that mutant S. boulardii survive and are taken up into gastrointestinal immune tissues on a similar level as WT S. boulardii. Mutant yeast recovered from gastrointestinal immune tissues furthermore retain expression of functional recombinant protein. These data show that auxotrophic mutant S. boulardii can safely express recombinant protein without antibiotic selection and can deliver recombinant protein to gastrointestinal immune tissues. These auxotrophic mutants of S. boulardii pave the way for future experiments to test the ability of S. boulardii to deliver therapeutics and mediate protection against gastrointestinal disorders.
Asunto(s)
Tracto Gastrointestinal/microbiología , Probióticos/administración & dosificación , Proteínas Recombinantes/metabolismo , Saccharomyces/genética , Animales , Tracto Gastrointestinal/efectos de los fármacos , RatonesRESUMEN
OBJECTIVE: HLA-B27 forms misfolded heavy chain dimers, which may predispose individuals to inflammatory arthritis by inducing endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). This study was undertaken to define the role of the UPR-induced ER-associated degradation (ERAD) pathway in the disposal of HLA-B27 dimeric conformers. METHODS: HeLa cell lines expressing only 2 copies of a carboxy-terminally Sv5-tagged HLA-B27 were generated. The ER stress-induced protein ER degradation-enhancing α-mannosidase-like protein 1 (EDEM1) was overexpressed by transfection, and dimer levels were monitored by immunoblotting. EDEM1, the UPR-associated transcription factor X-box binding protein 1 (XBP-1), the E3 ubiquitin ligase hydroxymethylglutaryl-coenzyme A reductase degradation 1 (HRD1), and the degradation-associated proteins derlin 1 and derlin 2 were inhibited using either short hairpin RNA or dominant-negative mutants. The UPR-associated ERAD of HLA-B27 was confirmed using ER stress-inducing pharamacologic agents in kinetic and pulse chase assays. RESULTS: We demonstrated that UPR-induced machinery can target HLA-B27 dimers and that dimer formation can be controlled by alterations to expression levels of components of the UPR-induced ERAD pathway. HLA-B27 dimers and misfolded major histocompatibility complex class I monomeric molecules bound to EDEM1 were detected, and overexpression of EDEM1 led to inhibition of HLA-B27 dimer formation. EDEM1 inhibition resulted in up-regulation of HLA-B27 dimers, while UPR-induced ERAD of dimers was prevented in the absence of EDEM1. HLA-B27 dimer formation was also enhanced in the absence of XBP-1, HRD1, and derlins 1 and 2. CONCLUSION: The present findings indicate that the UPR ERAD pathway can dispose of HLA-B27 dimers, thus presenting a potential novel therapeutic target for modulation of HLA-B27-associated inflammatory disease.
Asunto(s)
Degradación Asociada con el Retículo Endoplásmico/fisiología , Retículo Endoplásmico/fisiología , Antígeno HLA-B27/fisiología , Proteínas de la Membrana/fisiología , Pliegue de Proteína , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/efectos de los fármacos , Proteínas de Unión al ADN/fisiología , Células HeLa , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Factores de Transcripción del Factor Regulador X , Transducción de Señal/fisiología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/efectos de los fármacos , Factores de Transcripción/fisiología , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Ubiquitina-Proteína Ligasas/fisiología , Proteína 1 de Unión a la X-BoxRESUMEN
Major histocompatibility complex (MHC) class I molecules function to present pathogen-derived peptides to cytotoxic T cells or act as ligands for Natural Killer cells, thus alerting the immune system to the presence of invading pathogens. Furthermore MHC class I molecules can be strongly associated with autoimmune diseases. Therefore understanding not only the biosynthesis and the degradation pathways of MHC class I molecules has become important in determining their role in pathogen and autoimmune-related diseases. Here we describe how using epitope-tagged MHC class I molecules can aid in the analysis of MHC class I molecule biosynthesis and degradation and also complement studies using conventional conformationally specific antibodies. Coupled together with pharmacological manipulation which can target both biosynthetic and degradative pathways, this offers a powerful tool in analyzing MHC class I molecules.
Asunto(s)
Antígenos de Histocompatibilidad Clase I/biosíntesis , Antígenos de Histocompatibilidad Clase I/metabolismo , Proteolisis , Alelos , Electroforesis en Gel de Poliacrilamida , Epítopos/inmunología , Citometría de Flujo , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Immunoblotting , Inmunoprecipitación , Proteolisis/efectos de los fármacosRESUMEN
AIMS: The human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies (SpAs). The unusual biochemistry of HLA-B27 has been proposed to participate in disease development, especially the enhanced ability of HLA-B27 to form several heavy chain-dimer populations. HLA-B27 possesses three unpaired cysteine (C) residues at position 67, 308, and 325, in addition to the four conserved cysteine residues at p101, 164, 203, and 259. C67 was proposed to participate in dimer formation of recombinant HLA-B27 protein and in vivo heavy chain-dimers. However, the structurally conserved C164 was demonstrated to participate in endoplasmic reticulum (ER) resident heavy chain-dimer formation. We therefore wanted to determine whether these aggregates involve cysteines other than C164 and the basis for the difference between the observed heavy chain-dimer species. RESULTS: We determined that C164 and C101 can form distinct dimer structures and that the heterogenous nature of heavy chain-dimer species is due to differences in both redox status and conformation. Different HLA-B27 dimer populations can be found in physiologically relevant cell types derived from HLA-B27-positive patients with inflammatory arthritis. In addition, HLA-B27 dimer formation can be correlated with cellular stress induction. INNOVATION: The use of both mutagenesis and manipulating cellular redox environments demonstrates that HLA-B27 dimerization requires both specific cysteine?cysteine interactions and conformations with differing redox states. CONCLUSION: HLA-B27 heavy chain-dimerization is a complex process and these findings provide an insight into HLA-B27 misfolding and a potential contribution to inflammatory disease development.
Asunto(s)
Cisteína/metabolismo , Antígeno HLA-B27/química , Factor de Transcripción Activador 6/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Cisteína/genética , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Antígeno HLA-B27/genética , Antígeno HLA-B27/metabolismo , Humanos , Peso Molecular , Oxidación-Reducción , Multimerización de Proteína/fisiología , RatasRESUMEN
The major histocompatibility complex class I molecule human leukocyte antigen (HLA)-B27 is strongly associated with a group of inflammatory arthritic disorders known as the spondyloarthropathies. Many autoimmune diseases exhibit associations with major histocompatibility complex molecules encoded within the class II locus with defined immune responses either mediated by T or B-lymphocytes. Despite the association being known for over 30 years, no defined immune response and target autoantigens have been characterized for the spondyloarthropathies. Thus, the mechanism and role of HLA-B27 in disease pathogenesis remains undetermined. One hypothesis that has recently received much attention has focused around the enhanced propensity for HLA-B27 to misfold and the increased tendency of the heavy chain to dimerize. The misfolding of HLA-B27 has been associated with its redox status and this is postulated to be involved in disease development. Here we discuss the impact of the redox status on HLA-B27 biosynthesis and function.
Asunto(s)
Antígeno HLA-B27/metabolismo , Deficiencias en la Proteostasis/metabolismo , Espondiloartropatías/metabolismo , Cisteína/química , Disulfuros/química , Antígeno HLA-B27/química , Antígenos de Histocompatibilidad Clase II/inmunología , Prueba de Histocompatibilidad , Humanos , Linfocitos/inmunología , Oxidación-Reducción , Estrés Oxidativo/inmunología , Pliegue de Proteína , Multimerización de Proteína/inmunología , Microglobulina beta-2/metabolismoRESUMEN
The association between HLA-B27 and the group of autoimmune inflammatory arthritic diseases, the spondyloarthropathies (SpAs) which include ankylosing spondylitis (AS) and Reactive Arthritis (ReA), has been well established and remains the strongest association between any HLA molecule and autoimmune disease. The mechanism behind this striking association remains elusive; however animal model and biochemical data suggest that HLA-B27 misfolding may be key to understanding its association with the SpAs. Recent investigations have focused on the unusual biochemical structures of HLA-B27 and their potential role in SpA pathogenesis. Here we discuss how these unusual biochemical structures may participate in cellular events leading to chronic inflammation and thus disease progression.
RESUMEN
Proteins secreted by Trichinella spiralis have a potential role in remodelling host skeletal muscle. However, whilst many parasite-secreted proteins have been identified, it has rarely been demonstrated that these are secreted into the nurse cell. Using an informatics-based analysis, we have searched the T. spiralis expressed sequence tag (EST) datasets for cDNAs encoding potential secreted proteins. Here we describe the characterisation of three of the top candidates isolated from our analysis, termed secreted from muscle stage larvae (SML)-1, -2 and -3. All three proteins were demonstrated to be secreted by muscle stage larvae, and immunohistochemical analysis established that SML-1 and -2 are secreted into developing nurse cells. We also show that SML-2 is processed from a precursor into smaller peptides by a metalloprotease contained within T. spiralis-secreted products. With the identification of these and other secreted proteins, we now have molecules to test in functional assays designed to dissect molecular features of the developing nurse cell.
Asunto(s)
Proteínas del Helminto/metabolismo , Músculo Esquelético/parasitología , Trichinella spiralis/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting/métodos , Biología Computacional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Etiquetas de Secuencia Expresada , Femenino , Perfilación de la Expresión Génica/métodos , Proteínas del Helminto/genética , Larva/metabolismo , Larva/ultraestructura , Ratones , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Procesamiento Proteico-Postraduccional , Ratas , Ratas Sprague-Dawley , Trichinella spiralis/ultraestructuraRESUMEN
Parasitic nematodes that cause elephantiasis and river blindness threaten hundreds of millions of people in the developing world. We have sequenced the approximately 90 megabase (Mb) genome of the human filarial parasite Brugia malayi and predict approximately 11,500 protein coding genes in 71 Mb of robustly assembled sequence. Comparative analysis with the free-living, model nematode Caenorhabditis elegans revealed that, despite these genes having maintained little conservation of local synteny during approximately 350 million years of evolution, they largely remain in linkage on chromosomal units. More than 100 conserved operons were identified. Analysis of the predicted proteome provides evidence for adaptations of B. malayi to niches in its human and vector hosts and insights into the molecular basis of a mutualistic relationship with its Wolbachia endosymbiont. These findings offer a foundation for rational drug design.
Asunto(s)
Brugia Malayi/genética , Genoma de los Helmintos , Animales , Brugia Malayi/fisiología , Caenorhabditis/genética , Drosophila melanogaster/genética , Resistencia a Medicamentos/genética , Filariasis/parasitología , Humanos , Datos de Secuencia MolecularRESUMEN
The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.
Asunto(s)
Evolución Biológica , Secuencia Conservada , Nematodos/genética , Operón/genética , Trans-Empalme/genética , Animales , Emparejamiento Base , Secuencia de Bases , Genes de Helminto , Datos de Secuencia Molecular , Proteoma/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/metabolismo , ARN Lider Empalmado/genética , ARN Lider Empalmado/metabolismo , Ribosomas/metabolismoRESUMEN
A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis.
Asunto(s)
Proteínas del Helminto/fisiología , Onchocerca volvulus/química , Onchocerca volvulus/crecimiento & desarrollo , Inhibidores de Serina Proteinasa/fisiología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Catepsina G , Catepsinas/antagonistas & inhibidores , Quimotripsina/antagonistas & inhibidores , Regulación del Desarrollo de la Expresión Génica , Proteínas del Helminto/química , Proteínas del Helminto/genética , Larva/química , Datos de Secuencia Molecular , Elastasa Pancreática/antagonistas & inhibidores , Filogenia , Señales de Clasificación de Proteína , ARN Bicatenario/genética , ARN Mensajero/análisis , Proteínas Recombinantes/farmacología , Homología de Secuencia , Serina Endopeptidasas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Transcripción Genética/efectos de los fármacosRESUMEN
Cysteine proteinases are involved in a variety of important biological processes and have been implicated in molting and tissue remodeling in free living and parasitic nematodes. We show that in the lymphatic filarial nematode Brugia pahangi molting of third-stage larvae (L3) to fourth-stage larvae is dependent on the activity of a cathepsin L-like cysteine protease (CPL), which can be detected in the excretory/secretory (ES) products of molting L3. Directed cloning of a cysteine protease gene in B. pahangi and analysis of the expressed sequence tag (EST) and genomic sequences of the closely related human lymphatic filarial nematode Brugia malayi have identified a family of CPLs. One group of these enzymes, Bm-cpl-1, -4, -5 and Bp-cpl-4, is highly expressed in the B. malayi and B. pahangi infective L3 larvae. Immunolocalization indicates that the corresponding enzymes are synthesized and stored in granules of the glandular esophagus of L3 and released during the molting process. Functional analysis of these genes in Brugia and closely related CPL genes identified in the filarial nematode Onchocerca volvulus and the free living model nematode Caenorhabditis elegans indicate that these genes are also involved in cuticle and eggshell remodeling.
Asunto(s)
Brugia Malayi/enzimología , Brugia Malayi/genética , Brugia pahangi/enzimología , Brugia pahangi/genética , Catepsinas/genética , Genes de Helminto , Familia de Multigenes , Animales , Secuencia de Bases , Brugia Malayi/crecimiento & desarrollo , Brugia pahangi/efectos de los fármacos , Brugia pahangi/crecimiento & desarrollo , Catepsina L , Clonación Molecular , Cisteína Endopeptidasas/genética , Inhibidores de Cisteína Proteinasa/farmacología , ADN de Helmintos/genética , Cáscara de Huevo/crecimiento & desarrollo , Etiquetas de Secuencia Expresada , Larva/efectos de los fármacos , Larva/enzimología , Larva/crecimiento & desarrollo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Muda/efectos de los fármacos , Muda/fisiología , Filogenia , Especificidad de la EspecieRESUMEN
Immune responses to the intracellular Wolbachia bacteria of filarial nematodes are thought to contribute to the pathologic process of filarial infection. Here, we compare antibody responses of subjects living in an area where lymphatic filariasis is endemic with antibody responses elicited in a murine model of filarial infection, to provide evidence that the infective larval stage (L3), not adult nematodes, are the primary inducer of responses against Wolbachia. In human subjects, antibody responses to Brugia malayi Wolbachia surface protein (WSP) are most often correlated with antibody responses to the L3 stage of B. malayi. Analysis of anti-WSP responses induced in mice by different stages of the rodent filariae Litomosoides sigmodontis shows that the strongest anti-WSP response is elicited by the L3 stage. Although adult filarial nematode death may play a role in the generation of an anti-WSP response, it is the L3 stage that is the major source of immunogenic material, and incoming L3 provide a continual boosting of the anti-WSP response. Significant exposure to the endosymbiotic bacteria may occur earlier in nematode infection than previously thought, and the level of exposure to infective insect bites may be a key determinant of disease progression.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/inmunología , Brugia Malayi/fisiología , Filariasis/sangre , Wolbachia/inmunología , Animales , Anticuerpos Antihelmínticos/sangre , Brugia Malayi/inmunología , Brugia Malayi/microbiología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Filariasis/diagnóstico , Filarioidea/microbiología , Filarioidea/fisiología , Humanos , Larva/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Especificidad de la Especie , SimbiosisRESUMEN
BACKGROUND: Expressed sequence tags (ESTs) are single pass reads from randomly selected cDNA clones. They provide a highly cost-effective method to access and identify expressed genes. However, they are often prone to sequencing errors and typically define incomplete transcripts. To increase the amount of information obtainable from ESTs and reduce sequencing errors, it is necessary to cluster ESTs into groups sharing significant sequence similarity. RESULTS: As part of our ongoing EST programs investigating 'orphan' genomes, we have developed a clustering algorithm, CLOBB (Cluster on the basis of BLAST similarity) to identify and cluster ESTs. CLOBB may be used incrementally, preserving original cluster designations. It tracks cluster-specific events such as merging, identifies 'superclusters' of related clusters and avoids the expansion of chimeric clusters. Based on the Perl scripting language, CLOBB is highly portable relying only on a local installation of NCBI's freely available BLAST executable and can be usefully applied to > 95 % of the current EST datasets. Analysis of the Danio rerio EST dataset demonstrates that CLOBB compares favourably with two less portable systems, UniGene and TIGR Gene Indices. CONCLUSIONS: CLOBB provides a highly portable EST clustering solution and is freely downloaded from: http://www.nematodes.org/CLOBB
Asunto(s)
Biología Computacional/estadística & datos numéricos , Etiquetas de Secuencia Expresada , Alineación de Secuencia/métodos , Programas Informáticos/estadística & datos numéricos , Algoritmos , Animales , Secuencia de Bases , Benchmarking/estadística & datos numéricos , Caenorhabditis elegans/genética , Análisis por Conglomerados , Biología Computacional/métodos , Humanos , Lenguajes de Programación , Alineación de Secuencia/estadística & datos numéricos , Pez Cebra/genéticaRESUMEN
Cysteine proteases play critical biological roles in both intracellular and extracellular processes. We characterized Ce-cpl-1, a Caenorhabditis elegans cathepsin L-like cysteine protease. RNA interference with Ce-cpl-1 activity resulted in embryonic lethality and a transient delayed growth of larvae to egg producing adults, suggesting an essential role for cpl-1 during embryogenesis, and most likely during post-embryonic development. Cpl-1 gene (Ce-cpl-1:lacZ) is widely expressed in the intestine and hypodermal cells of transgenic worms, while the fusion protein (Ce-CPL-1::GFP) was expressed in the hypodermis, pharynx, and gonad. The CPL-1 native protein accumulates in early to late stage embryos and becomes highly concentrated in gut cells during late embryonic development. CPL-1 is also present near the periphery of the eggshell as well as in the cuticle of larval stages suggesting that it may function not only in embryogenesis but also in further development of the worm. Although the precise role of Ce-CPL-1 during embryogenesis is not yet clear it could be involved in the processing of nutrients responsible for synthesis and/or in the degradation of eggshell. Moreover, an increase in the cpl-1 mRNA is seen in the intermolt period approximately 4 h prior to each molt. During this process Ce-CPL-1 may act as a proteolytic enzyme in the processing/degradation of cuticular or other proteins. Similar localization of a related cathepsin L in the filarial nematode Onchocerca volvulus, eggshell and cuticle, suggests that some of the Ce-CPL-1 function during development may be conserved in other parasitic nematodes.