RESUMEN
The splicing of the microtubule-associated protein Tau is regulated during development and is found to be deregulated in a growing number of pathological conditions such as myotonic dystrophy type I (DM1), in which a reduced number of isoforms is expressed in the adult brain. DM1 is caused by a dynamic and unstable CTG repeat expansion in the DMPK gene, resulting in an RNA bearing long CUG repeats (n>50) that accumulates in nuclear foci and sequesters CUG-binding splicing factors of the muscle blind-like (MBNL) family, involved in the splicing of Tau pre-mRNA among others. However, the precise mechanism leading to Tau mis-splicing and the role of MBNL splicing factors in this process are poorly understood. We therefore used new Tau minigenes that we developed for this purpose to determine how MBNL1 and MBNL2 interact to regulate Tau exon 2 splicing. We demonstrate that an intronic region 250 nucleotides downstream of Tau exon 2 contains cis-regulatory splicing enhancers that are sensitive to MBNL and that bind directly to MBNL1. Both MBNL1 and MBNL2 act as enhancers of Tau exon 2 inclusion. Intriguingly, the interaction of MBNL1 and MBNL2 is required to fully reverse the mis-splicing of Tau exon 2 induced by the trans-dominant effect of long CUG repeats, similar to the DM1 condition. In conclusion, both MBNL1 and MBNL2 are involved in the regulation of Tau exon 2 splicing and the mis-splicing of Tau in DM1 is due to the combined inactivation of both.
Asunto(s)
Exones , Distrofia Miotónica/genética , Proteínas de Unión al ARN/fisiología , Elementos de Respuesta , Proteínas tau/genética , Secuencia de Bases , Línea Celular Tumoral , Humanos , Datos de Secuencia Molecular , Empalme del ARNRESUMEN
Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.
Asunto(s)
Mitocondrias/genética , Proteínas Mitocondriales/biosíntesis , Nucleoproteínas/fisiología , Biosíntesis de Proteínas , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/fisiología , Línea Celular Tumoral , ADN Mitocondrial/metabolismo , Células HEK293 , Humanos , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/fisiología , Proteínas Nucleares/fisiología , Prohibitinas , ARN/análisis , ARN/aislamiento & purificación , ARN Mensajero/análisis , ARN Mitocondrial , Proteínas Represoras/fisiología , Ribosomas/metabolismoRESUMEN
The bacterial homologue of C4orf14, YqeH, has been linked to assembly of the small ribosomal subunit. Here, recombinant C4orf14 isolated from human cells, co-purified with the small, 28S subunit of the mitochondrial ribosome and the endogenous protein co-fractionated with the 28S subunit in sucrose gradients. Gene silencing of C4orf14 specifically affected components of the small subunit, leading to decreased protein synthesis in the organelle. The GTPase of C4orf14 was critical to its interaction with the 28S subunit, as was GTP. Therefore, we propose that C4orf14, with bound GTP, binds to components of the 28S subunit facilitating its assembly, and GTP hydrolysis acts as the release mechanism. C4orf14 was also found to be associated with human mitochondrial nucleoids, and C4orf14 gene silencing caused mitochondrial DNA depletion. In vitro C4orf14 is capable of binding to DNA. The association of C4orf14 with mitochondrial translation factors and the mitochondrial nucleoid suggests that the 28S subunit is assembled at the mitochondrial nucleoid, enabling the direct transfer of messenger RNA from the nucleoid to the ribosome in the organelle.
Asunto(s)
ADN Mitocondrial/metabolismo , GTP Fosfohidrolasas/metabolismo , Mitocondrias/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Línea Celular Tumoral , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/aislamiento & purificación , Guanosina Trifosfato/metabolismo , Células HEK293 , HumanosRESUMEN
Mitochondrial DNA maintenance and segregation are dependent on the actin cytoskeleton in budding yeast. We found two cytoskeletal proteins among six proteins tightly associated with rat liver mitochondrial DNA: non-muscle myosin heavy chain IIA and ß-actin. In human cells, transient gene silencing of MYH9 (encoding non-muscle myosin heavy chain IIA), or the closely related MYH10 gene (encoding non-muscle myosin heavy chain IIB), altered the topology and increased the copy number of mitochondrial DNA; and the latter effect was enhanced when both genes were targeted simultaneously. In contrast, genetic ablation of non-muscle myosin IIB was associated with a 60% decrease in mitochondrial DNA copy number in mouse embryonic fibroblasts, compared to control cells. Gene silencing of ß-actin also affected mitochondrial DNA copy number and organization. Protease-protection experiments and iodixanol gradient analysis suggest some ß-actin and non-muscle myosin heavy chain IIA reside within human mitochondria and confirm that they are associated with mitochondrial DNA. Collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance.
Asunto(s)
Actinas/fisiología , ADN Mitocondrial/metabolismo , Cadenas Pesadas de Miosina/fisiología , Miosina Tipo IIA no Muscular/fisiología , Miosina Tipo IIB no Muscular/fisiología , Actinas/análisis , Actinas/antagonistas & inhibidores , Animales , Células Cultivadas , ADN Mitocondrial/química , ADN Mitocondrial/aislamiento & purificación , Silenciador del Gen , Humanos , Ratones , Mitocondrias/química , Mitocondrias/ultraestructura , Proteínas Mitocondriales/aislamiento & purificación , Cadenas Pesadas de Miosina/antagonistas & inhibidores , Miosina Tipo IIA no Muscular/análisis , Miosina Tipo IIA no Muscular/antagonistas & inhibidores , Miosina Tipo IIB no Muscular/antagonistas & inhibidores , RatasRESUMEN
The accessory subunit of mitochondrial DNA polymerase gamma, POLGbeta, functions as a processivity factor in vitro. Here we show POLGbeta has additional roles in mitochondrial DNA metabolism. Mitochondrial DNA is arranged in nucleoprotein complexes, or nucleoids, which often contain multiple copies of the mitochondrial genome. Gene-silencing of POLGbeta increased nucleoid numbers, whereas over-expression of POLGbeta reduced the number and increased the size of mitochondrial nucleoids. Both increased and decreased expression of POLGbeta altered nucleoid structure and precipitated a marked decrease in 7S DNA molecules, which form short displacement-loops on mitochondrial DNA. Recombinant POLGbeta preferentially bound to plasmids with a short displacement-loop, in contrast to POLGalpha. These findings support the view that the mitochondrial D-loop acts as a protein recruitment centre, and suggest POLGbeta is a key factor in the organization of mitochondrial DNA in multigenomic nucleoprotein complexes.
Asunto(s)
ADN Mitocondrial/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Subunidades de Proteína/metabolismo , Línea Celular Tumoral , ADN Polimerasa gamma , ADN Mitocondrial/análisis , ADN Mitocondrial/química , ADN Polimerasa Dirigida por ADN/genética , Humanos , Mitocondrias/enzimología , Mitocondrias/ultraestructura , Inhibidores de la Síntesis del Ácido Nucleico , Nucleoproteínas/metabolismo , Plásmidos/química , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Interferencia de ARNRESUMEN
Recombination intermediates containing four-way (Holliday) junctions are generated during DNA repair and replication in many systems, including yeast mitochondrial DNA (mtDNA). In contrast, convincing evidence for recombination in mammalian mtDNA is lacking. We have used two-dimensional agarose-gel electrophoresis to analyse non-linear forms of mtDNA in human heart muscle. Replication intermediates from both the coupled and strand-asynchronous mtDNA replication pathways were detected. An additional class of non-linear molecules, with the electrophoretic properties of four-way junctions, was also prominent. These molecules were insensitive to topoisomerase I or RNase H, but were diminished by branch migration or RuvC treatment. Junctional molecules were detected in all regions of the mitochondrial genome, were found in myocardial DNA from young and old adults, but were present at lower levels in skeletal muscle and placenta. We suggest that they could represent intermediates of mtDNA repair, given their prevalence in the oxyradical-rich environment of heart muscle mitochondria.
Asunto(s)
ADN Mitocondrial/genética , Miocardio/metabolismo , Recombinación Genética , Southern Blotting , ADN/metabolismo , Reparación del ADN , ADN Mitocondrial/biosíntesis , Electroforesis en Gel de Agar , Humanos , Músculo Esquelético/metabolismo , Placenta/metabolismoRESUMEN
X-linked Emery-Dreifuss muscular dystrophy is usually caused by absence of the nuclear membrane protein, emerin, due to nonsense mutations or deletions, but a few missense mutations also exist. A pathogenic g993t mutation causes a Q133H change in the nuclear targeting region of emerin, but it may also reduce emerin levels by affecting mRNA splicing. We have introduced the g993t mutation by in vitro mutagenesis and studied the effect of Q133H on nuclear targeting by transfection of COS-7 cells. No qualitative or quantitative differences in nuclear targeting were observed between normal and mutant emerin. Quantitative BIAcore analysis showed no significant change in lamin A binding to emerin when the mutation was present. We conclude that Q133 is not essential for nuclear targeting of emerin or its interaction with lamin A. Reduced emerin levels due to altered splicing or defective interaction with an unidentified binding partner remain possible pathogenic mechanisms.
Asunto(s)
Proteínas de la Membrana/genética , Distrofia Muscular de Emery-Dreifuss/genética , Mutación Puntual , Timopoyetinas/genética , Técnicas Biosensibles , Lamina Tipo A , Laminas , Proteínas de la Membrana/metabolismo , Distrofia Muscular de Emery-Dreifuss/etiología , Proteínas Nucleares/metabolismo , Unión Proteica , Proteínas Recombinantes/metabolismo , Timopoyetinas/metabolismoRESUMEN
The 2,160,837-base pair genome sequence of an isolate of Streptococcus pneumoniae, a Gram-positive pathogen that causes pneumonia, bacteremia, meningitis, and otitis media, contains 2236 predicted coding regions; of these, 1440 (64%) were assigned a biological role. Approximately 5% of the genome is composed of insertion sequences that may contribute to genome rearrangements through uptake of foreign DNA. Extracellular enzyme systems for the metabolism of polysaccharides and hexosamines provide a substantial source of carbon and nitrogen for S. pneumoniae and also damage host tissues and facilitate colonization. A motif identified within the signal peptide of proteins is potentially involved in targeting these proteins to the cell surface of low-guanine/cytosine (GC) Gram-positive species. Several surface-exposed proteins that may serve as potential vaccine candidates were identified. Comparative genome hybridization with DNA arrays revealed strain differences in S. pneumoniae that could contribute to differences in virulence and antigenicity.
Asunto(s)
Genoma Bacteriano , Análisis de Secuencia de ADN , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidad , Antígenos Bacterianos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Vacunas Bacterianas , Composición de Base , Metabolismo de los Hidratos de Carbono , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromosomas Bacterianos/genética , Biología Computacional , Elementos Transponibles de ADN , ADN Bacteriano/química , ADN Bacteriano/genética , Duplicación de Gen , Genes Bacterianos , Hexosaminas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Recombinación Genética , Secuencias Repetitivas de Ácidos Nucleicos , Especificidad de la Especie , Streptococcus pneumoniae/inmunología , Streptococcus pneumoniae/metabolismo , Virulencia , Operón de ARNrRESUMEN
Most pathogenic missense mutations in the lamin A/C gene identified so far cause autosomal-dominant dilated cardiomyopathy and/or Emery-Dreifuss muscular dystrophy. A few specific mutations, however, cause a disease with remarkably different clinical features: FPLD, or familial partial lipodystrophy (Dunnigan-type), which mainly affects adipose tissue. We have prepared lamin A with a known FPLD mutation (R482Q) by in vitro mutagenesis. Nuclear targeting of lamin A in transfected COS cells, human skeletal muscle cells or mouse adipocyte cell cultures (pre- and post-differentiation) was not detectably affected by the mutation. Quantitative in vitro measurements of lamin A interaction with emerin using a biosensor also showed no effect of the mutation. The results show that the loss of function of R482 in lamin A/C in FPLD does not involve loss of ability to form a nuclear lamina or to interact with the nuclear membrane protein, emerin.
Asunto(s)
Adipocitos/metabolismo , Núcleo Celular/metabolismo , Lipodistrofia/genética , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Nucleares/genética , Timopoyetinas/metabolismo , Animales , Secuencia de Bases , Células COS , Cardiomiopatía Dilatada/genética , Cartilla de ADN , Lamina Tipo A , Laminas , Distrofias Musculares/genética , Proteínas Nucleares/metabolismoRESUMEN
An essential component of functional genomics studies is the sequence of DNA expressed in tissues of interest. To provide a resource of bovine-specific expressed sequence data and facilitate this powerful approach in cattle research, four normalized cDNA libraries were produced and arrayed for high-throughput sequencing. The libraries were made with RNA pooled from multiple tissues to increase efficiency of normalization and maximize the number of independent genes for which sequence data were obtained. Target tissues included those with highest likelihood to have impact on production parameters of animal health, growth, reproductive efficiency, and carcass merit. Success of normalization and inter- and intralibrary redundancy were assessed by collecting 6000-23,000 sequences from each of the libraries (68,520 total sequences deposited in GenBank). Sequence comparison and assembly of these sequences was performed in combination with 56,500 other bovine EST sequences present in the GenBank dbEST database to construct a cattle Gene Index (available from The Institute for Genomic Research at http://www.tigr.org/tdb/tgi.shtml). The 124,381 bovine ESTs present in GenBank at the time of the analysis form 16,740 assemblies that are listed and annotated on the Web site. Analysis of individual library sequence data indicates that the pooled-tissue approach was highly effective in preparing libraries for efficient deep sequencing.
Asunto(s)
Biblioteca de Genes , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Animales , Bovinos , Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Femenino , Feto , Perfilación de la Expresión Génica/métodos , Especificidad de Órganos/genética , EmbarazoRESUMEN
While genome sequencing projects are advancing rapidly, EST sequencing and analysis remains a primary research tool for the identification and categorization of gene sequences in a wide variety of species and an important resource for annotation of genomic sequence. The TIGR Gene Indices (http://www.tigr.org/tdb/tgi. shtml) are a collection of species-specific databases that use a highly refined protocol to analyze EST sequences in an attempt to identify the genes represented by that data and to provide additional information regarding those genes. Gene Indices are constructed by first clustering, then assembling EST and annotated gene sequences from GenBank for the targeted species. This process produces a set of unique, high-fidelity virtual transcripts, or Tentative Consensus (TC) sequences. The TC sequences can be used to provide putative genes with functional annotation, to link the transcripts to mapping and genomic sequence data, to provide links between orthologous and paralogous genes and as a resource for comparative sequence analysis.
Asunto(s)
Bases de Datos Factuales , Etiquetas de Secuencia Expresada , Animales , Secuencia de Bases , Genes/genética , Humanos , Internet , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Mutations in human mitochondrial DNA are a well recognized cause of disease. A mutation at nucleotide position 8993 of human mitochondrial DNA, located within the gene for ATP synthase subunit 6, is associated with the neurological muscle weakness, ataxia, and retinitis pigmentosa (NARP) syndrome. To enable analysis of this mutation in control nuclear backgrounds, two different cell lines were transformed with mitochondria carrying NARP mutant mitochondrial DNA. Transformant cell lines had decreased ATP synthesis capacity, and many also had abnormally high levels of two ATP synthase sub-complexes, one of which was F(1)-ATPase. A combination of metabolic labeling and immunoblotting experiments indicated that assembly of ATP synthase was slowed and that the assembled holoenzyme was unstable in cells carrying NARP mutant mitochondrial DNA compared with control cells. These findings indicate that altered assembly and stability of ATP synthase are underlying molecular defects associated with the NARP mutation in subunit 6 of ATP synthase, yet intrinsic enzyme activity is also compromised.
Asunto(s)
ADN Mitocondrial/genética , Mutación , ATPasas de Translocación de Protón/genética , Adenosina Trifosfato/biosíntesis , Ataxia/genética , Humanos , Metionina/metabolismo , Debilidad Muscular/genética , Subunidades de Proteína , ATPasas de Translocación de Protón/química , Retinitis Pigmentosa/genéticaRESUMEN
Sublimons, originally identified in plant mitochondria, are defined as rearranged mtDNA molecules present at very low levels. We have analysed the primary structures of sublimons found in human cells and tissues and estimated their abundance. Each tissue of a given individual contains a wide range of different sublimons and the most abundant species differ between tissues in a substantially systematic manner. Sublimons are undetectable in rho(0) cells, indicating that they are bona fide derivatives of mtDNA. They are most prominent in post-mitotic tissue subject to oxidative stress. Rearrangement break-points, often defined by short direct repeats, are scattered, but hotspot regions are clearly identifiable, notably near the end of the D-loop. The region between the replication origins is therefore frequently eliminated. One other hotspot region is located adjacent to a known site of protein binding, suggesting that recombination may be facilitated by protein-protein interactions. For a given primary rearrangement, both deleted and partially duplicated species can be detected. Although each sublimon is typically present at a low level, at most a few copies per cell, sublimon abundance in a given tissue can vary over three orders of magnitude between healthy individuals. Collectively, therefore, they can represent a non-negligible fraction of total mtDNA. Their structures are very similar to those of the rearranged molecules found in pathological states, such as adPEO and MNGIE; therefore, we propose that, as in plants, human mtDNA sublimons represent a pool of variant molecules that can become amplified under pathological conditions, thus contributing to cellular dysfunction.
Asunto(s)
ADN Mitocondrial/genética , Mitocondrias/genética , Mitocondrias/patología , Recombinación Genética/genética , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/genética , Secuencia de Bases , Rotura Cromosómica/genética , Clonación Molecular , ADN Mitocondrial/química , ADN Mitocondrial/metabolismo , Electroforesis en Gel de Agar , Femenino , Fluorescencia , Dosificación de Gen , Duplicación de Gen , Humanos , Masculino , Persona de Mediana Edad , Mutagénesis/genética , Miocardio/metabolismo , Miocardio/patología , Conformación de Ácido Nucleico , Especificidad de Órganos , Estrés Oxidativo/genética , Reacción en Cadena de la Polimerasa , Eliminación de Secuencia/genética , Células Tumorales CultivadasRESUMEN
We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.
Asunto(s)
Electroforesis en Gel de Agar/métodos , Complejo Piruvato Deshidrogenasa/aislamiento & purificación , Humanos , Complejo Piruvato Deshidrogenasa/químicaRESUMEN
The vast body of Expressed Sequence Tag (EST) data in the public databases provide an important resource for comparative and functional genomics studies and an invaluable tool for the annotation of genomic sequences. We have developed a rigorous protocol for reconstructing the sequences of transcribed genes from EST and gene sequence fragments. A key element in developing this protocol has been the evaluation of a number of sequence assembly programs to determine which most faithfully reproduce transcript sequences from EST data. The TIGR Gene Indices constructed using this protocol for human, mouse, rat and a variety of other plant and animal models have demonstrated their utility in a variety of applications and are freely available to the scientific research community.
Asunto(s)
Etiquetas de Secuencia Expresada , Análisis de Secuencia de ADN/métodos , Algoritmos , Animales , Secuencia de Consenso , Bases de Datos Factuales , Humanos , Familia de Multigenes , RatasRESUMEN
Mutations of mitochondrial DNA are a significant cause of neuromuscular disease. Pathological mutant mitochondrial DNA has been studied in control nuclear backgrounds. These experiments entailed transfer of patient-derived mitochondria to rho(0) cells that lack mtDNA. A limitation of these studies has been the fact that the control nuclear backgrounds were unrelated to the affected tissues of patients. Therefore a rhabdomyosarcoma cell line that has 'muscle-like' properties was tested to determine whether it could be depleted of mtDNA. A human rhabdomyosarcoma cell line was treated with the DNA intercalating dye ethidium bromide (3, 8-diamino-5-ethyl-6-phenylphenanthridinium bromide) for 45 days. The treatment induced complete and permanent loss of mitochondrial DNA (rho(0)) in the rhabdomyosarcoma cells, as mtDNA remained undetectable after 8 months of growth in medium without drug. Crucially, the rhabdomyosarcoma rho(0) cells retained the ability to differentiate into myotubes with expression of muscle specific isoenzymes. The rhabdomyosarcoma rho(0) cell line provides a model system for studying pathological mutant mtDNA in cells that more closely resemble human muscle than the hitherto available human rho(0) cell lines.
Asunto(s)
ADN Mitocondrial/genética , Rabdomiosarcoma/genética , Rabdomiosarcoma/patología , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Creatina Quinasa/biosíntesis , ADN Mitocondrial/efectos de los fármacos , Etidio/farmacología , Humanos , Rabdomiosarcoma/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Although sequencing of the human genome will soon be completed, gene identification and annotation remains a challenge. Early estimates suggested that there might be 60,000-100,000 (ref. 1) human genes, but recent analyses of the available data from EST sequencing projects have estimated as few as 45,000 (ref. 2) or as many as 140, 000 (ref. 3) distinct genes. The Chromosome 22 Sequencing Consortium estimated a minimum of 45,000 genes based on their annotation of the complete chromosome, although their data suggests there may be additional genes. The nearly 2,000,000 human ESTs in dbEST provide an important resource for gene identification and genome annotation, but these single-pass sequences must be carefully analysed to remove contaminating sequences, including those from genomic DNA, spurious transcription, and vector and bacterial sequences. We have developed a highly refined and rigorously tested protocol for cleaning, clustering and assembling EST sequences to produce high-fidelity consensus sequences for the represented genes (F.L. et al., manuscript submitted) and used this to create the TIGR Gene Indices-databases of expressed genes for human, mouse, rat and other species (http://www.tigr.org/tdb/tgi.html). Using highly refined and tested algorithms for EST analysis, we have arrived at two independent estimates indicating the human genome contains approximately 120,000 genes.
Asunto(s)
Etiquetas de Secuencia Expresada , Genes , Genoma Humano , Algoritmos , Cromosomas Humanos Par 22/genética , Biología Computacional , Secuencia de Consenso/genética , Bases de Datos Factuales , Humanos , Internet , Mapeo Físico de Cromosoma , Reproducibilidad de los Resultados , Programas InformáticosRESUMEN
The np 3243 MELAS mtDNA mutation in tRNA(leu(UUR))has been variously proposed as a loss-of-function or as a gain-of-function mutation, based on apparently contradictory studies in cultured cell lines. A new report describing the molecular effects of the mutation in vivo now mirrors this variability. This should prompt a more systematic re-investigation of cells carrying the mutation, in order to separate primary from secondary and pathogenic from compensatory effects, all of which may contribute to disease phenotype. Nuclear genetic and developmental background, mitochondrial haplotype, and epigenetic effects may all influence the pathological outcome. Defects in both base-modification and aminoacylation of the mutant tRNA could play critical roles.