RESUMEN
Withstanding 3.5 billion years of genetic drift, the canonical genetic code remains such a fundamental foundation for the complexity of life that it is highly conserved across all three phylogenetic domains. Genome engineering technologies are now making it possible to rationally change the genetic code, offering resistance to viruses, genetic isolation from horizontal gene transfer, and prevention of environmental escape by genetically modified organisms. We discuss the biochemical, genetic, and technological challenges that must be overcome in order to engineer the genetic code.
Asunto(s)
Marcación de Gen/métodos , Código Genético , Ingeniería Metabólica/métodosRESUMEN
Aminoacyl-tRNA (transfer RNA) synthetases are essential components of the cellular translation machinery as they provide the ribosome with aminoacyl-tRNAs. Aminoacyl-tRNA synthesis is generally well understood. However, the mechanism of Cys-tRNACys formation in three methanogenic archaea ( Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus and Methanopyrus kandleri) is still unknown, since no recognizable gene for a canonical cysteinyl-tRNA synthetase could be identified in the genome sequences of these organisms. Here we review the different routes recently proposed for Cys-tRNACys formation and discuss its possible link with cysteine biosynthesis in these methanogenic archaea.
Asunto(s)
Archaea/metabolismo , Cisteína/biosíntesis , Genes Arqueales , Aminoacil-ARN de Transferencia/química , Sitios de Unión , Cisteína/química , Metano/química , Modelos Biológicos , Modelos Moleculares , Filogenia , Biosíntesis de Proteínas , Conformación Proteica , ARN/químicaRESUMEN
The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.
Asunto(s)
Proteínas Arqueales/metabolismo , Genoma Arqueal , Hidrógeno/metabolismo , Metano/metabolismo , Methanococcus/genética , Análisis de Secuencia de ADN , Proteínas Arqueales/genética , Methanococcus/metabolismo , Datos de Secuencia Molecular , ProteomaRESUMEN
To study the mitosis-specific phosphorylation of caldesmon (CaD), we generated a mutant of the C-terminal fragment (amino acids 244-538) of human fibroblast CaD (CaD39-6F), as well as a mutant of the full-length CaD (CaD-6F), in which all six potential phosphorylation sites for Cdc2 kinase were abolished. The mitotic CaD39-6F-overexpressing cells required more time to progress from anaphase start to 50% cytokinesis, exhibited larger size, and abnormally formed numerous small blebs. In contrast, overexpression of the wild-type C-terminal fragment of CaD (CaD39) did not result in abnormal bleb formation, but led to larger size and prolonged the time requirement between anaphase start and 50% cytokinesis. Similar abnormal blebs were also observed in the CaD-6F-overexpressing cells. CaD-6F-overexpressing cells did not show larger size but required more time to progress from anaphase start to 50% cytokinesis. These results suggest that mitosis-specific phosphorylation of CaD plays a role in inhibiting bleb formation and that the N-terminal fragment of CaD is required for cell size determination.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , División Celular , Membrana Celular/fisiología , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas de Unión a Calmodulina/genética , Tamaño de la Célula , Cricetinae , Fibroblastos , Expresión Génica , Humanos , Mutagénesis Sitio-Dirigida , Fosforilación , Mutación Puntual , Factores de Tiempo , Tropomiosina/análisis , Tropomiosina/aislamiento & purificaciónRESUMEN
Translation is the process by which ribosomes direct protein synthesis using the genetic information contained in messenger RNA (mRNA). Transfer RNAs (tRNAs) are charged with an amino acid and brought to the ribosome, where they are paired with the corresponding trinucleotide codon in mRNA. The amino acid is attached to the nascent polypeptide and the ribosome moves on to the next codon. Thus, the sequential pairing of codons in mRNA with tRNA anticodons determines the order of amino acids in a protein. It is therefore imperative for accurate translation that tRNAs are only coupled to amino acids corresponding to the RNA anticodon. This is mostly, but not exclusively, achieved by the direct attachment of the appropriate amino acid to the 3'-end of the corresponding tRNA by the aminoacyl-tRNA synthetases. To ensure the accurate translation of genetic information, the aminoacyl-tRNA synthetases must display an extremely high level of substrate specificity. Despite this highly conserved function, recent studies arising from the analysis of whole genomes have shown a significant degree of evolutionary diversity in aminoacyl-tRNA synthesis. For example, non-canonical routes have been identified for the synthesis of Asn-tRNA, Cys-tRNA, Gln-tRNA and Lys-tRNA. Characterization of non-canonical aminoacyl-tRNA synthesis has revealed an unexpected level of evolutionary divergence and has also provided new insights into the possible precursors of contemporary aminoacyl-tRNA synthetases.
Asunto(s)
Evolución Molecular , Genómica , Aminoacil-ARN de Transferencia/biosíntesis , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Filogenia , Biosíntesis de Proteínas , ARN Mensajero/genéticaRESUMEN
Post-transcriptional modifications in archaeal RNA are known to be phylogenetically distinct but relatively little is known of tRNA from the Methanococci, a lineage of methanogenic marine euryarchaea that grow over an unusually broad temperature range. Transfer RNAs from Methanococcus vannielii, Methanococcus maripaludis, the thermophile Methanococcus thermolithotrophicus, and hyperthermophiles Methanococcus jannaschii and Methanococcus igneus were studied to determine whether modification patterns reflect the close phylogenetic relationships inferred from small ribosomal subunit RNA sequences, and to examine modification differences associated with temperature of growth. Twenty-four modified nucleosides were characterized, including the complex tricyclic nucleoside wyosine characteristic of position 37 in tRNA(Phe) and known previously only in eukarya, plus two new wye family members of presently unknown structure. The hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, reported previously only in bacterial tRNA at the first position of the anticodon, was identified by liquid chromatography-electrospray ionization mass spectrometry in four of the five organisms. The ribose-methylated nucleosides, 2'-O-methyladenosine, N(2),2'-O-dimethylguanosine and N(2),N(2),2'-O-trimethylguanosine, were found only in hyperthermophile tRNA, consistent with their proposed roles in thermal stabilization of tRNA.
Asunto(s)
Procesamiento Postranscripcional del ARN , ARN de Archaea/metabolismo , ARN de Transferencia/metabolismo , Cromatografía Líquida de Alta Presión , Methanococcales/genética , Methanococcales/metabolismo , Nucleósidos/análisis , Nucleósidos/genética , Nucleótidos/genética , Nucleótidos/metabolismo , Filogenia , ARN de Archaea/genética , ARN de Transferencia/genética , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The methanogenic archaea Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus contain a dual-specificity prolyl-tRNA synthetase (ProCysRS) that accurately forms both prolyl-tRNA (Pro-tRNA) and cysteinyl-tRNA (Cys-tRNA) suitable for in vivo translation. This intriguing enzyme may even perform its dual role in organisms that possess a canonical single-specificity cysteinyl-tRNA synthetase (CysRS), raising the question as to whether this latter aminoacyl-tRNA synthetase is indeed required for cell viability. To test the postulate that all synthetase genes are essential, we disrupted the cysS gene (encoding CysRS) of Methanococcus maripaludis. The knockout strain was viable under normal growth conditions. Biochemical analysis showed that the pure M. maripaludis ProCysRS was capable of forming Cys-tRNA, implying that the dual-specificity enzyme compensates in vivo for the loss of CysRS. The canonical CysRS has a higher affinity for cysteine than ProCysRS, a reason why M. maripaludis may have acquired cysS by a late lateral gene transfer. These data challenge the notion that all twenty aminoacyl-tRNA synthetases are essential for the viability of a cell.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Methanococcus/enzimología , Aminoacil-ARNt Sintetasas/genética , Eliminación de Gen , Genes Arqueales , Methanococcus/genética , Methanococcus/crecimiento & desarrollo , FenotipoRESUMEN
Aminoacyl-tRNA is generally formed by aminoacyl-tRNA synthetases, a family of 20 enzymes essential for accurate protein synthesis. However, most bacteria generate one of the two amide aminoacyl-tRNAs, Asn-tRNA or Gln-tRNA, by transamidation of mischarged Asp-tRNA(Asn) or Glu-tRNA(Gln) catalyzed by a heterotrimeric amidotransferase (encoded by the gatA, gatB, and gatC genes). The Chlamydia trachomatis genome sequence reveals genes for 18 synthetases, whereas those for asparaginyl-tRNA synthetase and glutaminyl-tRNA synthetase are absent. Yet the genome harbors three gat genes in an operon-like arrangement (gatCAB). We reasoned that Chlamydia uses the gatCAB-encoded amidotransferase to generate both Asn-tRNA and Gln-tRNA. C. trachomatis aspartyl-tRNA synthetase and glutamyl-tRNA synthetase were shown to be non-discriminating synthetases that form the misacylated tRNA(Asn) and tRNA(Gln) species. A preparation of pure heterotrimeric recombinant C. trachomatis amidotransferase converted Asp-tRNA(Asn) and Glu-tRNA(Gln) into Asn-tRNA and Gln-tRNA, respectively. The enzyme used glutamine, asparagine, or ammonia as amide donors in the presence of either ATP or GTP. These results suggest that C. trachomatis employs the dual specificity gatCAB-encoded amidotransferase and 18 aminoacyl-tRNA synthetases to create the complete set of 20 aminoacyl-tRNAs.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Chlamydia trachomatis/genética , ARN Bacteriano/biosíntesis , ARN de Transferencia de Asparagina/biosíntesis , ARN de Transferencia de Glutamina/biosíntesis , Aminoacil-ARNt Sintetasas/aislamiento & purificación , Chlamydia trachomatis/enzimología , Electroforesis en Gel de Poliacrilamida , Genes BacterianosRESUMEN
The genetic code can be interpreted during translation as 21 amino acids and three termination signals. Recent advances at the interface of chemistry and molecular biology are extending the genetic code to allow assignment of new amino acids to existing codons, providing new functional groups for protein synthesis.
Asunto(s)
Biosíntesis de Proteínas , Proteínas/química , Aminoácidos/química , Aminoácidos/genética , Aminoacil-ARNt Sintetasas/genética , Aminoacil-ARNt Sintetasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Código Genético , Proteínas/genéticaRESUMEN
Five histone deacetylase genes (HDA1, RPD3, HOS1, HOS2, and HOS3) have been cloned from Candida albicans and characterized. Sequence analysis and comparison with 17 additional deacetylases resulted in a phylogenetic tree composed of three major groups. Transcription of the deacetylases HDA1 and RPD3 is down-regulated in the opaque phase of the white-opaque transition in strain WO-1. HOS3 is selectively transcribed as a 2.5-kb transcript in the white phase and as a less-abundant 2.3-kb transcript in the opaque phase. HDA1 and RPD3 were independently deleted in strain WO-1, and both switching between the white and opaque phases and the downstream regulation of phase-specific genes were analyzed. Deletion of HDA1 resulted in an increase in the frequency of switching from the white phase to the opaque phase, but had no effect on the frequency of switching from the opaque phase to the white phase. Deletion of RPD3 resulted in an increase in the frequency of switching in both directions. Deletion of HDA1 resulted in reduced white-phase-specific expression of the EFG1 3.2-kb transcript, but had no significant effect on white-phase-specific expression of WH11 or opaque-phase-specific expression of OP4, SAP1, and SAP3. Deletion of RPD3 resulted in reduced opaque-phase-specific expression of OP4, SAP1, and SAP3 and a slight reduction of white-phase-specific expression of WH11 and 3.2-kb EFG1. Deletion of neither HDA1 nor RPD3 affected the high level of white-phase expression and the low level of opaque-phase expression of the MADS box protein gene MCM1, which has been implicated in the regulation of opaque-phase-specific gene expression. In addition, there was no effect on the phase-regulated levels of expression of the other deacetylase genes. These results demonstrate that the two deacetylase genes HDA1 and RPD3 play distinct roles in the suppression of switching, that the two play distinct and selective roles in the regulation of phase-specific genes, and that the deacetylases are in turn regulated by switching.
Asunto(s)
Proteínas Fúngicas/fisiología , Histona Desacetilasas/fisiología , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Proteínas de Plantas , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe , Secuencia de Aminoácidos , Candida albicans/genética , Candida albicans/fisiología , Proteínas Portadoras/genética , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Expresión Génica , Histona Desacetilasas/genética , Proteína 1 de Mantenimiento de Minicromosoma , Datos de Secuencia Molecular , Mutagénesis , Homología de Secuencia de Aminoácido , Factores de Transcripción/genéticaRESUMEN
The gatC, gatA and gatB genes encoding the three subunits of glutamyl-tRNA(Gln) amidotransferase from Acidithiobacillus ferrooxidans, an acidophilic bacterium used in bioleaching of minerals, have been cloned and expressed in Escherichia coli. As in Bacillus subtilis the three gat genes are organized in an operon-like structure in A. ferrooxidans. The heterologously overexpressed enzyme converts Glu-tRNA(Gln) to Gln-tRNA(Gln) and Asp-tRNA(Asn) to Asn-tRNA(Asn). Biochemical analysis revealed that neither glutaminyl-tRNA synthetase nor asparaginyl-tRNA synthetase is present in A. ferrooxidans, but that glutamyl-tRNA synthetase and aspartyl-tRNA synthetase enzymes are present in the organism. These data suggest that the transamidation pathway is responsible for the formation of Gln-tRNA and Asn-tRNA in A. ferrooxidans.
Asunto(s)
Asparagina/genética , Aspartato-ARNt Ligasa , Gammaproteobacteria/enzimología , Glutamina/genética , Transferasas de Grupos Nitrogenados/metabolismo , Clonación Molecular , Codón/genética , Activación Enzimática/fisiología , Escherichia coli/genética , Escherichia coli/metabolismo , Gammaproteobacteria/genética , Transferasas de Grupos Nitrogenados/genética , Biosíntesis de Proteínas/fisiología , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Aminoacil-ARN de Transferencia/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido Nucleico , Especificidad por Sustrato/fisiologíaRESUMEN
Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.
Asunto(s)
Candida albicans/genética , Inhibidores de Histona Desacetilasas , Mutación , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Candida albicans/fisiología , División Celular/efectos de los fármacos , Ácidos Hidroxámicos/farmacología , Fenotipo , Regulación hacia ArribaRESUMEN
The role of tRNA as the adaptor in protein synthesis has held an enduring fascination for molecular biologists. Over four decades of study, taking in numerous milestones in molecular biology, led to what was widely held to be a fairly complete picture of how tRNAs and amino acids are paired prior to protein synthesis. However, recent developments in genomics and structural biology have revealed an unexpected array of new enzymes, pathways and mechanisms involved in aminoacyl-tRNA synthesis. As a more complete picture of aminoacyl-tRNA synthesis now begins to emerge, the high degree of evolutionary diversity in this universal and essential process is becoming clearer.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/biosíntesis , Aminoácidos/química , Aminoácidos/metabolismo , Aminoacil-ARNt Sintetasas/química , Animales , Archaea/genética , Archaea/metabolismo , Bacterias/genética , Bacterias/metabolismo , Evolución Molecular , Aminoacil-ARN de Transferencia/química , Aminoacil-ARN de Transferencia/genéticaRESUMEN
The synthesis of 5-aminolevulinic acid (ALA) is the rate-limiting step for the formation of all plant tetrapyrroles, including chlorophyll and heme, and regulation of ALA synthesis is therefore critical to plant development. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of this pathway and is encoded by a small family of nuclear HEMA genes. Here, we have used transgenic Arabidopsis (Arabidopsis thaliana L. Col) lines expressing chimeric HEMA1 promoter:gusA fusion genes, combined with RNA gel blot analyses, to characterise the light-mediated regulation of the Arabidopsis HEMA1 gene during de-etiolation. HEMA1 was expressed strongly, but not exclusively, in photosynthetic tissues and was shown to be light regulated at the transcriptional level by the phytochrome family of photoreceptors acting in both the far-red high irradiance and low fluence response modes. The HEMA2 gene, which is expressed only in roots of seedlings, was not light regulated. Analysis of truncated HEMA1 promoter constructs demonstrated that a -199/+252 promoter fragment was sufficient to confer full light-responsiveness to gusA expression. This fragment contained GT-1/I-box and CCA-1 binding sites that are implicated as the light-responsive cis elements. Both the full-length and truncated HEMA1 promoters required the presence of intact chloroplasts for full expression, consistent with previous indications that light and plastid factor signals converge to co-ordinately regulate expression of photosynthesis-related nuclear genes. These results provide the most comprehensive analysis to date of the light-regulation of a tetrapyrrole biosynthetic gene and support a direct link between regulation of HEMA1 transcription and chlorophyll accumulation during seedling de-etiolation.
Asunto(s)
Aldehído Oxidorreductasas/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Fitocromo/metabolismo , Plastidios/metabolismo , Aldehído Oxidorreductasas/metabolismo , Ácido Aminolevulínico/metabolismo , Arabidopsis/metabolismo , Northern Blotting , Genes Reporteros , Ácido Glucurónico/metabolismo , Hibridación in Situ , Datos de Secuencia Molecular , Fotoperiodo , Regiones Promotoras Genéticas , ARN de Planta/análisis , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A 2225 bp cDNA, designated RPA1, was isolated from an Oryza sativa cDNA library. Analysis revealed a 1761 bp coding sequence with 15 non-identical repeat units. The ORF encoded the A regulatory subunit of protein phosphatase 2A (PP2A-A) as ascertained by complementation of the yeast tpd3 mutant defective in this gene. The corresponding genomic DNA from a rice genome BAC library revealed that the gene contains eleven introns. The rice genome contains only a single copy of this gene as judged by Southern blot analysis. The PP2A protein is highly conserved in nature; the rice protein shows 88% amino acid identity with its counterparts in Arabidopsis or Nicotiana tabacum.
Asunto(s)
Genes de Plantas/genética , Oryza/genética , Fosfoproteínas Fosfatasas , Proteínas de Plantas/genética , Secuencia de Aminoácidos , ADN/química , ADN/genética , ADN Complementario/química , ADN Complementario/genética , Exones , Prueba de Complementación Genética , Intrones , Datos de Secuencia Molecular , Mutación , Oryza/enzimología , Proteína Fosfatasa 2 , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Bacterial tyrosyl-tRNA synthetases occur in two large subfamilies, TyrRS and TyrRZ, that possess about 25% amino acid identity. Their amino-terminal region, the active site domain, is more conserved (>36% identity). The carboxy-terminal segment of these enzymes includes the tRNA binding domain and contains only few conserved residues. Replacement of three of these residues in Acidithiobacillus ferrooxidans TyrRZ revealed that S356 and K395 play roles in tRNA binding, while H306, a residue at the junction of the catalytic and tRNA binding domains, stabilizes the Tyr-AMP:TyrRZ complex. The replacement data suggest that conserved amino acids in A. ferrooxidans TyrRZ and Bacillus stearothermophilus TyrRS play equivalent roles in enzyme function.
Asunto(s)
Adenosina Monofosfato/metabolismo , Proteínas Bacterianas/metabolismo , Gammaproteobacteria/enzimología , ARN de Transferencia/metabolismo , Tirosina-ARNt Ligasa/metabolismo , Tirosina/metabolismo , Adenosina Monofosfato/análogos & derivados , Proteínas Bacterianas/química , Clonación Molecular , Secuencia Conservada , Dimerización , Escherichia coli/genética , Gammaproteobacteria/genética , Expresión Génica , Prueba de Complementación Genética , Geobacillus stearothermophilus/enzimología , Mutagénesis Sitio-Dirigida , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Tirosina/análogos & derivados , Tirosina-ARNt Ligasa/química , Tirosina-ARNt Ligasa/genéticaRESUMEN
The availability of numerous complete microbial genome sequences has profoundly altered our understanding of a number of fundamental biological processes. For example the enzymes involved in aminoacyl-tRNA (AA-tRNA) synthesis, the key process responsible for the accuracy of protein synthesis, have been found to be highly species-specific. In particular, a number of pathogens contain certain pathways of AA-tRNA synthesis that are unrelated to those found in their mammalian hosts. Since AA-tRNA synthesis is indispensable for cell viability, the discovery of pathogen-specific pathways and enzymes presents novel therapeutic and diagnostic targets. Here we will review recent advances in the elucidation of AA-tRNA synthesis pathways and discuss the possible pharmaceutical exploitation of these discoveries. In particular, the integration of genomic and biochemical approaches to identify novel targets for the treatment of Chlamydial infections and the diagnosis and treatment of Lyme disease will be presented.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Grupo Borrelia Burgdorferi/genética , Chlamydia trachomatis/genética , Genoma Bacteriano , Aminoacil-ARNt Sintetasas/antagonistas & inhibidores , Aminoacil-ARNt Sintetasas/clasificación , Aminoacil-ARNt Sintetasas/metabolismo , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Grupo Borrelia Burgdorferi/enzimología , Grupo Borrelia Burgdorferi/patogenicidad , Infecciones por Chlamydia/tratamiento farmacológico , Chlamydia trachomatis/enzimología , Chlamydia trachomatis/patogenicidad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Enfermedad de Lyme/diagnóstico , Enfermedad de Lyme/tratamiento farmacológico , Proteoma/biosíntesis , Proteoma/efectos de los fármacos , ARN de Transferencia/biosíntesisRESUMEN
A variety of methods have emerged for genetic fingerprinting the infectious fungi. One of the most versatile is Southern blot hybridization with species-specific complex DNA probes that include sequences that identify hypervariable, moderately variable and invariant genomic sequences. These probes assess genetic relatedness at all the necessary levels including identical, highly related but non-identical, moderately related and unrelated. Methods are described for cloning complex probes, characterizing them and verifying their effectiveness at the different levels of resolution. The complex probes that have been developed for Candida albicans, C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis and Aspergillus fumigatus are described and discussed.
Asunto(s)
Candida/genética , Dermatoglifia del ADN/métodos , Sondas de ADN , Aspergillus fumigatus , Southern Blotting , Candida/clasificación , Candida/patogenicidad , Clonación Molecular , ADN de Hongos/genética , Variación Genética , Genoma Fúngico , Humanos , Secuencias Repetitivas de Ácidos Nucleicos , Especificidad de la EspecieRESUMEN
BACKGROUND: The initial stages of phagocytosis and cell motility resemble each other. The extension of a pseudopod at the leading edge of a migratory cell and the formation of a phagocytic cup are actin dependent, and each rely on the plasma membrane adhering to a surface during dynamic extension. RESULTS: A myosin VII null mutant exhibited a drastic loss of adhesion to particles, consistent with the extent of an observed decrease in particle uptake. Additionally, cell-cell adhesion and the adhesion of the leading edge to the substratum during cell migration were defective in the myosin VII null cells. GFP-myosin VII rescued the phagocytosis defect of the null mutant and was distributed in the cytosol and recruited to the cortical cytoskeleton, where it appeared to be enriched at the tips of filopods. It was also localized to phagocytic cups, but only during the initial stages of particle engulfment. During migration, GFP-myosin VII is found at the leading edge of the cell. CONCLUSIONS: Myosin VII plays an important role in mediating the initial binding of cells to substrata, a novel role for an unconventional myosin.