RESUMEN
In the course of our search for anti-dormant Mycobacterial substances, nybomycin (1) was re-discovered from the culture broth of a marine-derived Streptomyces sp. on the bioassay-guided separation. Compound 1 showed anti-microbial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG with the MIC of 1.0µg/mL under both actively growing aerobic conditions and dormancy inducing hypoxic conditions. Compound 1 is also effective to Mycobacterium tuberculosis including the clinically isolated strains. The mechanistic analysis indicated that 1 bound to DNA and induces a unique morphological change to mycobacterial bacilli leading the bacterial cell death.
Asunto(s)
Antituberculosos/farmacología , ADN Bacteriano/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Streptomyces/química , Antituberculosos/química , Antituberculosos/aislamiento & purificación , Organismos Acuáticos , Técnicas de Cultivo de Célula , Cósmidos/química , Cósmidos/metabolismo , ADN Bacteriano/química , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/genética , Mycobacterium bovis/metabolismo , Mycobacterium bovis/ultraestructura , Mycobacterium smegmatis/efectos de los fármacos , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/ultraestructura , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestructura , Quinolonas/química , Quinolonas/aislamiento & purificación , Quinolonas/farmacología , Streptomyces/metabolismoRESUMEN
Varicella zoster virus (VZV) ORF25 is a 156 amino acid protein belonging to the approximately 40 core proteins that are conserved throughout the Herpesviridae. By analogy to its functional orthologue UL33 in Herpes simplex virus 1 (HSV-1), ORF25 is thought to be a component of the terminase complex. To investigate how cleavage and encapsidation of viral DNA links to the nuclear egress of mature capsids in VZV, we tested 10 VZV proteins that are predicted to be involved in either of the two processes for protein interactions against each other using three independent protein-protein interaction (PPI) detection systems: the yeast-two-hybrid (Y2H) system, a luminescence based MBP pull-down interaction screening assay (LuMPIS), and a bioluminescence resonance energy transfer (BRET) assay. A set of 20 interactions was consistently detected by at least 2 methods and resulted in a dense interaction network between proteins associated in encapsidation and nuclear egress. The results indicate that the terminase complex in VZV consists of ORF25, ORF30, and ORF45/42 and support a model in which both processes are closely linked to each other. Consistent with its role as a central hub for protein interactions, ORF25 is shown to be essential for VZV replication.
Asunto(s)
Genes Virales , Herpesvirus Humano 3/química , Mapeo de Interacción de Proteínas/métodos , Proteínas Virales/química , Animales , Secuencia de Bases , Transferencia de Energía por Resonancia de Bioluminiscencia , Cápside/química , Núcleo Celular/química , Clonación Molecular/métodos , Cósmidos/química , Cósmidos/genética , ADN Viral/química , ADN Viral/genética , Escherichia coli/química , Escherichia coli/metabolismo , Eliminación de Gen , Células HeLa , Herpesvirus Humano 3/genética , Herpesvirus Humano 3/fisiología , Humanos , Sueros Inmunes/química , Sistemas de Lectura Abierta , Estructura Terciaria de Proteína , Conejos , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/genética , Replicación ViralRESUMEN
Past studies showed that Leishmania spp. promastigotes exhibit differential sensitivity to complement mediated lysis (CML) during development in vitro and in vivo. Leishmania chagasi promastigotes in cultures during logarithmic and stationary growth phases are CML-sensitive or CML-resistant when exposed to human serum, respectively, but only in cultures recently initiated with parasites from infected animals; serially passaged cultures become constitutively CML-sensitive regardless of growth phase. Building on these observations, a genetic screen was conducted to identify novel complement resistance factors of L. chagasi. A cosmid library containing genomic DNA was transfected into a promastigote line previously subjected to >50 serial passages. Selection with human serum for CML resistance yielded 12 transfectant clones. Cosmids isolated from 7 of these clones conferred CML resistance when transfected into an independent, high-passage promastigote culture; at 12% human serum, the mean survival of transfectants was 37% (+/- 11.6%), and that of control transfectants was about 1%. Inserts within the 7 cosmids were unique. Determination of the complete DNA sequence for 1 cosmid indicated that its 32-kilobase insert was 89% identical (overall) to a 31-kilobase region of Leishmania major chromosome 36, which is predicted to encode 6 genes, all of which encode hypothetical proteins.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , ADN Protozoario/análisis , Prueba de Complementación Genética , Leishmania infantum/genética , Leishmania infantum/inmunología , Animales , Cósmidos/química , Cósmidos/genética , Cricetinae , Vectores Genéticos , Humanos , Mesocricetus , Mapeo Restrictivo , Pase Seriado , TransfecciónRESUMEN
While current sequencing efforts consider the detection of alpha satellite repeats as logical end points for map construction, detailed maps of most pericentromeric regions are lacking to confirm this hypothesis. Here we identify the different alpha satellite families present at the pericentromeric region of chromosome 12. The order, size and location of these repeats is established using radiation hybrid analysis, pulsed field gel analysis and FISH and the maps are integrated with current sequence information. For the different classes of alpha satellites present at the chromosome 12 centromere the paralogs in the human genome were mapped by FISH. Unique sequences flanking the alpha satellite repeats were identified, some of which are not represented in the current draft sequence. This mapping effort localises the different alpha satellite repeats within the pericentromeric region and anchors them in the current maps. The novel sequences identified may serve as the end point for the ongoing sequencing efforts.
Asunto(s)
Centrómero , Cromosomas Humanos Par 12 , Southern Blotting , Aberraciones Cromosómicas , Cromosomas Humanos Par 12/ultraestructura , Cósmidos/análisis , Cósmidos/química , Cósmidos/aislamiento & purificación , Electroforesis en Gel de Campo Pulsado , Humanos , Hibridación Fluorescente in Situ , Repeticiones de Microsatélite , Mapeo de Híbrido por Radiación , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.
Asunto(s)
Adenosilhomocisteinasa/genética , Clonación Molecular , Trypanosoma cruzi/enzimología , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Secuencia de Consenso , Secuencia Conservada , Cósmidos/química , Cósmidos/genética , Electroforesis en Gel de Poliacrilamida , Regulación Enzimológica de la Expresión Génica , Biblioteca de Genes , Humanos , Leishmania donovani/enzimología , Leishmania donovani/genética , Datos de Secuencia Molecular , Peso Molecular , NAD/metabolismo , Ratas , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Trypanosoma cruzi/genéticaRESUMEN
Within the 80 kb aflatoxin pathway gene cluster characterized earlier, and between adhA and norA genes, we have identified an estA gene encoding an esterase from wild type strain Aspergillus parasiticus SRRC 143. The 1,500 bp genomic DNA and 945 bp cDNA sequences were determined for estA. Outside of the aflatoxin pathway gene cluster, an additional copy of the estA gene (named estA2) was also cloned from the same A. parasiticus strain. Comparison of the estA and estA2 sequences showed 9 substitutions within the 314 amino acid residues of their gene products, and no apparent defect was identified in the estA2. The estA gene is a homolog of the stcI gene identified in A. nidulans involved in the biosynthesis of sterigmatocystin and dihydro-sterigmatocystin for the conversion of versiconal hemiacetal acetate to versiconal. Reverse-transcriptase polymerase chain reaction (RT-PCR) experiments demonstrated that the estA is constitutively expressed. And only this estA gene, which is located within the aflatoxin pathway gene cluster, is expressed; no expression of the estA2 gene was detected under both aflatoxin conducive and non-conducive conditions. Possible reasons for the preferential expression of the estA over the estA2 gene have been discussed.
Asunto(s)
Aspergillus/enzimología , Hidrolasas de Éster Carboxílico/genética , Proteínas Fúngicas , Aflatoxina B1/biosíntesis , Aflatoxina B1/genética , Secuencia de Aminoácidos , Aspergillus/genética , Secuencia de Bases , Hidrolasas de Éster Carboxílico/biosíntesis , Cósmidos/química , Cósmidos/genética , ADN de Hongos/genética , ADN de Hongos/metabolismo , Expresión Génica , Datos de Secuencia Molecular , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de AminoácidoRESUMEN
The low molecular mass polypeptide (LMP2, LMP7, and MECL-1) genes code for beta-type subunits of the proteasome, a multimeric complex that degrades proteins into peptides as part of the MHC class I-mediated Ag-presenting pathway. These gene products are up-regulated in response to infection by IFN-gamma and replace the corresponding constitutively expressed subunits (X, Y, and Z) during the immune response. In humans, the LMP2 and LMP7 genes both reside within the class II region of the MHC (6p21.3), while MECL-1 is located at 16q22.1. In the present study, we have identified all three IFN-gamma-regulated beta-type proteasome subunits in Fugu, which are present as a cluster within the Fugu MHC class I region. We show that in this species, LMP7, LMP2, and MECL-1 are linked. Also within this cluster is an LMP2-like subunit (which seems specific to all teleosts tested to date) and a closely linked LMP7 pseudogene, indicating that within Fugu and potentially other teleosts, there has been an additional regional duplication involving these genes.
Asunto(s)
Cisteína Endopeptidasas/química , Cisteína Endopeptidasas/genética , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Familia de Multigenes , Animales , Cósmidos/química , Cósmidos/aislamiento & purificación , Exones , Peces Venenosos , Genes , Humanos , Intrones , Complejo Mayor de Histocompatibilidad/genética , Filogenia , Complejo de la Endopetidasa Proteasomal , Proteínas/química , Proteínas/genética , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/genéticaRESUMEN
One hundred and fourteen kilobase pairs (kb) of contiguous genomic sequence have been determined immediately distal to the his5 genetic marker located about 0.9 Mb from the centromere on the long arm of Schizosaccharomyces pombe chromosome 2. The sequence is contained in overlapping cosmid clones c16H5, c12D12, c24C6 and c19G7, of which 20 kb are identical to previously reported sequence from clone c21H7. The remaining 93 781 bp of sequence contains 10 known genes (cdc14, cdm1, cps1, gpa1, msh2, pck2, rip1, rps30-2, sad1 and ubl1), 32 open reading frames (ORFs) capable of coding for proteins of at least 100 amino acid residues in length, one 5S rRNA gene, one tRNA(Pro) gene, one lone Tf1-type long terminal repeat (LTR) and one lone Tf2-type LTR. There is a density of one protein-coding gene per 2.2 kb and 22 of the 42 ORFs (52%) incorporate one or more introns. Twenty-one of the novel ORFs show sequence similarities which suggest functions of their products, including a cyclin C, a MADS box transcription factor, mad2-like protein, telomere binding protein, topoisomerase II-associated protein, ATP-dependent DEAH box RNA helicase, G10 protein, ubiquitin-activating e1-like enzyme, nucleoporin, prolyl-tRNA synthetase, peptidylprolyl isomerase, delta-1-pyrroline-5-carboxylate dehydrogenase, protein transport protein, coatomer epsilon, TCP-1 chaperonin, beta-subunit of 6-phosphofructokinase, aminodeoxychorismate lyase, a phosphate transport protein and a thioredoxin.
Asunto(s)
Cromosomas Fúngicos/genética , Schizosaccharomyces/genética , Secuencia de Bases , Centrómero/química , Cromosomas Fúngicos/química , Cósmidos/química , Marcadores Genéticos , Intrones/genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Ribosómico/química , ARN Ribosómico/genética , Schizosaccharomyces/química , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Secuencias Repetidas Terminales/genéticaRESUMEN
From a cosmid clone of Streptomyces ambofaciens containing the dnaA and gyrAB genes, a 2.7-kb self-replicating DNA fragment containing the chromosome replication origin oriC was isolated. This cosmid was previously maped physically to a region near the middle of the 8-Mb linear chromosomal DNA. A pulsed-field gel electrophoresis time-course analysis revealed that sequences flanking oriC were overrepresented relative to the rest of the chromosomal DNA during rapid growth, indicating that this origin is active. In addition, the terminal regions of the chromosomal DNA showed a slight overrepresentation at the onset of stationary phase.
Asunto(s)
Replicación del ADN/genética , ADN Bacteriano/genética , Origen de Réplica/genética , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Clonación Molecular , Cósmidos/química , ADN Bacteriano/química , Densitometría , Electroforesis en Gel de Campo Pulsado , Electroporación , Dosificación de Gen , Biblioteca de Genes , Datos de Secuencia Molecular , Mapeo Restrictivo , Factores de TiempoRESUMEN
Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110,000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.
Asunto(s)
Pollos/genética , Mapeo Cromosómico/métodos , Cósmidos/química , Animales , Mapeo Cromosómico/veterinaria , ADN/química , Femenino , Biblioteca de Genes , Hibridación Fluorescente in Situ/veterinaria , MetafaseRESUMEN
Mutations in keratin genes account for a number of inherited keratodermas in humans. The groups of basic and acidic keratin genes are clustered on human chromosomes 12 and 17, respectively. The present authors have assigned the two putative keratin gene clusters to canine chromosomes using canine cosmid clones. Successful fluorescence in situ hybridization mapped the putative cluster of canine acidic genes to dog chromosome 20 and the putative cluster of basic keratin genes to a small autosome not yet included in the partial canine standard karyotype.
Asunto(s)
Mapeo Cromosómico/veterinaria , Perros/genética , Queratinas/genética , Familia de Multigenes , Animales , Bandeo Cromosómico/veterinaria , Cósmidos/química , Enfermedades de los Perros/genética , Humanos , Hibridación Fluorescente in Situ/veterinaria , Queratinas/química , Queratodermia Palmoplantar/genética , Queratodermia Palmoplantar/veterinaria , Masculino , Hibridación de Ácido Nucleico , Reacción en Cadena de la Polimerasa/veterinaria , Análisis de Secuencia de ADNAsunto(s)
Repeticiones de Dinucleótido/genética , Visón/genética , Polimorfismo Genético/genética , Alelos , Animales , Mapeo Cromosómico/veterinaria , Cósmidos/química , Cartilla de ADN/química , Marcadores Genéticos , Hibridación Fluorescente in Situ/veterinaria , Masculino , Reacción en Cadena de la Polimerasa/veterinariaAsunto(s)
Bovinos/genética , Factores de Crecimiento Nervioso/genética , Proteínas Tirosina Quinasas Receptoras/genética , Receptores de Factor de Crecimiento Nervioso/genética , Receptores de Neurotransmisores/genética , Animales , Secuencia de Bases , Mapeo Cromosómico/veterinaria , Secuencia Conservada , Cósmidos/química , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ/veterinaria , Ratones , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Proteínas Tirosina Quinasas Receptoras/química , Receptor de Factor Neurotrófico Ciliar , Receptores de Factor de Crecimiento Nervioso/química , Receptores de Neurotransmisores/química , Análisis de Secuencia de ADN/veterinariaRESUMEN
We previously reported the isolation of alphoid satellite clones from a human genomic library using a DNA immunoprecipitation with centromere protein B (CENP-B). Here, we have characterized the distribution of CENP-B-binding sites on the 3-kb BamHI repeats of the cos2 clone. Using in situ hybridization, this alphoid satellite was located primarily at the centromeric region of chromosome 6. The functional binding sites were mapped by precipitating the restriction fragments with recombinant CENP-B in vitro. One repeat (2B3-11) consisted of 19 copies of alphoid monomer, eight of which possessed the binding sites, while another (2B3-9) consisted of 18 copies of the monomer, seven of which possessed the binding sites. The distribution of the sites was well conserved between them, except for the terminus. A similar analysis with the remaining 6-kb region suggested the presence of a continuous 1-kb region with regular spacing of EcoRI sites and the CENP-B-binding sites. When the nucleotide sequence of 2B3-11 was compared with that of another chromosome 6-specific alphoid repeat (p308) that had been described previously, this 1-kb region was highly conserved between them. The distribution of the CENP-B binding sites and the order of alphoid monomers might define the folding of alphoid repeats in the centromeric region.
Asunto(s)
Autoantígenos , Centrómero , Proteínas Cromosómicas no Histona/fisiología , Cromosomas Humanos Par 6/genética , Secuencia Conservada , Proteínas de Unión al ADN/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuencia de Bases , Sitios de Unión/genética , Sitios de Unión/fisiología , Proteína B del Centrómero , Mapeo Cromosómico , Cósmidos/química , Cósmidos/genética , ADN Satélite/química , ADN Satélite/genética , Humanos , Homología de Secuencia de Ácido NucleicoRESUMEN
We have isolated and characterized Tgmr, a copia-like retrotransposon, linked tightly to the Rps1-k allele that confers race-specific resistance of soybean to the the fungal pathogen Phytophthora sojae. Southern analysis followed by PCR and sequence analyses, using primers based on sequences flanking the insertion site confirmed that the element was inserted in the neighboring region of Rps1-k but not in that of the other four Rps1 alleles. This implies that Tgmr was transposed into the Rps1-k flanking site after the divergence of Rps1 alleles. Southern analysis of a series of diverse soybean cultivars revealed a high level of polymorphism of Tgmr-related sequences. These results indicate that this low copy retroelement family could have been active in the soybean genome in the recent past. Tgmr contains long terminal repeats (LTR) and four non-overlapping open reading frames (ORF), presumably originating from mutations leading to stop codons of a single ORF. The conserved domains for gag, protease, integrase, reverse transcriptase and RNaseH are present in the internal portion of the element. However, the protease, reverse transcriptase and RNaseH of this element are non-functional due to the presence of several stop codons. Possible transactivation of Tgmr and application of this element in insertional mutagenesis for soybean are discussed.
Asunto(s)
Alelos , Genes de Plantas , Ligamiento Genético , Glycine max/genética , Phytophthora/patogenicidad , Retroelementos , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Cósmidos/química , Marcadores Genéticos , Genoma de Planta , Inmunidad Innata , Datos de Secuencia Molecular , Phytophthora/genética , Polimorfismo de Longitud del Fragmento de Restricción , Glycine max/química , Especificidad de la EspecieRESUMEN
We developed a new cycle condition method optimized for direct sequencing LAWRIST16 cosmid clones based on dye terminator and dye primer cycle sequencing chemistries. We report a direct comparison of the two most widely used sequencing polymerase enzymes (AmpliTaq FS and Thermosequenase). The sequencing data obtained is of high quality and we believe this method could be routinely used in large scale sequencing facilities.
Asunto(s)
Cósmidos/química , Cósmidos/genética , Análisis de Secuencia de ADN/métodos , Redes de Comunicación de Computadores , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , Análisis de Secuencia de ADN/instrumentaciónRESUMEN
As a part of the Bacillus subtilis genome sequencing project, we have determined a 25-kb sequence covering the 17 degrees-19 degrees region. This region contains 26 complete open reading frames (ORFs) including the alkA and adaA/B operon, which encode genes for adaptive response to DNA alkylation. A homology search for the newly identified 21 ORFs revealed that 4 of them exhibit a significant similarity to known proteins, e.g., methicillin-resistant Staphylococcus aureus (MRSA) protein homolog, proteins involved in chloramphenicol resistance, glucosamine synthase and an ABC transporter protein. The remaining 17 ORFs did not show any significant sequence similarities to known gene products in the database.
Asunto(s)
Bacillus subtilis/química , Bacillus subtilis/genética , Cromosomas Bacterianos/química , Cromosomas Bacterianos/genética , ADN Bacteriano/química , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cósmidos/química , Sistemas de Lectura Abierta , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The nucleotide sequence of a 45,137-bp segment covering the 19 degrees and 23 degrees region in the 360 degrees map of the Bacillus subtilis genome was determined. This region contained 45 open reading frames (ORFs) including 7 which corresponded to the products of genes with known functions that had been previously sequenced. The known genes were: glpT and glpQ for glycerol utilization pathway; purT for a part of the purine synthesis pathway; mpr for an extracellular metalloprotease; pss and psd for the parts of the phospholipid synthesis pathway; and gltP for a glutamine transporter. Deduced amino acid sequences of the 22 newly identified ORFs showed significant homologies to known gene products in the database such as a Methicillin-resistant Staphylococcus aureus (MRSA) gene which is related to drug resistance, a two-component response regulator, a series of amino acid permeases, transcriptional regulators, beta-lactamase, the phosphotransferase system (PTS) enzyme II for sugar uptake, and the eukaryotic ECA39 gene which is associated with cancer and apoptosis, etc. The remaining 16 ORFs did not show any significant sequence similarities to known gene products in the database.