RESUMO
Current therapies fail to cure most metastatic or recurrent bone cancer. We explored the efficacy and the pathways involved in natural killer (NK) cells' elimination of osteosarcoma (OS) cells, including tumor initiating cells (TICs), which are responsible for chemotherapy resistance, recurrence, and metastasis. The expression of ligands for NK cell receptors was studied in primary OS cell lines by flow cytometry. In vitro cytotoxicity of activated and expanded NK (NKAE) cells against OS was tested, and the pathways involved explored by using specific antibody blockade. NKAE cells' ability to target OS TICs was analyzed by flow cytometry and sphere formation assays. Spironolactone (SPIR) was tested for its ability to increase OS cells' susceptibility to NK cell lysis in vitro and in vivo. We found OS cells were susceptible to NKAE cells' lysis both in vivo and in vitro, and this cytolytic activity relied on interaction between NKG2D receptor and NKG2D ligands (NKG2DL). SPIR increased OS cells' susceptibility to lysis by NKAE cells, and could shrink the OS TICs. Our results show NKAE cells target OS cells including the TICs compartment, supporting the use of NK-cell based immunotherapies for OS.
Assuntos
Neoplasias Ósseas/terapia , Proliferação de Células , Imunoterapia Adotiva/métodos , Células Matadoras Ativadas por Linfocina/transplante , Ativação Linfocitária , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Células-Tronco Neoplásicas/metabolismo , Osteossarcoma/terapia , Animais , Antineoplásicos/farmacologia , Neoplasias Ósseas/imunologia , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Comunicação Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Citotoxicidade Imunológica , Humanos , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Ativadas por Linfocina/metabolismo , Ligantes , Camundongos Endogâmicos NOD , Camundongos SCID , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/patologia , Osteossarcoma/imunologia , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transdução de Sinais , Espironolactona/farmacologia , Fatores de TempoRESUMO
Genetic variation in the catechol-O-methyltransferase (COMT) gene may influence the susceptibility to schizophrenia and the response to neuroleptic treatment. The authors tested for an association between a COMT haplotype and schizophrenia-spectrum disorders and for an eventual influence of a specific COMT genotype in the clinical outcome and in the response to treatment. The genotypes for single nucleotide polymorphisms rs737865, rs4633, rs6267, rs4680 (Val 158 Met) and rs165599 were determined in 207 patients with schizophrenia-spectrum disorders and 204 paired controls. Statistical tests for linkage disequilibrium and for case-control differences in haplotype frequencies were performed using log-linear modelling embedded within the expectation-maximization algorithm. P-values based on permutations were calculated using the software UNPHASED, and odds ratios were estimated using the SHEsis platform. The response to neuroleptic treatment was assessed by the Global Assessment of Functioning scale and the severity of psychotic symptoms by the positive and negative syndrome scale (PANSS) scale. The overall disease status was significantly associated with the T-G (Val) diplotype for rs4633-rs4680 (P=0.0049). A significant association was observed between schizophrenia, but not other related disorders, and genotypes GG (Val/Val) for rs4680 and TT for rs4633. Val/Val patients with schizophrenia showed a higher severity of the psychotic symptoms and a worse response to the neuroleptic treatment. COMT genetic variation seems to be involved in the psychotic symptomatology of the schizophrenia-spectrum disorders and specifically in the narrow schizophrenia phenotype. Our results show an influence of the Val 158 Met polymorphism on the severity of psychotic symptoms and on the response to treatment.
Assuntos
Antipsicóticos/uso terapêutico , Catecol O-Metiltransferase/genética , Polimorfismo de Nucleotídeo Único , Esquizofrenia/tratamento farmacológico , Adulto , Estudos de Casos e Controles , Feminino , Frequência do Gene , Predisposição Genética para Doença , Haplótipos , Humanos , Desequilíbrio de Ligação , Masculino , Razão de Chances , Fenótipo , Estudos Prospectivos , Escalas de Graduação Psiquiátrica , Fatores de Risco , Esquizofrenia/enzimologia , Esquizofrenia/genética , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
We have analyzed the regulation and expression of ASPP members, genes implicated in the regulation of the apoptotic function of the TP53 tumor-suppressor gene, in acute lymphoblastic leukemia (ALL). Expression of ASPP1 was significantly reduced in ALL and was dependent on hypermethylation of the ASPP1 gene promoter. Abnormal ASPP1 expression was associated with normal function of the tumor-suppressor gene TP53 in ALL. The analyses of 180 patients with ALL at diagnosis showed that the ASPP1 promoter was hypermethylated in 25% of cases with decreased mRNA expression. Methylation was significantly higher in adult ALL vs childhood ALL (32 vs 17%, P = 0.03) and T-ALL vs B-ALL (50 vs 9%, P = 0.001). Relapse rate (62 vs 44%, P = 0.05) and mortality (59 vs 43%, P = 0.05) were significantly higher in patients with methylated ASPP1. DFS and OS were 32.8 and 33.7% for patients with unmethylated ASPP1 and 6.1 and 9.9% for methylated patients (P < 0.001 y P < 0.02, respectively). On the multivariate analysis, methylation of the ASPP1 gene promoter was an independent poor prognosis factor in ALL patients. Our results demonstrate that decreased expression of ASPP1 in patients with ALL is due to an abnormal methylation of its promoter and is associated with a poor prognosis.
Assuntos
Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Criança , Pré-Escolar , Feminino , Perfilação da Expressão Gênica , Genes p53 , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Leucemia-Linfoma Linfoblástico de Células Precursoras/fisiopatologia , Prognóstico , Regiões Promotoras Genéticas , Recidiva , SobrevidaRESUMO
Human genetic integrity is compromised by the intense industrial activity, which emphasizes the importance to determine an "acceptable" genetic damage level and to carry out routine genotoxicity assays in the populations at risk. Micronuclei are cytoplasmatic bodies of nuclear origin which correspond to genetic material that is not correctly incorporated in the daughter cells in the cellular division; they reflect the existence of chromosomal aberrations and are originated by chromosomal breaks, replication errors followed by cellular division of the DNA and/or exposure to genotoxic agents. There are several factors able to modify the number of micronuclei present in a given cell, among them are age, gender, vitamins, medical treatments, daily exposure to genotoxic agents, etc. The cytogenetic assay for the detection of micronuclei (CBMN: cytokinesis-block micronucleus) is based on the use of a chemical agent, cytochalasin-B, which is able to block cytocinesis but allowing the nuclear division, therefore yielding binucleated and monodivided cells. The micronuclei scoring is performed on 1000 binucleated cells and the starting sample may vary, although most studies are performed on peripheral blood lymphocytes. The micronuclei assay is considered a practical, universally validated and technically feasible protocol which is useful to evaluate the genetic instability induced by genotoxic agents.
Assuntos
Aberrações Cromossômicas , Instabilidade Genômica , Testes para Micronúcleos/métodos , Mutagênicos/toxicidade , Adulto , Fatores Etários , Antioxidantes/farmacologia , Instabilidade Cromossômica , Citocalasina B/farmacologia , Citocinese , Dano ao DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Sensibilidade e Especificidade , Fatores Sexuais , Fumar , Fatores de TempoRESUMO
La integridad genética de la población humana se encuentra comprometida por la gran actividad industrial; por lo que es importante determinar qué se conoce como un nivel aceptable de daño genético y realizar ensayos de genotoxicidad de manera rutinaria en poblaciones de riesgo. Los micronúcleos son cuerpos citoplasmáticos de naturaleza nuclear, se corresponden con material genético no incorporado correctamente a las células hijas durante la división celular, reflejan aberraciones cromosómicas y se originan por roturas cromosómicas, por errores durante la replicación y posterior división celular del ADN y/o por la exposición a agentes genotóxicas. Existen factores capaces de influir o modificar el número de micronúcleos presentes en una célula (edad, género, vitaminas, tratamientos médicos, exposición diaria a agentes genotóxicos, etc.).El ensayo citogenético para la detección de micronúcleos (CBMN: cytokinesis-block micronucleus) se basa en la utilización de un agente químico, denominado citocalasina-B capaz de impedir la citocinesis permitiendo la división nuclear proporcionando a las células un aspecto de células binucleadas monodivididas. El recuento de micronúcleos se realiza sobre 1.000 células binucleadas y la muestra de partida puede variar aunque lo óptimo es el uso de linfocitos aislados de sangre periférica.El ensayo de micronúcleos está considerado como un ensayo práctico, universalmente validado y accesible tecnológicamente, útil para evaluar la inestabilidad genética inducida por agentes genotóxicos
Human genetic integrity is compromised by the intense industrial activity, which emphasizes the importance to determine an acceptable genetic damage level and to carry out routine genotoxicity assays in the populations at risk. ;;Micronuclei are cytoplasmatic bodies of nuclear origin which correspond to genetic material that is not correctly incorporated in the daughter cells in the cellular division; they reflect the existence of chromosomal aberrations and are originated by chromosomal breaks, replication errors followed by cellular division of the DNA and/or exposure to genotoxic agents. There are several factors able to modify the number of micronuclei present in a given cell, among them are age, gender, vitamins, medical treatments, daily exposure to genotoxic agents, etc. ;;The cytogenetic assay for the detection of micronuclei (CBMN: cytokinesis-block micronucleus) is based on the use of a chemical agent, cytochalasin-B, which is able to block cytocinesis but allowing the nuclear division, therefore yielding binucleated and monodivided cells. The micronuclei scoring is performed on 1000 binucleated cells and the starting sample may vary, although most studies are performed on peripheral blood lymphocytes. ;;The micronuclei assay is considered a practical, universally validated and technically feasible protocol which is useful to evaluate the genetic instability induced by genotoxic agents
Assuntos
Adulto , Humanos , Aberrações Cromossômicas , Instabilidade Genômica , Mutagênicos/toxicidade , Testes para Micronúcleos/métodos , Fatores Etários , Antioxidantes/farmacologia , Instabilidade Cromossômica , Citocalasina B/farmacologia , Divisão Celular , Dano ao DNA , Sensibilidade e Especificidade , Fatores Sexuais , Tabagismo , Fatores de TempoRESUMO
Homologs of the XerCD enzymes, which in Escherichia coli have been shown to be responsible for resolving chromosomal multimers prior to chromosome segregation, were identified in the genomes of Staphylococcus aureus and Streptococcus pneumoniae. Phylogenetic and conservation pattern analysis suggests that the S. aureus gene products are orthologs of XerC and D. A S. aureus xerC null mutant displayed in vitro characteristics consistent with the segregation defect reported for E. coli xer mutants, and was found to be attenuated in a murine infection model. Strikingly, the S. aureus xerD gene appears to be absolutely required for viability, and may therefore be the first example of an essential gene of the lambda integrase family. In contrast, phylogenetic and conservation pattern analysis show that the S. pneumoniae gene products are more closely related to phage integrases than to XerCD. S. pneumoniae xer1, 2 and 3 null mutants were each found to be attenuated in a murine infection model, suggesting that they may control processes which affect virulence.
Assuntos
DNA Nucleotidiltransferases/genética , Proteínas de Escherichia coli , Integrases , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Streptococcus pneumoniae/enzimologia , Streptococcus pneumoniae/genética , Alelos , Sequência de Aminoácidos , Animais , Cromossomos Bacterianos/genética , Sequência Conservada , Escherichia coli/enzimologia , Escherichia coli/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Mutação , Filogenia , Infecções Pneumocócicas/etiologia , Recombinases , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Infecções Estafilocócicas/etiologia , Staphylococcus aureus/patogenicidade , Streptococcus pneumoniae/patogenicidade , Virulência/genéticaRESUMO
The tylosin producer Streptomyces fradiae contains four known resistance genes, two of which (tlrA and tlrD) encode methyltransferases that act on ribosomal RNA at a common site. Expression of tlrA is regulated via transcriptional attenuation. A short transcript, only 411 nucleotides long, terminates 27 nucleotides into the methylase-coding sequence in the uninduced state. Induction of tlrA is proposed to involve a ribosome-mediated conformational change within the mRNA leader that allows transcription to continue beyond the attenuation site, resulting in a transcript about 1450 nucleotides long. Transplantation of tlrD and/or tlrA into Streptomyces albus revealed that the induction specificity of tlrA depends upon the state of the ribosomes and is significantly altered in strains also expressing tlrD.
Assuntos
Genes Bacterianos , Metiltransferases/genética , Streptomyces/efeitos dos fármacos , Streptomyces/genética , Tilosina/farmacologia , Sequência de Bases , Sítios de Ligação , Sequência de Carboidratos , Mapeamento Cromossômico , Primers do DNA/genética , DNA Bacteriano/genética , Resistência Microbiana a Medicamentos/genética , Metiltransferases/metabolismo , Dados de Sequência Molecular , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Streptomyces/metabolismo , Transcrição Gênica , Tilosina/química , Tilosina/metabolismoRESUMO
Nucleotide sequence analysis of a 5,000-bp region of the bialaphos antibiotic production (bap) gene cluster defined five open reading frames (ORFs) which predicted structural genes in the order bah, ORF1, ORF2, and ORF3 followed by the regulatory gene, brpA (H. Anzai, T. Murakami, S. Imai, A. Satoh, K. Nagaoka, and C.J. Thompson, J. Bacteriol. 169:3482-3488, 1987). The four structural genes were translationally coupled and apparently cotranscribed from an undefined promoter(s) under the positive control of the brpA gene product. S1 mapping experiments indicated that brpA was transcribed by two promoters (brpAp1 and brpAp2) which initiate transcription 150 and 157 bp upstream of brp A within an intergenic region and at least one promoter further upstream within the bap gene cluster (brpAp3). All three transcripts were present at low levels during exponential growth and increased just before the stationary phase. The levels of the brpAp3 band continued to increase at the onset of stationary phase, whereas brpAp1-and brpAp2-protected fragments showed no further change. BrpA contained a possible helix-turn-helix motif at its C terminus which was similar to the C-terminal regulatory motif found in the receiver component of a family of two-component transcriptional activator proteins. This motif was not associated with the N-terminal domain conserved in other members of the family. The structural gene cluster sequenced began with bah, encoding a bialaphos acetylhydrolase which removes the N-acetyl group from bialaphos as one of the final steps in the biosynthetic pathway. The observation that Bah was similar to a rat and to a bacterial (Acinetobacter calcoaceticus) lipase probably reflects the fact that the ester bonds of triglycerides and the amide bond linking acetate to phosphinothricin are similar and hydrolysis is catalyzed by structurally related enzymes. This was followed by two regions encoding ORF1 and ORF2 which were similar to each other (48% nucleotide identity, 31% amino acid identity), as well as to GrsT, a protein encoded by a gene located adjacent to gramicidin S synthetase in Bacillus brevis, and to vertebrate (mallard duck and rat) thioesterases. The amino acid sequence and hydrophobicity profile of ORF3 indicated that it was related to a family of membrane transport proteins. It was strikingly similar to the citrate uptake protein encoded by the transposon Tn3411.
Assuntos
Amidoidrolases/genética , Antibacterianos/biossíntese , Genes Bacterianos , Genes Reguladores , Família Multigênica , Compostos Organofosforados/metabolismo , Streptomyces/genética , Tioléster Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Fases de Leitura Aberta , Conformação Proteica , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Streptomyces/metabolismoRESUMO
In addition to tlrA, tlrB and tlrC, which were previously cloned by others, a fourth antibiotic-resistance gene (tlrD) has been isolated from Streptomyces fradiae, a producer of tylosin (Ty), and cloned in Streptomyces lividans. Like tlrA, tlrD encodes an enzyme that methylates the N6-amino group of the A2058 nucleoside within 23S ribosomal RNA. However, whereas the tlrA protein dimethylates that nucleoside, the tlrD product generates N6-monomethyladenosine. The genes also differ in their mode of expression: tlrA is inducible, whereas tlrD is apparently expressed constitutively, and it has been confirmed that the tlrA-encoded enzyme can add a second methyl group to 23S rRNA that has already been monomethylated by the tlrD-encoded enzyme. Presumably, that is what happens in S. fradiae.
Assuntos
Metiltransferases/genética , RNA Ribossômico 23S/metabolismo , Streptomyces/genética , Tilosina/metabolismo , Southern Blotting , Clonagem Molecular , Resistência Microbiana a Medicamentos/genética , Expressão Gênica , Genes Bacterianos , Metilação , Metiltransferases/metabolismo , Mapeamento por Restrição , Streptomyces/efeitos dos fármacos , Streptomyces/metabolismo , Tilosina/farmacologiaRESUMO
A resistance gene, carB, originally isolated from the carbomycin-producing organism, Streptomyces thermotolerans, confers on Streptomyces lividans high-level resistance to the drug. However, ribosomes from S. lividans expressing carB show only moderate resistance to this macrolide in vitro, although they are highly resistant to the action of lincosamide antibiotics. The carB product monomethylates the amino group of the adenosine residue located at position 2058 in 23S ribosomal RNA. In contrast, ribosomes from S. lividans expressing ermE, in which 23S RNA is dimethylated at this same position, are much more highly resistant to macrolides and insensitive to lincosamides.
Assuntos
Resistência Microbiana a Medicamentos/genética , Leucomicinas/biossíntese , RNA Bacteriano/genética , RNA Ribossômico 23S/genética , RNA Ribossômico/genética , Streptomyces/genética , Proteínas de Bactérias/biossíntese , Clonagem Molecular , Metilação , Testes de Sensibilidade Microbiana , Fenótipo , RNA Bacteriano/metabolismo , RNA Ribossômico 23S/metabolismo , Mapeamento por Restrição , Streptomyces/metabolismoRESUMO
Streptomyces lividans TK21 possesses inducible ribosomal RNA methylase activity that confers high-level resistance to lincomycin and lower levels of resistance to certain macrolides. The methylase gene (designated lrm) is inducible by erythromycin and other macrolides and also by celesticetin (a lincosamide) but not by lincomycin. The lrm enzyme monomethylates the N6-amino group of adenosine at position 2058 within 23S-like ribosomal RNA.
Assuntos
Lincomicina/farmacologia , RNA Ribossômico/metabolismo , Streptomyces/efeitos dos fármacos , Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos , Indução Enzimática/efeitos dos fármacos , Macrolídeos , Metilação , RNA Bacteriano/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , tRNA Metiltransferases/biossínteseRESUMO
Ribosomes from Streptomyces griseofuscus expressing tlrA, a resistance gene isolated from the tylosin producer Streptomyces fradiae, are resistant to macrolide and lincosamide antibiotics in vitro. The tlrA product was found to be a methylase that introduces two methyl groups into a single base within 23S rRNA, generating N6,N6-dimethyladenine at position 2058. This activity is therefore similar to the ermE resistance mechanism in Saccharopolyspora erythraea (formerly Streptomyces erythraeus).
Assuntos
Leucomicinas/farmacologia , Metiltransferases/metabolismo , RNA Ribossômico 23S/genética , RNA Ribossômico/genética , Streptomyces/genética , Resistência Microbiana a Medicamentos/genética , Cinética , Metilação , Plasmídeos , Biossíntese de Proteínas , RNA Ribossômico 23S/metabolismo , Ribossomos/metabolismo , Streptomyces/efeitos dos fármacos , Transcrição Gênica , TilosinaRESUMO
Nucleotide sequence of a 906-bp fragment of Streptomyces alboniger DNA containing the gene (pac), which encodes a puromycin N-acetyltransferase (PAC), has been determined. The pac gene contains a 600-nt open reading frame, starting with an ATG codon, which encodes a polypeptide of Mr 21,531; this is consistent with the 23 +/- 1.5 kDa size of the PAC enzyme. High-resolution S1 mapping indicates that transcription starts at or next to a C residue 35 bp upstream from the putative ATG start codon. A 263-bp DNA fragment from the 5' region of the pac gene has promoter activity in the promoter-probe plasmid pIJ486. Its -35 and -10 regions show significant structural homology to the corresponding regions of the hyg gene promoter, but they are different from the promoter sequences of other Streptomyces and Escherichia coli genes.
Assuntos
Acetiltransferases/genética , Genes Bacterianos , Streptomyces/genética , Acetiltransferases/biossíntese , Sequência de Aminoácidos , Sequência de Bases , Códon/genética , DNA Bacteriano , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos , Regiões Promotoras Genéticas , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Streptomyces/enzimologia , Transcrição GênicaRESUMO
The 5' region of the SGA and STA2 genes, encoding the intra- and extracellular glucoamylases, respectively, from Saccharomyces cerevisiae have been sequenced. In addition, the transcription initiation sites have been determined. Four distinct short elements (named I to IV) were found in both genes. Element III has the consensus sequence PuCATTTAPiG with a bilateral symmetry around the central T, and is present in both genes as a direct repeat. This motive seems responsible for the coregulation of STA2 and SGA by the repressor STA10 gene of S. cerevisiae.
Assuntos
Genes Fúngicos , Genes , Glucana 1,4-alfa-Glucosidase/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/enzimologia , Transcrição GênicaRESUMO
A 207 bp DNA fragment from the 5' region of the hyg gene of Streptomyces hygroscopicus was located preceding a DNA sequence encoding the mature form of human interferon alpha 2. This gene fusion, inserted in the Streptomyces vector pIJ702, expressed interferon activity in Streptomyces lividans indicating that the 207 bp sequence has promoter activity. The transcription initiation site was located. No significant homology with previously described Streptomyces promoters could be found. It appears therefore, it represents a novel class of Streptomyces promoters.
Assuntos
Genes Bacterianos , Fosfotransferases/genética , Regiões Promotoras Genéticas , Streptomyces/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Endonucleases , Interferon Tipo I/genética , Canamicina Quinase , Plasmídeos , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico , Endonucleases Específicas para DNA e RNA de Cadeia Simples , Transcrição Gênica , Transformação BacterianaRESUMO
The Streptomyces hygroscopicus hyg gene encoding a hygromycin B phosphotransferase has been introduced into different sites of both the Escherichia coli plasmid pBR322 and the Escherichia coli-Saccharomyces cerevisiae shuttle vector YRp7. When this gene was inserted into the BamHI site of pBR322 and then cloned in E. coli phosphorylating activity was not detected, indicating that the hyg gene promoter was not functional in this bacterium. However, when the hyg gene was inserted into either the unique PstI site of pBR322 or into each of the two PstI sites of YRp7, phosphotransferase activity was observed. Analysis of the translation products from these constructions by coupled in vitro transcription-translation systems suggested that in all cases transcrition was regulated by a promoter not provided by the inserted hyg gene and that the synthesized polypeptide was identical to that present in S. hygroscopicus.
Assuntos
Clonagem Molecular , Escherichia coli/genética , Genes Bacterianos , Genes , Fosfotransferases/genética , Streptomyces/genética , Sequência de Bases , Enzimas de Restrição do DNA , Canamicina Quinase , Plasmídeos , Biossíntese de Proteínas , Streptomyces/enzimologia , Transcrição GênicaRESUMO
A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.
Assuntos
Fosfotransferases/isolamento & purificação , Streptomyces/enzimologia , Canamicina Quinase , Cinética , Peso Molecular , Fosforilação , Fosfotransferases/metabolismoRESUMO
The nucleotide sequence of a 1467 bp fragment of Streptomyces hygroscopicus DNA containing the gene (hyg) encoding a hygromycin B phosphotransferase (HPH) has been determined. The N-terminal amino acid sequence of HPH determined by automated Edman degradation has allowed the coding sequence of the hyg gene to be identified. The translation initiation triplet is GTG and 5 bp preceding it there is a sequence complementary to the 3'-end of 16S rRNA from S. lividans. The transcriptional start and termination sites have been determined; the presumptive promoter region has only partial homology to that of the Streptomyces vinaceus vph gene and is different to the promoter sequences of other Streptomyces genes.
Assuntos
Fosfotransferases/genética , Streptomyces/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Códon , DNA Bacteriano/genética , Genes , Genes Bacterianos , Vetores Genéticos , Canamicina Quinase , Conformação de Ácido Nucleico , Streptomyces/genética , Transcrição GênicaRESUMO
The gene encoding the phosphotransferase enzyme that modifies hygromycin B in its producing organism Streptomyces hygroscopicus, has been cloned in the Streptomyces vector pIJ41. Two plasmids, pFM4 and pFM6, containing 2.1 and 19.6 kb inserts of Streptomyces hygroscopicus DNA, respectively, which express the modifying enzyme, have been isolated. A 3.1 kb PstI restriction fragment from pFM4 was inserted in the Streptomyces vector pIJ350 and the resulting plasmids, pMZ11.1 and pMZ11.2, express the hygromycin B-resistance phenotype. The utility of this dominant marker for cloning experiments is discussed in the text.
Assuntos
Clonagem Molecular , Genes Bacterianos , Genes , Fosfotransferases/genética , Streptomyces/genética , Bacillus subtilis/genética , Enzimas de Restrição do DNA , Canamicina Quinase , Fenótipo , Fosfotransferases/isolamento & purificação , Plasmídeos , Especificidade da Espécie , Streptomyces/enzimologiaRESUMO
Bouvardin is an antitumor drug that inhibits protein synthesis in intact eukaryotic cells and cell-free systems. Our present studies have shown that bouvardin acts at the level of the 80 S ribosome in a site somehow involved with the interaction of EF1 and EF2. Indeed bouvardin inhibits EF1-dependent binding of aminoacyl-tRNA and EF2-dependent translocation of peptidyl-tRNA but does not affect the nonenzymic translocation since this reaction does not require EF2. The site of the 80 S ribosome involved in the interaction with bouvardin appears to be independent from the cycloheximide and the cryptopleurine binding sites since yeast mutants resistant to cycloheximide or cryptopleurine are sensitive to bouvardin.