RESUMO
The influence of tumor necrosis factor alpha (TNF-alpha) on the processes of sphingomyelin cycle activation and intensity of peroxidation in animal brain in vivo has been studied. Alterations in activity of sphingomyelinase, a key sphingomyelin cycle enzyme and in sphingomyelin, ceramide content as well as accumulation of the products of lipid peroxidation (diene conjugates and diene ketons) were measured in the cortex, the cerebellum and the hippocampus of rats 5, 15, 30 min, 1, 2 and 5 hours after TNF-alpha intraperitoneal injection in dosage 100 mkg per animal. It is shown that 2 hours after the injection, TNF-alpha initiated an accumulation of the products of lipid peroxidation, which intensively developed in the cerebellum and the hippocampus. Sphingomyelinase activation was found in the same brain structures. At the initial stage of TNF-alpha action, an increase of lipid peroxidation products correlated with sphingomyelinase activation in the cerebellum and the hippocampus suggesting an interaction of two cell signal systems of sphingomyelin cycle and oxidative system.
Assuntos
Encéfalo/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Encéfalo/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Injeções Intraperitoneais , Masculino , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Espectrofotometria , Fator de Necrose Tumoral alfa/administração & dosagem , Fator de Necrose Tumoral alfa/farmacocinéticaAssuntos
Peptídeos beta-Amiloides/efeitos adversos , Esfingomielina Fosfodiesterase/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Doença de Alzheimer/metabolismo , Animais , Apoptose/efeitos dos fármacos , Ceramidas/biossíntese , Cerebelo/efeitos dos fármacos , Cerebelo/enzimologia , Cromatografia em Camada Fina/instrumentação , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
An oligomeric chimeric protein DB-2 was constructed, to design drugs with antitumor activity and to develop highly sensitive immunospecific tests for the diagnostics of a wide variety of malignant epithelial cells in humans. DB-2 contains an immunodominant site of tetanus toxin and a fragment of the locus of the human tumor-associated antigen MUC1 with a variable number of tandem repeats. A pentameric superhelical fragment of the human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and its main biochemical properties were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
Assuntos
Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Repetições Minissatélites , Mucina-1/química , Neoplasias/imunologia , Proteínas Recombinantes de Fusão/química , Sequência de Bases , Biopolímeros , Proteína de Matriz Oligomérica de Cartilagem , Primers do DNA , Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Humanos , Proteínas Matrilinas , Mucina-1/genética , Mucina-1/imunologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
The oligomeric recombinant protein DB-1 containing the immunodominant sites of the circumsporozoite protein of Plasmodium falciparum and tetanus toxin was constructed to optimize the schemes of presentation of B-cell epitopes during vaccination with chimeric proteins without the use of adjuvants. A fragment of the pentameric coiled-coil human cartilage oligomeric matrix protein was used as an oligomerization matrix. The expression of the protein in Escherichia coli cells was studied, a method for its purification was developed, and it was biochemically characterized. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.
Assuntos
Proteínas da Matriz Extracelular/química , Glicoproteínas/química , Epitopos Imunodominantes/química , Plasmodium falciparum/química , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Sequência de Bases , Biopolímeros , Proteína de Matriz Oligomérica de Cartilagem , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Proteínas da Matriz Extracelular/genética , Glicoproteínas/genética , Humanos , Epitopos Imunodominantes/imunologia , Proteínas Matrilinas , Dados de Sequência Molecular , Conformação Proteica , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/genéticaRESUMO
To study the structure-function relationship of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), genes were constructed that encode its three deletion mutants: D1, a mutant with the deletion of six amino acid residues (37-42) some of which are a part of a beta-structural region; D2, a mutant with the deletion of the unstructured six-aa sequence of a loop (45-50); and D3, a mutant with the deletion of 14 aa residues (37-50) corresponding to the A-B loop and encoded by the second exon of the gmcsf gene. The expression products of these genes in E. coli were accumulated in a fraction of insoluble proteins. The secondary structures of the mutant proteins were similar to that of the full-size GM-CSF, but the biological activity of the deletion mutants was 130 times lower than that of the GM-CSF: they stimulated the proliferation of the TF-1 cell line at 3 ng/ml concentration. The resulting proteins displayed antagonistic properties toward the full-size GM-CSF, with the inhibition degree of its colony-stimulating activity being 27%. A decrease in the mutant activity in the row D2 > D1 > D3 implies the importance of the conserved hydrophobic residues involved in the formation of the beta-structure for the formation of the GM-CSF functional conformation.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Deleção de Sequência , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Genes Sintéticos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Humanos , Dados de Sequência Molecular , Proteínas Recombinantes , Mapeamento por RestriçãoRESUMO
A convenient method of regioselective introduction of 1-beta-D-galactopyranosylthymine into oligonucleotides was developed and the substrate properties of the modified oligonucleotides were investigated in the enzymic reaction of formation and hydrolysis of internucleotide bonds. The English version of the paper.
Assuntos
Bioquímica/métodos , Galactose/química , Galactose/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Timina/química , Timina/metabolismo , Desoxirribonuclease EcoRI/química , Desoxirribonuclease EcoRI/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Galactose/análogos & derivados , Hidrólise , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Relação Estrutura-Atividade , Timina/análogos & derivadosRESUMO
The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF-alpha secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-alpha, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF-alpha detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF-alpha is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.
Assuntos
Apoptose , Fígado/patologia , Traumatismo por Reperfusão , Esfingomielinas/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Ceramidas/metabolismo , DNA/metabolismo , Fragmentação do DNA , Peroxidação de Lipídeos , Fígado/metabolismo , Ratos , Ratos Wistar , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/farmacologia , Fatores de TempoRESUMO
The Yersinia pestis (causative agent of plague) capsule antigen is a homopolymer of Caf1 protein. Export of the subunits is mediated by the periplasmic chaperone Caf1M. To study the mechanism of Caf1M activity, two hybrid genes including coding sequences for the Caf1 signal peptide, human granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-1 (IL-1) receptor antagonist, and mature Caf1 were constructed and expressed in Escherichia coli. We have shown that in the absence of Caf1M the majority of Caf1 moieties within the hybrid proteins undergo proteolysis in the periplasmic space, presumably by the DegP protease. The coexpression of a gene for chaperone Caf1M significantly increased the amount of full-size hybrid proteins in the periplasm, probably as a result of stabilization of the subunits spatial structure within the hybrid. This effect was not observed in JCB571 cells, which lack periplasmic disulfide isomerase DsbA, essential for Caf1M activity.
Assuntos
Proteínas de Bactérias/genética , Chaperonas Moleculares/genética , Proteínas Recombinantes de Fusão/genética , Yersinia pestis/genética , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Regulação Bacteriana da Expressão Gênica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Proteína Antagonista do Receptor de Interleucina 1 , Chaperonas Moleculares/química , Proteínas Recombinantes de Fusão/química , Sialoglicoproteínas/genética , Yersinia pestis/químicaRESUMO
F1 antigen (Caf1) of Yersinia pestis is assembled via the Caf1M chaperone/Caf1A usher pathway. We investigated the ability of this assembly system to facilitate secretion of full-length heterologous proteins fused to the Caf1 subunit in Escherichia coli. Despite correct processing of a chimeric protein composed of a modified Caf1 signal peptide, mature human interleukin-1beta (hIL-1beta), and mature Caf1, the processed product (hIL-1beta:Caf1) remained insoluble. Coexpression of this chimera with a functional Caf1M chaperone led to the accumulation of soluble hIL-1beta:Caf1 in the periplasm. Soluble hIL-1beta:Caf1 reacted with monoclonal antibodies directed against structural epitopes of hIL-1beta. The results indicate that Caf1M-induced release of hIL-1beta:Caf1 from the inner membrane promotes folding of the hIL-1beta domain. Similar results were obtained with the fusion of Caf1 to hIL-1beta receptor antagonist or to human granulocyte-macrophage colony-stimulating factor. Following coexpression of the hIL-1beta:Caf1 precursor with both the Caf1M chaperone and Caf1A outer membrane protein, hIL-1beta:Caf1 could be detected on the cell surface of E. coli. These results demonstrate for the first time the potential application of the chaperone/usher secretion pathway in the transport of subunits with large heterogeneous N-terminal fusions. This represents a novel means for the delivery of correctly folded heterologous proteins to the periplasm and cell surface as either polymers or cleavable monomeric domains.
Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Proteína Antagonista do Receptor de Interleucina 1 , Interleucina-1/genética , Interleucina-1/metabolismo , Chaperonas Moleculares/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , SolubilidadeRESUMO
Various methods have been investigated for the isolation and purification of fusion proteins of precursors of human insulin in the form of S-sulfonates, from the biomass of transformed Escherichia coli cells. Fusion proteins were prepared with different sizes and structures of the leader peptide and the poly-His position (inserted for purification by metal chelate affinity chromatography). The fusion proteins contained an IgG-binding B domain of protein A from Staphylococcus aureus at the N-terminus and an Arg residue between the leader peptide of the molecule and the proinsulin sequence, for trypsin cleavage of the leader peptide. Six residues of Cys in proinsulin allow the chemical modification of the protein as a (Cys-S-SO(-)(3))(6) derivative (S-sulfonate), which increases its polyelectrolytic properties and improves the efficiency of its isolation. Various methods of oxidative sulfitolysis were compared with catalysis by sodium tetrathionate or cystine and Cu2+ or Ni2+ ions. An optimum scheme for the isolation and purification of S-sulfonated fusion proteins was developed by the combination of metal-chelating affinity and ion-exchange chromatography. Highly purified (95%) S-sulfonated fusion protein was recovered which was 85% of the fusion protein contained in the biomass of E. coli cells. Folding of fusion protein S-sulfonate occurred with high yield (up to 90-95%). We found that the fusion protein-S-sulfonate has proinsulin-like secondary structure. This structure causes highly efficient fusion protein folding.
Assuntos
Insulina/isolamento & purificação , Proinsulina/isolamento & purificação , Sequência de Aminoácidos , Biomassa , Biotecnologia/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia por Troca Iônica/métodos , Dicroísmo Circular , Clonagem Molecular , Escherichia coli , Humanos , Corpos de Inclusão/química , Corpos de Inclusão/metabolismo , Insulina/química , Insulina/genética , Dados de Sequência Molecular , Plasmídeos , Proinsulina/química , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Ácidos SulfônicosRESUMO
Genes encoding fragments of polypeptide chains of murine lymphotoxins (LT), namely, LT-alpha truncated from the N-terminus and the LT-beta extracellular domain, containing N-terminal hepta- and hexahistidine epitopes, respectively, were expressed in E. coli cells. The recombinant proteins purified by metallochelate chromatography were used to obtain polyclonal antibodies that specifically recognize murine LT.
Assuntos
Anticorpos/isolamento & purificação , Epitopos/genética , Escherichia coli/genética , Expressão Gênica , Linfotoxina-alfa/genética , Animais , Clonagem Molecular , Epitopos/imunologia , Histidina/genética , Histidina/imunologia , Immunoblotting , Linfotoxina-alfa/imunologia , Camundongos , PlasmídeosRESUMO
A set of seven hybridomas producing monoclonal antibodies (MAbs) to the human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was obtained. The properties of the monoclonal antibodies were characterized, and pairs of MAbs specific to different non-overlapping epitopes of GM-CSF were identified. A sensitive and simple method of two-site ELISA for GM-CSF was developed on the basis of two MAbs. According to this method, one MAb is absorbed onto a microtiter plate and another is labeled with biotin and used for the detection of GM-CSF bound to the first MAb. MAb labeled with biotin, in its turn, was visualized with the streptavidin-horseradish peroxidase conjugate. The sensitivity of this test was no less than 0.5 ng/ml, and a linear dose-response relationship was observed within a concentration interval from 0.5 to 32 ng/ml. No cross-reactivity was found with human tumor necrosis factor-alpha, granulocyte colony-stimulating factor, interleukin-2, or interleukin-3 in this test system.
Assuntos
Anticorpos Monoclonais/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Animais , Reações Cruzadas , Fator Estimulador de Colônias de Granulócitos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Humanos , Interleucina-2/imunologia , Interleucina-3/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Sensibilidade e Especificidade , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Two schemes for efficient and productive isolation for mutant human recombinant tumor necrosis factor-alpha (TNF-alpha R32H) from Escherichia coli cells were developed. The methods include membrane filtration, ion-exchange chromatography and gel filtration, and centrifugation with subsequent free-flow electrophoresis as an alternative procedure. The target product was obtained as active trimer with total yield more than 50% and greater than 98% purity according to PAGE, size-exclusion chromatography, HPLC, and HPCE.
Assuntos
Citocinas/isolamento & purificação , Proteínas Recombinantes/isolamento & purificação , Fator de Necrose Tumoral alfa/isolamento & purificação , Cromatografia/métodos , Eletroforese/métodos , Escherichia coli/genética , Humanos , Mutação/genética , Análise de Sequência , Fator de Necrose Tumoral alfa/genéticaRESUMO
TNF-alpha induced sphingomyelin hydrolysis by sphingomyelinase and both sphingosine and ceramide generation have been reported to be implicated in a number of TNF-alpha responses, including cytotoxicity and apoptosis. We found that sphingosine, a highly cytotoxic product of enzymatic degradation of sphingomyelin, is accumulated in liver of mice treated with TNF-alpha. To determine the role of sphingosine in TNF-alpha toxicity, TNF-alpha mutants differing in their cytotoxicity to L929 cells as well as haemorrhagic tumor necrosis, tumor regression and lethal toxicity in mice were used in our experiments. The mutants with highest toxicity and tumor-necrotizing activity caused accumulation of sphingosine exceeded its control level 5,5 times in murine liver cells. TNF-alpha variants which caused moderate increase in sphingosine content were significantly less toxic. The observed relationship between toxicity of TNF-alpha mutants, the toxicity of sphingosine, and the extent of its accumulation in murine liver provides evidence to suggest that this sphingomyelin metabolite may be mediator of TNF-alpha-induced cell damage and death.
Assuntos
Mutagênese , Esfingosina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular , Sobrevivência Celular , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Proteínas Recombinantes/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Relação Estrutura-Atividade , Fator de Necrose Tumoral alfa/química , Fator de Necrose Tumoral alfa/genéticaRESUMO
A number of hybrid proteins containing amino acid sequences of human lymphotoxin and human and mouse tumor necrosis factors were obtained by means of genetic engineering. By using these proteins, an antigenic determinant in the molecule of human tumor necrosis factor for monoclonal antibodies E7H2 (MAb E7H2), previously developed against this factor was localized. It was demonstrated by Western blot analysis that the MAb E7H2-binding site is located in the sequence region 37-49 of human tumor necrosis factor and includes the sequence Val41GluLeuArg44 directly interacting with MAb E7H2.
Assuntos
Epitopos/imunologia , Proteínas Recombinantes de Fusão/imunologia , Fator de Necrose Tumoral alfa/imunologia , Animais , Especificidade de Anticorpos , Mapeamento de Epitopos , Engenharia Genética , Humanos , Camundongos , Mutação , Proteínas Recombinantes de Fusão/genéticaRESUMO
Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.
Assuntos
Genes , Óperon Lac , RNA Mensageiro/genética , Ribossomos/genética , Plasmídeos , Biossíntese de ProteínasRESUMO
Using recombinant DNA technology, construction and bacterial expression of genes was carried out which code for hybrid proteins, human tumor necrosis factor and hepatitis B core protein fused to immunogenic epitopes of foot-and-mouth disease virus, strains A22 and O1-194. Hybrids of tumor necrosis factor with foot-and-mouth disease antigenic determinants protected laboratory animals against the experimental challenge with a homologous strain of foot-and-mouth disease virus. Hybrid protein that contained immunogenic regions of two strains, A22 and O1-194, protected animals against infection with both A and O serotypes. Hybrid proteins based on hepatitis B virus core antigen retained the ability to assemble into core-like particles.
Assuntos
Aphthovirus/genética , Epitopos/biossíntese , Antígenos do Núcleo do Vírus da Hepatite B/genética , Fator de Necrose Tumoral alfa/genética , Animais , Aphthovirus/imunologia , Sequência de Bases , DNA Complementar/genética , Epitopos/genética , Cobaias , Humanos , Immunoblotting , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Human hepatocarcinoma HepG2 cells are known to be insensitive to tumor necrosis factor (TNF) cytotoxicity. In this report, preliminary washing of HepG2 cells with serum-free medium to remove endogenous and exogenous alpha-fetoprotein (AFP) from the cultivation medium transfers cells from the TNF-resistant to the TNF-sensitive state without addition of any transcriptional inhibitors. HepG2 cells sensitized to by washing again became TNF-resistant after their treatment with exogenous AFP. Protective AFP activity against TNF-induced cytotoxicity directly depends on the AFP/TNF concentration ratio, demonstrating biphasic AFP activity. Our data show that 0.2 mg/ml of AFP acts synergistically to enhance cytotoxicity of suboptimal TNF doses. In contrast, the same AFP dose significantly attenuates the cytotoxicity of high TNF doses. It is concluded that AFP can function as a protective factor against TNF cytotoxicity in human hepatoma cells. These observations suggest that AFP secretion by certain tumor cells allows a highly flexible regulation of TNF cytotoxicity, dependent on the amount of endogenous AFP.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Fator de Necrose Tumoral alfa/toxicidade , alfa-Fetoproteínas/farmacologia , alfa-Fetoproteínas/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Humanos , Células Tumorais CultivadasRESUMO
The isolation of single-stranded (ss) phagemid DNA using standard protocols often results in impure preparations, which contain undesirable quantities of chromosomal and/or double-stranded (ds) phagemid DNA. Here we report a simple and efficient method for elimination of virtually all dsDNA by incubation of phagemid viral particles with deoxyribonuclease I. In addition to analyzing the ratio of linear-to-circular topological forms of ssDNA after deoxyribonuclease I treatment, we verified that no decrease in transformation efficiency occurred and demonstrated that ssDNA molecules covered by capsid proteins remained intact following such treatment.
Assuntos
DNA de Cadeia Simples/isolamento & purificação , DNA Viral/isolamento & purificação , Desoxirribonucleases/química , Bacteriófagos/genética , Eletroforese em Gel de Ágar , Escherichia coli/enzimologia , Escherichia coli/genética , Desnaturação de Ácido Nucleico , Plasmídeos , Transformação GenéticaRESUMO
A model for virion RNA of the poliomyelitis virus, which does not pass the stage of DNA copies during biogenesis, demonstrates that Taq DNA polymerase is capable of synthesizing 960-bp cDNA with the specific primer. When comparing the nucleotide sequence of the starting virion RNA and recombinant DNAs, isolated from several independent clones, copying and amplification of virion RNA appear accurate (one substitution per 960 bp). A comparison of Taq and Tth DNA polymerases in RT/PCR indicates that the sensitivity of Taq polymerase seems to be two orders of magnitude higher than that of Tth polymerase. The RNA detection level under the chosen conditions approached 10(4) RNA copies per test. The present investigation indicates the great versatility of Taq polymerase, which promoted the reverse transcription reaction of RNA, cDNA amplification, screening of recombinant clones as well as sequencing of recombinant DNA. Thus application of Taq polymerase is rather promising not only to detect nucleic acids in biological samples, but also for isolating and cloning individual genes, encoded on DNA and/or on RNA templates.