RESUMEN
We have devised a sensitive and rapid method for the detection of several bacterial pathogens in clinical specimens using PCR. This method has been named Direct Labeling and Detection Procedure (DLDP) and is based on the direct incorporation of a nonradioactive digoxigenin label (DIG-11-dUTP) into a microbial species-specific gene fragment during amplification. Following amplification, the resulting PCR products are cleansed of nonincorporated DIG-11-dUTP, spotted onto a nylon membrane, fixed by UV-crosslinking and the labeled DNA is visualized by digoxigenin detection reagents. Using cultivated reference bacteria (Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa) we were able to demonstrate a rapid and sensitive detection of < 20 CFU of bacteria in human secretions (sputum, urine, mucous). The present study suggests that DLDP can be used as a reliable method for indication of bacteria in clinical or environmental specimens with the proviso that the selected corresponding oligonucleotide primers provide amplification of strong species-specific genes.
Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Reacción en Cadena de la Polimerasa/métodos , Infecciones Bacterianas/orina , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Humanos , Resistencia a la Meticilina , Moco/microbiología , Pseudomonas aeruginosa/aislamiento & purificación , Reproducibilidad de los Resultados , Esputo/microbiología , Staphylococcus aureus/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
In the search for the prevalence and distribution pattern of Gs-alpha gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at "hot-spot" codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing. The prevailing type of single-base substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys). The fragments of the Gs-alpha gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned in Escherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43%. Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred. There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-alpha gene and the level of Gs-alpha protein expression in the corresponding thyroid tumors.
Asunto(s)
Proteínas de Unión al GTP/genética , Mutación , Neoplasias de la Tiroides/genética , Adulto , Anciano , Secuencia de Bases , Clonación Molecular , Codón , ADN/química , Escherichia coli/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia MolecularRESUMEN
Point mutations occurring at codon 201 of the gene coding for the alpha subunit of the stimulatory G protein impair intrinsic GTPase activity and lead to a constitutive activation of adenylate cyclase. We have examined thyroid follicular and papillary carcinomas and follicular adenomas and found samples that bear this mutation at codon 201 of the Gs alpha gene. Both mutation-positive and mutation-negative tissue samples were investigated for the level of Gs alpha expression relative to a pool of normal thyroid tissue, using immunoblotting against two (mid-region-specific and C-end-specific) antipeptide antibodies. Using 8000 g and 100,000 g membrane fractions of homogenized tissues we have demonstrated that the Gs alpha proteins in normal ad neoplastic thyroid tissues are represented by three isoforms: 43 kDa, 45 kDa and 52 kDa. We have quantified and compared the amount of Gs alpha protein and find it is overexpressed in mutation-bearing tissue samples.
Asunto(s)
Carcinoma Papilar Folicular/genética , Carcinoma Papilar/genética , Proteínas de Unión al GTP/genética , Mutación Puntual , Neoplasias de la Tiroides/genética , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Membrana Celular/química , Codón , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/inmunología , Expresión Génica , Humanos , Immunoblotting , Sustancias Macromoleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Conejos , Fracciones Subcelulares , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiología , Neoplasias de la Tiroides/metabolismoAsunto(s)
Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Plásmidos , Tiroiditis Autoinmune/inmunología , Yersinia enterocolitica/genética , Secuencia de Aminoácidos , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Yersinia enterocolitica/inmunologíaRESUMEN
We propose a simple and reliable method to increase the sensitivity of mutation specific oligonucleotide hybridization (MSOH) at least 2.5 times, when it is used to detect mutations in samples of DNA from tumor tissues. The method is based on using single stranded (ss) DNA, amplified by asymmetric PCR, as a target for MSOH analysis. During the first step, genomic DNA, isolated from tissue samples, has to be amplified by "standard", symmetric PCR, with sense and antisense primers in equimolar concentration. This amplification can be performed in a diminished volume of reaction mixture. In the second step obtained double stranded (ds) PCR DNA-product can be used as a template for asymmetric PCR, using only a single primer. The ss DNA must be complementary to the set of mutation specific oligonucleotides. By this innovation we have been able to clarify questionable results of MSOH using ds DNA as a target. Comparing MSOH from ss DNA to that from ds DNA, the observed rate of Gs-alpha mutations in thyroid tumor tissue samples increased to 16.7% (14/66) from 6% (4/66).
Asunto(s)
Codón/genética , Cartilla de ADN , ADN de Neoplasias/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Neoplasias de la Tiroides/genética , Secuencia de Bases , ADN de Neoplasias/aislamiento & purificación , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Humanos , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Oligonucleótidos AntisentidoRESUMEN
The influence of the brucella OMP preparations obtained by the genetic engineering technique (18, 38 and 18 + 38 kD) on the formation of the specific protection and progress of infectious process in brucellosis in the in vivo experiments has been studied. The OMPs synthesized in Escherichia coli cells GSE579 and having mol mass 18 and 38 kD are common antigens for a number of brucella species. The 18 kD OMP was found to protect 66.7% of experimental animals against brucellosis, while the protection by the commercial live vaccine was 78.0%. The 38 kD OMP did not possess the protective activity with the index of infectivity in the experimental group of animals being higher than the one in the control group (77.9% and 53.4% respectively). The indexes of colony forming units in the mice spleen were also higher in the experimental group of animals. The obtained results suggest that the 38 kD OMP may be a factor of brucella virulence.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/inmunología , Brucella abortus/inmunología , Brucella melitensis/inmunología , Escherichia coli , Expresión Génica , Animales , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Brucella abortus/patogenicidad , Brucella melitensis/patogenicidad , Brucelosis/inmunología , Brucelosis/microbiología , Cobayas , Ratones , Ratones Endogámicos BALB C , Virulencia/genéticaRESUMEN
A non-isotopic amplification system was used to identify and indicate Brucella. The terminal sequences of a protein gene fragment in Brucella outer membrane were identified and direct and reverse primers were chosen for a polymerase chain reaction. (PCR). PCR amplifies a specific DNA fragment, 700 kb in size, only in representatives of the Brucella genus. A probe was design, which is the central part of the amplified DNA fragment, 550 kb in size. Single Brucella cells were detectable with an unlabelled probe in the analyzed samples during hybridization reactions. The system can be recommended for a rapid and reliable analysis in medical and veterinary practice.
Asunto(s)
Brucella/genética , ADN Bacteriano/análisis , Hibridación de Ácido Nucleico/métodos , Secuencia de Bases , Sondas de ADN , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
The homogeneous preparations of the brucella protein antigens were isolated from the hybrid producer strains Escherichia coli 6SE579 and 6SE800 by the cold osmotic shock technique and further purification on immunosorbents. The 18 + 38 and 38 kDa antigens were obtained. The antiserum specific to brucella 38 kDa antigen was obtained and used for isolation of the 18 kDa antigen from the producer strain 6SE579 synthesizing two brucella antigens. The immunosorbent developed on the basis of BrCn-agarose conjugated with antibodies from the serum has permitted isolation of 18 kDa protein antigen preparation. Thus, the combined technique of cold osmotic shock and affinity chromatography on immunosorbents permits one to isolate highly purified individual antigens of brucella from Escherichia coli K12 producer cells.
Asunto(s)
Antígenos Bacterianos/aislamiento & purificación , Brucella/inmunología , Escherichia coli/genética , Antígenos Bacterianos/genética , Western Blotting , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Focalización IsoeléctricaRESUMEN
The possibility of preparing B. abortus vaccine strain 19-BA with multiresistance to antibiotics was shown. The strain was obtained by the spontaneous induction of resistance to rifampicin with the subsequent transformation of nonconjugative hybrid plasmid pOVI which, in addition to rifampicin resistance, governed the resistance of brucellae to tetracycline, doxycycline, ampicillin, and streptomycin. Experiments on guinea pigs demonstrated the immunization with both multiresistant vaccine strain GSA1 and B. abortus initial vaccine strain 19-BA.
Asunto(s)
Vacuna contra la Brucelosis/inmunología , Brucella abortus/inmunología , Animales , Brucella/efectos de los fármacos , Brucella/genética , Brucella/inmunología , Brucella/patogenicidad , Vacuna contra la Brucelosis/genética , Brucella abortus/efectos de los fármacos , Brucella abortus/genética , Brucella abortus/patogenicidad , Brucelosis/patología , Brucelosis/prevención & control , Conjugación Genética/efectos de los fármacos , Conjugación Genética/genética , Farmacorresistencia Microbiana/genética , Farmacorresistencia Microbiana/inmunología , Cobayas , Inmunización , Plásmidos/efectos de los fármacos , Plásmidos/genética , Recombinación Genética/efectos de los fármacos , Recombinación Genética/genética , Factores de Tiempo , Virulencia/efectos de los fármacos , Virulencia/genética , Virulencia/inmunologíaRESUMEN
The sensitizing properties of brucella 31 kD and 31+15 kD protein antigens produced by Escherichia coli cell carrying and expressing the corresponding brucella genes were compared. In experiments evaluating the test of mouse feet oedema in CBA line animals the property of the 31 kD antigen preparation to induce the specific oedema effect was demonstrated on the level comparable with the one produced by the brucella outer membrane proteins preparation. The obtained data were confirmed in the experiments on adoptive transfer of prolonged type hypersensitivity via the spleen cells from the sensitized donors to intact recipient animals. The future of molecular cloning technique usage for obtaining the homogeneous stable preparations of brucella antigens with low reactivity and high specificity is discussed.
Asunto(s)
Antígenos Bacterianos/biosíntesis , Brucella/inmunología , Escherichia coli/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Escherichia coli/metabolismo , Hipersensibilidad Tardía , Ratones , Ratones Endogámicos CBA , Sensibilidad y EspecificidadRESUMEN
The libraries of Brucella melitensis 565 and Brucella abortus 99 in Escherichia coli cells have been constructed. Some clones of Escherichia coli producing the specific brucella antigens have been found in immunological tests with brucella antiserum. Two strains producing antigens have been characterized, one being from Brucella melitensis 565 and another from Brucella abortus 99 clone libraries . Both strains synthesize two antigens that were studied by immunoelectrophoresis, immunoblotting after treatment of antigen preparations with different physical and chemical agents substrate specific enzymes. Both strains are found to synthesize the specific brucella antigens of protein nature. One of them has the mol mass about 15 kD, another--31-32 kD. The 31-32 kD antigen can be, evidently, referred to as the main protein of an outer membrane of brucella.
Asunto(s)
Proteínas Bacterianas/química , Brucella/genética , Escherichia coli/genética , Genes Bacterianos , Antígenos Bacterianos/química , Antígenos Bacterianos/genética , Western Blotting , Brucella/inmunología , Clonación Molecular , ADN Bacteriano/química , Electroforesis , Epítopos/genética , Peso MolecularRESUMEN
The birepliconed plasmid pOV13 possesses all the properties of a vector for DNA cloning in a broad host range of bacterial cells. pOV13 is transfered by transformation and stably inherited by Escherichia coli, Brucella, Pseudomonas cells determining the resistance to streptomycin, tetrocycline and kanamycin in these bacteria. The plasmid pOV13 is a multicopy plasmid optimal in replication capacity (23kb). The plasmid carries single sites for some restriction endonucleases that are used for DNA cloning, including some restriction sites in antibiotic resistance genes. The examples of DNA cloning with the selection of recombinant clones by the insertional inactivation of kanamycin or tetracycline resistance and expression of the cloned DNAs are presented.
Asunto(s)
Clonación Molecular , Bacterias Gramnegativas/genética , Plásmidos , ADN/genética , Recombinación Genética , Mapeo Restrictivo , Transformación GenéticaRESUMEN
The hybrid plasmid pOV13 proposed as a potential vector for DNA cloning in a broad bacterial host range has been constructed on the basis of the broad host range plasmid RSF1010 and a shortened derivative of RP4, the plasmid pVZ115 serving a marker DNA fragment. The plasmid pOV13 contains the genes for streptomycin, kanamycin and tetracycline resistance and single cleavage sites for restriction endonucleases BamHI, BgIII, SalI, SmaI, PvuII, XhoI, as well as double cleavage sites for restriction endonucleases PstI and HindIII permitting one to clone DNA with insertional inactivation of genes. The physicogenetical map of the birepliconed plasmid pOV13 is presented.
Asunto(s)
Clonación Molecular , ADN Bacteriano/genética , Vectores Genéticos , Plásmidos , Recombinación Genética , Escherichia coli/genética , Hibridación de Ácido Nucleico , Mapeo Restrictivo , Transformación BacterianaRESUMEN
The mobilizing activity of the plasmid R68,45 in Escherichia coli and Brucella abortus has been studied. The plasmid R68,45 has been found to lose its ability to mobilize the chromosome in Brucella suis cells. The experiments on the conjugational transfer of R68,45 were confirmed by restriction analysis of the plasmid DNA. R68,45 has been shown to lose Cma in brucella cells via deletion. The molecular mechanisms of deletion process in brucella and other bacteria are discussed.
Asunto(s)
Brucella/genética , Plásmidos , Conjugación Genética , Escherichia coli/genética , Genes BacterianosRESUMEN
A recA-like gene from Pseudomonas aeruginosa was cloned and identified by means of interspecific complementation of gene recA repair defect in Escherichia coli. The gene was mapped in the PvuII-HindIII Ps. aeruginosa chromosome fragment of 1.5 kbp in length. Having been recloned in pUC18 or 19 plasmids in either of possible orientations, this fragment was shown to complement three different defects of E. coli recA mutants: in repair, recombination and SOS functions.
Asunto(s)
Clonación Molecular , Genes Bacterianos , Pseudomonas aeruginosa/genética , Rec A Recombinasas/genética , Bacteriófago lambda/genética , Mapeo Cromosómico , Enzimas de Restricción del ADN , Escherichia coli/genética , Plásmidos , Respuesta SOS en GenéticaRESUMEN
We have chosen the process of F'-plasmid formation as a model for a study of the illegitimate, recA-independent recombination. Our genetic data showed that F'-formation was under genetic control of the Hfr donor cells. To determine the role of recipient cells in the process of F-prime formation we compared the pattern of transconjugant formation in the crosses of Hfr and F' donors with the recA-nalA+ and recA-nalA- recipients, nalA- mutation in the recipient lead to the sharp decrease of the total yield of transconjugants selected for proximal markers and decrease the fraction of conjugative plasmids in progeny. The plasmids inherited in the nalA- recipient in HfrxF- crosses possess only proximal selected markers while the appropriate distribution of proximal and more distal markers took place in the nalA+ recipient. The homogeneity for the sensitivity of nalA- progeny to the male specific phages in comparison with the nalA+ transconjugants was also observed. The data obtained show that the nalA- mutation has a strong influence on the process of final inheritance and probably formation of F'-plasmids in the recipient cells. The extent of the influence of the nalA- mutation on the pattern of F'-plasmid inheritance was dependent upon the primary F' structure formed (or existed) in the donor cell.
Asunto(s)
Escherichia coli/genética , Factor F , Recombinación Genética , Conjugación Genética , MutaciónRESUMEN
Isolation and properties of the mutant of HfrH with altered frequency and specificity, of F'-plasmid formation are described. This mutant, designated fpf-5, showed significant reduction in the total yield of transconjugants selected for proximal markers in the cross with the F- recA- recipient. The crosses between the fpf-5 mutant donor and recA-nalA- recipient were sterile. The fpf-5 mutation resulted in the marked changes of the frequency of recombination exchanges involving definite chromosomal regions. The mutation reduced the frequency of the recA-independent recombination in the chromosomal fragment flanked by the leu and proA, sharply decreased the frequency of the recA-dependent and completely blocked recA-independent recombination in the chromosomal regions bordered by the genes proA and gal. Moreover the fpf-5 mutation affected the process of conjugative F'-plasmid formation suggesting effect of the mutation on recombinational exchange between the chromosome and integrated F-plasmid. The data obtained confirmed our earlier conclusion about fundamental role of the Hfr donor cell in the determination of the frequency and specificity of recombination leading to the F'-plasmid formation, and showed that the recombination events initiating F'-formation process occur in the Hfr donor cell.
Asunto(s)
Escherichia coli/genética , Factor F , Conjugación Genética , Cruzamientos Genéticos , Escherichia coli/aislamiento & purificación , Genes , Mutación , Especificidad de la EspecieRESUMEN
Assuming the similarity of the processes of illegitimate recombination, such as deletion formation, with the process of F' plasmid formation, we have undertaken the study of the influence of recA- and seg- alleles of Hfr donor on the F' plasmid formation. The data obtained demonstrate the strong influence of donor genotype on the frequency of F' plasmid formation and on the nature of F' plasmids formed, thus demonstrating that the most of F' plasmids have been formed via recombination in Hfr donor cells. The recA- mutation decreased the total yield of F' plasmids selected using both proximal and distal Hfr markers and affected drastically the distribution of the F' plasmids inheriting different proximal unselected markers. The existence of recA-dependent and recA-independent modes of F' plasmid formation was demonstrated. The Escherichia coli chromosome contains regions which involve preferentially in recA-dependent (between proA and gal, and clockwise from gal) or recA-independent (between leu and proA, and the region counterclockwise from argE) recombination. The seg-2 mutation causes only partial block of both recA-dependent and recA-independent recombination pathways, however it causes dramatic decrease of genetic exchanges leading to the formation of the type II F' plasmids. Both seg- and recA- mutations decrease the frequency of the formation of Tra+ F' transconjugants. The percent of Tra- transconjugants, which remain sensitive to MS2 and Q beta donor specific phages, also drops significantly under the influence of the recA- and seg- alleles. Thus, the recombination involving the F structure in wild type strains and seg- mutants occures preferentially in the points of F outside the regions essential for transfer and sensitivity to male specific phages, while in recA- and recA-ges- strains the points inside these regions (tra operon) frequently involved in F' plasmid looping out. There exist more strict correlation between the fertility and sensitivity to phage Q beta than to phage MS2.