RESUMEN
OBJECTIVES: The purpose of this study was to determine whether the QT interval dynamics that precede torsade de pointes are consistent with the initiation of this arrhythmia by early afterdepolarization-induced triggered activity. BACKGROUND: Early afterdepolarization-induced triggered activity has been suggested as an electrophysiologic mechanism for torsade de pointes. Consequently, the initiation of torsade de pointes should involve time- and rate-dependent alterations of ventricular repolarization similar to those known to modulate the development of early afterdepolarizations. METHODS: RR and QT intervals were measured in digitized 24-h ambulatory electrocardiographic recordings obtained from seven patients with acquired prolongation of ventricular repolarization. Each patient had one or more episodes of torsade de pointes. The relation between RR and QT intervals was determined before, during and after multiple episodes of torsade de pointes. RESULTS: In patients with multiple episodes of ventricular arrhythmias, the onset of the arrhythmias was associated with a critical prolongation of the QT interval. In some episodes, prolongation of the QT interval was associated with sudden prolongation of the sinus cycle length, whereas in other episodes, the QT interval prolonged progressively at a constant cycle length. CONCLUSIONS: The association between a critically prolonged QT interval and the onset of ventricular arrhythmias suggests that the initial complex of torsade de pointes is an early afterdepolarization-induced triggered response. However, prolongation of the QT interval itself was not sufficient to account for the initiation of torsade de pointes, suggesting that other, as yet unidentified factors are required.
Asunto(s)
Frecuencia Cardíaca , Síndrome de QT Prolongado/fisiopatología , Torsades de Pointes/fisiopatología , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Síndrome de QT Prolongado/complicaciones , Masculino , Persona de Mediana Edad , Factores de Tiempo , Torsades de Pointes/etiologíaRESUMEN
OBJECTIVES: The age-dependence of the development of ventricular arrhythmias was studied in German shepherd dogs with inherited ventricular arrhythmias and sudden death. BACKGROUND: A colony of German shepherd dogs has been established that exhibit inherited ventricular arrhythmias and sudden death. The incidence of arrhythmias increases with age. Because ventricular tachycardia is associated with bradycardia, it was hypothesized that the increased incidence of arrhythmias was related to age-dependent slowing of heart rate. METHODS: Arrhythmia counts and RR intervals were measured from serial ambulatory ECG recordings obtained in 71 dogs (1-48 weeks). In addition, 19 dogs were challenged with phenylephrine (10 micrograms/kg i.v.) at 15, 28, and 45 weeks of age, 10 dogs were challenged with epinephrine (1 microgram/kg i.v.) at 3, 5, 7, 9, 11, 18, and 28 weeks of age, and 10 dogs were challenged at 28 weeks with epinephrine (2.5 micrograms/kg i.v.), before and after propranolol (0.5 mg/kg i.v.). RESULTS: The incidence and severity of ventricular arrhythmias increased between 7 and 28 weeks of age and decreased between 28 and 44 weeks of age. The age-dependent increase in the incidence of ventricular tachycardia was associated with age-dependent reductions in sinus rate. Baroreflex-mediated slowing of the heart rate unmasked arrhythmias in young animals that did not spontaneously display arrthythmias and exacerbated existing arrhythmias in older animals. However, the magnitude of baroreflex-induced bradycardia was similar from 7-18 weeks of age, yet the incidence of arrhythmias increased progressively. Moreover, the waning of ventricular arrhythmias in older animals was not associated with more rapid sinus rates. CONCLUSION: The risk for sudden death in dogs with inherited ventricular arrhythmias increases with age in part because of age-dependent slowing of heart rate and in part because of other heart-rate-independent factors. The correspondence between the development of ventricular tachycardia and sinus pauses is consistent with the hypothesis that ventricular arrhythmias are initiated by early afterdepolarization-induced triggered activity.
Asunto(s)
Envejecimiento , Arritmias Cardíacas/etiología , Muerte Súbita Cardíaca , Enfermedades de los Perros/etiología , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/fisiopatología , Enfermedades de los Perros/genética , Enfermedades de los Perros/fisiopatología , Perros , Electrocardiografía Ambulatoria , Epinefrina , Corazón/fisiopatología , Frecuencia Cardíaca , Incidencia , Fenilefrina , Factores de Riesgo , SimpatomiméticosRESUMEN
Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.
Asunto(s)
ADN de Hongos/genética , ADN Ribosómico/genética , Vectores Genéticos , Kluyveromyces/genética , ARN Ribosómico/genética , Recombinación Genética , Northern Blotting , Southern Blotting , Amplificación de Genes , Humanos , Mitosis , Muramidasa/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transformación GenéticaRESUMEN
A bla(VIM-2) metallo-beta-lactamase determinant, identical to that previously identified in Pseudomonas aeruginosa COL-1 isolate from a French hospital, was detected on a 28-kb plasmid carried by a nosocomial isolate of P. aeruginosa from Verona, Italy. In this plasmid the bla(VIM-2) determinant was inserted into a class 1 integron of original structure, named In72, that contains a partially deleted intI1 integrase gene and two gene cassettes. The first cassette carries an aacA4 aminoglycoside acetyl transferase determinant. The second cassette carries a bla(VIM-2) determinant followed by a partially deleted attC site. The structure of In72 was notably different from that of In56, the bla(VIM-2)-containing integron found in the COL-1 isolate, revealing the existence of molecular heterogeneity among bla(VIM-2)-containing integrons in clinical isolates of P. aeruginosa from Europe.
Asunto(s)
Elementos Transponibles de ADN , Plásmidos , Pseudomonas aeruginosa/genética , Recombinación Genética , beta-Lactamasas/genética , Acetiltransferasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Integrasas/genética , Datos de Secuencia MolecularRESUMEN
Patients infected with human immunodeficiency virus type 1 (HIV-1) are being treated with a number of different combinations of antiretroviral compounds that target the essential viral enzymes reverse transcriptase and protease. Different sets of HIV-1 mutations that confer drug resistance have been well defined; they allow reasonable prediction of the drug sensitivity pattern from analysis of the HIV-1 genotype in vivo. Since periodical monitoring of genotypic resistance is expected to improve clinical management in a large number of infected patients, practical and cost-effective methods are highly desirable to set at least medium-scale sequencing in clinical diagnostic settings. We present a complete protocol for direct sequencing of HIV-1 reverse transcriptase and protease-coding regions. Features making the system amenable to routine clinical use include: 1. Highly robust presequencing steps (plasma RNA extraction, reverse transcription, and nested PCR); 2. Direct use of the crude unpurified PCR product as the sequencing template; and 3. Use of infrared-labeled sequencing primers consistently allowing long reads, thus obviating the need for sequencing of both DNA strands.
Asunto(s)
Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Reacción en Cadena de la Polimerasa/métodos , Inhibidores de Proteasas/farmacología , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Farmacorresistencia Microbiana , Electroforesis/métodos , Proteasa del VIH/efectos de los fármacos , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/efectos de los fármacos , VIH-1/genética , Humanos , Datos de Secuencia Molecular , ARN Viral/sangre , ARN Viral/aislamiento & purificación , Análisis de Secuencia de ADN/instrumentación , Moldes GenéticosRESUMEN
The photomutagenicity of the furochromone khellin was tested in Ames Salmonella strains using 8-methoxypsoralen (8-MOP) and 4,5', 8-trimethylpsoralen (TMP) as positive controls. When khellin was assayed with strain TA1537, mutation induction was not detectable; in the same strain, an equitoxic dose (52-56% level of survival) of TMP (used at a concentration 12-fold lower than khellin and with a UVA dose 83-fold lower than that used with khellin) yielded an increase in revertants/plate 3-fold above the spontaneous background. In strain TA102, khellin plus UVA treatment yielded a 2-fold increase in revertants/plate above the spontaneous background (79% survival). 8-MOP, however, used at a concentration 8-fold lower than khellin with a UVA dose 13-fold lower than khellin, yielded an increase in revertants/plate about 14-fold above background (66% survival) in the same strain. These data show that khellin has a weak photomutagenic potential and, along with the previously reported low photogenotoxic potential in eukaryotic cell systems, support the notion that khellin may be safer than bifunctional psoralens for clinical use.
Asunto(s)
Khellin/toxicidad , Mutágenos/toxicidad , Enfermedades de la Piel/tratamiento farmacológico , Khellin/uso terapéutico , Pruebas de Mutagenicidad , Fotoquímica , Salmonella typhimurium/efectos de los fármacosRESUMEN
We report the nucleotide sequence of the 2.2-kb 5'-flanking region of the human lysozyme gene. Four Alu repeats are located within this upstream region. Classification and dating of these four Alu elements, as well as of the four Alu elements present within the human lysozyme structural gene, were performed. Transposition of the eight Alu repeats found in the human lysozyme locus has apparently occurred at four different times during the primate genome evolution. Considering that Alu repeats are interspersed throughout human DNA with an average spacing of 4 kb, the presence of eight such repeats within the 8-kb lysozyme gene region and, in particular, of four of them in the 2.2-kb region upstream of the structural gene, appears quite unusual.
Asunto(s)
Genes , Hominidae/genética , Muramidasa/genética , Regiones Promotoras Genéticas , Secuencias Repetitivas de Ácidos Nucleicos , Animales , Secuencia de Bases , Evolución Biológica , ADN/genética , Genoma Humano , Humanos , Datos de Secuencia Molecular , Primates/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , TATA BoxRESUMEN
A new plasmid vector, pCRP, allowing the expression of human recombinant monoclonal antibody Fab fragments fused with a bacterial acid phosphatase has been constructed. pCRP can accept heavy- and light-chain cDNAs cloned from combinatorial antibody libraries displayed on filamentous phages with the pCombIII system and is able to direct expression to soluble Fabs in which the carboxy-terminus of the heavy chain is fused to the amino-terminus of the mature PhoN nonspecific acid phosphatase of Providencia stuartii. Using the pCRP vector, we expressed two different human recombinant Fabs cloned from combinatorial libraries (one anti-tetenus toxoid and the other anti-HIV-1 gp120) fused with the acid phosphatase. In both cases chimeric antibodies were obtained which retained the antigen-binding ability and the enzymatic activity. Similar Fab-enzyme fusions can be successfully used, even unpurified, in enzyme immunoassays.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Fosfatasa Ácida/genética , Fosfatasa Ácida/metabolismo , Anticuerpos Monoclonales/genética , Secuencia de Bases , Biblioteca de Genes , Genes Bacterianos/genética , Vectores Genéticos/genética , Proteína gp120 de Envoltorio del VIH/inmunología , VIH-1 , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Datos de Secuencia Molecular , Providencia/enzimología , Toxoide Tetánico/inmunologíaRESUMEN
A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.
Asunto(s)
Fosfatasa Ácida/genética , Clonación Molecular/métodos , Vectores Genéticos/genética , Plásmidos/genética , Escherichia coli/genética , Genes Bacterianos/genética , VIH-1/genética , Reacción en Cadena de la Polimerasa , Providencia/enzimología , Origen de Réplica/genética , beta-Lactamasas/genéticaAsunto(s)
Enfermedades de los Perros/diagnóstico , Taquicardia Ventricular/veterinaria , Factores de Edad , Animales , Cruzamiento , Enfermedades de los Perros/genética , Enfermedades de los Perros/fisiopatología , Enfermedades de los Perros/terapia , Perros , Electrocardiografía/veterinaria , Electrocardiografía Ambulatoria/veterinaria , Femenino , Frecuencia Cardíaca , Masculino , Linaje , Pronóstico , Taquicardia Ventricular/diagnóstico , Taquicardia Ventricular/genética , Taquicardia Ventricular/fisiopatología , Taquicardia Ventricular/terapiaRESUMEN
To test whether hyperkalemia suppresses ventricular fibrillation (VF) by reducing the slope of the action potential duration (APD) restitution relation, we determined the effects of the extracellular K(+) concentration ([K(+)](o)) ([KCl] = 2.7-12 mM) on the restitution of APD and maximum upstroke velocity (V(max)) the magnitude of APD alternans and spatiotemporal organization during VF in isolated canine ventricle. As [KCl] was increased incrementally from 2.7 to 12 mM, V(max) was reduced progressively. Increasing [KCl] from 2.7 to 10 mM decreased the slope of the APD restitution relation at long, but not short, diastolic intervals (DI), decreased the range of DI over which the slope was >/=1, and reduced the maximum amplitude of APD alternans. At [KCl] = 12 mM, the range of DI over which the APD restitution slope was >/=1 increased, and the maximum amplitude of APD alternans increased. For [KCl] = 4-8 mM, the persistence of APD alternans at short DI was associated with maintenance of VF. For [KCl] = 10-12 mM, the spontaneous frequency during VF was reduced, and activation occurred predominantly at longer DI. The lack of APD alternans at longer DI was associated with conversion of VF to a periodic rhythm. These results provide additional evidence for the importance of APD restitution kinetics in the development of VF.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Cloruro de Potasio/farmacología , Fibrilación Ventricular/tratamiento farmacológico , Fibrilación Ventricular/fisiopatología , Potenciales de Acción/fisiología , Animales , Diástole/fisiología , Perros , Endocardio/fisiología , Espacio Extracelular/metabolismo , Femenino , Análisis de Fourier , Ventrículos Cardíacos/efectos de los fármacos , Hiperpotasemia/inducido químicamente , Hiperpotasemia/fisiopatología , Masculino , Marcapaso Artificial , Función VentricularRESUMEN
Ventricular arrhythmias and the proclivity for sudden death have been identified in German shepherd dogs. This disorder is inherited, and affected animals can be consistently produced from an established colony. The arrhythmias are most prevalent in young dogs between 22 and 26 weeks of age, with death most frequent at this same age. Death occurs most frequently during presumed sleep or at rest after exercise or excitement. The QT interval is not prolonged; however, more frequent notching of the T wave exists in affected dogs compared to control dogs. Polymorphic rapid nonsustained ventricular tachycardia occurs most frequently following long RR intervals. Accordingly, perturbations that decrease the heart rate or enhance sinus arrhythmia increase the incidence of ventricular arrhythmias. Because the arrhythmias are age, behavior, and heart rate dependent, the autonomic nervous system may play a role in their generation. As determined by metaiodobenzyl-guanidine scintigraphy and immunocytochemical staining of tyrosine hydroxylase, cardiac sympathetic innervation is regionally deficient in affected dogs. Evidence suggests that initiation of the ventricular arrhythmias is caused by early afterdepolarization (EAD)-induced triggered activity originating from left ventricular Purkinje fibers. Alpha 1-adrenergic stimulation provokes EADs in the Purkinje fibers and ventricular arrhythmias in the dogs. The development of EADs may be related to heterogeneity of repolarizing currents (Ito in particular) in affected dogs. From this canine model of spontaneous ventricular arrhythmias, the opportunity exists to investigate the interplay between abnormal development of cardiac innervation and the genesis of lethal ventricular arrhythmias.
Asunto(s)
Arritmias Cardíacas/fisiopatología , Muerte Súbita Cardíaca , Modelos Animales de Enfermedad , Animales , PerrosRESUMEN
Despite recent advances in our understanding of the mechanism for ventricular fibrillation (VF), important electrophysiological aspects of the development of VF still are poorly defined. It has been suggested that the onset of VF involves the disintegration of a single spiral wave into many self-perpetuating waves. It has been further suggested that such a process requires that the slope of the electrical restitution relation be >/=1. The same theory anticipates that a single spiral wave will be stable (not disintegrate) if the maximum slope of the restitution relation is <1. We have shown previously that the slope of the restitution relation during rapid pacing and during VF is >/=1 in canine ventricle. We now show that drugs that reduce the slope of the restitution relation (diacetyl monoxime and verapamil) prevent the induction of VF and convert existing VF into a periodic rhythm. In contrast, a drug that does not reduce the slope of the restitution relation (procainamide) does not prevent the induction of VF, nor does it regularize VF. These results indicate that the kinetics of electrical restitution is a key determinant of VF. Moreover, they suggest novel approaches to preventing the induction or maintenance of VF.
Asunto(s)
Fibrilación Ventricular/fisiopatología , Potenciales de Acción/efectos de los fármacos , Animales , Diacetil/análogos & derivados , Diacetil/farmacología , Perros , Femenino , Masculino , Procainamida/farmacología , Especificidad de la Especie , Fibrilación Ventricular/etiología , Verapamilo/farmacologíaRESUMEN
The restitution kinetics of action potential duration (APD) were investigated in paced canine Purkinje fibers (P; n = 9) and endocardial muscle (M; n = 9), in isolated, perfused canine left ventricles during ventricular fibrillation (VF; n = 4), and in endocardial muscle paced at VF cycle lengths (simulated VF; n = 4). Restitution was assessed with the use of two protocols: delivery of a single extrastimulus after a train of stimuli at cycle length = 300 ms (standard protocol), and fixed pacing at short cycle lengths (100-300 ms) that induced APD alternans (dynamic protocol). The dynamic protocol yielded a monotone increasing restitution function with a maximal slope of 1.13 +/- 0.13 in M and 1.14 +/- 0.17 in P. Iteration of this function reproduced the APD dynamics found experimentally, including persistent APD alternans. In contrast, the standard protocol yielded a restitution relation with a maximal slope of 0.57 +/- 0.18 in M and 0.84 +/- 0.20 in P, and iteration of this function did not reproduce the APD dynamics. During VF, the restitution kinetics at short diastolic interval were similar to those determined with the dynamic protocol (maximal slope: 1.72 +/- 0.47 in VF and 1.44 +/- 0.49 in simulated VF). Thus APD dynamics at short coupling intervals during fixed pacing and during VF were accounted for by the dynamic, but not the standard, restitution relation. These results provide further evidence for a strong relationship among the kinetics of electrical restitution, the occurrence of APD alternans, and complex APD dynamics during VF.
Asunto(s)
Potenciales de Acción , Ramos Subendocárdicos/fisiología , Fibrilación Ventricular/fisiopatología , Animales , Perros , Estimulación Eléctrica , Femenino , Ventrículos Cardíacos/inervación , Ventrículos Cardíacos/fisiopatología , MasculinoRESUMEN
The performance of oligonucleotide primers containing deoxyinosine (dl) at all ambiguous positions for polymerase chain reaction, based on ambiguous sequence information derived either from compilations of consensus nucleotide sequences or from amino acid sequences, has been evaluated in two model systems represented respectively by amplification of conserved genomic regions from different types of human papillomavirus and by amplification of a region of the human lysozyme cDNA on the basis of the protein amino acid sequence. In both instances the dl-containing primers obtained the expected amplification products. When using short primers or primers with very high dl contents, however, peculiar reaction conditions had to be adopted to obtain successful amplification and, in the latter case, performance remained suboptimal. Comparison of results with those obtained using corresponding degenerate primers showed that the use of dl-containing primers can be advantageous in terms of both specificity and yield of the amplification product. Sequence analysis of amplification products showed that dG residues are always found at positions corresponding to the dl residues of the primers.
Asunto(s)
Cartilla de ADN/análisis , ADN Viral/genética , Inosina/análogos & derivados , Papillomaviridae/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Viral/análisis , Amplificación de Genes , Genoma Viral , Immunoblotting , Inosina/análisis , Datos de Secuencia Molecular , Muramidasa/genética , Reacción en Cadena de la PolimerasaRESUMEN
A DNA probe is described that can be used for identification of Providencia stuartii by means of filter hybridization assays. The probe, which is a fragment of the P. stuartii phoN gene coding for an acid phosphatase, appeared to be able to recognize only P. stuartii strains in slot-blot hybridization experiments performed with total DNA extracted from 545 strains of 64 different Gram-negative bacterial species, including all the major representatives of the family Enterobacteriaceae. Owing to the problems that may be often encountered for correct identification of P. stuartii at the species level when using commercial identification systems, this probe may result useful for fast and reliable identification of P. stuartii strains for taxonomical, epidemiological and diagnostic studies.
Asunto(s)
Sondas de ADN , ADN Bacteriano , Providencia/aislamiento & purificación , Fosfatasa Ácida/genética , Proteínas Bacterianas/genética , ADN Bacteriano/genética , Enterobacteriaceae/genética , Infecciones por Enterobacteriaceae/diagnóstico , Infecciones por Enterobacteriaceae/microbiología , Genes Bacterianos , Hibridación de Ácido Nucleico , Providencia/genética , Especificidad de la EspecieRESUMEN
An Achromobacter xylosoxydans strain showing broad-spectrum resistance to beta-lactams (including carbapenems) and aminoglycosides was isolated at the University Hospital of Verona (Verona, Italy). This strain was found to produce metallo-beta-lactamase activity and to harbor a 30-kb nonconjugative plasmid, named pAX22, carrying a bla(VIM-1) determinant inserted into a class 1 integron. Characterization of this integron, named In70, revealed an original array of four gene cassettes containing, respectively, the bla(VIM-1) gene and three different aminoglycoside resistance determinants, including an aacA4 allele, a new aph-like gene named aphA15, and an aadA1 allele. The aphA15 gene is the first example of an aph-like gene carried on a mobile gene cassette, and its product exhibits close similarity to the APH(3')-IIa aminoglycoside phosphotransferase encoded by Tn5 (36% amino acid identity) and to an APH(3')-IIb enzyme from Pseudomonas aeruginosa (38% amino acid identity). Expression of the cloned aphA15 gene in Escherichia coli reduced the susceptibility to kanamycin and neomycin as well as (slightly) to amikacin, netilmicin, and streptomycin. Characterization of the 5' and 3' conserved segments of In70 and of their flanking regions showed that In70 belongs to the group of class 1 integrons associated with defective transposon derivatives originating from Tn402-like elements. The structure of the 3' conserved segment indicates the closest ancestry with members of the In0-In2 lineage. In70, with its array of cassette-borne resistance genes, can mediate broad-spectrum resistance to most beta-lactams and aminoglycosides.
Asunto(s)
Alcaligenes/efectos de los fármacos , Elementos Transponibles de ADN , Genes Bacterianos , Kanamicina Quinasa/genética , Resistencia betalactámica , beta-Lactamasas/genética , Alcaligenes/enzimología , Alcaligenes/genética , Secuencia de Aminoácidos , Aminoglicósidos , Antibacterianos/farmacología , Secuencia de Bases , Orden Génico , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Plásmidos/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
A system for expression cloning of bacterial phosphatase-encoding genes has been developed, and its potential has been investigated. The system is based on histochemical screening of bacterial genomic libraries, constructed in an Escherichia coli multicopy plasmid vector, for phosphatase-producing clones using an indicator medium (named TPMG) made of Tryptose-Phosphate agar supplemented with the phosphatase substrate phenolphthalein diphosphate and the stain methyl green. To test the performance of this system, three genomic libraries were constructed from bacterial strains of different species which showed different patterns of phosphatase activity, and were screened using the TPMG medium. Following a partial screening, three different phosphatase-encoding genes (respectively encoding a class A non-specific acid phosphatase, an acid-hexose phosphatase and a non-specific alkaline phosphatase) were shotgun-cloned from the above libraries, indicating that the TPMG-based expression cloning system can be useful for rapid isolation of different bacterial phosphatase-encoding genes.
Asunto(s)
Fosfatasa Ácida/genética , Bacterias/enzimología , Clonación Molecular/métodos , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Proteínas Recombinantes de Fusión/metabolismo , Bacterias/efectos de los fármacos , Bacterias/aislamiento & purificación , Técnicas Bacteriológicas , Colorantes/metabolismo , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Biblioteca de Genes , Vectores Genéticos , Verde de Metilo/metabolismo , Fenolftaleínas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Coloración y EtiquetadoRESUMEN
A metallo-beta-lactamase determinant was cloned from a genomic library of Legionella (Fluoribacter) gormanii ATCC 33297(T) constructed in the plasmid vector pACYC184 and transformed into Escherichia coli DH5alpha, by screening for clones showing a reduced susceptibility to imipenem. The product of the cloned determinant, named FEZ-1, contains a 30-kDa polypeptide and exhibits an isoelectric pH of 7.6. Sequencing revealed that FEZ-1 is a molecular-class B beta-lactamase which shares the closest structural similarity (29.7% of identical residues) with the L1 enzyme of Stenotrophomonas maltophilia, being a new member of the highly divergent subclass B3 lineage. All the residues that in L1 are known to be directly or indirectly involved in coordination of the zinc ions were found to be conserved also in FEZ-1, suggesting that the geometry of zinc coordination in the active site of the latter enzyme is identical to that of L1. Unlike L1, however, FEZ-1 appeared to be monomeric in gel permeation chromatography experiments and exhibited a distinctive substrate specificity with a marked preference for cephalosporins and meropenem. The properties of FEZ-1 overall resembled those of a beta-lactamase previously purified from the same strain of L. gormanii (T. Fujii, K. Sato, K. Miyata, M. Inoue, and S. Mitsuhashi, Antimicrob. Agents Chemother. 29:925-926, 1986) and are as yet unique among class B enzymes, reinforcing the notion that considerable functional heterogeneity can be encountered among members of this class. A system for overexpression of the bla(FEZ-1) gene in E. coli, based on the T7 phage promoter, was also developed.