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COVID-19 , Coinfección , Costo de Enfermedad , Salud Global/tendencias , Manejo de Atención al Paciente , Tuberculosis Pulmonar , COVID-19/epidemiología , COVID-19/terapia , COVID-19/transmisión , Coinfección/economía , Coinfección/epidemiología , Coinfección/terapia , Infecciones Comunitarias Adquiridas/epidemiología , Infecciones Comunitarias Adquiridas/prevención & control , Países en Desarrollo , Recursos en Salud , Humanos , Pandemias , Manejo de Atención al Paciente/métodos , Manejo de Atención al Paciente/organización & administración , Manejo de Atención al Paciente/tendencias , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/terapia , Tuberculosis Pulmonar/transmisiónRESUMEN
We earlier documented the involvement of novel Sp-family-like protein factors in transcription from the Autographa californica nucleopolyhedrovirus (AcNPV) polyhedrin (polh) gene promoter [Ramachandran et al. (2001) J. Biol. Chem. 276: 23440-23449]. These zinc-dependent Sp-like factors bind to two putative Sp-factor-binding motifs, present within the AcSp sequence upstream of the polh promoter, with very high affinity (K(d) = 2.1 x 10(-12) M). Like other polh-promoter-associated host transcription factors, these Sp-like factors display tolerance to high ion concentrations up to even 3 M NaCl. An electrophoretic mobility shift assay demonstrated a probable cross-talk between the Spodoptera frugiperda (Sf9) Sp-family-like proteins and the TFIID complex. In complementary experiments, specific replacements of the Sp-factor-binding motifs with TATA-like elements resulted in expression of a luciferase reporter gene to almost the same level as that obtained with a wild-type native construct. Our results point to the possibility of the involvement of TFIID and Sf9 Sp protein interaction in transcription from the baculovirus polyhedrin promoter.
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Proteínas de Insectos , Nucleopoliedrovirus/genética , Factor de Transcripción Sp1/fisiología , Spodoptera/virología , Proteínas Virales/genética , Animales , Secuencia de Bases , Sitios de Unión , Extractos Celulares/análisis , Células Cultivadas , Clonación Molecular , Reactivos de Enlaces Cruzados/metabolismo , ADN Viral/metabolismo , Relación Dosis-Respuesta a Droga , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Genes Virales , Vectores Genéticos , Luciferasas/metabolismo , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión , Plásmidos , Unión Proteica , Cloruro de Sodio/farmacología , Spodoptera/citología , Spodoptera/metabolismo , Rayos Ultravioleta , Proteínas Estructurales Virales/metabolismoRESUMEN
Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.
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Transportadoras de Casetes de Unión a ATP/efectos de los fármacos , Adenosina Trifosfatasas/efectos de los fármacos , Proteínas Bacterianas/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Mycobacterium phlei/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Micobacterias no Tuberculosas/efectos de los fármacos , Tuberculosis Resistente a Múltiples Medicamentos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Humanos , Pruebas de Sensibilidad Microbiana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Mycobacterium phlei/genética , Mycobacterium phlei/fisiología , Mycobacterium tuberculosis/genética , Micobacterias no Tuberculosas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tuberculosis Resistente a Múltiples Medicamentos/genéticaRESUMEN
Primary congenital glaucoma (PCG) has been associated with CYP1B1 gene (2p21), with a predominantly autosomal recessive mode of inheritance. Our earlier studies attributed CYP1B1 mutations to only 40% of Indian PCG cases. In this study, we included 72 such PCG cases where CYP1B1 mutations were detected in only 12 patients in heterozygous condition, implying involvement of other gene(s). On screening these patients for mutations in myocilin (MYOC), another glaucoma-associated gene, using denaturing high-performance liquid chromatography followed by sequencing, we identified a patient who was double heterozygous at CYP1B1 (c.1103G>A; Arg368His) and MYOC (c.144G>T; Gln48His) loci, suggesting a digenic mode of inheritance of PCG. In addition, we identified the same MYOC mutation, implicated for primary open angle glaucoma, in three additional PCG patients who did not harbor any mutation in CYP1B1. These observations suggest a possible role of MYOC in PCG, which might be mediated via digenic interaction with CYP1B1 and/or an yet unidentified locus associated with the disease.
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Proteínas del Ojo/genética , Glaucoma/congénito , Glaucoma/genética , Glicoproteínas/genética , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP1B1 , Proteínas del Citoesqueleto , Femenino , Haplotipos , Humanos , Masculino , Mutación PuntualRESUMEN
BACKGROUND: One mechanism proposed for drug resistance in Mycobacterium tuberculosis (MTB) is by efflux of the drugs by membrane located pumps. We report a novel and definite association between drug resistance and transcription levels of a tap-like pump (Rv1258c) in a multi-drug resistant MTB patient isolate (ICC154) which possesses a unique genotypic signature. MATERIALS AND METHODS: The isolate ICC154 was tested for drug sensitivity. Over-expression of Rv1258c as a function of drug pressure was analyzed by RT-PCR and the strain was typed using fluorescent amplified fragment length polymorhism (FAFLP). RESULT: In the presence of rifampicin and ofloxacin, this isolate shows increased transcription of the gene Rv1258c. Genotypic fingerprinting revealed the presence of unique FAFLP markers. CONCLUSION: A clear association between drug resistance and overexpression of an efflux protein is evident from our studies. The presence of specific markers has implications in rapid identification of MDR clinical isolates and consequent disease management.
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Antibacterianos/farmacología , Proteínas Bacterianas , Proteínas Portadoras/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , ADN Bacteriano/análisis , Farmacorresistencia Bacteriana/genética , Regulación de la Expresión Génica , Genoma , Humanos , Pruebas de Sensibilidad Microbiana , Bombas de Protones/metabolismo , Muestreo , Sensibilidad y Especificidad , Tuberculosis Resistente a Múltiples Medicamentos/diagnóstico , Tuberculosis Resistente a Múltiples Medicamentos/tratamiento farmacológicoRESUMEN
The recent sequencing of the human genome, resulting from two independent global efforts, is poised to revolutionize all aspects of human health. This landmark achievement has also vindicated two differeint methodologies that can now be used to target other important large genomes. The human genome sequence has revealed several novel/surprising features notably the probable presence of a mere 30-35,000 genes. In depth comparisons have led to classification of protein families and identification of several orthologues and paralogues. Information regarding non-protein coding genes as well as regulatory regions has thrown up several new areas of research. Although still incomplete, the sequence is poised to become a boon to pharmaceutical companies with the promise of delivering several new drug targets. Several ethical concerns have also been raised and need to be addressed in earnest. This review discusses all these aspects and dwells on the possible impact of the human genome sequence on human health, medicine and also health care delivery system.
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Genoma Humano , Proyecto Genoma Humano , Biología Molecular , Animales , Secuencia de Bases , Mapeo Cromosómico/métodos , Bases de Datos de Ácidos Nucleicos , Proyecto Genoma Humano/economía , Proyecto Genoma Humano/ética , Humanos , Biología Molecular/ética , Biología Molecular/tendencias , Farmacogenética , Polimorfismo Genético , Proteoma/análisis , Factores de TiempoRESUMEN
CaMDR1 encodes a major facilitator superfamily (MFS) protein in Candida albicans whose expression has been linked to azole resistance and which is frequently encountered in this human pathogenic yeast. In this report we have overexpressed CaMdr1p in Sf9 insect cells and demonstrated for the first time that it can mediate methotrexate (MTX) and fluconazole (FLC) transport. MTX appeared to be a better substrate for CaMdr1p among these two tested drugs. Due to severe toxicity of these drugs to insect cells, further characterization of CaMdr1p as a drug transporter could not be done with this system. Therefore, as an alternative, CaMdr1p and Cdr1p, which is an ABC protein (ATP binding cassette) also involved in azole resistance in C. albicans, were independently expressed in a common hypersensitive host JG436 of Saccharomyces cerevisiae. This allowed a better comparison between the functionality of the two export pumps. We observed that while both FLC and MTX are effluxed by CaMdr1p, MTX appeared to be a poor substrate for Cdr1p. JG436 cells expressing Cdr1p thus conferred resistance to other antifungal drugs but remained hypersensitive to MTX. Since MTX is preferentially transported by CaMdr1p, it can be used for studying the function of this MFS protein.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Candida albicans/metabolismo , Farmacorresistencia Fúngica Múltiple/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antifúngicos/metabolismo , Antifúngicos/farmacología , Antimetabolitos Antineoplásicos/metabolismo , Antimetabolitos Antineoplásicos/farmacología , Sitios de Unión , Transporte Biológico , Candida albicans/efectos de los fármacos , Candida albicans/genética , Línea Celular , Clonación Molecular , Fluconazol/metabolismo , Fluconazol/farmacología , Humanos , Metotrexato/metabolismo , Metotrexato/farmacología , Transformación GenéticaRESUMEN
We earlier documented the involvement of a cellular factor, polyhedrin (polh) promoter-binding protein, in transcription from the Autographa californica nuclear polyhedrosis virus polh gene promoter. Sequences upstream of the polh promoter were found to influence polh promoter-driven transcription. Analysis of one such region, which could partially compensate for the mutated polh promoter and also activate transcription from the wild-type promoter, revealed a sequence (AcSp) containing a CACCC motif and a loose GC box resembling the binding motifs of the transcription factor Sp1. AcSp and the consensus Sp1 sequence (cSp) specifically bound factor(s) in HeLa and Spodoptera frugiperda (Sf9) insect cell nuclear extracts to generate identical binding patterns, indicating the similar nature of the factor(s) interacting with these sequences. The AcSp and cSp oligonucleotides enhanced in vivo expression of a polh promoter-driven luciferase gene. In vivo mopping of these factor(s) significantly reduced transcription from the polh promoter. Recombinant viruses carrying deletions in the upstream AcSp sequence confirmed the requirement of these factor(s) in polh promoter-driven transcription in the viral context. We demonstrate for the first time DNA-protein interactions involving novel members of the Sp family of proteins in adult insect cells and their involvement in transcription from the polh promoter.
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Proteínas de Insectos , Nucleopoliedrovirus/genética , Factor de Transcripción Sp1/fisiología , Spodoptera/virología , Proteínas Virales/genética , Animales , Secuencia de Bases , Unión Competitiva , Extractos Celulares/análisis , Núcleo Celular/metabolismo , Secuencia de Consenso , ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Genes Virales , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Proteínas de la Matriz de Cuerpos de Oclusión , Regiones Promotoras Genéticas , Spodoptera/metabolismo , Transcripción Genética , Proteínas Estructurales Virales , Zinc/metabolismoRESUMEN
In this paper we report the successful expression of the winged bean basic agglutinin (WBA I) in insect cells infected with a recombinant baculovirus carrying the WBA I gene and its characterization in terms of its carbohydrate binding properties. The expressed protein appears to have a lower molecular weight than the native counterpart which is consistent with the lack of glycosylation of the former. Moreover, the expressed protein maintains its dimeric nature. Hence, a role for glycosylation in modulation of dimerization of WBA I is ruled out unlike Erythrina corallodendron (EcorL). Despite this the protein is active, with its sugar specificity unaltered.
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Baculoviridae/genética , Células Eucariotas/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Vectores Genéticos , Lectinas/genética , Lectinas de Plantas , Spodoptera/genética , Animales , Sitios de Unión/genética , Metabolismo de los Hidratos de Carbono , Células Cultivadas , Dimerización , Ingeniería Genética/métodos , Glicosilación , Lectinas/metabolismo , Peso Molecular , Regiones Promotoras Genéticas/fisiología , Unión Proteica/genética , Proteínas Recombinantes de Fusión/genética , Spodoptera/metabolismo , TransfecciónRESUMEN
The HBe negative phenotype, a natural precore mutant (G1896A/G1897A) of HBV with aborted HBeAg expression is known to cause chronic hepatitis. The destabilized C : G base-pairing in the lower stem of epsilon-hairpin due to G1896A substitution is reportedly compensated by a second C1858T mutation and suggested to play an important role in enhanced selection of the HBe negative variant. We undertook to investigate presence of such compensatory mutations at other positions by analyzing epsilon-sequences (nts. 1847-1907) as well as to look for their effect(s), if any, on the consensus sequence of the overlapping core-initiator of HBe negative HBV variants in CLD patients. Three of the 5 HBe negative patients had classical G1896A mutation having a second compensatory mutation at nt. 1858. One patient showed an additional G1897A substitution, presenting as a novel precore stop codon mutation (UGG-->UAA), followed by a compensatory mutation at position 1857. In the third patient, a G1899A substitution was seen which compensated the impaired U at position 1855. Other substitution and deletion mutations were also observed in the remaining epsilon-hairpin, which however, did not produce any compensatory mutation. Further, all the precore variants showed a conserved G at position 1904, important for the optimal context of their core-initiator which however, remained impaired with A (nt. 1850). Our results suggest that the nts. 1851-1859 and nts. 1895-1904 in the lower stem, and restoration of authentic base-pairings therein, maintain the structural integrity and stability of the epsilon-hairpin. This may have a role in the enhanced selection of the HBe negative variants and persistence of HBV infection in chronic liver disease patients.
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Antígenos e de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Mutación , Emparejamiento Base , Secuencia de Bases , Codón sin Sentido , ADN Viral , Hepatitis B/virología , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ARNRESUMEN
We investigated IS6110 polymorphism in clinical isolates of Mycobacterium tuberculosis from patients attending the outpatient department at various hospitals in northern India. DNA fingerprinting of 126 clinical isolates of M. tuberculosis was carried out using restriction fragment length polymorphism (RFLP) associated with the IS6110 element in M. tuberculosis genomes. A substantive degree of polymorphism was evident in the MDR M. tuberculosis isolates. The number of bands in the fingerprints varied from 0 to 19. However, the lack of common bands made it difficult to cluster the majority of these isolates. We were also unable to associate drug resistance with IS6110 copy number. Specific regions of the gyrA and katG genes from a representative number of these isolates were sequenced to determine the genotype. The majority of the isolates analyzed were found to belong to Group 1, indicating that these strains were evolutionarily older. We find no evidence of the W strain, prevalent in the US, in our study. The epidemiological patterns of the various strains in India seem to be very complex, as reflected by the presence of a large number of different strains (types) within north India.
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Antituberculosos/farmacología , Elementos Transponibles de ADN/genética , Farmacorresistencia Bacteriana Múltiple/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Polimorfismo Genético/genética , Tuberculosis/microbiología , Dermatoglifia del ADN , Evolución Molecular , Humanos , India , Mycobacterium tuberculosis/efectos de los fármacos , Fenotipo , Filogenia , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
Phosphorylation of serine 51 residue on the alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha) inhibits the guanine nucleotide exchange (GNE) activity of eIF2B, presumably, by forming a tight complex with eIF2B. Inhibition of the GNE activity of eIF2B leads to impairment in eIF2 recycling and protein synthesis. We have partially purified the wild-type (wt) and mutants of eIF2alpha in which the serine 51 residue was replaced with alanine (51A mutant) or aspartic acid (51D mutant) in the baculovirus system. Analysis of these mutants has provided novel insight into the role of 51 serine in the interaction between eIF2 and eIF2B. Neither mutant was phosphorylated in vitro. Both mutants decreased eIF2alpha phosphorylation occurring in hemin and poly(IC)-treated reticulocyte lysates due to the activation of double-stranded RNA-dependent protein kinase (PKR). However, addition of 51D, but not 51A mutant eIF2alpha protein promoted inhibition of the GNE activity of eIF2B in hemin-supplemented rabbit reticulocyte lysates in which relatively little or no endogenous eIF2alpha phosphorylation occurred. The 51D mutant enhanced the inhibition in GNE activity of eIF2B that occurred in hemin and poly(IC)-treated reticulocyte lysates where PKR is active. Our results show that the increased interaction between eIF2 and eIF2B protein, occurring in reticulocyte lysates due to increased eIF2alpha phosphorylation, is decreased significantly by the addition of mutant 51A protein but not 51D. Consistent with the idea that mutant 51D protein behaves like a phosphorylated eIF2alpha, addition of this partially purified recombinant subunit, but not 51A or wt eIF2alpha, increases the interaction between eIF2 and 2B proteins in actively translating hemin-supplemented lysates. These findings support the idea that phosphorylation of the serine 51 residue in eIF2alpha promotes complex formation between eIF2alpha(P) and eIF2B and thereby inhibits the GNE activity of eIF2B.
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Factor 2B Eucariótico de Iniciación/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Serina/metabolismo , Alanina/genética , Animales , Ácido Aspártico/genética , Baculoviridae/genética , Sistema Libre de Células/metabolismo , Factor 2 Eucariótico de Iniciación/biosíntesis , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/inmunología , Factor 2B Eucariótico de Iniciación/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/metabolismo , Hemina/metabolismo , Humanos , Masculino , Nucleopoliedrovirus/genética , Nucleopoliedrovirus/metabolismo , Fosforilación , Poli I-C/metabolismo , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Reticulocitos/metabolismo , Spodoptera/genética , Spodoptera/metabolismoRESUMEN
Genomic changes are a hallmark of the neoplastic process. These range from alterations at specific loci and defined karyotypic changes which influence tumor behavior to generalized alterations exemplified by microsatellite instability. Generalized genomic changes within a tumor would be evidence in favor of the mutator hypothesis which postulates a role for such extensive changes during tumorigenesis. In this report, we have used the DNA fingerprinting technique of randomly amplified polymorphic DNA (RAPD) analysis to study genomic alterations within primary human astrocytic tumors (gliomas) in a locus non-specific manner. The RAPD fingerprinting profile of consecutive segments of tumors 2 mm across was studied; 17 astrocytic (high- and low-grade) tumors were sectioned end to end. Tissue from 50 consecutive sections, 40 microm thick (total 2 mm across), was pooled and taken to be a tumor compartment. DNA was subjected to RAPD amplification by 15 random 10-mer primers. A tumor segment was taken to have a DNA fingerprinting pattern different from others in the same specimen when its RAPD profile differed from others by at least one band of one RAPD reaction. All but one of the tumors showed compartments with a unique genetic profile, indicating genomic instability leading to widespread intra-tumor genetic heterogeneity. Eight tumors were also studied for loss of heterozygosity (LOH) of the p53 and D17S379 loci in the different segments as examples of alteration of specific tumor influencing loci. Three showed LOH of p53, which was limited to only one compartment of each tumor. The extensive intra-tumor genetic instability detected in this study is suggestive of the overall high rate of change in the genomes of tumors including those of a lower grade. It is hypothesized that some of these altered clones, which manifest as zones of heterogeneity in a solid tumor, may accumulate changes at loci known to influence tumor behavior, and thus clinical outcome.
Asunto(s)
Neoplasias Encefálicas/genética , Heterogeneidad Genética , Glioblastoma/genética , Dermatoglifia del ADN , Humanos , Pérdida de Heterocigocidad , Técnica del ADN Polimorfo Amplificado Aleatorio , Proteína p53 Supresora de Tumor/genéticaRESUMEN
BACKGROUND/AIMS: HBV-related chronic liver disease patients often present with hepatic decompensation and are not eligible for interferon therapy. Whether long-term lamivudine is effective in these patients was prospectively evaluated. METHODS: Eighteen patients with HBV-related decompensated cirrhosis, all with quantitative DNA +ve and 10 HBeAg +ve, were given lamivudine 150 mg/d. RESULTS: Each patient received at least 9 months (mean 17.9) of lamivudine. Three HBeAg+ve patients (30%) seroconverted to anti-HBe and one lost HBsAg during the follow-up. An improvement from baseline in the aspartate aminotransferase (130 vs. 72 IU/l, p<0.04); alanine aminotransferase (111 vs. 58 IU/l, p<0.01) and Child-Pugh score (8.3 vs 6.7, p<0.013) was seen. Lamivudine had no significant side-effects. HBV DNA became undetectable in all patients by 8 weeks of therapy. In three (17%) patients, HBV DNA again became positive at 9, 9 and 27 months. YMDD mutant was, however, detected in only one (6%). A significant reduction was noted in the morbidity and hospitalizations for complications of liver disease before and after starting lamivudine (1.5+/-0.7 vs. 0.6+/-0.7, p<0.002). CONCLUSIONS: In decompensated HBV-related cirrhosis, lamivudine: i) is effective in suppressing HBV DNA and seroconversion to anti-HBe (30%), ii) can achieve significant improvement in clinical and biochemical status of liver functions.
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Hepatitis B Crónica/complicaciones , Lamivudine/administración & dosificación , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/etiología , Inhibidores de la Transcriptasa Inversa/administración & dosificación , Administración Oral , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
The identification of potential baculovirus origins of replication (ori) has involved the generation and characterization of defective interfering particles that contain major genomic deletions yet retain their capability to replicate by testing the replication ability of transiently transfected plasmids carrying viral sequences in infected cells. So far, there has not been any evidence to demonstrate the actual utilization of these putative origins in Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) replication. By using the method of origin mapping by competitive PCR, we have obtained quantitative data for the ori activity of the HindIII-K region and the ie-1 promoter sequence in AcMNPV. We also provide evidence for differential activity of the two ori in the context of the viral genome through the replication phase of viral infection. Comparison of the number of molecules representing the HindIII-K and ie-1 origins vis-à-vis the non-ori polH region in a size-selected nascent DNA preparation revealed that the HindIII-K ori is utilized approximately 14 times more efficiently than the ie-1 region during the late phase of infection. HindIII-K also remains the more active ori through the early and middle replication phases. Our results provide in vivo evidence in support of the view that AcMNPV replication involves multiple ori that are activated with vastly different efficiencies during the viral infection cycle.
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ADN Viral , Proteínas de Unión al ADN , Nucleopoliedrovirus/genética , Origen de Réplica , Replicación Viral , Animales , Línea Celular , Desoxirribonucleasa HindIII , Genoma Viral , Proteínas Inmediatas-Precoces/genética , Mariposas Nocturnas/virología , Nucleopoliedrovirus/fisiología , Proteínas de la Matriz de Cuerpos de Oclusión , Reacción en Cadena de la Polimerasa/métodos , Spodoptera/citología , Transactivadores/genética , Proteínas Virales/genética , Proteínas Estructurales ViralesRESUMEN
A synthetic gene encoding twelve B cell epitopes, six T-cell proliferative epitopes, and three cytotoxic T lymphocyte (CTL) epitopes from nine stage-specific antigens, representing the sporozoite, liver stage, asexual blood-stage, and sexual-stage antigens of Plasmodium falciparum, was constructed by assembling overlapping oligonucleotides followed by PCR extension and annealing. A three-step PCR protocol using twelve long oligonucleotides was employed to generate a 1053 base-pair synthetic gene, the identity of which was confirmed by sequencing. This synthetic gene, named CDC/NII MAL VAC-1, was cloned, and the recombinant protein was expressed in the Baculovirus Expression Vector System (BEVS). The selection of malarial epitopes for inclusion in this vaccine construct was based on immunoepidemiological studies in malaria endemic area, in vitro, and in vivo protection studies in model systems. The 41 kDa BEVS-expressed recombinant protein reacted with mouse antibodies specific for individual B cell epitopes in the vaccine construct and with sera from clinically immune Kenyan adults. An immunization study in three strains of mice that differ at the H-2 locus demonstrated that the BEVS-expressed recombinant protein is immunogenic; the candidate vaccine antigen induced high titer antibodies, and lymphocyte proliferative and IFN-gamma responses. These results demonstrate that individual B and T cell epitopes can be assembled to create synthetic genes that encode proteins capable of eliciting specific antibody and T cell responses.