RESUMEN
Deinococcus radiodurans is extremely resistant to ionizing radiation. How this bacterium can grow under chronic gamma radiation [50 grays (Gy) per hour] or recover from acute doses greater than 10 kGy is unknown. We show that D. radiodurans accumulates very high intracellular manganese and low iron levels compared with radiation-sensitive bacteria and that resistance exhibits a concentration-dependent response to manganous chloride [Mn(II)]. Among the most radiation-resistant bacterial groups reported, Deinococcus, Enterococcus, Lactobacillus, and cyanobacteria accumulate Mn(II). In contrast, Shewanella oneidensis and Pseudomonas putida have high iron but low intracellular manganese concentrations and are very sensitive. We propose that Mn(II) accumulation facilitates recovery from radiation injury.
Asunto(s)
Deinococcus/efectos de la radiación , Manganeso/fisiología , Tolerancia a Radiación/fisiología , Medios de Cultivo , Reparación del ADN , ADN Bacteriano , Deinococcus/fisiología , Deinococcus/ultraestructura , Hierro/fisiología , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
PURPOSE: Antisense radiotherapy is an approach based on the targeting of mRNA of specific genes by complementary oligonucleotide probes labelled with an Auger-electron-emitting radioisotope. Decay of the Auger emitter should specifically destroy the targeted mRNA while producing minimal damage to the rest of mRNA pool and the nuclear DNA. The feasibility of this approach was investigated by using full-length human multidrug-resistance gene (mdr1) mRNA as a target. MATERIALS AND METHODS: Antisense oligonucleotides were labelled with [125I] I-dCTP by primer extension and annealed to target mRNA. Breaks in the target mRNA were analysed by denaturing polyacrylamide gel electriphoresis. RESULTS: The efficiency of 125I-labelled antisense oligonucleotides in producing RNA strand breaks was tested on short synthetic RNA and DNA targets. The position and specificity of 125I-induced breaks in the full-length mRNA were then tested and compared with the cleavage of the target by RNase H. The distribution of the breaks in the longer mRNA is different from that in the short RNA targets, most likely due to a complex folding of RNA strands in the full-length mRNA. CONCLUSIONS: The authors posit that 125I-labelled antisense probes could be useful not only for targeting mRNA, but also as probes for mRNA folding in vivo.
Asunto(s)
Nucleótidos de Desoxicitosina/química , Marcación de Gen/métodos , Radioisótopos de Yodo/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , ARN sin Sentido/química , ARN Mensajero/química , ARN Mensajero/efectos de la radiación , Nucleótidos de Desoxicitosina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Estudios de Factibilidad , Silenciador del Gen/efectos de la radiación , Radioisótopos de Yodo/administración & dosificación , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de la radiación , Estabilidad del ARN/efectos de la radiación , ARN sin Sentido/administración & dosificación , ARN Mensajero/genética , Radiofármacos/administración & dosificación , Radiofármacos/químicaRESUMEN
The aim of this study was to evaluate the possibility of detecting nonsense and frame-shift mutations in exon 11 of brca1 gene by constructing fusion open reading frame (ORF) "exon 11 ORF-alpha-peptide of beta-galactosidase". The ability/inability of this newly constructed ORF to cause alpha-complementation in E. coli delta M15gal cells transformed by the plasmid with the ORF may reflect the absence/presence of nonsense and frame-shift mutations in the studied fragment. A single ORF fragment of exon 11 of brca1 gene--LacZ' gene was designed in pGEN7Zf plasmid, the plasmid was shown to cause Lac+ phenotype in E. coli delta M15gal. Four frame-shift deletion mutations were introduced into exon 11 sequence in the plasmid. Surprisingly, the frame-shift deletion mutations did not influence the ability of plasmids to induce Lac+ phenotype in E. coli delta M15gal in 3 cases and only one deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal. We suppose that the phenomenon can be explained by the alpha-peptide translation reinitiation from inframe ATG codons situated within the exon 11 sequence. Seven inframe ATG sequences were found in exon 11, at least two in-frame ATG-containing fragments were demonstrated to cause reinitiation. On the other hand, the only deletion mutation resulted in inability of the plasmid to form Lac+ phenotype in E. coli delta M15gal did not leave LacZ' in-frame ATG in econ 11 sequence. We conclude that it is possible to detect frame-shift mutations by in-frame cloning with the LacZ' reporter gene, but this possibility is strongly impeded by the reinitiation of alpha-peptide translation from the in-frame ATG codons within the studied sequence.
Asunto(s)
Proteína BRCA1/genética , Codón sin Sentido , Análisis Mutacional de ADN/métodos , Mutación del Sistema de Lectura , Proteínas Recombinantes de Fusión/genética , beta-Galactosidasa/genética , Escherichia coli/genética , Exones , Humanos , Plásmidos , Biosíntesis de ProteínasRESUMEN
Selective targeting of therapeutic and diagnostic agents improves their efficacy and minimizes potentially adverse side effects. Existing methods for selective targeting are based on chemical conjugation of therapeutics and diagnostics, or their carriers, to cell-specific targeting molecules (e.g., growth factors, antibodies). These methods are limited by potential damage to targeting molecules that can be inflicted by the conjugation procedure. In addition, conjugation procedures have to be developed on a case-by-case basis. In order to avoid these problems we have developed a new approach to constructing molecular vehicles for target-mediated delivery of therapeutics and diagnostics. In this approach, the targeting molecule is expressed as a fusion protein containing a recognition tag. The recognition tag is defined as a peptide or protein that can bind non-covalently another peptide or protein (adapter). In turn, the adapter is chemically conjugated to a carrier of therapeutics or diagnostics. The assembled molecular delivery vehicle contains a carrier-adapter conjugate bound non-covalently to a recognition tag fused to the targeting protein. The advantages of this technology are: (i) no chemical modification of targeting molecules, and (ii) universal, 'off-the-shelf' carrier-adapter constructs that can be combined with different fusion targeting proteins. To obtain a proof-of-principle we have constructed VEGF fusion proteins containing a 15-aa S-peptide fragment of RNase A as a recognition tag. Using the S-protein fragment of RNase A as an adapter and polyethylenimine as a DNA carrier we have achieved selective gene delivery to cells overexpressing VEGFR-2.
Asunto(s)
Sistemas de Liberación de Medicamentos , Factores de Crecimiento Endotelial/administración & dosificación , Linfocinas/administración & dosificación , Neoplasias/diagnóstico , Neoplasias/terapia , Animales , Factores de Crecimiento Endotelial/farmacocinética , Terapia Genética , Humanos , Luciferasas/biosíntesis , Luciferasas/genética , Linfocinas/farmacocinética , Polietileneimina , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de Factores de Crecimiento/genética , Receptores de Factores de Crecimiento/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/farmacocinética , Ribonucleasa Pancreática/metabolismo , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
Phenobarbital-dependent protein binding was shown to occur to DNA fragments from the coding region of the cytochrome P450BM-3 gene from Bacillus megaterium. Incubation of the DNA fragments from the coding region of the gene with total cell extract from Bacillus megaterium revealed two DNA regions with protein-binding capacity: +237/+318 and +319/+425 considering 'O' as the start of cytochrome P450BM-3 translation. DNaseI footprint analysis of the fragment +319/+425 with the total cell extract showed that some protein(s) protected DNA stretches from the position +373 up to the position +389 on the transcribed strand and from the position +378 up to the position +398 on the non-transcribed strand. DNaseI footprint analysis of the fragment +237/+318 revealed the protection in the region +262/+277 on the non-transcribed strand. Three regions protected by cell extract protein(s) from DNaseI hydrolysis (+262/+277, +373/+389 and +378/+398) appeared to be strongly homologous to the Barbie box sequence. Barbie-box-like sequences were found in the majority of regulatory regions of phenobarbital-inducible genes whose regulatory sequences had been reported (Fulco et al., 1994). Our results suggest that a functional role of Barbie box sequence takes place not only in regulatory but also in the coding region of the gene. In line with that hypothesis we analyzed all cytochrome P450 genes in respect to the presence of Barbie box-like sequences in their coding parts. At least one cytochrome P450 gene (CYP6A1, phenobarbital-inducible gene from Musca domestica) was shown to contain Barbie box sequence in the coding part of the gene.
Asunto(s)
Bacillus megaterium/genética , Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Genes Bacterianos/genética , Oxigenasas de Función Mixta/genética , Fenobarbital/farmacología , Bacillus megaterium/metabolismo , Huella de ADN , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , NADPH-Ferrihemoproteína Reductasa , Unión Proteica , Secuencias Reguladoras de Ácidos Nucleicos/genética , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Protein-binding DNA-regions inside and in the vicinity of the phenobarbital-inducible P450bm-3 gene from Bacillus megaterium were characterized by gel-retardation technique and foot-printing analysis. Regions with induction-dependent protein binding capacity were shown to be situated in -279bp/-215bp, -215bp/+83bp and +236bp/+425bp fragments. Precise localization of DNA-protein contacts was established by foot-printing analysis for region -215bp/+83bp. The data obtained are discussed in line with previously reported information on the regulatory regions of various phenobarbital-regulated genes.
Asunto(s)
Bacillus megaterium/enzimología , Bacillus megaterium/genética , Proteínas Bacterianas , Sistema Enzimático del Citocromo P-450/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Oxigenasas de Función Mixta/genética , Sitios de Unión , ADN Bacteriano/aislamiento & purificación , Desoxirribonucleasa I , Genes Bacterianos , NADPH-Ferrihemoproteína Reductasa , Mapeo Restrictivo , Transcripción GenéticaRESUMEN
Two genomic clones, pC1.2 and p20D (containing inserts of 2.0 and 1.6 kb, respectively) were isolated from the A2b region to polytene chromosome IV of Chironomus thummi thummi salivary gland cells. Upon in situ hybridization to polytene chromosomes of C. thummi thummi and C. thummi piger, p20D DNA hybridized mainly over the A2b region of chromosome IV, whereas pC1.2 DNA hybridized to at least 90 sites distributed over all the chromosomes. A partial nucleotide sequence analysis showed that these clones were very similar and allowed the detection of a 596 bp insert in the pC1.2 clone. This insert possesses all of the essential features of a Class II transposable element and was called MEC. It carries a nearly perfect 107 bp terminal inverted repeat containing one mismatch and is flanked by a 5 bp direct repeat. The 372 bp central region contains a short open reading frame with a coding capacity of 58 amino acids.