RESUMEN

One osmotolerant strain from among 44 yeast isolates was selected based on its growth abilities in media containing high concentrations of sucrose. This selected strain, named SKENNY, was identified as Meyerozyma guilliermondii by sequencing the internal transcribed spacer regions and partial D1/D2 large-subunit domains of the 26S ribosomal RNA. SK-ENNY was utilized to produce high-fructose glucose syrup (HFGS) from sucrose-containing biomass. Conversion rates to HFGS from 310-610 g/l of pure sucrose and from 75-310 g/l of sugar beet molasses were 73.5-94.1% and 76.2-91.1%, respectively. In the syrups produced, fructose yields were 89.4-100% and 96.5-100% and glucose yields were 57.6-82.5% and 55.3-79.5% of the theoretical values for pure sucrose and molasses sugars, respectively. This is the first report of employing M. guilliermondii for production of HFGS from sucrose-containing biomass.

Asunto(s)

Biomasa , Fermentación , Microbiología de Alimentos/métodos , Jarabe de Maíz Alto en Fructosa , Sacarosa/análisis , Levaduras/fisiología , Beta vulgaris/química , Medios de Cultivo , Microbiología de Alimentos/economía , Fructosa/análisis , Glucosa/análisis , Melaza/microbiología , ARN Ribosómico , Sacarosa/metabolismo , Levaduras/genética

RESUMEN

The human antiretroviral factor APOBEC3G (A3G) deaminates the newly synthesized minus strand of the human immunodeficiency virus 1 (HIV-1), which results in the abolition of the infectivity of virus-infectivity-factor (Vif)-deficient HIV-1 strains.1-6 A unique property of A3G is that it deaminates a CCC hot spot that is located close to the 5' end more effectively than one that is less close to the 5' end. However, the mechanism of this process is elusive as it includes nonspecific binding of A3G to DNA and sliding of A3G along the DNA strand. Therefore, this process cannot be analyzed by existing methods using the Michaelis-Menten theory. A new real-time NMR method has been developed to examine the nonspecific binding and the sliding processes explicitly, and it was applied to the analysis of the deamination by A3G. As a result, the location-dependent deamination can be explained by a difference in the catalytic rates that depend on the direction of the approach of A3G to the target cytidine. Real-time NMR experiments also showed that A3G deaminates CCCC tandem hotspots with little redundancy, which suggests that A3G efficiently mutates many CCC hotspots that are scattered throughout the HIV-1 genome.

Asunto(s)

Citidina Desaminasa/metabolismo , ADN de Cadena Simple/metabolismo , Resonancia Magnética Nuclear Biomolecular/métodos , Desaminasa APOBEC-3G , Secuencia de Bases , ADN de Cadena Simple/química , Desaminación , Infecciones por VIH/metabolismo , VIH-1/fisiología , Interacciones Huésped-Patógeno , Humanos , Unión Proteica

RESUMEN

The addressable DNA nanostructures offer ideal platforms to construct organized assemblies of multiple protein molecules. Sequence-specific DNA binding proteins that target defined sites on DNA nanostructures would act as orthogonal adaptors to carry individual protein molecules to the programmed addresses. We have recently developed a protein-based adaptor by utilizing the sequence-specific DNA binding zinc finger protein to locate a monomeric protein of interest at specific positions on DNA origami, which serves as a molecular switchboard. We herein report a new adaptor to locate a protein dimer on the DNA origami scaffold based on a homodimeric basic-leucine zipper protein GCN4. Specific binding of GCN4 to programmed addresses on DNA origami and orthogonal targeting by GCN4- and zinc finger protein-based adaptors to the respective addresses on DNA origami were confirmed by gel electrophoretic and AFM analyses. Furthermore, a GCN4-fused homodimeric enzyme showed even higher activity than the wild type enzyme, and exhibited avid reactivity when assembled at the specific site of DNA origami. Thus, GCN4 serves as an ideal adaptor to locate homodimeric proteins in the functional form on DNA origami.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Ácidos Nucleicos Inmovilizados/química , Sitios de Unión , Microscopía de Fuerza Atómica , Nanoestructuras/química , Unión Proteica

RESUMEN

The xylose-fermenting recombinant Saccharomyces cerevisiae and its improvement have been studied extensively. The redox balance between xylose reductase (XR) and xylitol dehydrogenase (XDH) is thought to be an important factor in effective xylose fermentation. Using protein engineering, we previously successfully reduced xylitol accumulation and improved ethanol production by reversing the dependency of XDH from NAD(+) to NADP(+). We also constructed a set of novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis. In the present study, we constructed a set of recombinant S. cerevisiae carrying a novel set of mutated strictly NADPH-dependent XR and NADP(+)-dependent XDH genes with overexpression of endogenous xylulokinase (XK) to study the effects of complete NADPH/NADP(+) recycling on ethanol fermentation and xylitol accumulation. All mutated strains demonstrated reduced xylitol accumulation, ranging 34.4-54.7% compared with the control strain. Moreover, compared with the control strain, the two strains showed 20% and 10% improvement in ethanol production.

Asunto(s)

Aldehído Reductasa/genética , Biocombustibles , D-Xilulosa Reductasa/genética , Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Aldehído Reductasa/metabolismo , Biotecnología/métodos , D-Xilulosa Reductasa/metabolismo , Etanol/análisis , Fermentación , Glucosa/metabolismo , NADP/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Xilitol/metabolismo , Xilosa/metabolismo

RESUMEN

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP(C)) of PrP into an abnormal form (PrP(Sc)) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP(C) is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP(Sc) level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP(C), resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.

Asunto(s)

Aptámeros de Nucleótidos/química , Proteínas PrPC/química , Animales , Aptámeros de Nucleótidos/farmacología , Bovinos , Línea Celular , Ratones , Modelos Moleculares , Péptidos/química , Proteínas PrPSc/análisis

RESUMEN

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially (15)N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [(1)H, (15)N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR=1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.

Asunto(s)

Péptido Hidrolasas/química , Proteínas no Estructurales Virales/química , Virus Relacionado con el Virus Xenotrópico de la Leucemia Murina/enzimología , Carbamatos/química , Descubrimiento de Drogas , Furanos , Inhibidores de la Proteasa del VIH/química , VIH-1/química , VIH-1/enzimología , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Sulfonamidas/química

RESUMEN

The structure of r(GGAGGAGGAGGA) (R12) changes from a single-stranded form to a compact quadruplex one in response to K(+). In a hammerhead ribozyme, two portions of the catalytic core are linked with the stem and are located in close proximity in order to exert activity. In this study, the stem was replaced by R12 (or R11, which lacks the terminal A residue) with or without linker residues. One of the newly constructed ribozymes exhibited enhanced activity in response to K(+), and we suggest that quadruplex formation restored the active catalytic core. Other ribozymes exhibited repressed activity in response to K(+), suggesting that formation of the active core was prevented. Thus, we have succeeded in developing 'intelligent' ribozymes whose activity is either repressed or enhanced in response to K(+). This switching capability may have therapeutic applications because of the differences between intra- and extracellular K(+) concentrations.

Asunto(s)

Conformación de Ácido Nucleico/efectos de los fármacos , Potasio/farmacología , ARN Catalítico/química , ARN Catalítico/metabolismo , Secuencia de Bases , Dominio Catalítico , Cinética , Modelos Moleculares , Datos de Secuencia Molecular

RESUMEN

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only.

Asunto(s)

D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismo , Aerobiosis , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , D-Xilulosa Reductasa/biosíntesis , D-Xilulosa Reductasa/genética , Fermentación , Expresión Génica , Genes Fúngicos , Ingeniería Metabólica/métodos , Mutación/genética , NAD/genética , NAD/metabolismo , NADP/genética , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Pichia/enzimología , Pichia/genética , Pichia/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Transaldolasa/genética , Transaldolasa/metabolismo , Xilitol/genética , Xilitol/metabolismo , Xilosa/genética

RESUMEN

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD(+) to NADP(+). In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.

Asunto(s)

Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Pichia/enzimología , Ingeniería de Proteínas , Xilosa/metabolismo , Clonación Molecular , D-Xilulosa Reductasa/metabolismo , Fermentación , Mutagénesis Sitio-Dirigida , Mutación

RESUMEN

Oxanine (Oxa), generated from guanine (Gua) by NO- or HNO(2)-induced nitrosative oxidation, has been thought to cause mutagenic problems in cellular systems. In this study, the response of Oxa to different enzymatic functions was explored to understand how similarly it can participate in biomolecular reactions compared to the natural base, Gua. The phosphorylation efficiency of the T4 polynucleotide kinase was highest when Oxa was located on the 5'-end of single stranded DNAs compared to when other nucleobases were in this position. The order of phosphorylation efficiency was as follows; Oxa>Gua>adenine (Ade) approximately thymine (Thy)>cytosine (Cyt). Base-pairing of Oxa and Cyt (Oxa:Cyt) between the ligation fragment and template was found to influence the ligation performance of the T4 DNA ligase to a lesser degree compared to Gua:Cyt. In addition, EcoRI and BglII showed higher cleavage activities on DNA substrates containing Oxa:Cyt than those containing Gua:Cyt, while BamHI, HindIII and EcoRV showed lower cleavage activity; however, this decrease in activity was relatively small.

Asunto(s)

ADN Ligasas/química , Enzimas de Restricción del ADN/química , ADN de Cadena Simple/química , Polinucleótido 5'-Hidroxil-Quinasa/química , Nucleósidos de Purina/química , Emparejamiento Base , Citosina/química , División del ADN , ADN de Cadena Simple/genética , Fosforilación , Nucleósidos de Purina/genética

RESUMEN

Bioethanol production from xylose is important for utilization of lignocellulosic biomass as raw materials. The research on yeast conversion of xylose to ethanol has been intensively studied especially for genetically engineered Saccharomyces cerevisiae during the last 20 years. S. cerevisiae, which is a very safe microorganism that plays a traditional and major role in industrial bioethanol production, has several advantages due to its high ethanol productivity, as well as its high ethanol and inhibitor tolerance. However, this yeast cannot ferment xylose, which is the dominant pentose sugar in hydrolysates of lignocellulosic biomass. A number of different strategies have been applied to engineer yeasts capable of efficiently producing ethanol from xylose, including the introduction of initial xylose metabolism and xylose transport, changing the intracellular redox balance, and overexpression of xylulokinase and pentose phosphate pathways. In this review, recent progress with regard to these studies is discussed, focusing particularly on xylose-fermenting strains of S. cerevisiae. Recent studies using several promising approaches such as host strain selection and adaptation to obtain further improved xylose-utilizing S. cerevisiae are also addressed.

Asunto(s)

Etanol/metabolismo , Ingeniería Genética , Microbiología Industrial/métodos , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismo , Fermentación , Saccharomyces cerevisiae/genética

RESUMEN

High stability of the oligonucleotides immobilized on the glass is essential for the reliable DNA microarray analysis. In the present study, effect of end-capping of the unreacted silanol, remaining after the surface amine-functionalization, was explored: (1) Cy3-NHS (N-hydorxysuccinincimide) dye was spotted on the surface and change in the fluorescent intensity was measured. (2) DNA probes were immobilized by the reactivity of oxanine linked at the 5'-end, the complementary oligonucleotides with Cy5-fluorescence at the 5'-end was hybridized, and the time-dependence of the fluorescence intensity was observed. Both the systems showed improved stability of the immobilized molecules, indicative of the stabilization by end-capping.

Asunto(s)

Sondas de ADN/química , ADN/química , Vidrio , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Silanos/química , Estructura Molecular

RESUMEN

Hypoxanthine (Hyp), a deaminated base of adenine (Ade), can be employed as a good probe molecule to reveal the significance of the minor groove of guanine (Gua) in biomolecular interactions because Hyp possesses a similar structure to Gua lacking its 2-amino group. In this study, we examined cleavage efficiencies of restriction endonuclease enzymes on DNA substrates with Hyp in their recognition sequences. As a substrate for BglII, EcoRI and BamHI, 24-mer DNA oligomer with Hyp (in place of Gua) was prepared together with its complementary sequences with cytosine (Cyt) or thymine (Thy) as the counter base. At 37 degrees C incubation for 1 h, BglII and EcoRI showed higher DNA cleavage reactivity on Hyp-containing DNA substrates than on normal ones, whereas BamHI showed lower values on Hyp-containing substrates. Such high cleavage performance of BglII and EcoRI on Hyp-containing DNA substrates is in contrast to the results obtained 20 years ago, in which short DNA substrates (8- or 10-mer) and low reaction temperatures (15-20 degrees C) were employed. These new results suggest that the lack of the exocyclic 2-amino group of Gua could contribute to enhanced recognition access of BglII and EcoRI to DNA substrates.

Asunto(s)

Proteínas Bacterianas/química , División del ADN , Enzimas de Restricción del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/química , Hipoxantina/química , Secuencia de Bases , Enzimas de Restricción del ADN/química , Desoxirribonucleasa BamHI/química , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa EcoRI/química , Desoxirribonucleasa EcoRI/metabolismo , Electroforesis en Gel de Poliacrilamida , Enlace de Hidrógeno , Cinética , Datos de Secuencia Molecular , Estructura Molecular

RESUMEN

The recombinant industrial Saccharomyces cerevisiae strain MA-R5 was engineered to express NADP(+)-dependent xylitol dehydrogenase using the flocculent yeast strain IR-2, which has high xylulose-fermenting ability, and both xylose consumption and ethanol production remarkably increased. Furthermore, the MA-R5 strain produced the highest ethanol yield (0.48 g/g) from nonsulfuric acid hydrolysate of wood chips.

Asunto(s)

D-Xilulosa Reductasa/genética , D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Cromosomas Fúngicos/genética , NADP/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Madera/metabolismo , Xilosa/metabolismo

RESUMEN

Xanthine (Xan) and oxanine (Oxa) are major damage products from guanine (Gua) by NO- or HNO(2)-induced nitrosative deamination. Xan- and Oxa-containing oligodeoxynucleotides are essential substrates for the biochemical studies to reveal genotoxicity or mutagenesis raised by nitrosative oxidation. In previous study, we have developed chemical synthesis method for obtaining Oxa-ODN. Here, we proposed an efficient preparation method of Xan-ODN by incubating of Oxa-ODN in N(alpha)-acetyl-(L)-histidine (Ac-His) for 3 days at mild condition.

Asunto(s)

Histidina/análogos & derivados , Oligodesoxirribonucleótidos/síntesis química , Nucleósidos de Purina/química , Xantina/química , Catálisis , Histidina/química , Oligodesoxirribonucleótidos/química

RESUMEN

Oxanine (Oxa), a mutagenic lesion generated from guanine by nitrosative oxidation, can make a covalent bonding with -NH(2) or -SH group since Oxa possesses O-acylisourea conformation in the base-ring structure. We employed such unique reactive functionality of Oxa for fabrication of DNA-immobilized system. When DNA oligomer with Oxa at 5'-end (Oxa-end DNA) is spotted on amine-functionalized surface, the DNA probe can be immobilized without any involvement of activation step or any treatment of chemical linker. We further optimized the fabrication conditions for efficient immobilization of Oxa-end DNA.

Asunto(s)

Sondas de ADN/química , Análisis de Secuencia por Matrices de Oligonucleótidos , Nucleósidos de Purina/química , Humedad , Concentración de Iones de Hidrógeno , Temperatura

RESUMEN

Pterin is an electron transfer compound in biological systems. Among the analogs, 6-formylpterin (6FP) has been demonstrated to have many marked physiological and pharmacological activities. In previous study, we have elucidated that 6FP derivatives in which the 3-position is modified possess reactive oxygen species (ROS), which are involved in the modulation of a variety of cell functions, generation activities through the oxidation of NADH to NAD(+) in the dark at neutral pH. In the present study, we have demonstrated that the ROS generation activity by 6FP derivative is enhanced in the presence of 3-methyl-1-phenyl-2-pyrazolin-5-one. In this reaction, 3-methyl-1-phenyl-2-pyrazolin-5-one is reacted with the formyl group on the 6-position of 6FP derivative to give the activated product. The present results would be helpful for designing pharmaceutical ROS generation system in vivo.

Asunto(s)

NAD/química , Pteridinas/química , Especies Reactivas de Oxígeno/química , Antipirina/análogos & derivados , Antipirina/química , Edaravona , Oxidación-Reducción , Pterinas/química , Especies Reactivas de Oxígeno/metabolismo

RESUMEN

A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD(+)-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP(+). In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP(+)-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP(+)-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain.

Asunto(s)

D-Xilulosa Reductasa/genética , D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismo , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , NADP/metabolismo , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

We constructed a set of recombinant Saccharomyces cerevisiae strains with xylose-fermenting ability. A recombinant S. cerevisiae strain D-XR/ARSdR/XK, in which protein engineered NADP(+)-dependent XDH was expressed, showed 40% increased ethanol production and 23% decrease in xylitol excretion as compared with the reference strain D-XR/XDH/XK expressing the wild-type XDH.

Asunto(s)

D-Xilulosa Reductasa/metabolismo , Etanol/metabolismo , NADP/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/enzimología , Xilosa/metabolismo , D-Xilulosa Reductasa/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Xilitol/metabolismo

RESUMEN

BACKGROUND: A promoter that is activated by ionizing radiation may be a useful tool for cancer therapy since, with such a promoter, the therapeutic gene can be expressed only in cancer tissues by irradiation. An artificially constructed promoter is advantageous as natural promoters may have physiological limitations. However, reasonably designing a promoter is hampered by shortage of information about the relationship between the structure and properties of a promoter DNA. MATERIALS AND METHODS: Binding sites of four transcription factors that were activated by radiation were randomly ligated and linked to a TATA-box sequence to control the luciferase gene located downstream. Transiently transfected cancer cells with such a vector were exposed to X-ray irradiation and enhancement of luciferase expression was assessed. To improve promoter sensitivity, mutations were randomly introduced into a constructed promoter by error-prone polymerase chain reaction (epPCR). RESULTS: Of the 11 promoters constructed, the clone 11 promoter (clone 11 + TATA-box) showed a 5-fold enhancement 6 h after the 10 Gy X-ray irradiation in HeLa cells. A mutant designated the clone 11-9-37 promoter generated through two steps of epPCR showed a sensitivity 4.8 times higher than the clone 11 promoter to the 10 Gy X-rays, showing 21.6-fold enhancement of luciferase expression. Clone 11 was composed of 16 cis-acting elements, and the clone 11-9-37 promoter carried six point mutations. CONCLUSION: A sensitively responsive promoter to radiation could be constructed using this method, possibly leading to the construction of a promoter of interest that could be applied for clinical use.

Asunto(s)

Reacción en Cadena de la Polimerasa/métodos , Regiones Promotoras Genéticas/efectos de la radiación , Línea Celular Tumoral , Elementos de Facilitación Genéticos , Humanos , TATA Box , Factores de Transcripción/genética