RESUMEN
We have examined the association, dissociation, and exchange of the 5S specific transcription factor (TFIIIA) with somatic- and oocyte-type 5S DNA. The factor associates faster with somatic than with oocyte 5S DNA, and the rate of complex formation is accelerated by vector DNA. Once formed, the TFIIIA-5S DNA complex is stable for greater than 4 h in the absence of free 5S DNA, and its dissociation is identical for somatic and for oocyte 5S DNA. In the presence of free 5S DNA, the factor transfers promptly from the complex to the free 5S DNA site. Unexpectedly, the direct exchange of factor between 5S DNA sites leads to proteolysis at the C-terminal arm of TFIIIA.
Asunto(s)
ADN Ribosómico/metabolismo , Regulación de la Expresión Génica , Factores de Transcripción/metabolismo , Animales , ADN Recombinante/metabolismo , Femenino , Conformación de Ácido Nucleico , Oocitos , Plásmidos , Unión Proteica , ARN Ribosómico/genética , Factor de Transcripción TFIIIA , Xenopus laevisRESUMEN
A chimeric oligonucleotide composed of DNA and modified RNA residues was used to direct correction of the mutation in the hemoglobin betaS allele. After introduction of the chimeric molecule into lymphoblastoid cells homozygous for the betaS mutation, there was a detectable level of gene conversion of the mutant allele to the normal sequence. The efficient and specific conversion directed by chimeric molecules may hold promise as a therapeutic method for the treatment of genetic diseases.
Asunto(s)
Anemia de Células Falciformes/genética , Conversión Génica , Hemoglobina Falciforme/genética , Oligodesoxirribonucleótidos/genética , Oligorribonucleótidos/genética , Transfección , Alelos , Anemia de Células Falciformes/terapia , Secuencia de Bases , Células Cultivadas , Terapia Genética , Globinas/genética , Humanos , Linfocitos , Datos de Secuencia Molecular , Mutación Puntual , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de RestricciónRESUMEN
The myostatin gene, known as Growth Differentiation Factor 8 (GDF8), located at chromosome 2 (BTA2) in cattle, is specifically expressed during embryo development and in the adult skeletal muscle. Molecular analysis of this gene reveals the presence of three exons and two introns. Several cattle breeds, such as Piedmontese, Belgian Blue, Blond'Aquitaine, among others, show polymorphisms in this gene, which are directly related to double muscling phenotype. Piedmontese cattle shows a nucleotide transition G --> A (G938A) at exon 3, resulting in the substitution of cysteine to tyrosine, leading to a protein structure change, which determines myostatin inactivation and consequent muscular hypertrophy. The objective of this work was to implant the polymorphism G938A, naturally existent in Piedmontese breed, into in vitro propagated foetal myoblasts, from Nellore cattle. Single strand DNA (ssDNA) oligonucleotides were used to direct the same nucleotidic transition (G938A) to exon 3. Two transfection protocols (cationic lipid solution and electroporation) were tested and, 48 hours after transfection, RNA and DNA were extracted from myoblasts. Reverse transcription and polymerase chain reaction (PCR) were performed, using primers flanking the mutation region. The PCR products were cloned and analyzed by DNA sequencing, and it was possible to detect the nucleotidic CT transition at position 938, in the electroporated myoblasts. The existence of a positive signal in the transfection indicates that ssDNA oligonucleotides can be used to introduce this point mutation in specific functional gene sites.
Asunto(s)
Bovinos/genética , ADN de Cadena Simple/genética , Mioblastos/metabolismo , Miostatina/genética , Mutación Puntual , Animales , Secuencia de Bases , Diferenciación Celular , Línea Celular , Femenino , Regulación de la Expresión Génica , Ingeniería Genética , Datos de Secuencia Molecular , Mioblastos/citología , Miostatina/metabolismo , Polimorfismo Genético , ARN/genética , TransfecciónRESUMEN
The relationship between RNA synthesis and homologous pairing in vitro, catalyzed by RecA protein, was examined by using an established strand transfer assay system. When a short DNA duplex is mixed with single-stranded circles, RecA protein promotes the transfer of the minus strand of the duplex onto the complementary region of the plus-strand circle, with the displacement of the plus strand of the duplex. However, if minus-strand RNA is synthesized from the duplex pairing partner, joint molecules containing the RNA transcript, the plus strand of the DNA duplex, and the plus-strand circle are also observed to form. This reaction, which is dependent on RNA polymerase, sequence homology, and RecA protein, produces a joint molecule that can be dissolved by treatment with RNase H but not RNase A. Under these reaction conditions, product molecules form even when the length of shared homology between duplex and circle is reduced to 15 bp.
Asunto(s)
ADN Circular/química , ADN de Cadena Simple/química , ARN/química , Rec A Recombinasas/metabolismo , Recombinación Genética , Transcripción Genética , Técnicas In Vitro , Homología de Secuencia de Ácido NucleicoRESUMEN
RecA protein catalyzes the homologous pairing of a single-stranded circular DNA and a linear duplex DNA molecule. When the duplex is packaged into chromatin, formation of homologously paired complexes is blocked. We have established a system for studying the RecA-promoted reaction by using a duplex fragment containing a single-phased nucleosome. Under these conditions there is no reaction leading to formation of joint molecule complexes. However, transcription on the chromatin template activates the formation of complexes. Reaction is dependent on RNA synthesis and DNA sequence homology and proceeds regardless of the direction of transcription.
Asunto(s)
Nucleosomas/fisiología , Rec A Recombinasas/metabolismo , Recombinación Genética , Transcripción Genética , Sistema Libre de Células , Cromatina/ultraestructura , ARN Polimerasas Dirigidas por ADN/metabolismo , Técnicas In Vitro , Moldes GenéticosRESUMEN
Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.
Asunto(s)
Exodesoxirribonucleasas/genética , Proteínas Fúngicas/genética , Genes Fúngicos , Ustilago/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Reparación del ADN/genética , ADN de Hongos/química , ADN de Hongos/genética , ADN de Hongos/metabolismo , Exodesoxirribonucleasa V , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Ácidos Nucleicos Heterodúplex/metabolismo , Hibridación de Ácido Nucleico , ARN de Hongos/química , ARN de Hongos/metabolismo , Ustilago/metabolismoRESUMEN
muREC2/RAD51L1 is a radiation-inducible gene that regulates cell cycle progression. To elucidate the biological function of muREC2/RAD51L1, the gene was disrupted in embryonic stem cells by homologous recombination. Mice heterozygous for muREC2/RAD51L1 appear normal and fertile; however, no homozygous pups were born after interbreeding of heterozygous mice. Timed pregnancy studies showed that homozygous mutant embryos were severely retarded in growth as early as ca. 5 days gestation (E5.5) and were completely resorbed by E8.5. Mutant blastocyst outgrowth was also severely impaired in a double-knockout embryo, but embryonic development did progress further in a p53-null background. These results suggest that muREC2/RAD51L1 plays a role in cell proliferation and early embryonic development, perhaps through interaction with p53.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas/fisiología , Proteína p53 Supresora de Tumor/fisiología , Animales , Blastómeros/citología , Células Cultivadas , Proteínas de Unión al ADN/genética , Desarrollo Embrionario y Fetal , Femenino , Eliminación de Gen , Marcación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Fenotipo , Embarazo , Proteínas/genética , Recombinasa Rad51 , Recombinación Genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Chimeric RNA/DNA oligonucleotides have been used successfully to correct point and frameshift mutations in cells as well as in animal and plant models. This approach is one of several nucleic acid repair technologies that will help elucidate the function of newly discovered genes. Understanding the mechanisms by which these different technologies direct gene alteration is essential for progress in their application to functional genomics.
Asunto(s)
Reparación del ADN , Genoma , Ácidos Nucleicos/química , ADN/química , Daño del ADN , Modelos Genéticos , Oligonucleótidos/metabolismo , Plásmidos/metabolismo , ARN/química , Proteínas Recombinantes de Fusión/química , Saccharomyces cerevisiae/metabolismoRESUMEN
Synthetic oligonucleotides have been used to direct base exchange and gene repair in a variety of organisms. Among the most promising vectors is chimeric oligonucleotide (CO), a double-stranded, RNA-DNA hybrid molecule folded into a double hairpin conformation: by using the cell's DNA repair machinery, the CO directs nucleotide exchange as episomal and chromosomal DNA. Systematic dissection of the CO revealed that the region of contiguous DNA bases was the active component in the repair process, especially when the single-stranded ends were protected against nuclease attack. Here, the utility of this vector is expanded into Saccharomyces cerevisiae. An episome containing a mutated fusion gene encoding hygromycin resistance and eGFP expression was used as the target for repair. Substitution, deletion and insertion mutations were corrected with different frequencies by the same modified single-stranded vector as judged by growth in the presence of hygromycin and eGFP expression. A substitution mutation was repaired the most efficiently followed by insertion and finally deletion mutants. A strand bias for gene repair was also observed; vectors designed to direct the repair of nucleotide on the non-transcribed (non-template) strand displayed a 5-10-fold higher level of activity. Expanding the length of the oligo-vector from 25 to 100 nucleotides increases targeting frequency up to a maximal level and then it decreases. These results, obtained in a genetically tractable organism, contribute to the elucidation of the mechanism of targeted gene repair.
Asunto(s)
Cinamatos , Reparación del ADN , ADN/genética , Mutación del Sistema de Lectura , Higromicina B/análogos & derivados , Oligonucleótidos/genética , Mutación Puntual , ARN/genética , Secuencia de Bases , Recuento de Colonia Microbiana , Electroporación , Marcación de Gen , Proteínas Fluorescentes Verdes , Higromicina B/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Chimeric oligonucleotides (chimeras), consisting of RNA and DNA bases folded by complementarity into a double hairpin conformation, have been shown to alter or repair single bases in plant and animal genomes. An uninterrupted stretch of DNA bases within the chimera is known to be active in the sequence alteration while RNA residues aid in complex stability. In this study, the two strands were separated in the hope of defining the role each plays in conversion. Using a series of single-stranded oligonucleotides, comprised of all RNA or DNA residues and various mixtures, several new structures have emerged as viable molecules in nucleotide conversion. When extracts from mammalian and plant cells and a genetic readout assay in bacteria are used, single-stranded oligonucleotides, containing a defined number of thioate backbone modifications, were found to be more active than the original chimera structure in the process of gene repair. Single-stranded oligonucleotides containing fully modified backbones were found to have low repair activity and in fact induce mutation. Molecules containing various lengths of modified RNA bases (2'-O-methyl) were also found to possess low activity. Taken together, these results confirm the directionality of nucleotide conversion by the DNA strand of the chimera and further present a novel, modified single-stranded DNA molecule that directs conversion in plant and animal cell-free extracts.
Asunto(s)
Reparación del ADN/genética , ADN Recombinante/metabolismo , ADN de Cadena Simple/metabolismo , Conversión Génica/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Plantas , ARN/metabolismo , Animales , Secuencia de Bases , Extractos Celulares , Línea Celular , Sistema Libre de Células , ADN Recombinante/genética , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Electroporación , Resistencia a la Kanamicina/genética , Ratones , Proteína 2 Homóloga a MutS , Proteína 3 Homóloga de MutS , Mutación/genética , Ácidos Nucleicos Heterodúplex/genética , Ácidos Nucleicos Heterodúplex/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Células Vegetales , Plantas/genética , Plásmidos/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , ARN/genética , Resistencia a la Tetraciclina/genética , Transformación BacterianaRESUMEN
HsRec2/Rad51B is a 350-amino acid protein with a molecular mass of 38,300 Da that appears to be involved in cell cycle regulation and UV-induced apoptosis. The mouse and human genes were isolated based on their homology to a recombinational repair gene from Ustilago maydis and contain functional domains to hRAD51 and hLIM 15 (M. C. Rice et al., Proc. Natl. Acad. Sci. USA, 94: 7417-7422, 1997). Here, we report the results of studies on the behavior of CHO cells containing a plasmid encoding a wild-type hsRec2/Rad51B, a full-length protein with a single mutation at residue 163, which lies in the putative src site, and a truncated version of hsRec2/Rad51B, containing only the first 100 amino acids at the NH2 terminus. Using fluorescence-activated cell sorting analysis to follow the progression of cells through the cell cycle, we find that stable transfectants constitutively overexpressing the wild-type human Rec2/Rad51B protein exhibit a G1 delay. In addition, when irradiated with UV at a dose of 15 J/m2, CHO cells transfected with the various hREC2/RAD51B vectors exhibited different responses. Cells expressing the wild-type human Rec2/Rad51B underwent apoptosis, with the greatest cell death occurring 24 h after irradiation. The control cells, which contained an empty vector, and the cells expressing truncated hsRec2/Rad51B or the full-length Rec2 with a mutation at residue 163 did not. In summary, these findings of cell cycle slowing and UV-induced apoptosis in CHO cells constitutively expressing the human Rec2/Rad51B protein suggest that hsRec2/Rad51B plays a role in a DNA damage surveillance pathway.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/genética , ADN/efectos de la radiación , Fase G1 , Proteínas/genética , Animales , Apoptosis , Células CHO , Cricetinae , Humanos , Ratones , Recombinasa Rad51 , Relación Estructura-Actividad , Transfección , Rayos UltravioletaRESUMEN
The DNA binding properties of a protein from the lower eukaryote Ustilago maydis have been characterized. Using both filter binding and gel retention assays, we demonstrate that this protein, termed UBP1 (Ustilago binding protein one), binds preferentially to DNA molecules lacking chain interruptions. The introduction of DNA breaks by a restriction enzyme or a purified nuclease, from Ustilago maydis, causes the dissociation of protein-DNA complexes. UBP1 stimulates the relaxation of negatively supercoiled DNA, mediated by Ustilago type I topoisomerase, through a mechanism most likely involving the association of UBP1 with the DNA rather than with the topoisomerase. The prebinding of UBP1 to DNA templates, subsequently assembled into minichromosomes, results in the development of a disorganized nucleosomal array. Possible roles for UBP1 in processes that involve changes in DNA topology, such as chromatin assembly, are discussed.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/metabolismo , Ustilago/metabolismo , ADN/química , ADN/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/farmacología , Nucleasa Microcócica , Nucleosomas , Moldes GenéticosRESUMEN
Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.
Asunto(s)
ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas del Grupo de Alta Movilidad/fisiología , Histonas/fisiología , Ustilago/enzimología , ADN Superhelicoidal/metabolismo , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/metabolismo , CinéticaRESUMEN
The structural gene HMP1 encoding a cruciform DNA binding protein from Ustilago maydis has been cloned. Gene isolation was enabled by a polymerase chain reaction procedure using primers designed from amino acid sequence obtained from the purified protein. DNA sequence determination has revealed that the gene encodes a protein containing 98 amino acids with a calculated molecular weight of 10151. Comparison of the cDNA and genomic sequences indicated the presence of a single intron in the 5' coding region of the gene. The gene was over-expressed as a translational fusion with a hexahistidine leader sequence enabling affinity purification of the protein on an immobilized metal matrix. Protein isolated after over-expression exhibited cruciform binding activity, conforming earlier purified native protein results. Sequence analysis indicated that no HMG box was present and very little homology to other known cruciform binding proteins was found. It is plausible that HMP1 represents a novel class of proteins that recognize such secondary structures.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Fúngicos , Ustilago/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas Fúngicas , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Ustilago/metabolismoRESUMEN
A gene encoding a Ustilago maydis Rad51 orthologue has been isolated, rad51-1, a mutant constructed by disrupting the gene, was as sensitive to killing by ultraviolet light and gamma radiation as the rec2-1 mutant and slightly more sensitive to killing by methyl methanesulfonate. There was no suppression of killing by ultraviolet light when a rec2-1 strain was transformed with a multicopy plasmid containing RAD51, nor was there suppression when rad51-1 was transformed with a multicopy plasmid containing REC2. Recombination proficiency as measured by a gap repair assay was diminished in both rec2-1 and rad51-1 strains. In rec2-1 the frequency of recombination was decreased, but the spectrum of events was similar to that observed in wild type, while in rad51-1 the frequency as well as the spectrum of recombination events were different. Studies with the rec2-1 rad51-1 double mutant indicated that there was epistasis in the action of REC2 and RAD51 in certain repair and recombination functions, but some measure of independent action in other functions.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas Fúngicas/genética , Ustilago/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromosomas Fúngicos , ADN de Hongos , Rayos gamma , Regulación de la Expresión Génica , Genes Fúngicos , Mitosis , Datos de Secuencia Molecular , Plásmidos , Recombinasa Rad51 , Recombinación Genética , Rayos UltravioletaRESUMEN
Determining the function of newly discovered genes is at the center of the evolving field of genomics. With the elucidation of the human DNA sequence, the importance of single base changes to gene function has become apparent. In some cases, nucleotide alteration accounts for inherited disorders, but in other cases, subtle, even conservative, base changes can influence the function of a gene and its product. To identify how critical genetic changes alter function, molecular tools such as synthetic vectors have been created to direct nucleotide exchange. Some of these vectors, including chimeric RNA/DNA oligonucleotides and modified single-stranded oligonucleotides, have shown promise in the specific alteration of a single base at an exact position within the gene. Here, we describe the activity of the synthetic vectors in a mammalian cell system. The episomal target contains a mutation in the neomycin resistance gene fused to a reporter ligand-binding domain. Correction of the mutated base enables translation of the normal fusion product. This protein can now bind a ligand, resulting in the expression of the fusion protein visualized by green fluorescence. Hence, the activity of any similar vector can be measured easily (and in real time) using confocal microscopy. The system provides the basis for examining the effectiveness of new targeting molecules for creating or repairing single base alterations. In addition, genes suspected of affecting the frequency of repair can be tested through their expression in cells harboring the mutated target plasmid. Once the frequency of exchange in cells is established, the use of these vectors will become commonplace in a process designed to generate specific single base changes in genes involved in signal transduction. Such changes should help define functional domains within these proteins.
Asunto(s)
Reparación del ADN/genética , ADN de Cadena Simple/genética , ADN/genética , Marcación de Gen/métodos , Vectores Genéticos/genética , Ácidos Nucleicos Heterodúplex/genética , Oligonucleótidos/genética , ARN/genética , Animales , Marcación de Gen/instrumentación , Terapia Genética/instrumentación , Terapia Genética/métodos , Células HeLa/química , Células HeLa/metabolismo , Humanos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Ratas , Ratas Gunn , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/genética , Transfección/métodos , Células Tumorales CultivadasRESUMEN
Gene conversion of genetically inherited point mutations is a fundamental methodology for treating a variety of diseases. We tested the feasibility of a new approach using an RNA/DNA chimeric oligonucleotide. The beta-globin gene was targeted at the point mutation causing sickle cell anemia. The chimera is designed to convert an A residue to a T after creating a mismatched basepair. In a CD34+-enriched population of normal cells a 5-11% conversion rate was measured using restriction enzyme polymorphism and direct DNA sequence analyses. The closely related delta-globin gene sequence appeared unchanged despite successful conversion at the beta-globin locus.
Asunto(s)
Antígenos CD34/genética , ADN/genética , Conversión Génica , Marcación de Gen , Células Madre Hematopoyéticas/metabolismo , Mutagénesis Sitio-Dirigida/genética , Oligonucleótidos Antisentido/genética , ARN/genética , Marcación de Gen/métodos , Vectores Genéticos/síntesis química , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Análisis de Secuencia de ADNRESUMEN
In vitro transcription experiments using a Xenopus laevis cell-free extract have demonstrated that a DNA fragment containing a glucocorticoid response element (GRE) significantly enhances the expression of a methionine tRNA gene. This stimulation can be observed when the element is located in cis or trans. In the cis configuration, the element can be located 2900 basepairs from the gene and still display transcriptional enhancement. In trans, the enhancement can occur at a low element to template molecular ratio. The nucleosome positioning and chromatin structure near the tRNA gene appear to be unaffected by the presence of the long terminal repeat of the mouse mammary tumor virus which contains the GRE. Taken together, these data suggest that GREs can stimulate transcription of hormonally unresponsive genes, perhaps by providing a "molecular sink" to which inhibitors may bind. Further experimentation may reveal a correlation between these in vitro studies and in vivo gene regulation.
Asunto(s)
ADN/metabolismo , Elementos de Facilitación Genéticos , ARN de Transferencia de Metionina/genética , Receptores de Glucocorticoides/metabolismo , Transcripción Genética , Animales , Virus del Sarcoma Aviar/genética , Secuencia de Bases , Sitios de Unión , ADN Superhelicoidal/metabolismo , ADN Viral/genética , VIH/genética , Virus del Tumor Mamario del Ratón/genética , Conformación de Ácido Nucleico , Oocitos/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Moldes Genéticos , Xenopus laevisRESUMEN
A number of cellular transformations are due, in large part, to a single base mutation that alters the function of the expressed protein. Similarly, alterations in the DNA sequence of a gene involved in cell proliferation can have a significant effect on the viability of particular cells, Thus, the capacity to modulate the base sequence of such a gene would be a useful tool for cancer therapeutics. We have developed an experimental strategy that centers around site-specific DNA base mutation or correction using a unique chimeric oligonucleotide. This chimeric molecule has demonstrated higher recombinogenic activities than identical oligonucleotides containing only DNA residues, both in vitro and in vivo. The chimeric molecule is designed to hybridize to a target site within the genome and induce a single base mismatch at the residue targeted for mutation. The DNA structure created at this site is recognized by the host cell's repair system which mediates the correction reaction. The bcr-abl fusion gene, the product of a translocation between human chromosomes 9 and 22, and the cause of chronic myelogenous leukemia (CML) can be targeted for gene correction. This fusion gene is a good choice because (1) it is a unique target in CML; (2) it is a single copy target; (3) the DNA sequence of the fusion gene is unique. The goal of such experiments is to knock-out the fusion gene by changing a glutamine or lysine codon into a stop codon through a chimeric directed DNA repair system.
Asunto(s)
Terapia Genética/métodos , Neoplasias/terapia , Apoptosis , Secuencia de Bases , ADN de Neoplasias/genética , Proteínas de Fusión bcr-abl/genética , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/genéticaRESUMEN
We have successfully uncoupled the previously interlocked activities of chromatin assembly and in vitro transcription promoted by the Xenopus oocyte S-150 cell-free extract. Our isolated fraction catalyzes extensive chromatin assembly measured both by changes in DNA topology and Micrococcal nuclease digestions. The assembly of chromatin is slowed by the exogenous addition of ATP. In the absence of exogenously added ATP, the fraction forms a chromatin template that is transcriptionally inert. Addition of small amounts of the HeLa cell extract (S-100) converts these templates into transcriptionally active ones without disrupting the chromatin structure. Our protocol defines a method for the isolation of a fraction from the Xenopus cell free extract that catalyzes the assembly of transcriptionally inactive chromatin. We characterize this reaction and establish conditions for the transcriptional activation of these inactive minichromosomes.