RESUMEN

A new route to regioselectively dialkoxy-functionalized benzo[b]furan derivatives has been developed from 3-halo-2-iodoanisoles bearing an additional methoxy group, which have been accessed through an ortho-zincation/iodination reaction. Two palladium-catalyzed processes, namely a Sonogashira coupling followed by a tandem hydroxylation/cyclization sequence, give rise to new and interesting dimethoxy-substituted benzo[b]furans.

RESUMEN

2,3-Dihaloanilines have been proved as useful starting materials for synthesizing 4-halo-1H-indoles. Subsequent or in situ functionalization of the prepared haloindoles allows the access to a wide variety of 2,4- or 2,3,4-regioselectively functionalized indoles in good overall yields. As no efficient synthetic routes to 2,3-dihaloanilines have been described in the literature, different approaches to the preparation of these 1,2,3-functionalized aromatic precursors are now presented. The most general one involves a Smiles rearrangement from the corresponding 2,3-dihalophenols and allows the preparation of 2,3-dihaloanilides in a straightforward and synthetically useful manner.

Asunto(s)

Compuestos de Anilina/química , Compuestos de Anilina/síntesis química , Halógenos/química , Indoles/química , Estereoisomerismo , Especificidad por Sustrato

RESUMEN

The Agrocybe aegerita mitochondrial genome possesses two polB genes with linear plasmid origin. The cloning and sequencing of the regions flanking Aa-polB P1 revealed two large inverted repeats (higher than 2421 nt) separated by a single copy region of 5834 nt. Both repeats contain identical copies of the nad4 gene. The single copy region contains two disrupted genes with plasmid origin Aa-polB P1 and a small ORF homologous to a small gene described in two basidiomycete linear plasmids. The phylogenetic analyses argue in favor of a same plasmid origin for both genes but, surprisingly, these genes were separated by a mitochondrial tRNA-Met. Both strands of the complete region containing the two nad4 inverted copies and the tRNA-Met appear to be transcribed on large polycistronic mRNAs. A model summarizing the events that would have occurred is proposed: (1) capture of the tRNA by the plasmid before its integration in the mtDNA or acquisition of the tRNA gene by recombination after the plasmid integration, (2) integration of the plasmid in the mtDNA, accompanied by a large duplication containing the nad4 gene and (3) erosion of the plasmid sequences by large deletions and mutations.

Asunto(s)

Agaricales/genética , Complejo I de Transporte de Electrón/genética , Genoma Mitocondrial/genética , Plásmidos/genética , Agaricales/clasificación , Secuencia de Bases , ADN Mitocondrial/genética , Evolución Molecular , Proteínas Fúngicas/genética , Duplicación de Gen , Proteínas Mitocondriales/genética , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , ARN de Transferencia/genética , Análisis de Secuencia de ADN

RESUMEN

Full details are provided for a recently invented method to couple indoles and pyrroles to carbonyl compounds. The reaction is ideally suited for structurally complex substrates and exhibits high levels of chemoselectivity (functional group tolerability), regioselectivity (coupling occurs exclusively at C-3 of indole or C-2 of pyrrole), stereoselectivity (substrate control), and practicality (amenable to scaleup). In addition, quaternary stereocenters are easily and predictably generated. The reaction has been applied to a number of synthetic problems including total syntheses of members of the hapalindole family of natural products, ketorolac, acremoauxin A, and oxazinin 3. Mechanistically, this coupling protocol appears to operate by a single electron-transfer process requiring generation of an electron-deficient radical adjacent to a carbonyl which is then intercepted by an indole or pyrrole anion.

Asunto(s)

Carbono/química , Indoles/química , Morfolinas/síntesis química , Oxazinas/química , Pirroles/química , Química Orgánica/métodos , Dimerización , Electrones , Indoles/síntesis química , Ketorolaco/química , Modelos Químicos , Conformación Molecular , Oxígeno/química , Fosfinas/química , Estereoisomerismo

RESUMEN

An efficient and regioselective synthesis of 4- and 7-alkoxyindoles has been developed from commercially available starting materials such as 3-halophenols and 3-chloroanisole. Directed ortho-metalation followed by two palladium-catalyzed processes, a Sonogashira coupling and a tandem amination/cyclization reaction, allows the synthesis of regiochemically pure 4- and 7-substituted indoles. This strategy has been successfully applied to the preparation of 2-[3-(2-amino-2-oxoacetyl)-1-benzyl-2-ethyl-1H-indol-4-yloxy]acetic acid (LY315920), a known inhibitor of phospholipase A2.

Asunto(s)

Alcanos/síntesis química , Anisoles/química , Inhibidores Enzimáticos/síntesis química , Indoles/síntesis química , Fenoles/química , Fosfolipasas A/antagonistas & inhibidores , Alquilación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Éteres/química , Indoles/química , Estructura Molecular , Oxígeno/química , Fosfolipasas A/metabolismo , Fosfolipasas A2 , Estereoisomerismo

RESUMEN

The treatment of 2-fluorophenyl 2-iodophenylamines, ether, and thioether, easily prepared from commercially available products, with 3.3 equiv of t-BuLi and further reaction with selected electrophiles gives rise to functionalized carbazole, dibenzofuran, and dibenzothiophene derivatives in a direct and regioselective manner. The process involves an anionic cyclization on a benzyne-tethered aryllithium intermediate.

RESUMEN

Tandem Sonogashira coupling/5-endo-dig cyclization reactions on 2,3-dihalophenols suppose a straightforward entry to 4-halobenzo[b]furans, which can be easily transformed into 4-functionalized benzo[b]furans, that are difficult to synthesize by other procedures. On the other hand, the starting 2,3-dihalophenols are efficiently prepared from commercially available 3-halophenols, via their N,N-diethyl carbamates by selective lithiation at the 2-positions by treatment with s-BuLi/TMEDA or LDA at low temperature and reaction with halogen electrophilic reagents.

Asunto(s)

Benceno/química , Furanos/química , Furanos/síntesis química , Hidrocarburos Halogenados/química , Carbamatos/química , Hidrólisis , Estructura Molecular

RESUMEN

Canned truffle products labeled Tuber melanosporum, the famous Perigord truffle, may contain other less tasty and cheaper truffle species. To protect consumers from fraud, a PCR DNA-based method was used to unequivocally identify the nature of the product. Several rapid and simple cell lysis procedures, used in conjunction with a commercially available DNA purification kit, were evaluated for their effectiveness in recovering DNA from canned truffle. In parallel, a marker for T. melanosporum was tested on the mitochondrial rDNA. These two techniques were then combined to differentiate T. melanosporum from other truffle species like T. aestivum, T. brumale or T. indicum up to the legal threshold in canned products. These findings not only allow a comparison of the effectiveness of the different DNA extraction methods but also provide a preliminary indication of the specificity and sensitivity of the detection with the mitochondrial marker that might be attainable for truffle species in a quantitative PCR-based analysis method.

Asunto(s)

Ascomicetos/clasificación , ADN de Hongos/genética , Contaminación de Alimentos/análisis , Reacción en Cadena de la Polimerasa/métodos , Ascomicetos/genética , ADN Mitocondrial/genética , ADN Ribosómico/genética , Contaminación de Alimentos/prevención & control , Conservación de Alimentos , Tecnología de Alimentos , Polimorfismo de Longitud del Fragmento de Restricción , Sensibilidad y Especificidad

RESUMEN

We have developed new DNA extraction and purification procedures for investigation of mycorrhized seedlings and canned truffles. Use of these procedures on approximately 100 mg initial material enabled good sample representation. For mycorrhized seedlings, Taq polymerase inhibitors were discarded irrespective of tree species. In routine analysis we systematically used consensus primers ITS1/ITS4 to check the absence of Taq polymerase inhibitors and the presence of fungus DNA. Positive response with ITS validates other positive or negative PCR results. Absence of amplification with ITS prevents validation of other results. For canned truffles, DNA harvested from ascocarps sterilized for one and a half hours at 115 degrees C was amplified with specific primers. We have developed consensus primers, named R12/F12, to check for the presence of amplifiable fungus DNA and the absence of Taq polymerase inhibitors. Here also, positive response with consensus R12/F12 validates other positive or negative PCR results. We have developed one primer pair specific for T. brumale and another specific for T. melanosporum. We can then characterize these two taxa, which enables the use of "truffle or truffled" French designations. We can also characterize T. indicum, the Asiatic black truffle that might fraudulently be sold as T. melanosporum and T. brumale. These three specific primer pairs were used independently of DNA extraction from tree seedlings or canned truffles. Our process is specific, sensitive, convenient, and quick.

Asunto(s)

ADN de Hongos/química , Micorrizas/química , Plantones/química , Plantones/microbiología , Árboles , ADN de Hongos/aislamiento & purificación , Micorrizas/clasificación , Reacción en Cadena de la Polimerasa/métodos , Sensibilidad y Especificidad , Especificidad de la Especie , Propiedades de Superficie , Árboles/microbiología

RESUMEN

The NS5B protein, or RNA-dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3' end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3' UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3' end of the HCV minus-strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3' end of the minus-strand RNA: (a) the presence of a C residue as the 3' terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42-nucleotide stretch. These results indicate that the 3' end of the minus-strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.

Asunto(s)

Hepacivirus/enzimología , ARN Viral/biosíntesis , ARN Polimerasa Dependiente del ARN/metabolismo , Secuencia de Bases , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Hepacivirus/genética , Técnicas In Vitro , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteínas no Estructurales Virales/aislamiento & purificación , Proteínas no Estructurales Virales/metabolismo

RESUMEN

In the filamentous fungi, cell fusion between unlike individuals generally triggers a cell-death reaction known as vegetative incompatibility. In Podospora anserina, it was shown that, during this cell-death reaction, there is a strong increase in proteolytic activity. Here, we report the purification of a 36-kDa protease that is induced during the incompatibility reaction. An internal peptide of this protein displayed a strong similarity with the PEPC subtilisin-like serine protease from Aspergillus niger. This led us to use the pepC gene as a probe to clone the homologous gene from P. anserina, which we called pspA. The expression of pspA is upregulated at the transcript level during the progress of the incompatibility reaction. This induction is diminished in strains bearing suppressors of the vegetative incompatibility reaction. The fact that pspA is homologous to PrB, a vacuolar protease involved in autophagy in yeast, suggests that the incompatibility cell-death reaction and autophagy might be related processes.

Asunto(s)

Ascomicetos/genética , Proteínas Bacterianas/genética , Proteínas de Choque Térmico/genética , Serina Endopeptidasas/genética , Subtilisinas/genética , Secuencia de Aminoácidos , Ascomicetos/química , Ascomicetos/crecimiento & desarrollo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN de Hongos/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/genética , Proteínas de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Mutación , Fenotipo , Filogenia , ARN/metabolismo , Serina Endopeptidasas/metabolismo , Regulación hacia Arriba

RESUMEN

A His-tag recombinant carboxyl half part of the HTLV-I surface envelope glycoprotein was overexpressed in E. coli as a secreted form in order to study its biochemical properties and to determine its three-dimensional structure by X-ray crystallography. Starting from several hundred milliliters of culture, a centrifugation was used to eliminate the cells. After solubilization and centrifugation, the protein was then purified by a one-step chromatographic purification procedure. Immobilized Metal Affinity Chromatography (IMAC) was performed by evaluating the tri-dentate iminodiacetic acid (IDA) chelating group with chelating Sepharose fast flow, and the tetra-dendate nitrilotriacetic acid (NTA) chelating group with NTA-agarose. The latter was the most suitable gel for our protein. This expression system and the use of affinity chromatography is a rapid technique to obtain a soluble protein for use in structural studies to further understand the mechanisms of HTLV-1 entry into target cells.

Asunto(s)

Cromatografía de Afinidad/métodos , Productos del Gen env/química , Histidina/química , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Oncogénicas de Retroviridae/química , Quelantes/química , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación

RESUMEN

Hepatocellular carcinoma (HCC), the main type of primary liver cancer, is characterized by a high rate of intra-hepatic invasion. The stroma of HCC is infiltrated by myofibroblasts. We have previously shown that hepatocyte growth factor (HGF) secreted by human liver myofibroblasts greatly increased the in vitro invasiveness of 3 human HCC cell lines. In this study we show that the conditioned medium (CM) from the same HCC cell lines dose-dependently stimulates HGF secretion by myofibroblasts. This effect was post-transcriptional as no increase in HGF mRNA was observed. We show that the effect of CM is not due to IL-1, IL-6, IGF-1, bFGF or PDGF, previously shown to stimulate HGF synthesis in other models. Our data demonstrate that HCC cells increase HGF secretion by liver myofibroblasts in a paracrine way that could act to enhance invasion.

Asunto(s)

Fibroblastos/efectos de los fármacos , Factor de Crecimiento de Hepatocito/metabolismo , Hígado/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Carcinoma Hepatocelular/patología , Medios de Cultivo Condicionados/farmacología , Relación Dosis-Respuesta a Droga , Fibroblastos/citología , Fibroblastos/metabolismo , Factor de Crecimiento de Hepatocito/genética , Humanos , Factor I del Crecimiento Similar a la Insulina/farmacología , Interleucina-1/farmacología , Interleucina-6/farmacología , Hígado/citología , Hígado/metabolismo , Músculo Liso/citología , Músculo Liso/metabolismo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas

RESUMEN

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.

Asunto(s)

Complejo Piruvato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/enzimología , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Mitocondrias/enzimología , Mutación , Complejo Piruvato Deshidrogenasa/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura

RESUMEN

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) initiates the synthesis of DNA from the 3' end of its specific primer, tRNALys3. The regions of tRNALys3 in close contact with RT are well known, while a precise knowledge of the RT regions interacting with tRNALys3 is not yet available. To address this question we cross-linked the heterodimeric p66/p51 RT to tRNALys3 using cis-aquahydroxydiammino-platinum. Ribonucleoprotein complexes of molecular masses higher than the p66 subunit were obtained. After RNase A digestion of the RT-tRNA complex, a labeled oligoribonucleotide (ORN) was mainly found associated to the p66 subunit. This labeled p66-ORN complex was then proteolyzed with Staphylococcus aureus V8 protease. A highly purified radioactive peptide was obtained after two chromatographic purification steps. Its N-terminal sequence corresponded with amino acid residues 241VQPI244. Using the crystallographic structure of HIV-1 RT, this peptide was localized at the beta14-sheet end, near to the hairpin formed by beta12 and beta13-sheets ("primer grip") and the alphaH-helix. The so called "VQPI peptide" is in the border of the thumb and the palm subdomains of the p66 subunit. This study palliates the absence of a three- dimensional structure of the RT-tRNA complex and led to a peptide in interaction with tRNALys3 present in all HIV-1 RT isolates.

Asunto(s)

Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , ARN de Transferencia de Lisina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Reactivos de Enlaces Cruzados , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/genética , Humanos , Modelos Moleculares , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , ARN/química , ARN/metabolismo , ARN de Transferencia de Lisina/química

Asunto(s)

Agonistas alfa-Adrenérgicos/efectos adversos , Clonidina/análogos & derivados , Dermatitis Alérgica por Contacto/etiología , Agonistas alfa-Adrenérgicos/uso terapéutico , Anciano , Anciano de 80 o más Años , Clonidina/efectos adversos , Clonidina/uso terapéutico , Oftalmopatías/tratamiento farmacológico , Femenino , Humanos , Pruebas del Parche

RESUMEN

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.

Asunto(s)

ADN Bacteriano/química , Factores de Elongación de Péptidos/genética , Proteínas Ribosómicas/genética , Spiroplasma/genética , Spiroplasma/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Cromatografía de Afinidad , Mapeo Cromosómico , Cromosomas Bacterianos , Clonación Molecular , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Genes Bacterianos , Datos de Secuencia Molecular , Familia de Multigenes , Sondas de Oligonucleótidos , Operón , Factores de Elongación de Péptidos/aislamiento & purificación , Factores de Elongación de Péptidos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Proteínas Ribosómicas/aislamiento & purificación , Proteínas Ribosómicas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido

RESUMEN

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase beta (pol beta). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol beta-type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol beta sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol beta. Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a beta-type DNA polymerase in plants.

Asunto(s)

ADN Polimerasa beta/genética , ADN Polimerasa Dirigida por ADN/genética , Proteínas de Plantas/genética , Triticum/enzimología , Triticum/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , ADN de Plantas/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Ratas , Homología de Secuencia de Aminoácido , Triticum/embriología

RESUMEN

A cDNA encoding the proliferating cell nuclear antigen (PCNA) from Brassica napus (oilseed rape) was shown to complement the lethal deletion mutation in the PCNA gene (delta POL30) of Saccharomyces cerevisiae. We provide unequivocal evidence that the B. napus PCNA can perform all the essential functions of the yeast PCNA in DNA replication, although some species-specific differences may exist. In addition, the B. napus PCNA expressed as a fusion polypeptide with glutathione S-transferase (GST) was shown to stimulate the activity and processivity of two delta-like DNA polymerases from wheat in vitro. These experiments provide direct biochemical evidence that the B. napus PCNA may function as an auxiliary factor in plant cell DNA replication.

Asunto(s)

Brassica/genética , Replicación del ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , ADN Polimerasa III , Eliminación de Gen , Prueba de Complementación Genética , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética

RESUMEN

In eukaryotic cells, there is much evidence to indicate that the replication of the mitochondrial genome is carried out by a specific DNA polymerase named DNA polymerase gamma. In the yeast S. cerevisiae, a DNA polymerase gamma has been partially purified and the gene encoding the catalytic subunit identified. The characteristics of this enzyme are the same as those found in higher eukaryotes, except for the requirement for a higher magnesium concentration. During a purification procedure of yeast mitochondrial DNA polymerase, we have isolated a second DNA polymerase activity. Using different approaches ive have ruled out the possibility of nuclear contamination or a product of proteolysis. From its properties, this new DNA polymerase activity seems to be different from any yeast DNA polymerase. This new mitochondrial DNA polymerase activity provides evidence that the animal model of mitochondrial DNA replication cannot be generalized. The presence of two DNA polymerases in yeast mitochondria could reflect a different replication or repair mechanism.

Asunto(s)

ADN Polimerasa Dirigida por ADN/metabolismo , Mitocondrias/enzimología , Saccharomyces cerevisiae/enzimología , Fraccionamiento Celular/métodos , ADN Polimerasa Dirigida por ADN/aislamiento & purificación