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1.
Nat Genet ; 17(1): 100-3, 1997 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-9288107

RESUMEN

Werner syndrome (WS) is an uncommon autosomal recessive disorder characterized by premature aging. The clinical manifestations of WS, including atherosclerosis and osteoporosis, appear early in adulthood, and death in the fourth to sixth decade commonly ensues from myocardial infarction or cancer. In accord with the aging phenotype, cells from WS patients have a reduced replicative life span in culture. Genomic instability is observed at the cytogenetic level in the form of chromosome breaks and translocations and at the molecular level by multiple large deletions. The Werner syndrome gene (WRN) has recently been cloned. The predicted product is a 1,432-amino-acid protein whose central domain is homologous to members of the RecQ family of DNA helicases. Such homology does not necessarily mean that WRN encodes an active helicase. For example, the Saccharomyces cerevisiae RAD26 gene protein and the human transcription-repair coupling factor CSB (Cockayne syndrome 8) are highly homologous to known helicases, yet neither encodes an active helicase. Moreover, the Bloom's syndrome gene (BLM), discovered before WRN, is also homologous to the RecQ family of DNA helicases, though we still await demonstration that it encodes an active helicase. Here we report that the WS protein does indeed catalyze DNA unwinding.


Asunto(s)
ADN Helicasas/genética , Mutación Puntual , Síndrome de Werner/enzimología , Síndrome de Werner/genética , Adulto , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , ADN Helicasas/aislamiento & purificación , ADN Helicasas/metabolismo , Humanos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Spodoptera , Transfección
2.
J Exp Med ; 131(6): 1190-9, 1970 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-4246140

RESUMEN

Australia antigen [Au(1)], a particle associated with viral hepatitis, was isolated from the plasma of a patient with chronic anicteric hepatitis and leukemia who had received radioactive phosphorus. We have found that the immunoreactivity and appearance of Au(1) in the electron microscope were not altered by treatment with enzymes including trypsin, pronase, lipase, phospholipase C, ribonuclease, deoxyribonuclease, amylase, and neuraminidase. In contrast, other serum constituents were degraded by these enzymes. Therefore, treatment of the patient's plasma with many enzymes was exploited as an initial step for the isolation of Au(1). Subsequently, Au(1) was purified from the enzyme-treated (32)P-labeled plasma by gel filtration through Sephadex G-200 and centrifugation through sucrose and in cesium chloride gradients. There were no detectable human serum components in the purest fractions, as tested by immunoelectrophoresis and immunodiffusion. The density of the purified Au(1) was 1.21 in CsCl. The particle measured about 200 A in diameter, was predominantly spherical in shape and appeared to be composed of subunits. Nucleic acids were not detected by spectrophotometric, radiochemical, and chemical analyses. Immunoreactivity of purified Au(1) was destroyed by heating for 1 hr at 85 degrees C but was stable at 56 degrees C. Treatment with Carnoy's solution (3 parts ethanol:1 part glacial acetic acid) followed by pronase disrupted the particles as seen with the electron microscope. These findings, combined with other published information on Australia antigen and viral hepatitis, suggest that the bulk of Australia antigen in the blood of this patient is an incomplete virus or virus capsid.


Asunto(s)
Antígenos/aislamiento & purificación , Virus de la Hepatitis B/inmunología , Amilasas/farmacología , Centrifugación , Centrifugación por Gradiente de Densidad , Cesio , Cromatografía en Gel , Desoxirribonucleasas/farmacología , Síndrome de Down/inmunología , Hepatitis A/inmunología , Virus de la Hepatitis B/clasificación , Humanos , Inmunoelectroforesis , Leucemia/inmunología , Lipasa/farmacología , Microscopía Electrónica , Neuraminidasa/farmacología , Fosfolipasas/farmacología , Isótopos de Fósforo , Ribonucleasas/farmacología , Tripsina/farmacología
3.
J Cell Biol ; 58(3): 594-601, 1973 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-4747918

RESUMEN

The requirement for Zn(++) in DNA replication by phytohemagglutinin-stimulated human lymphocytes was studied. When 6 microM o-phenanthroline, a chelator with a high affinity for Zn(++), is added to cultures of stimulated lymphocytes a nearly complete inhibition of thymidine incorporation results within a few hours. In contrast, the incorporation of uridine is only slightly reduced and the incorporation of leucine is unaffected. m-Phenanthroline, a nonchelating analogue, does not alter the rate of thymidine incorporation even when present in 10-fold greater amounts than o-phenanthroline. The inhibition of thymidine incorporation by o-phenanthroline could be entirely reversed by the addition of Zn(++) to the cultures, or could be prevented by the prior addition of either Zn(++) or Ni(++). All other divalent cations tested were incapable of reversing the o-phenanthroline inhibition of thymidine incorporation.


Asunto(s)
Replicación del ADN , Activación de Linfocitos , Zinc/fisiología , Proteínas Sanguíneas/biosíntesis , Cationes Bivalentes , Células Cultivadas , Desoxicitidina/metabolismo , Humanos , Lectinas , Leucina/metabolismo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Níquel/farmacología , Fenantrolinas/farmacología , ARN/biosíntesis , Timidina/metabolismo , Factores de Tiempo , Tritio , Uridina/metabolismo , Zinc/farmacología
4.
Science ; 241(4866): 703-5, 1988 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-2456617

RESUMEN

DNA can form structures other than the Watson-Crick double helix. The potential contributions to gene regulation from one such structure have been investigated by assembling a promoter capable of adopting cruciform base-pairing. Transcription from this promoter by RNA polymerase in vitro was repressed as the cruciform was extruded by increasing negative DNA supercoiling. Transcription in vivo was induced as supercoiling was relaxed by growth in conditions that inhibit DNA gyrase. A DNA conformational change is therefore capable of regulating the initiation of transcription.


Asunto(s)
ADN Bacteriano/genética , ADN Superhelicoidal/genética , Escherichia coli/genética , Conformación de Ácido Nucleico , Regiones Promotoras Genéticas , Transcripción Genética , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis
5.
Science ; 226(4676): 833-5, 1984 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-6436977

RESUMEN

DNA polymerase-alpha is the major replicative DNA polymerase in animal cells. The gene coding for a mutant DNA polymerase-alpha was transferred from one cell to another by transfection of DNA from mutant cells. The DNA was isolated from a mutant hamster cell line resistant to aphidicolin, a specific inhibitor of DNA polymerase-alpha, and transferred into an aphidicolin-sensitive cell line. The resulting transfectants exhibited increased survival in the presence of aphidicolin and contained an aphidicolin-resistant DNA polymerase-alpha.


Asunto(s)
ADN Polimerasa II/genética , Transfección , Animales , Afidicolina , Línea Celular , Células Clonales , Cricetinae , Cricetulus/genética , Diterpenos/farmacología , Escherichia coli/genética , Humanos , Ratones , Mutación , Salmón/genética
6.
Science ; 194(4272): 1434-6, 1976 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-1006310

RESUMEN

Thirty-one metal salts have been tested for their ability to affect the accuracy of DNA synthesis in vitro. All ten salts of metal carcinogens decreased the fidelity of DNA synthesis. Of the three metals which beforehand were considered to be possible mutagens or carcinogens, only one decreased fidelity. In contrast, 17 noncarcinogenic metal salts did not affect fidelity even when present at concentrations that were clearly inhibitory.


Asunto(s)
Carcinógenos , Replicación del ADN/efectos de los fármacos , Metales/farmacología , Mutágenos , Carcinógenos/farmacología , Relación Dosis-Respuesta a Droga , Métodos , Mutágenos/farmacología
7.
Science ; 213(4509): 765-7, 1981 Aug 14.
Artículo en Inglés | MEDLINE | ID: mdl-6454965

RESUMEN

The fidelity of copying natural DNA in vitro with each of the three classes of eukaryotic DNA polymerases has been determined. DNA polymerases-beta and -gamma are highly inaccurate, catalyzing noncomplementary single-base substitution at a frequency between 1/3000 and 1/8000. DNA polymerase-alpha is substantially more accurate, with an error rate of 1/30,000. When the error rates of these DNA polymerases are considered in the context of the accuracy of DNA replicative processes in vivo, it seems likely that other factors must exist in mammalian cells which are involved in the accurate replication and maintenance of the genetic information.


Asunto(s)
Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Bacteriófago phi X 174/genética , Bovinos , Núcleo Celular/enzimología , ADN/biosíntesis , Humanos , Ratones , Mitocondrias/enzimología , Ratas , Especificidad por Sustrato , Moldes Genéticos
8.
Science ; 242(4882): 1168-71, 1988 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-2460924

RESUMEN

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.


Asunto(s)
ADN/biosíntesis , VIH/enzimología , ADN Polimerasa Dirigida por ARN/metabolismo , Virus de la Mieloblastosis Aviar/enzimología , Bacteriófago phi X 174/genética , ADN Polimerasa II/metabolismo , ADN Viral/biosíntesis , Electroforesis en Gel de Poliacrilamida , VIH/genética , Cinética , Virus de la Leucemia Murina de Moloney/enzimología , Mutación , Nucleótidos/metabolismo
10.
Trends Genet ; 16(5): 213-20, 2000 May.
Artículo en Inglés | MEDLINE | ID: mdl-10782115

RESUMEN

Werner syndrome (WS) is an autosomal recessive genetic disorder that is manifested by genetic instability and premature onset of age-related diseases, including atherosclerosis and cancer. The gene that is mutated in WS cells (WRN) has been identified recently. Characterizations of the WRN gene product indicate that WRN encodes both a 3'-->5' DNA helicase, belonging to the Escherichiacoli RecQ helicase family, and a 3'-->5' DNA exonuclease. Studies to define the molecular mechanism of WRN-DNA transactions are currently underway in many laboratories. Preliminary results indicate that WRN functions as a key factor in resolving aberrant DNA structures that arise from DNA metabolic processes such as replication, recombination and/or repair, to preserve the genetic integrity in cells.


Asunto(s)
ADN Helicasas/genética , ADN Helicasas/metabolismo , Síndrome de Werner/genética , Adenosina Trifosfatasas/genética , Animales , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Humanos , Ratones , RecQ Helicasas , Recombinación Genética , Proteína de Replicación A , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Helicasa del Síndrome de Werner
11.
J Clin Invest ; 49(1): 161-9, 1970 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-4188267

RESUMEN

The ability of peripheral blood lymphocytes to respond to phytohemagglutinin (PHA) in vitro was studied in patients with Down's syndrome. The response was measured by the increase in DNA polymerase activity and the rate of incorporation of tritiated thymidine by the cultured lymphocytes. These activities were significantly lower in PHA-stimulated lymphocytes from patients with Down's syndrome compared with age- and sex-matched, mentally retarded patients without Down's syndrome from the same institution and the normal healthy volunteers. The impairment in response to PHA does not seem to be related to the presence of Australia antigen in patients with Down's syndrome or to institutionalization itself. In contrast to DNA polymerase activity and thymidine-(3)H uptake, there was no significant difference in the percentage of blast transformation in the three groups studied. The poor response of the lymphocytes from patients with Down's syndrome to a mitogenic stimulus could reflect an impairment of cellular immune functions in these patients which may be one of the factors contributing to the vulnerability of these patients to repeated or persistent infections.


Asunto(s)
ADN Nucleotidiltransferasas/metabolismo , Síndrome de Down/sangre , Lectinas/farmacología , Linfocitos/efectos de los fármacos , Adolescente , Adulto , Antígenos/análisis , Proteínas Sanguíneas/análisis , Síndrome de Down/enzimología , Síndrome de Down/inmunología , Femenino , Humanos , Discapacidad Intelectual/sangre , Activación de Linfocitos , Linfocitos/enzimología , Linfocitos/inmunología , Linfocitos/metabolismo , Masculino , Timidina/metabolismo , Tritio , gammaglobulinas/análisis
12.
Mol Cell Biol ; 9(2): 469-76, 1989 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-2469002

RESUMEN

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.


Asunto(s)
ADN Viral/biosíntesis , ADN Polimerasa Dirigida por ARN/metabolismo , Retroviridae/enzimología , Virus de la Mieloblastosis Aviar/enzimología , Virus de la Mieloblastosis Aviar/genética , Secuencia de Bases , Codón/genética , ADN Viral/genética , Genes Virales , Datos de Secuencia Molecular , Virus de la Leucemia Murina de Moloney/enzimología , Virus de la Leucemia Murina de Moloney/genética , Pruebas de Mutagenicidad , Mutación , ADN Polimerasa Dirigida por ARN/genética , Retroviridae/genética
13.
Nat Biotechnol ; 17(2): 143-7, 1999 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-10052349

RESUMEN

New techniques now make it feasible to tailor enzymes for cancer gene therapy. Novel enzymes with desired properties can be created and selected from vast libraries of mutants containing random substitutions within catalytic domains. In this review, we first consider genes for the ablation of tumors, namely, genes that have been mutated (or potentially can be mutated) to afford enhanced activation of prodrugs and increased sensitization of tumors to specific chemotherapeutic agents. We then consider genes that have been mutated to provide better protection of normal host tissues, such as bone marrow, against the toxicity of specific chemotherapeutic agents. Expression of the mutant enzyme could render sensitive tissues, such as bone marrow, more resistant to specific cytotoxic agents.


Asunto(s)
Terapia Enzimática , Terapia Genética , Neoplasias/terapia , Médula Ósea , Evolución Molecular Dirigida , Enzimas/genética , Humanos , Profármacos/metabolismo
14.
Nucleic Acids Res ; 29(11): 2418-26, 2001 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-11376161

RESUMEN

We have used random sequence mutagenesis to generate mutants of DNA polymerase beta in an effort to identify amino acid residues important for function, catalytic efficiency and fidelity of replication. A library containing 100 000 mutants at residues 274-278 in the N-helix of the thumb subdomain of the polymerase was constructed and screened for polymerase activity by genetic complementation. The genetic screen identified 4000 active pol beta mutants, 146 of which were sequenced. Each of the five positions mutagenized tolerated substitutions, but residues G274 and F278 were only found substituted in combination with mutations at other positions. The least conserved residue, D276, was replaced by a variety of amino acids and, therefore, does not appear to be essential for function. Steady-state kinetic analysis, however, demonstrated that D276 may be important for catalytic efficiency. Mutant D276E exhibited a 25-fold increase in catalytic efficiency over the wild-type enzyme but also a 25-fold increase in G:T misincorporation efficiency. We present a structural model that can account for the observations and we discuss the implications of this study for the question of enzyme optimization by natural selection.


Asunto(s)
ADN Polimerasa beta/genética , ADN Polimerasa beta/metabolismo , Sustitución de Aminoácidos , Animales , Biblioteca de Genes , Prueba de Complementación Genética , Cinética , Mutagénesis , Mutación , Nucleótidos/metabolismo , Plásmidos/genética , Ratas
15.
Nucleic Acids Res ; 28(17): 3260-8, 2000 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-10954593

RESUMEN

Werner syndrome (WS) is an autosomal recessive disease characterized by early onset of many features of aging, by an unusual spectrum of cancers, and by genomic instability. The WS protein (WRN) possesses 3'-->5' DNA helicase and associated ATPase activities, as well as 3'-->5' DNA exonuclease activity. Currently, WRN is the only member of the widely distributed RecQ DNA helicase family with documented exonuclease activity. It is not known whether deficiency of the exonuclease or helicase/ATPase activities of WRN, or all of them, is responsible for various elements of the WS phenotype. WRN exonuclease has limited homology to Escherichia coli RNaseD, a tRNA processing enzyme. We show here that WRN preferentially degrades synthetic DNA substrates containing alternate secondary structures, with an exonucleolytic mode of action suggestive of RNaseD. We present evidence that structure-dependent binding of WRN to DNA requires ATP binding, while DNA degradation requires ATP hydrolysis. Apparently, the exonuclease and ATPase act in concert to catalyze structure-dependent DNA degradation. We propose that WRN protein functions as a DNA processing enzyme in resolving aberrant DNA structures via both exonuclease and helicase activities.


Asunto(s)
ADN Helicasas/metabolismo , ADN/química , ADN/metabolismo , Exonucleasas/metabolismo , Conformación de Ácido Nucleico , Síndrome de Werner/enzimología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , ADN/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Activación Enzimática , Exodesoxirribonucleasas , Humanos , RecQ Helicasas , Especificidad por Sustrato , Moldes Genéticos , Helicasa del Síndrome de Werner
16.
Nucleic Acids Res ; 27(18): 3702-11, 1999 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-10471740

RESUMEN

Fluoropyrimidines, such as 5-fluorouracil (5-FU), are used extensively in cancer therapy. In the cell, 5-FU is metabolized to 5-fluorodeoxyuridylate (5-FdUMP), a tight binding covalent inhibitor of thymidylate synthase (TS). In order to create 5-FdUMP resistant enzymes to protect chemosensitive normal cells and further understand mechanisms of 5-FdUMP resistance, we have randomized four residues within the active site of TS. Our previous studies identified alterations in residues which produce active TS with enhanced resistance to 5-fluorouridine (5-FdUR). By remutagenizing a subset of the 13 previously targeted residues (A197, L198, C199 and V204), an unbiased random library can be created allowing for extensive testing of all possible amino acid substitutions at each of the sites. Using genetic complementation and selection in Escherichia coli, we identified the spectrum of substitutions that yield active TS as well as those that resulted in 5-FdUR resistant mutants of TS. The 5-FdUR resistant TS were found to share several structural features including hydrophobic substitutions at residue 197, retention of the wild-type leucine 198, the alteration C199L (present in 64% of the drug-resistant library), and polar alterations of valine 204. The catalytic activity of mutants with these features was approximately equal to that of the wild-type TS.


Asunto(s)
Sustitución de Aminoácidos , Antimetabolitos Antineoplásicos/farmacología , Floxuridina/farmacología , Mutagénesis , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismo , Antimetabolitos Antineoplásicos/metabolismo , Sitios de Unión , Catálisis , Clonación Molecular , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Floxuridina/metabolismo , Biblioteca de Genes , Prueba de Complementación Genética , Humanos , Modelos Moleculares , Mutación , Selección Genética , Análisis de Secuencia de ADN , Distribuciones Estadísticas , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química
17.
Cancer Res ; 61(8): 3230-9, 2001 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-11309271

RESUMEN

We have proposed that an early step in tumor progression is the expression of a mutator phenotype resulting from mutations in genes that normally function in the maintenance of genetic stability. There is new and strong experimental evidence that supports the concept of a mutator phenotype in cancer. As technologies for chromosomal visualization and DNA advance, there are increasing data that human cancer cells contain large numbers of mutations. First, I will review the concept of a mutator phenotype. Second, I will present the recent evidence that individual cancer cells contain thousands of mutations. Third, I will explore potential target genes that are required for maintenance of genetic stability in normal cells and ask if they are mutated in cancer cells. Fourth, I will address the timing of a mutator phenotype; is it an early event during tumor progression? Do tumors already contain cells that harbor mutations rendering them resistant to most chemotherapeutic agents? Lastly, I will speculate on the theoretical and practical implication of a mutator phenotype in cancer and consider the possibility of cancer prevention by delay, i.e., a reduction in mutation rates early during carcinogenesis might slow the progression of tumors.


Asunto(s)
Mutación/fisiología , Neoplasias/genética , Animales , Humanos , Fenotipo
18.
Cancer Res ; 52(5): 1082-6, 1992 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-1310639

RESUMEN

An important source of endogenous oxygen radicals are phagocytic cells such as neutrophils and macrophages. The human leukemia cell line HL-60 can be induced to differentiate into a neutrophil-like cell population. Among the properties of these differentiated cells is the ability to produce reactive oxygen species when stimulated by tumor promoters. Mutagenesis induced by HL-60-generated free radicals was assessed using the M13mp2 forward mutation assay. Single-stranded M13mp2 DNA was coincubated with phorbol ester-stimulated HL-60 cells, after which mutations were scored by transfecting the DNA into SOS-induced Escherichia coli. The mutation frequency was increased 6-fold above background in DNA incubated with HL-60 cells. The majority of the mutations were single-base substitutions. However, approximately 6% of the mutations were tandem double substitutions that occurred in runs of adjacent cytidines. Overall, the mutations were clustered at apparent "hot spots," many of which were similar to sites seen using iron to generate oxygen radicals. These results suggest that human cells able to produce oxygen radicals in response to tumor promoters might play a significant role in the generation of tumors.


Asunto(s)
ADN Bacteriano/efectos de los fármacos , ADN de Cadena Simple/efectos de los fármacos , Peróxido de Hidrógeno/metabolismo , Leucemia/genética , Leucemia/metabolismo , Superóxidos/metabolismo , Transfección , ADN Bacteriano/metabolismo , ADN de Cadena Simple/metabolismo , Escherichia coli/genética , Humanos , Pruebas de Mutagenicidad , Acetato de Tetradecanoilforbol , Células Tumorales Cultivadas
19.
Cancer Res ; 36(2 Pt 1): 516-23, 1976 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-1260750

RESUMEN

The in vitro accuracy of DNA replication has been investigated through the measurement of the frequency with which noncomplementary nucleotides were incorporated during polynucleotide replication. The effect of beta-propiolactone treatment of deoxynucleotide templates, ribopolynucleotide templates, and the DNA polymerase from avian myeloblastosis virus was determined. Treatment of the deoxynucleotide template, poly(dA) (see article) oligo(dT) 12-18, by beta-propiolactone resulted in an increased frequency of noncomplementary nucleotide incorporation during DNA polymerization. Carcinogen treatment of the ribonucleotide templates, poly(rA) (see article) oligo(dT) 12-18, and poly(rC) (see article) oligo(dG) 12-18, and carcinogen treatment of avian myeloblastosis virus DNA polymerase did not alter the frequency of noncomplementary nucleotide incorporation. This suggested that carcinogen-induced error incorporation during DNA synthesis was restricted solely to the treatment of a deoxynucleotide template.


Asunto(s)
Carcinógenos/farmacología , Replicación del ADN/efectos de los fármacos , Lactonas/farmacología , Propiolactona/farmacología , Virus de la Mieloblastosis Aviar/enzimología , ADN Nucleotidiltransferasas , Oligodesoxirribonucleótidos , Oligorribonucleótidos , Poli A , Poli C , Polidesoxirribonucleótidos , Moldes Genéticos
20.
Cancer Res ; 49(8): 2141-6, 1989 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-2649235

RESUMEN

We have analyzed colon carcinomas by a combination of histological enrichment, cell sorting, polymerase chain reaction, and direct sequencing of the c-Ki-ras-2 gene. DNA was chemically extracted from 50-microns sections of paraffin-embedded colon carcinomas and amplified in vitro, and mutations were documented directly by DNA sequencing. Enrichment for tumor cells was obtained histologically and by sorting nuclei on the basis of DNA content differences. Mutations in codon 12 were present in both aneuploid and diploid subpopulations of sorted carcinomas, suggesting that these mutations precede ploidy alterations in the progression of these neoplasms. We have demonstrated the feasibility of utilizing DNA from tissues treated with different fixatives, including methyl carnoys, formalin, and Hollande's solution. This procedure allows one to retrospectively reconstruct the temporal relationship between the occurrence of mutations and sequential morphological changes during tumorigenic progression.


Asunto(s)
Neoplasias del Colon/genética , ADN de Neoplasias/análisis , ADN Polimerasa Dirigida por ADN/farmacología , Genes ras , Mutación , Secuencia de Bases , Neoplasias del Colon/patología , Citometría de Flujo , Humanos
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