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1.
Sci Adv ; 6(5): eaax2659, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32064334

RESUMO

Accumulating somatic mutations have been implicated in age-related cellular degeneration and death. Because of their random nature and low abundance, somatic mutations are difficult to detect except in single cells or clonal cell lineages. Here, we show that in single hepatocytes from human liver, an organ exposed to high levels of genotoxic stress, somatic mutation frequencies are high and increase substantially with age. Considerably lower mutation frequencies were observed in liver stem cells (LSCs) and organoids derived from them. Mutational spectra in hepatocytes showed signatures of oxidative stress that were different in old age and in LSCs. A considerable number of mutations were found in functional parts of the liver genome, suggesting that somatic mutagenesis could causally contribute to the age-related functional decline and increased incidence of disease of human liver. These results underscore the importance of stem cells in maintaining genome sequence integrity in aging somatic tissues.


Assuntos
Envelhecimento , Diferenciação Celular/genética , Genoma Humano , Hepatócitos , Fígado , Análise de Célula Única , Células-Tronco , Envelhecimento/genética , Envelhecimento/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Estresse Oxidativo/genética , Células-Tronco/citologia , Células-Tronco/metabolismo
2.
Nature ; 537(7620): 427-431, 2016 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-27556946

RESUMO

Mice deficient in the DNA excision-repair gene Ercc1 (Ercc1∆/-) show numerous accelerated ageing features that limit their lifespan to 4-6 months. They also exhibit a 'survival response', which suppresses growth and enhances cellular maintenance. Such a response resembles the anti-ageing response induced by dietary restriction (also known as caloric restriction). Here we report that a dietary restriction of 30% tripled the median and maximal remaining lifespans of these progeroid mice, strongly retarding numerous aspects of accelerated ageing. Mice undergoing dietary restriction retained 50% more neurons and maintained full motor function far beyond the lifespan of mice fed ad libitum. Other DNA-repair-deficient, progeroid Xpg-/- (also known as Ercc5-/-) mice, a model of Cockayne syndrome, responded similarly. The dietary restriction response in Ercc1∆/- mice closely resembled the effects of dietary restriction in wild-type animals. Notably, liver tissue from Ercc1∆/- mice fed ad libitum showed preferential extinction of the expression of long genes, a phenomenon we also observed in several tissues ageing normally. This is consistent with the accumulation of stochastic, transcription-blocking lesions that affect long genes more than short ones. Dietary restriction largely prevented this declining transcriptional output and reduced the number of γH2AX DNA damage foci, indicating that dietary restriction preserves genome function by alleviating DNA damage. Our findings establish the Ercc1∆/- mouse as a powerful model organism for health-sustaining interventions, reveal potential for reducing endogenous DNA damage, facilitate a better understanding of the molecular mechanism of dietary restriction and suggest a role for counterintuitive dietary-restriction-like therapy for human progeroid genome instability syndromes and possibly neurodegeneration in general.


Assuntos
Envelhecimento/genética , Restrição Calórica , Reparo do DNA/genética , Dieta Redutora , Instabilidade Genômica , Animais , Encéfalo/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Endonucleases/deficiência , Endonucleases/genética , Feminino , Masculino , Camundongos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/prevenção & controle , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Transcriptoma
3.
Oncogene ; 31(50): 5172-9, 2012 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22349820

RESUMO

Observations that genome-wide DNA hypomethylation induces genome instability and tumors in animals caution against the indiscriminate use of demethylating agents, such as 5-aza-2'-deoxycytidine (5-Aza-dC). Using primary mouse embryonic fibroblasts harboring a lacZ mutational reporter construct that allows the quantification and characterization of a wide range of mutational events, we found that, in addition to demethylation, treatment with 5-Aza-dC induces γ-H2AX expression, a marker for DNA breaks, and both point mutations and genome rearrangements. To gain insight into the source of these mutations, we first tested the hypothesis that the mutagenic effect of 5-Aza-dC may be directly mediated through the DNA methyltransferase 1 (DNMT1) covalently trapped in 5-Aza-dC-substituted DNA. Knockdown of DNMT1 resulted in increased resistance to the cytostatic effects of 5-Aza-dC, delayed onset of γ-H2AX expression and a significant reduction in the frequency of genome rearrangements. There was no effect on the 5-Aza-dC-induced point mutations. An alternative mechanism for 5-Aza-dC-induced demethylation and genome rearrangements via activation-induced cytidine deaminase (AID) followed by base excision repair (BER) was found not to be involved. That is, 5-Aza-dC treatment did not significantly induce AID expression and inhibition of BER did not reduce the frequency of genome rearrangements. Thus, our results indicate that the formation of DNMT1 adducts is the prevalent mechanism of 5-Aza-dC-induced genome rearrangements, although hypomethylation per se may still contribute. As the therapeutic effects of 5-Aza-dC greatly depend on the presence of DNMT1, the expression level of DNA methyltransferases in tumors may serve as a prognostic factor for the efficacy of 5-Aza-dC treatment.


Assuntos
Azacitidina/análogos & derivados , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Rearranjo Gênico/efeitos dos fármacos , Animais , Antimetabólitos Antineoplásicos/farmacologia , Azacitidina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citidina Desaminase/genética , Citidina Desaminase/metabolismo , DNA/efeitos dos fármacos , DNA/genética , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1 , Dano ao DNA , Metilação de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Decitabina , Regulação para Baixo/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Mutagênese/efeitos dos fármacos , Mutagênese/genética , Mutação/efeitos dos fármacos , Mutação/genética
4.
Neuroscience ; 145(4): 1341-7, 2007 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-17137723

RESUMO

Genome instability has been implicated as a major cause of both cancer and aging. Using a lacZ-plasmid transgenic mouse model we have shown that mutations accumulate with age in a tissue-specific manner. Genome rearrangements, including translocations and large deletions, are a major component of the mutation spectrum in some tissues at old age such as heart. Such large mutations were also induced by hydrogen peroxide (H2O2) in lacZ-plasmid mouse embryonic fibroblasts (MEFs) and demonstrated to be replication-independent. This was in contrast to ultraviolet light-induced point mutations, which were much more abundant in proliferating than in quiescent MEFs. To test if large rearrangements could adversely affect patterns of gene expression we PCR-amplified global mRNA content of single MEFs treated with H2O2. Such treatment resulted in a significant increase in cell-to-cell variation in gene expression, which was found to parallel the induction and persistence of genome rearrangement mutations at the lacZ reporter locus. Increased transcriptional noise was also found among single cardiomyocytes from old mice as compared with similar cells from young mice. While these results do not directly indicate a cause and effect relationship between genome rearrangement mutations and transcriptional deregulation, they do underscore the stochastic nature of genotoxic effects on cells and tissues and could provide a mechanism for age-related cellular degeneration in postmitotic tissue, such as heart or brain.


Assuntos
Envelhecimento/genética , Regulação da Expressão Gênica/genética , Instabilidade Genômica/genética , Mutação/genética , Animais , Dano ao DNA/genética , Genes Reporter/genética , Humanos , Camundongos , Modelos Animais , Mutação/efeitos dos fármacos , Mutação/efeitos da radiação , Elementos Reguladores de Transcrição/genética
6.
J Med Genet ; 38(12): 824-33, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11748305

RESUMO

While sequence analysis is considered by many to be the most sensitive method of detecting unknown mutations in large genes such as BRCA1, most published estimates of the prevalence of mutations in this gene have been derived from studies that have used other methods of gene analysis. In order to determine the relative sensitivity of techniques that are widely used in research on BRCA1, a set of blinded samples containing 58 distinct mutations were analysed by four separate laboratories. Each used one of the following methods: single strand conformational polymorphism analysis (SSCP), conformation sensitive gel electrophoresis (CSGE), two dimensional gene scanning (TDGS), and denaturing high performance liquid chromatography (DHPLC). Only the laboratory using DHPLC correctly identified each of the mutations. The laboratory using TDGS correctly identified 91% of the mutations but produced three apparent false positive results. The laboratories using SSCP and CSGE detected abnormal migration for 72% and 76% of the mutations, respectively, but subsequently confirmed and reported only 65% and 60% of mutations, respectively. False negatives therefore resulted not only from failure of the techniques to distinguish wild type from mutant, but also from failure to confirm the mutation by sequence analysis as well as from human errors leading to misreporting of results. These findings characterise sources of error in commonly used methods of mutation detection that should be addressed by laboratories using these methods. Based upon sources of error identified in this comparison, it is likely that mutations in BRCA1 and BRCA2 are more prevalent than some studies have previously reported. The findings of this comparison provide a basis for interpreting studies of mutations in susceptibility genes across many inherited cancer syndromes.


Assuntos
Frequência do Gene/genética , Genes BRCA1 , Testes Genéticos/métodos , Mutação/genética , Cromatografia Líquida de Alta Pressão , Análise Mutacional de DNA/economia , Análise Mutacional de DNA/métodos , Éxons/genética , Genes BRCA2 , Predisposição Genética para Doença/genética , Testes Genéticos/economia , Humanos , Desnaturação de Ácido Nucleico , Polimorfismo Conformacional de Fita Simples , Reprodutibilidade dos Testes , Projetos de Pesquisa , Sensibilidade e Especificidade , Método Simples-Cego , Temperatura
7.
Genomics ; 78(1-2): 83-90, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11707076

RESUMO

Two-dimensional gene scanning (TDGS) is a method for analyzing multiple DNA fragments in parallel for all possible sequence variations, using extensive multiplex PCR and two-dimensional electrophoretic separation on the basis of size and melting temperature. High throughput application of TDGS is limited by the prolonged time periods necessary to complete the second-dimension electrophoretic separation step--denaturing gradient gel electrophoresis--and the current need for gel staining. To address these problems, we constructed a high-voltage, automatic, two-dimensional electrophoresis system and used this in combination with thinner gels to reduce two-dimensional electrophoresis time about 80%. Instead of gel staining, we used three different fluorophores to simultaneously analyze three samples in the same gel. These improvements greatly increase TDGS speed and throughput and make the method highly suitable for large-scale single-nucleotide polymorphism discovery and genetic testing.


Assuntos
DNA/análise , Eletroforese em Gel Bidimensional/métodos , Reação em Cadeia da Polimerase/métodos , Proteínas Adaptadoras de Transdução de Sinal , Proteína BRCA1/genética , Proteínas de Transporte , DNA/genética , Humanos , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Proteínas Nucleares , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/genética
8.
Mech Ageing Dev ; 122(15): 1915-8, 2001 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11557289

RESUMO

The Ames dwarf mouse phenotype is based on a homozygous single gene mutation in the Prop-1 gene that markedly extends life span. Since its discovery, interest in breeding these mice as a model to study retardation of aging has increased dramatically. However, the homozygous Prop-1 mutants are infertile, which necessitates breeding heterozygotes. Heterozygotes cannot be distinguished from the wildtype, while the homozygote dwarf phenotype only becomes apparent after about 3 weeks. Hence, there is a need for a simple test to genotype individual animals at an early stage for the absence or presence of one or two copies of the Prop-1 mutant gene. Here we present a Prop-1 genotyping protocol, based on a PCR reaction followed by a PflMI digestion.


Assuntos
Nanismo/genética , Proteínas de Homeodomínio/genética , Mutação , Fatores de Transcrição/genética , Animais , Feminino , Genótipo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes
9.
Mech Ageing Dev ; 122(12): 1321-31, 2001 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-11438122

RESUMO

Mutations arise in the germline by errors of replication, recombination and repair, and the movement of transposable elements. Transgenic mice bearing reporter genes such as lacZ have proven useful for measurements of spontaneous and induced mutation frequencies, as well as studies of the effects of ageing. In this study, testicular DNA from lacZ transgenic mice was examined for age-related effects on mutation frequency and type. The recovered transgene was tested for simple substitutions and rearrangements including transposition of endogenous mobile elements. There was no evidence for either an age-related accumulation of mutations, or for the insertion of retrotransposons into the lacZ reporter gene in the testis. We conclude that the frequency of retrotransposition of several mouse mobile elements into the lacZ reporter gene is less than 3.73x10(-8). This is significantly less than the known frequency of approximately 7% of all spontaneous mutations in the mouse being due to retrotransposition of these elements.


Assuntos
Envelhecimento/fisiologia , Mutação , Túbulos Seminíferos/fisiologia , Animais , DNA/genética , Elementos de DNA Transponíveis , Frequência do Gene , Óperon Lac/genética , Masculino , Camundongos , Camundongos Transgênicos/genética , Retroelementos/genética
10.
Arterioscler Thromb Vasc Biol ; 21(7): 1131-8, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11451741

RESUMO

Transgenic mice overexpressing Cu/Zn superoxide dismutase (hSod1Tg(+/0)) or catalase (hCatTg(+/0)) and knockout mice underexpressing manganese superoxide dismutase (Sod2(+/)(-)) or glutathione peroxidase-1 (Gpx1(-/-)) were used to study the effect of antioxidant enzymes on cell-mediated low density lipoprotein (LDL) oxidation and oxidized LDL (oxLDL)-induced apoptosis. Incubation of LDL with mouse aortic segments or smooth muscle cells (SMCs) resulted in a significant increase in LDL oxidation. However, LDL oxidation was significantly reduced when LDL was incubated with aortic segments and SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with those obtained from wild-type mice. In contrast, LDL oxidation was significantly increased when LDL was incubated with aortic segments and SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. CuSO(4)-oxidized LDL increased DNA fragmentation and caspase activities in the primary cultures of mouse aortic SMCs. However, oxLDL-induced DNA fragmentation and caspase activities were reduced 50% in SMCs obtained from hSod1Tg(+/0) and hCatTg(+/0) mice compared with wild-type control mice. In contrast, oxLDL-induced DNA fragmentation and caspase activities were significantly increased in SMCs obtained from Sod2(+/)(-) and Gpx1(-/-) mice. These findings suggest that overexpression of Cu/Zn superoxide dismutase or catalase reduces cell-mediated LDL oxidation and oxLDL-induced apoptosis, whereas underexpression of manganese superoxide dismutase or glutathione peroxidase-1 increases cell-mediated LDL oxidation and oxLDL-induced apoptosis.


Assuntos
Aorta/citologia , Aorta/enzimologia , Apoptose , Lipoproteínas LDL/biossíntese , Lipoproteínas LDL/farmacologia , Músculo Liso Vascular/enzimologia , Animais , Antioxidantes/metabolismo , Caspases/metabolismo , Catalase/genética , Catalase/metabolismo , Catalase/fisiologia , Células Cultivadas , Técnicas de Cultura , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/fisiologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Oxirredução , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase/fisiologia , Glutationa Peroxidase GPX1
11.
Ann N Y Acad Sci ; 928: 85-96, 2001 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-11795532

RESUMO

With the ample gene sequence information that has become available with the human genome project virtually completed, it has become possible to identify functional gene variants and their frequencies in elderly populations with different aging-related characteristics. Such a genetic epidemiological approach could lead to new insights with respect to the basic mechanisms of aging and longevity as well as the identification of new targets to prevent or retard some of the late-age adverse effects. Using our recently developed two-dimensional gene scanning (TDGS) technology platform we demonstrate the feasibility of this approach by screening two different populations of centenarians for polymorphic variation in the BRCA1 breast cancer susceptibility gene, one of the many genes involved in genome maintenance. The initial results obtained with this approach suggest differences in BRCA1 genotype frequencies between the centenarian populations and controls.


Assuntos
Análise Mutacional de DNA/métodos , Genes BRCA1 , Longevidade/genética , Polimorfismo Genético , Idoso , Idoso de 80 Anos ou mais , Apolipoproteínas E/genética , Pareamento de Bases , Neoplasias da Mama/epidemiologia , Neoplasias da Mama/genética , DNA/genética , Eletroforese em Gel Bidimensional , Estudos de Viabilidade , Frequência do Gene , Predisposição Genética para Doença , Variação Genética , Genótipo , Haplótipos/genética , Humanos , Mutação , Desnaturação de Ácido Nucleico , Fenótipo , Risco , Sensibilidade e Especificidade
12.
Free Radic Biol Med ; 29(7): 664-73, 2000 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11033419

RESUMO

Two types of transgenic mice were generated to evaluate the role of hydrogen peroxide in the formation of nuclear DNA damage. One set of lines overexpresses wild-type human catalase cDNA, which is localized to peroxisomes. The other set overexpresses a human catalase construct that is targeted to the nucleus. Expression of the wild-type human catalase transgene was found in liver, kidney, skeletal muscle, heart, spleen, and brain with muscle and heart exhibiting the highest levels. Animals containing the nuclear-targeted construct had a similar pattern of expression with the highest levels in muscle and heart, but with lower levels in liver and spleen. In these animals, immunofluorescence detected catalase present in the nuclei of kidney, muscle, heart, and brain. Both types of transgenic animals had significant increases of catalase activities compared to littermate controls in most tissues examined. Despite enhanced activities of catalase, and its presence in the nucleus, there were no changes in levels of 8OHdG, a marker of oxidative damage to DNA. Nor were there differences in mutant frequencies at a Lac Z reporter transgene. This result suggests that in vivo levels of H(2)O(2) may not generate 8OHdG or other types of DNA damage. Alternatively, antioxidant defenses may be optimized such that additional catalase is unable to further protect nuclear DNA against oxidative damage.


Assuntos
Catalase/metabolismo , Núcleo Celular/enzimologia , Dano ao DNA , Desoxiguanosina/análogos & derivados , 8-Hidroxi-2'-Desoxiguanosina , Animais , Biomarcadores/análise , Encéfalo/enzimologia , Catalase/genética , Cruzamentos Genéticos , DNA Complementar , Desoxiguanosina/análise , Humanos , Rim/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Transgênicos , Músculo Esquelético/enzimologia , Miocárdio/enzimologia , Estresse Oxidativo , Peroxissomos/enzimologia
13.
Proc Natl Acad Sci U S A ; 97(15): 8403-8, 2000 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-10900004

RESUMO

Somatic mutation accumulation has been implicated as a major cause of cancer and aging. By using a transgenic mouse model with a chromosomally integrated lacZ reporter gene, mutational spectra were characterized at young and old age in two organs greatly differing in proliferative activity, i.e., the heart and small intestine. At young age the spectra were nearly identical, mainly consisting of G. C to A.T transitions and 1-bp deletions. At old age, however, distinct patterns of mutations had developed. In small intestine, only point mutations were found to accumulate, including G.C to T.A, G.C to C.G, and A.T to C.G transversions and G.C to A.T transitions. In contrast, in heart about half of the accumulated mutations appeared to be large genome rearrangements, involving up to 34 centimorgans of chromosomal DNA. Virtually all other mutations accumulating in the heart appeared to be G.C to A.T transitions at CpG sites. These results suggest that distinct mechanisms lead to organ-specific genome deterioration and dysfunction at old age.


Assuntos
Envelhecimento/genética , Coração , Intestino Delgado , Mutação , Animais , Linhagem Celular Transformada , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação Puntual
15.
Mutat Res ; 450(1-2): 167-80, 2000 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-10838141

RESUMO

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA(-/-) mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA(-/-) mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA(-/-) mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.


Assuntos
Cocarcinogênese , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Mutagênese , Proteínas de Ligação a RNA/genética , 2-Acetilaminofluoreno/toxicidade , Animais , Sequência de Bases , Benzo(a)pireno/toxicidade , DNA de Neoplasias/genética , Humanos , Hipoxantina Fosforribosiltransferase/genética , Óperon Lac , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Modelos Genéticos , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Induzidas por Radiação/genética , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Raios Ultravioleta/efeitos adversos , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo , Proteína de Xeroderma Pigmentoso Grupo A
17.
Oncogene ; 19(16): 2060-6, 2000 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-10803467

RESUMO

Somatic mutations in mtDNA have recently been identified in colorectal tumours. Studies of oncocytic tumours have led to hypotheses which propose that defects in oxidative phosphorylation may result in a compensatory increase in mitochondrial replication and/or gene expression. Mutational analysis of mtDNA in thyroid neoplasia, which is characterised by increased numbers of mitochondria and is also one of the most common sites of oncocytic tumours. has been limited to date. Using the recently developed technique of two-dimensional gene scanning, we have successfully examined 21 cases of thyroid tumours, six cases of non-neoplastic thyroid pathology, 30 population controls, nine foetal thyroid tissues and nine foetal tissues of non-thyroid origin, either kidney or liver. We have identified three different somatic mutations (23%) in papillary thyroid carcinomas. In addition, we have found significant differential distributions of mtDNA sequence variants between thyroid carcinomas and controls. Interestingly, these variants appear to be more frequent in the genes which encode complex I of the mitochondrial electron transport chain compared to normal population controls. These findings suggest first, that somatic mtDNA mutations may be involved in thyroid tumorigenesis and second, that the accumulation of certain non-somatic variants may be related to tumour progression in the thyroid.


Assuntos
Carcinoma Papilar/genética , DNA Mitocondrial/genética , Mutação , Neoplasias da Glândula Tireoide/genética , Adenoma/genética , Estudos de Casos e Controles , DNA de Neoplasias/genética , Variação Genética , Humanos , Dados de Sequência Molecular , NADH Desidrogenase/genética , Glândula Tireoide/embriologia , Glândula Tireoide/patologia
18.
Mutat Res ; 447(1): 117-35, 2000 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-10686308

RESUMO

Aging has been explained in terms of an accumulation of mutations in the genome of somatic cells, leading to tissue atrophy and neoplasms, as well as increased loss of function. Recent advances in transgenic mouse modeling and genomics technology have created, for the first time, the opportunity to begin testing this theory. In this paper the existing evidence for a possible role of somatic mutation accumulation in aging will be re-evaluated on the basis of the evolutionary logic of aging and recent insights in genome structure and function. New strategies for investigating the relationship between genome instability, mutation accumulation and aging will be discussed.


Assuntos
Envelhecimento/genética , Mutação , Senilidade Prematura/genética , Animais , Evolução Biológica , Morte Celular/genética , Transformação Celular Neoplásica/genética , Senescência Celular/genética , Citogenética , Reparo do DNA/genética , DNA Mitocondrial/genética , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Telomerase/genética
19.
J Med Genet ; 36(10): 747-53, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10528853

RESUMO

The hereditary breast and ovarian cancer syndrome is associated with a high frequency of BRCA1 mutations. However, the widespread use of BRCA1 testing has been limited to date by three principal concerns: the fear of loss of health and life insurance, the uncertain clinical value of a positive test result, and the current lack of an inexpensive and sensitive screening test for BRCA1 mutations. We have developed an inexpensive system for gene mutational scanning, based on a combination of extensive multiplex PCR amplification and two dimensional electrophoresis. The efficiency of this system, as a screening test for BRCA1 mutations, was evaluated in a panel of 60 samples from high risk women, 14 of which contained a previously identified mutation in BRCA1. All 14 mutations were identified, as well as an additional five that had previously escaped detection. In addition to the 19 mutations, a total of 15 different polymorphic variants were scored, most of which were recurring. All were confirmed by nucleotide sequencing. The cost of screening per sample was calculated to be approximately US$70 for the manual technique used in this study, and may be reduced to approximately US$10 with the introduction of commercially available PCR robotics and fluorescent imaging. Implementation of this method of mutation screening in the research and clinical setting should permit rapid accrual of quantitative data on genotype-phenotype associations for the evaluation of diagnostic testing.


Assuntos
Genes BRCA1/genética , Testes Genéticos/métodos , Neoplasias da Mama/genética , Análise Mutacional de DNA/economia , Análise Mutacional de DNA/métodos , Eletroforese em Gel Bidimensional , Éxons , Feminino , Testes Genéticos/economia , Humanos , Masculino , Modelos Genéticos , Neoplasias/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Reprodutibilidade dos Testes , Análise de Sequência de DNA
20.
Environ Mol Mutagen ; 34(2-3): 112-20, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10529734

RESUMO

Transgenic animal models harboring chromosomally integrated shuttle vectors with bacterial reporter genes are now widely used to measure in vivo mutant frequencies. The lacZ-plasmid transgenic mouse model has a unique sensitivity to large rearrangements compared to systems using bacteriophage lambda vectors, which mainly detect point mutations and small deletions or insertions. In this study, the background mutant frequencies and spectra in the lacZ-plasmid transgenic mouse model were investigated. While the majority of the recovered lacZ-mutants appeared to have originated in the mouse, a subset of mutants are likely to represent artifacts, and occur with a frequency of about 1.3 x 10(-5), irrespective of the total mutant frequency. Galactose-insensitive host cells, due to galE back mutations or galK or galT forward mutations, grow through the positive selection system and cause a small subset of the background. When using HindIII to excise the plasmids from genomic DNA, the largest contribution to the background, (1.1 +/- 0.3) x 10(-5), appeared to be caused by star activity, i.e., cleavage at nucleotide sequences other than the HindIII restriction enzyme recognition sequence, during the recovery procedure. Finally, a total of 10 polymorphic sites in different copies of the lacZ-plasmid cluster in founder line 60 were discovered.


Assuntos
Óperon Lac , Plasmídeos , Mutação Puntual , Polimorfismo Genético , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Escherichia coli/genética , Camundongos , Camundongos Transgênicos
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