RESUMO
AIMS: To describe the evolution of proficiency testing for molecular diagnostic pathology with respect to determining unambiguously the patient identity of tissue samples by microsatellite analysis. METHOD: Four rounds of quality control exchanges of samples from different patients were sent with the purpose of identifying the correct origin of these samples. The samples were either paraffin wax embedded sections on glass, sections in tubes, or isolated DNA. Blinded samples were distributed to all participating laboratories. No restrictions to the method and short tandem repeat markers used for identification were imposed. RESULTS: In four subsequent rounds the number of participating laboratories increased from three to 10. The numbers of samples tested increased in time from five to 12. The microsatellite markers used by the different laboratories showed little overlap. In the first three rounds, in which isolated DNA was provided, all samples were accurately classified irrespective of the microsatellite markers used. In the last round, which also included paraffin wax embedded sections, a small number of laboratories experienced problems, either with amplification or incorrect classification of a few samples. CONCLUSION: Proficiency testing was useful, and showed country wide high quality and correct identification of (patient) samples with molecular techniques for diagnostic purposes.
Assuntos
Técnicas Genéticas/normas , Patologia Clínica/normas , DNA/análise , Marcadores Genéticos , Humanos , Laboratórios/normas , Repetições de Microssatélites , Países Baixos , Inclusão em Parafina , Controle de Qualidade , Sequências de Repetição em TandemRESUMO
Recently, we and others reported instability in the (C)8 repeat in exon 5 of MSH6 as a preferential target for somatic mutations in tumours from MSH6 germline mutation carriers. Here, we report that in 45% of tumours from MLH1, MSH2 and MSH6 germline mutation carriers no sequence change in the (C)8 repeat of MSH6 was found upon DNA sequencing analysis of PCR products with a shift in electrophoresis mobility. Using "standard" PCR primers a high frequency of instability (50-86%) of the (C)8 repeat was found, but using a modified PCR reverse primer, accomplishing modulation of non-templated addition of adenine during in vitro PCR amplification by the Taq polymerase, a markedly lower frequency of instability was found in tumours from MLH1, MSH2 and MSH6 mutation carriers (6, 13 and 40%, respectively). Furthermore, a significant difference of the frequency of instability of the (C)8 repeat in tumours from MSH6 mutation carriers was found compared to MLH1, MSH2 mutation carriers. These results might have important implications for the detection of instability of other short mononucleotide repeats, e.g. TGFbetaRII, BAX, IGFRII, PTEN, BRCA2.
Assuntos
Proteínas de Ligação a DNA , Proteínas Fúngicas/genética , Proteínas de Saccharomyces cerevisiae , Expansão das Repetições de Trinucleotídeos , Proteínas Adaptadoras de Transdução de Sinal , Viés , Proteínas de Transporte , Análise Mutacional de DNA , Primers do DNA/metabolismo , Éxons , Deleção de Genes , Humanos , Repetições de Microssatélites , Proteína 1 Homóloga a MutL , Proteína 2 Homóloga a MutS , Mutação , Proteínas de Neoplasias/genética , Proteínas Nucleares , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas/genética , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Hereditary non-polyposis colorectal cancer (HNPCC) is the most common genetic susceptibility syndrome for colorectal cancer. HNPCC is most frequently caused by germline mutations in the DNA mismatch repair (MMR) genes MSH2 and MLH1. Recently, mutations in another MMR gene, MSH6 (also known as GTBP), have also been shown to result in HNPCC. Preliminary data indicate that the phenotype related to MSH6 mutations may differ from the classical HNPCC caused by defects in MSH2 and MLH1. Here, we describe an extended Dutch HNPCC family not fulfilling the Amsterdam criteria II and resulting from a MSH6 mutation. Overall, the penetrance of colorectal cancer appears to be significantly decreased (p<0.001) among the MSH6 mutation carriers in this family when compared with MSH2 and MLH1 carriers (32% by the age of 80 v >80%). Endometrial cancer is a frequent manifestation among female carriers (six out of 13 malignant tumours). Transitional cell carcinoma of the urinary tract is also relatively common in both male and female carriers (10% of the carriers). Moreover, the mean age of onset of both colorectal cancer (MSH6 v MSH2/MLH1 = 55 years v 44/41 years) and endometrial carcinomas (MSH6 v MSH2/MLH1 = 55 years v 49/48 years) is delayed. As previously reported, we confirm that the pattern of microsatellite instability, in combination with immunohistochemical analysis, can predict the presence of a MSH6 germline defect. The detailed characterisation of the clinical phenotype of this kindred contributes to the establishment of genotype-phenotype correlations in HNPCC owing to mutations in specific mismatch repair genes.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Proteínas de Ligação a DNA/genética , Mutação em Linhagem Germinativa/genética , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Pareamento Incorreto de Bases/genética , Carcinoma de Células de Transição/epidemiologia , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/patologia , Neoplasias Colorretais Hereditárias sem Polipose/epidemiologia , Neoplasias Colorretais Hereditárias sem Polipose/patologia , Análise Mutacional de DNA , Reparo do DNA/genética , Diagnóstico Diferencial , Neoplasias do Endométrio/epidemiologia , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Mutação da Fase de Leitura/genética , Predisposição Genética para Doença/genética , Genótipo , Humanos , Imuno-Histoquímica , Masculino , Repetições de Microssatélites/genética , Pessoa de Meia-Idade , Países Baixos , Linhagem , Penetrância , Neoplasias Urológicas/epidemiologia , Neoplasias Urológicas/genética , Neoplasias Urológicas/patologiaRESUMO
Instability of microsatellite repeat sequences has been observed in colorectal carcinomas and in extracolonic malignancies, predominantly endometrial tumours, occurring in the context of hereditary non-polyposis colorectal cancer (HNPCC). Microsatellite instability (MSI) as a feature of human DNA mismatch repair (MMR)-driven tumourigenesis of the uterine mucosa has been studied primarily in sporadic tumours showing predominantly somatic hypermethylation of MLH1. The present study shows that all endometrial carcinomas (n=12) from carriers of MLH1 and MSH2 germline mutations demonstrate an MSI-high phenotype involving all types of repeat markers, while in endometrial carcinomas from MSH6 mutation carriers, only 36% (4 out of 11) demonstrate an MSI-high phenotype. Interestingly, an MSI-high phenotype was found in endometrial hyperplasias from MSH2 mutation carriers, in contrast to hyperplasias from MLH1 mutation carriers, which exhibited an MSI-stable phenotype. Instability of only mononucleotide repeat markers was found in both endometrial carcinomas and hyperplasias from MSH6 mutation carriers. In 29 out of 31 (94%) endometrial tumour foci, combined MSI and immunohistochemical analysis of MLH1, MSH2, and MSH6 could predict the identified germline mutation. The observation of MSI in endometrial hyperplasia and of altered protein staining for the MMR genes supports the idea that inactivation of MMR genes is an early event in endometrial tumourigenesis. A correlation was found between the variation in the extent and level of MSI and the age of onset of carcinoma, suggesting differences in the rate of tumour progression. A high frequency of MSI in hyperplasias, found only in MSH2 mutation carriers, might indicate a more rapid tumour progression, correlating with an earlier age of onset of carcinoma. The present study indicates that assessment of altered protein staining combined with MSI analysis of endometrial tumours might direct the mutational analysis of MMR genes.
Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo do DNA/genética , Hiperplasia Endometrial/genética , Neoplasias do Endométrio/genética , Repetições de Microssatélites/genética , Adulto , Idade de Início , Neoplasias Colorretais Hereditárias sem Polipose/complicações , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Progressão da Doença , Hiperplasia Endometrial/etiologia , Hiperplasia Endometrial/metabolismo , Neoplasias do Endométrio/etiologia , Neoplasias do Endométrio/metabolismo , Feminino , Mutação em Linhagem Germinativa , Humanos , Pessoa de Meia-Idade , Fenótipo , Valor Preditivo dos TestesRESUMO
Specific defects in DNA repair pathways are reflected by DNA microsatellite instability (MSI) and play an important role in carcinogenesis. Reported frequencies in gastric non-Hodgkin's lymphomas (NHL) vary from 14% to as high as 90%. Another form of genetic instability in tumours is allelic imbalance (AI) due to loss or gain of genetic material at a specific chromosomal region. This might point to the presence of a tumour suppressor gene or oncogene. We examined both MSI and AI in 26 gastric lymphomas (10 low-grade and 13 high-grade MALT lymphomas and three cases lacking MALT features and categorised as diffuse large B cell lymphoma (DLCL)). Tumour components and normal cells (epithelium, muscle) were microdissected from paraffin-embedded resection samples. Contrary to other studies we did not observe frequent MSI when investigating 18 different loci distributed over 12 chromosomes. Microsatellite instability of a single locus was found in 1/10 (10%) low-grade MALT lymphomas and 2/13 (15%) high-grade MALT lymphomas. These data indicate that DNA mismatch repair genes do not play a role in the pathogenesis of these lymphomas. Allelic imbalance was detected in 60% (6/10) of low-grade MALT lymphomas, in 62% (8/13) of high-grade MALT lymphoma and in 67% (2/3) of DLCL. In high-grade lymphomas more loci showed AI (one to seven loci, with a mean of 2.5 loci per case) than in the low-grade lymphomas (one to two loci, with a mean of 1.3 loci per case), possibly reflecting an increased genomic instability.
Assuntos
Perda de Heterozigosidade/genética , Linfoma/genética , Repetições de Microssatélites/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Neoplasias Gástricas/genética , Expansão das Repetições de Trinucleotídeos/genética , Análise Mutacional de DNA , Epitélio/metabolismo , Genes myc/genética , Marcadores Genéticos/genética , Genótipo , Humanos , Linfoma de Zona Marginal Tipo Células B/classificação , Linfoma de Zona Marginal Tipo Células B/genética , Linfoma Difuso de Grandes Células B/genética , Linfoma não Hodgkin/genética , Músculos/metabolismo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Proteínas Proto-Oncogênicas/genética , Proteína X Associada a bcl-2RESUMO
Two-dimensional (2-D) DNA fingerprinting was used to investigate genomic changes in human low-grade gliomas of different subtypes. DNA variations were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2-D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)-amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus-specific probe for the rehybridization of the 2-D blots. One of these probes detected a spot loss in 7 of 28 low-grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus-specific probes that may be instrumental in the discovery of the critical early events of glioma pathogenesis.
Assuntos
Impressões Digitais de DNA , Glioma/genética , Repetições Minissatélites , Mapeamento Cromossômico , Cromossomos Humanos Par 11 , Clonagem Molecular , Eletroforese em Gel Bidimensional , Humanos , Hibridização In Situ , Hibridização de Ácido Nucleico , Reação em Cadeia da PolimeraseRESUMO
In breast cancer, inactivating point mutations in the E-cadherin gene are frequently found in invasive lobular carcinoma (ILC) but never in invasive ductal carcinoma (IDC). Lobular carcinoma in situ (LCIS) adjacent to ILC has previously been shown to lack E-cadherin expression, but whether LCIS without adjacent invasive carcinoma also lacks E-cadherin expression and whether the gene mutations present in ILC are already present in LCIS is not known. We report here that E-cadherin expression is absent in six cases of LCIS and present in 150 cases of ductal carcinoma in situ (DCIS), both without an adjacent invasive component. Furthermore, using mutation analysis, we could demonstrate the presence of the same truncating mutations and loss of heterozygosity (LOH) of the wild-type E-cadherin in the LCIS component and in the adjacent ILC. Our results indicate that E-cadherin is a very early target gene in lobular breast carcinogenesis and plays a tumour-suppressive role, additional to the previously suggested invasion-suppressive role.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Caderinas/metabolismo , Carcinoma in Situ/metabolismo , Carcinoma Lobular/metabolismo , Carcinoma in Situ/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/genética , Deleção Cromossômica , DNA/análise , Heterozigoto , Humanos , Imuno-HistoquímicaRESUMO
Loss of expression of the intercellular adhesion molecule E-cadherin frequently occurs in invasive lobular breast carcinomas as a result of mutational inactivation. Expression patterns of E-cadherin and the molecules comprising the cytoplasmic complex of adherens junctions, alpha-, beta- and gamma-catenin, were studied in a series of 38 lobular breast carcinomas with known E-cadherin mutation status. The effect of loss of E-cadherin by mutational inactivation (or other mechanisms) on the expression of catenins was investigated. Complete loss of plasma membrane-associated E-cadherin expression was observed in 32 out of 38 invasive lobular carcinomas, for which in 21 cases a mutation was found in the extracellular domain of E-cadherin. In total, 15 frameshift mutations of small deletions or insertions, ranging from 1 to 41 bp, three non-sense mutations, and three splice mutations were identified. Mutations were scattered over the whole coding region and no hot spots could be detected. In all cases, simultaneous loss of E-cadherin and alpha- and beta-catenin expression was found; in 50 per cent of these cases, additional loss of gamma-catenin was observed. In six invasive lobular carcinomas, expression of both E-cadherin and catenins was retained. In none of these carcinomas was an E-cadherin mutation detected. Lobular carcinoma in situ adjacent to invasive lobular carcinoma showed simultaneous loss of E-cadherin and catenins in all the cases studied--remarkably, also, in four cases positive for E-cadherin and catenin expression in the invasive component. These results indicate that simultaneous loss of E-cadherin and alpha-, beta- and gamma-catenin may be an important step in the formation of lobular carcinoma in situ, as a precursor of invasive lobular breast cancer. Events additional to E-cadherin inactivation must be involved in the transition of lobular carcinoma in situ to invasive lobular carcinoma.
Assuntos
Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transativadores , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Carcinoma in Situ/genética , Carcinoma in Situ/metabolismo , Carcinoma in Situ/patologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Análise Mutacional de DNA , Desmoplaquinas , Feminino , Humanos , Invasividade Neoplásica , alfa Catenina , beta Catenina , gama CateninaRESUMO
The origin of malignant mixed Müllerian tumours (MMMTs) has long been debated, due to the indefinite relationship between epithelial and mesenchymal malignant cells. In order to obtain insight into the clonal relationship between the two components of these tumours, molecular genetic changes were investigated at the level of loss of heterozygosity (LOH) in both cells types. LOH was studied in a series of six cases with 74 polymorphic microsatellite markers mapping to 19 different chromosomes. The epithelial and the mesenchymal neoplastic cells were separately microdissected from formalin-fixed, paraffin-embedded tissue, prior to DNA isolation. LOH was observed for 35 different markers mapping to chromosomes 3, 6, 8, 11, 15, 16, 17, 18, 21, and X. The most frequently involved chromosomes were 17p, 17q, 11q, 15q, and 21q. LOH was observed in five out of six cases and identical alleles were lost in the epithelial and in the mesenchymal cells. No genetic differences were observed between the two cell types for any of the informative markers. Immunohistochemistry (IHC) and TP53 mutation analysis revealed involvement of TP53 in all cases. Mutations were identified in five MMMTs. In four tumours, of which three had a missense mutation, strong nuclear staining for p53 was observed. In the remaining two cases, the mutation resulted in a stop codon, with no nuclear staining for p53 by IHC. The results support a monoclonal origin of MMMTs, with the absence of genetic changes uniquely associated with either of the phenotypes. The latter finding is compatible with current opinion that these neoplasms should be considered as metaplastic carcinomas and supports the conversion hypothesis.
Assuntos
Deleção Cromossômica , Neoplasias do Endométrio/genética , Tumor Mulleriano Misto/genética , Neoplasias Ovarianas/genética , Idoso , Idoso de 80 Anos ou mais , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Feminino , Genes p53 , Heterozigoto , Humanos , Técnicas Imunoenzimáticas , Pessoa de Meia-Idade , Tumor Mulleriano Misto/metabolismo , Tumor Mulleriano Misto/patologia , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Reação em Cadeia da Polimerase , Proteína Supressora de Tumor p53/metabolismoRESUMO
We have analysed a series of 49 human breast cancers for mutations in the entire coding region plus flanking intron sequences of the E-cadherin gene. The tumours included 41 infiltrating lobular carcinomas, two infiltrating ducto-lobular carcinomas and six infiltrative ductal carcinomas. In the lobular carcinomas 23 different somatic mutations were detected, of which seven were insertions, 11 deletions, two nonsense mutations and three splice site mutations. The other tumours showed no detectable E-cadherin mutations. All the frameshift and nonsense mutations are expected to generate a secreted E-cadherin fragment instead of a transmembrane protein with cell adhesion activity. The majority of the mutations (21 of 23) were found in combination with loss of heterozygosity of the wild type E-cadherin locus (16q22.1), a hallmark of classical tumour suppressor genes. The mutations were scattered over the whole coding region and no hot spots could be identified. All mutations described here were previously unreported. In conclusion, we have identified up to now E-cadherin mutations in 27 of 48 (56%) infiltrating lobular breast carcinomas and in 0 of 50 breast cancers of other histopathological subtypes. These data provide strong evidence that frequent E-cadherin mutations are involved in the particular etiology of sporadic lobular breast cancers.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Mutação , Sítios de Ligação , Neoplasias da Mama/patologia , Caderinas/metabolismo , Carcinoma Lobular/genética , Carcinoma Lobular/patologia , Cromossomos Humanos Par 16 , Análise Mutacional de DNA , Heterozigoto , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Análise de Sequência de DNARESUMO
Compelling experimental evidence exists for a potent invasion suppressor role of the cell-cell adhesion molecule E-cadherin. In addition, a tumour suppressor effect has been suggested for E-cadherin. In human cancers, partial or complete loss of E-cadherin expression correlates with malignancy. To investigate the molecular basis for this altered expression we developed a comprehensive PCR/SSCP mutation screen for the human E-cadherin gene. For 49 breast cancer patients the occurrence of tumour-specific mutations in the E-cadherin gene was examined. No relevant DNA changes were encountered in any of 42 infiltrative ductal or medullary breast carcinoma samples. In contrast, four out of seven infiltrative lobular breast carcinomas harboured protein truncation mutations (three nonsense and one frameshift) in the extracellular part of the E-cadherin protein. Each of the four lobular carcinomas with E-cadherin mutations showed tumour-specific loss of heterozygosity of chromosomal region 16q22.1 containing the E-cadherin locus. In compliance with this, no E-cadherin expression was detectable by immunohistochemistry in these four tumours. These findings offer a molecular explanation for the typical scattered tumour cell growth in infiltrative lobular breast cancer.
Assuntos
Neoplasias da Mama/genética , Caderinas/genética , Carcinoma Lobular/genética , Genes Supressores de Tumor , Mutação , Sequência de Bases , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Caderinas/fisiologia , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Carcinoma Lobular/etiologia , Carcinoma Lobular/metabolismo , Cromossomos Humanos Par 16/genética , Primers do DNA/genética , DNA de Neoplasias/genética , Feminino , Heterozigoto , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Gene mutations have been implicated in the etiology of cancer, developmental anomalies, genetic disease and aging. Many different methods for mutation detection have been developed and applied to obtain a more fundamental insight in the chain of molecular events that ultimately lead to mutations. Most of these methods, however, can only be applied to cultured cells and therefore do not allow comparative analysis of mutations in various organs and tissues in an intact organism. The main difficulty in studying mutagenesis in chromosomal DNA is to identify and isolate mutated genes with a high efficiency. Here we describe the development and application of LacZ transgenic mouse models for studying, in different organs and tissues, spontaneous or induced mutations. Such models allow study of the induction of DNA damage, repair, mutagenesis and carcinogenesis in one animal system. Accordingly, results obtained may ultimately provide greater insight into the chain of events from in vivo exposure to genotoxic agents to mutations and their ultimate physiological endpoints. In addition to their use in fundamental research, transgenic animal mutation models find a major application in the field of genetic toxicology testing, in particular with respect to organ specificity.
Assuntos
Óperon Lac , Camundongos Transgênicos/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Mutação , Animais , Bacteriófago lambda/genética , Elementos de DNA Transponíveis , Escherichia coli/genética , Vetores Genéticos , Camundongos , Testes de Mutagenicidade/métodos , Especificidade de ÓrgãosRESUMO
We have recently used two-dimensional DNA typing to detect genetic alterations in breast tumours. This method, which is based on size separation in neutral gels and sequence separation in denaturing gradient gels followed by hybridisation analysis with mini- and microsatellite core probes, allows the simultaneous analysis of hundreds of allelic fragments in a very short time. Here we demonstrate the potency of this method for total genome scanning of the tumour genome by analysing a small series of breast cancers. Comparison of tumour and normal DNA from ten breast cancer patients, using two-dimensional DNA typing with four core probes, revealed a considerable number of genomic alterations. In contrast, with Southern blot analysis only a few alterations were observed using the same probes. Most of the changes observed (74%) were deletions (absence of spots in the tumour) while 20% corresponded to amplifications (spots of higher intensity in the tumour) and 5% were new spots (gains). About 10% of the genomic changes detected appeared to occur in the tumours of more than one patient.
Assuntos
Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , DNA de Neoplasias/análise , DNA/análise , Sequência de Bases , Biomarcadores Tumorais/análise , Southern Blotting , Neoplasias da Mama/sangue , Carcinoma Ductal de Mama/sangue , Sondas de DNA , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Eletroforese em Gel Bidimensional , Feminino , Genoma Humano , Humanos , Valor Preditivo dos Testes , PrognósticoRESUMO
Transgenic mice with integrated shuttle vectors containing the LacZ mutational target gene were used to study spontaneous mutational events in vivo. The transgenic mouse strain used carries the LacZ transgene on the X chromosome and was previously found to be characterized by approximately 25-fold higher spontaneous mutation frequency in liver and brain compared with at least three other transgenic mouse strains. To determine the nature of in vivo spontaneous mutational events, 35 mutant LacZ genes isolated from liver and brain of mice from strain 35.5 were analyzed at the DNA sequence level. The results obtained indicate that single base-pair changes were predominant in both liver and brain. However, in liver the majority of mutations were transitions whereas in brain transversions were predominantly observed. Six mutants appeared to contain multiple dispersed mutations, separated by as much as 44 bp. Mutations were generally located within a 500 bp region encoding the active site of the beta-galactosidase protein. Our results indicate that spontaneous mutations at the LacZ transgene are tissue specific and dependent on the chromosomal position of the LacZ transgene.
Assuntos
DNA Bacteriano , DNA , Genes Bacterianos , Mutação , Cromossomo X , beta-Galactosidase/genética , Animais , Sequência de Bases , Encéfalo/enzimologia , Feminino , Vetores Genéticos , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Dados de Sequência MolecularRESUMO
A method for the efficient rescue of lac operator containing plasmids from transgenic mouse genomic DNA is described. The method is based on the high affinity of the LacI repressor protein for the lac operator sequence. Using the LacI repressor protein conjugated to magnetic beads, more than 95% of plasmid sequences could be purified from restriction enzyme digested genomic DNA. After circularization, the plasmids were introduced into Escherichia coli by means of electroporation. Since the plasmid was cloned into a bacteriophage lambda vector, the efficiency of plasmid rescue could easily be compared with in vitro packaging. Our results indicate that plasmid rescue is about 25 times more efficient. Application of this method should be especially useful with transgenic mouse models harboring LacZ plasmid shuttle vectors for studying spontaneous or induced mutations in vivo.
Assuntos
DNA Recombinante/isolamento & purificação , Proteínas de Escherichia coli , Técnicas Genéticas , Plasmídeos/isolamento & purificação , Animais , Proteínas de Bactérias , Bacteriófago lambda/genética , Biotecnologia , DNA Recombinante/genética , Escherichia coli/genética , Estudos de Avaliação como Assunto , Vetores Genéticos , Óperon Lac , Repressores Lac , Magnetismo , Camundongos , Camundongos Transgênicos , Plasmídeos/genética , Proteínas RepressorasAssuntos
Mapeamento Cromossômico , Doença das Coronárias/patologia , Vasos Coronários/patologia , Transplante de Coração , Músculo Liso Vascular/patologia , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Adulto , Sequência de Bases , Linhagem Celular , Mapeamento Cromossômico/métodos , Angiografia Coronária , Doença das Coronárias/etiologia , Doença das Coronárias/cirurgia , Humanos , Masculino , Sondas Moleculares/genética , Dados de Sequência Molecular , Complicações Pós-OperatóriasRESUMO
To study spontaneous and induced mutagenesis in vivo we recently constructed a series of transgenic mice harboring different numbers of bacteriophage lambda shuttle vectors, provided with a LacZ mutational target gene, integrated in their genome. The transgenic mice enabled analysis of spontaneous and induced mutation frequencies in postmitotic tissues like liver and brain. The obtained data indicated spontaneous mutation frequencies in the order of 10(-5)-10(-6). Here we report a 25-100 times higher spontaneous mutation frequency in liver and brain DNA of mice from strain 35.5, with the lambda-gt10LacZ concatemer integrated on the X-chromosome. These results indicate the presence of a mutational 'hot spot' in the mammalian somatic genome in vivo.