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J Vijg

Publications and source records attributed to J Vijg.

At least 19 recordsLinked to original sources
PubMed1998

Mutational scanning of mitochondrial DNA by two-dimensional electrophoresis.

An expedient, accurate, and cost-efficient test was developed to scan critical regions of the mitochondrial genome for all possible mutations by two-dimensional DNA electrophoresis. The test involves a two-step multiplex PCR amplification: a long-distance PCR to amplify almost the entire mitochondrial genome, which then serves as template for the amplification of 25 short PCR fragments in two multiplex groups corresponding to regions implicated in human diseases. The mixture of fragments was subsequently subjected to two-dimensional electrophoretic separation, first by size in a nondenaturant polyacrylamide gel and then on the basis of basepair sequence in a denaturing gradient polyacrylamide gel. This latter process of denaturing gradient gel electrophoresis is a most accurate form of mutation detection on the basis of differences in melting behavior of mutant and wildtype fragments. Evaluation of the method using samples with known homoplasmic and heteroplasmic mutations, as well as CEPH pedigrees to study segregation of polymorphic variants, indicated a very high accuracy; none of the previously identified mutations and polymorphisms escaped detection, and no erroneous segregation patterns of polymorphic variants were observed. In addition, two variants were found to be novel mutations when analyzed by sequence analysis. One of these novel mutations was a heteroplasmic mutation in the COXIII gene that was found to segregate to homoplasmy in the next generation. Heteroplasmic mutations as low as 1% of mtDNA could still be detected.

DNA Mutational Analysis↗
PubMed1998

Transgenic assays for mutations and cancer: current status and future perspectives.

Transgenic mouse modelling has proved to be a powerful approach to explore the various steps involved in spontaneous and induced carcinogenesis. Some of the multitude of models currently available have the potential to become a substitute for the expensive, long-term rodent bioassay to predict carcinogenicity of environmental compounds. Here, we review the progress in the development and use of transgenic mouse models specifically for the purpose of carcinogenicity and mutagenicity testing.

Animals↗
PubMed1998

Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests.

With the current rapid pace at which human disease genes are identified there is a need for practical, cost-efficient genetic screening tests. Two-dimensional electrophoretic separation of PCR-amplified gene fragments on the basis of size and base pair sequence, in non-denaturing and denaturing gradient polyacrylamide gels respectively, provides a rapid parallel approach to gene mutational scanning. Accuracy of the denaturing gradient gel electrophoresis (DGGE) component of this system strongly depends on the design of the PCR primers and the melting characteristics of the fragments they encompass. We have developed a fully automated generally applicable procedure to generate optimal two-dimensional test designs at a minimum amount of time and effort. Designs were generated for the RB1 , TP53 , MLH1 and BRCA1 genes that can be readily implemented in research and clinical laboratories as low cost genetic screening tests.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Comprehensive mutational scanning of the p53 coding region by two-dimensional gene scanning.

A comprehensive mutational scanning test for the p53 coding region based on multiplex PCR and two-dimensional DNA electrophoresis was designed and evaluated. In a 2-step multiplex PCR, the p53 coding region (exons 2-11) was amplified as a single 8646-bp fragment by long-distance PCR in step one. This fragment served as a template for the subsequent co-amplification of the individual exons in two multiplex groups in step two. The multiplex products were then separated, first on the basis of size in non-denaturant polyacrylamide gels and then on the basis of sequence by denaturing gradient gel electrophoresis (DGGE). Primers for optimal PCR, melting behavior and 2-D gel distribution were designed using a recently developed computer program. The resulting two-dimensional gene scanning (TDGS) test was evaluated by screening, in a blinded fashion, 29 coded DNA samples from Li-Fraumeni syndrome patients with previously identified germline mutations. All mutations were correctly detected. This assay provides an accurate, cost-effective and non-radioactive method for simultaneous mutational scanning of all p53 coding exons.

Base Sequence↗
PubMed1998

Critical factors in the performance and cost of two-dimensional gene scanning: RB1 as a model.

Two-dimensional (2-D) gene scanning (TDGS) is a method for mutation detection based on the electrophoretic separation of PCR-amplified DNA fragments according to size and base pair sequence. The use of denaturing gradient gel electrophoresis (DGGE) as the second separation step provides virtually 100% sensitivity, while the 2-D format allows the inspection of multiple gene fragments simultaneously. Analysis of many exons in parallel is greatly facilitated by extensive PCR multiplexing based on preamplification by long-distance PCR. Recently, TDGS has been applied to detect mutations in the retinoblastoma tumor suppressor gene RB1. Using RB1 as a model, we have now analyzed each step of the protocol, presenting overall improvements and a detailed cost analysis, where the total cost of the assay is found to be about $40 (US). An overall picture of TDGS cost-performance, as compared to direct sequencing, is provided as a function of the number of target fragments.

Costs and Cost Analysis↗
PubMed1997

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recovery the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Aging↗
PubMed1997

Exclusion of PTEN and 10q22-24 as the susceptibility locus for juvenile polyposis syndrome.

Juvenile polyposis syndrome (JPS; MIM 174900) is an autosomal dominant condition with incomplete penetrance characterized by hamartomatous polyps of the gastrointestinal tract and a risk of gastrointestinal cancer. Gastrointestinal hamartomatous polyps are also present in Cowden syndrome (CS; MIM 158350) and Bannayan-Zonana syndrome (BZS; also called Ruvalcaba-Myhre-Smith syndrome; MIM 153480). The susceptibility locus for both CS and BZS has recently been identified as the novel tumor suppressor gene PTEN, encoding a dual specificity phosphatase, located at 10q23.3. A putative JPS locus, JP1, which most likely functions as a tumor suppressor, had previously been mapped to 10q22-24 in both familial and sporadic juvenile polyps. Given the shared clinical features of gastrointestinal hamartomatous polyps among the three syndromes and the coincident mapping of JP1 to the region of PTEN, we sought to determine whether JPS was allelic to CS and BZS by mutation analysis of PTEN and linkage approaches. Microsatellite markers spanning the CS/BZS locus (D10S219, D10S551, D10S579, and D10S541) were used to compute multipoint lod scores in eight informative families with JPS. Lod scores of < -2.0 were generated for the entire region, thus excluding PTEN and any genes within the flanking 20-cM interval as candidate loci for familial JPS under our statistical models. In addition, analysis of PTEN using a combination of denaturing gradient gel electrophoresis and direct sequencing was unable to identify a germline mutation in 14 families with JPS and 11 sporadic cases. Therefore, at least a proportion of JPS cases are not caused by germline PTEN alteration or by an alternative locus at 10q22-24.

Chromosomes, Human, Pair 10↗
PubMed1997

Denaturing gradient gel electrophoresis (DGGE) increases resolution and informativity of Alu-directed inter-repeat PCR.

By inter-repeat PCR, multiple polymorphic loci can be targeted in parallel. To improve resolution and extend the number of detectable polymorphisms, Alu-directed inter-repeat PCR products from two large pedigrees of the Centre d'Etude du Polymorphisme Humain (CEPH) were electrophoretically resolved in non-denaturing polyacrylamide gels and, separately, on the basis of sequence content by denaturing gradient gel electrophoresis (DGGE). The resolution in DGGE gels was found to be superior to that in non-denaturing gels and a higher number of fragments was detected separately. The number of polymorphic bands detected by DGGE alone, however, was lower than that after size separation. This is ascribed to the fact that because of complete melting, small polymorphic fragments can run off the gel. With three Alu-specific primers, 18 and 16 polymorphic bands per individual were detected by size separation in pedigrees 1200 and 6600, respectively. In the same two pedigrees, seven and 15 polymorphic bands, respectively, were detected by DGGE. Segregation analysis of polymorphisms in the CEPH pedigrees indicated that most polymorphisms detected by DGGE were different from those detected by size separation.

DNA↗
PubMed1997

Transgenic mouse models for studying mutations in vivo: applications in aging research.

To study mutation accumulation in the DNA of somatic cells and tissues during aging in vivo, a transgenic mouse model has been constructed. The model harbors plasmid vectors, containing the lacZ reporter gene, integrated head to tail at various chromosomal locations. Procedures have been worked out to efficiently recover the plasmids into E. coli host cells. A positive selection system, permitting only E. coli cells with a lacZ mutated plasmid to grow, allows for the accurate determination of mutation frequencies as the ratio of mutant colonies versus the total number of transformants, i.e., the total number of plasmid copies recovered. Results obtained from a life span study of plasmid mice with vector clusters on chromosome 3 and 4 indicated age-related mutation accumulation in the liver, but not in the brain. Comparison of the mutational spectra revealed a significantly larger proportion of large size-change mutations in liver than in brain.

Aging↗
PubMed1997

Rapid accumulation of genome rearrangements in liver but not in brain of old mice.

Somatic mutations have long been considered a possible cause of ageing. To directly study mutational events in organs and tissues of ageing mammals, a transgenic mouse model has been generated that harbours lacZ reporter genes as part of chromosomally integrated plasmids. Using this model, we determined spontaneous mutant frequencies and spectra in mouse liver and brain as a function of age. In the liver, mutant frequencies increased with age from birth to 34 months; in the brain, an increase was observed only between birth and 4-6 months. Molecular characterization of the mutations showed that a substantial portion involved genome rearrangement events, with one breakpoint in a reporter gene and the other in the mouse flanking sequence. In the liver, these genome rearrangements did not increase with age until after 27 months, when they increased rapidly. In brain, the frequency of genome rearrangements was lower than in liver and did not increase with age.

Aging↗
PubMed1997

Induction of DNA adducts and mutations in spleen, liver and lung of XPA-deficient/lacZ transgenic mice after oral treatment with benzo[a]pyrene: correlation with tumour development.

We were interested to study the relationship between DNA lesions, DNA repair, mutation fixation, and tumour development. Therefore, mice harbouring lacZ reporter genes and being either wild-type or defective in the DNA excision repair gene XPA, were treated with the genotoxic carcinogen benzo[a]pyrene at an oral dose of 13 mg/kg b.w. (3 times/week). At different time points, i.e. 1, 5, 9 or 13 weeks after start of the oral administration, levels of BPDE-N2-dG adducts (the major formed DNA adduct by benzo[a]pyrene in mice), and lacZ mutation frequencies were measured both in target (spleen) and non-target (lung and liver) tissues. Both in wild-type and XPA-deficient mice, benzo[a]pyrene treatment resulted in increased BPDE-N2-dG adduct levels in all three tissues analysed. In XPA-deficient mice, BPDE-N2-dG adduct levels still increased up to 13 weeks of oral benzo[a]pyrene treatment, whereas in DNA repair proficient mice steady-state levels were reached after 5 weeks of treatment. After 13 weeks, the BPDE-N2-dG adduct levels observed in XPA-/- mice, were 2- to 3-fold higher than the steady state levels observed in XPA+/+ mice in the same tissues. Mutation frequencies in the lacZ reporter gene were the same in wild-type and XPA-deficient mice that were treated with the solvent only. Oral benzo[a]pyrene treatment resulted in an increase in mutation frequency in the lacZ marker gene in all three tissues, but this increase was most profound in the spleen. After 13 weeks of treatment, a 7-fold increase in lacZ mutation frequency was detected in the spleen of wild-type mice as compared to mutation frequencies in control mice. At the same time point, a 15-fold increase in lacZ mutation frequency was observed in the spleen of XPA-deficient mice. The data presented here show, that a defect in NER mainly results in enhanced mutation frequencies in lymphocytic cells after oral treatment with the genotoxic compound benzo[a]pyrene. Interestingly, as we established in a previously performed carcinogenicity assay, the same oral treatment with benzo[a]pyrene induced lymphomas residing in the spleen of XPA-deficient mice.

Animals↗
PubMed1997

Mapping of 34 minisatellite loci resolved by two-dimensional DNA typing.

Two-dimensional (2-D) DNA typing is based on electrophoretic separation of genomic DNA fragments in two dimensions according to independent criteria (size and base-pair sequence), followed by hybridization analysis using multilocus probes. The technique allows simultaneous visualization of several hundred loci as spots in a 2-D pattern. The majority of the loci resolved are polymorphic. Using linkage analysis in a large CEPH family, this study reports the mapping of 34 loci detected by the minisatellite core probe 33.6. By multipoint linkage analysis, regional chromosome positions of the 33.6 loci could be deduced, showing no evidence of clustering. In the analysis of spot patterns, use was made of a computerized image analysis system specifically designed for 2-D DNA typing. Since experimental variations between different separation patterns were automatically corrected for with this program, rapid and reliable scorings could be obtained. The results presented demonstrate the availability of reliable genetic information throughout the 2-D separation pattern. Adding the use of semiautomated computerized pattern analysis, this study further substantiates the applicability of 2-D DNA typing in genome scanning, not only in theoretical but also in practical terms. Moreover, it can be anticipated that this method will have a specific advantage in studies that scan for trinucleotide repeat expansions and somatic instability, where the repeat sequences detected by appropriate core probes are of particular interest.

Chromosome Mapping↗
PubMed1997

Sources of variability in mutant frequency determinations in different organs of lacZ plasmid-based transgenic mice: experimental features and statistical analysis.

Sources of variability that may affect the measurement of mutant frequencies in lacZ plasmid-based transgenic mice, including rescue-to-rescue, animal-to-animal (within organ), and organ-to-organ, were studied in the context of some of the experimental features that distinguish this model from other currently used systems. Statistical analysis of repeated determinations of DNA from kidney, spleen, liver, lung, and brain indicated that 350,000 colony-forming units from each of three animals should be analyzed per treatment group in order to detect a 50% difference in mutant frequencies. Consideration is given to some of the experimental features of the transgenic assay system, including its positive selection system, its rescue efficiency, the ability to detect large deletions in the lacZ target genes, the contribution of Escherichia coli-derived mutations to the spontaneous mutant frequencies observed in vivo, and cost effectiveness of mutant frequency determinations.

Animals↗
PubMed1996

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis.

The large number of possible disease-causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two-dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2-dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported.

Cystic Fibrosis↗
PubMed1996

Mutational scanning of large genes by extensive PCR multiplexing and two-dimensional electrophoresis: application to the RB1 gene.

With the rapid increase in the number of identified human disease genes, the development of accurate and cost-efficient mutation tests has become opportune. Here we present a combination of extensive PCR multiplexing and two-dimensional (2-D) DNA electrophoresis to screen for mutations in 26 exons of the retinoblastoma (RB1) tumor suppressor gene. In 2-D electrophoresis, fragments are separated according to size and base pair sequence in non-denaturing and denaturing gradient gels, respectively. All target fragments, designed to have optimal melting characteristics, were prepared in a two-step PCR (a 6-plex long-PCR pre-amplification and a subsequent 25-plex short-PCR) followed by heteroduplexing. The mixture of PCR amplicons was then subjected to 2-D electrophoresis under a single set of experimental conditions. With this design, 35 previously identified mutations in 18 different exons were detected in 33 bilateral retinoblastoma patients. These results suggest that 2-D electrophoresis in this format provides a generally applicable, practical and fast way to diagnose with high accuracy large genes for a broad spectrum of possible disease-causing mutations.

DNA Mutational Analysis↗
PubMed1996

Evaluation of a plasmid-based transgenic mouse model for detecting in vivo mutations.

To study in vivo somatic mutations a C57BL/6 transgenic mouse model was constructed harboring multiple chromosomally integrated copies of the plasmid pUR288, which carried the lacZ reporter gene as the mutational target. We previously demonstrated that lacZ-containing plasmids could be rescued from their integrated state efficient enough to detect mutations in lacZ by positive selection. The smaller size of the plasmid vector, as compared with our earlier transgenic mouse model based on bacteriophage lambda vectors, should offer considerable advantages in terms of rescue efficiency and sensitivity to large size alterations in the lacZ gene. To evaluate the plasmid-based mouse model for its suitability to detect in vivo mutations, we determined mutant frequencies in different organs of untreated and ethyl nitrosourea (ENU)-treated animals using a new, improved protocol. The rescue efficiencies obtained were as high as 200,000/micrograms genomic DNA; millions of transformants could be obtained in one single experiment. The average spontaneous mutant frequency in four different organs of 4- to 8-week-old mice ranged from 4.41 to 6.82 x 10(-5), compared with a mutant frequency of the same plasmid grown in Escherichia coli of approximately 1 x 10(-5) or less. Single treatments with 100 and 250 mg ENU/kg body wt resulted in a 7- and 14-fold increase, respectively, in spleen mutant frequency at 14 days after i.p. administration of the alkylating agent. Restriction enzyme analysis showed that a considerable portion of spontaneous mutants were size changes varying from approximately 100 to 3000 bp. Some mutant plasmids contained mouse genomic sequences, which is indicative of large genetic rearrangement events involving the 3' flanking regions of the transgene cluster. Among the ENU-induced mutants, size changes comprised only a minor fraction of the total, which is in keeping with the known ENU mutation spectra in vitro and in vivo. The high rescue efficiency of this plasmid-based model, in combination with its sensitivity to a broad spectrum of mutations, including large deletions, makes it very suitable as a general in vivo mutagenicity test system.

Animals↗