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At least 271 records · Page 15Linked to original sources
PubMed2003

Mutational analysis of p53 in human tumors: direct DNA sequencing and SSCP.

This chapter describes the techniques necessary to identify p53 genomic mutations in either frozen or paraffin-embedded tumor samples. DNA is extracted from the tumor samples and then used as a template to amplify the p53 coding sequences by polymerase chain reaction (PCR). The PCR products are characterized by agarose gel electrophoresis. Subsequently, mutations can be detected by direct DNA sequencing of the PCR products, followed by acrylamide gel electrophoresis and autoradiography. Alternatively, PCR products containing mutations can be identified by their aberrant mobility on denaturing acrylamide gels, using single-stranded conformation polymorphism (SSCP).

DNA Mutational Analysis↗
PubMed1998

Genomic organization and mutational analysis of HERG, a gene responsible for familial long QT syndrome.

Familial long QT syndrome (LQTS) is characterized by prolonged ventricular repolarization. Clinical symptoms include recurrent syncopal attacks, and sudden death may occur as a result of ventricular tachyarrhythmias. Three genes responsible for this syndrome (KVLQT1, HERG, and SCN5A) have been identified so far, and mutations have been reported on the basis of partially characterized genomic organization. To optimize the search for HERG mutations, we have determined the genomic structure of HERG and investigated mutations in LQTS families. Human genomic clones containing the HERG gene were isolated from a human genomic library by using reverse-transcribed polymerase chain reaction (RT-PCR) products from this gene as probes. We determined exon/intron boundaries and flanking intronic sequences by using primers synthesized on the basis of the HERG cDNA sequence available in the DNA database. HERG was shown to consist of 15 exons spanning approximately 19 kb on chromosome 7q35. Subsequently, we synthesized oligonucleotide primers to cover the entire coding region and searched for mutations in 36 Japanese LQTS families. When genomic DNA from each proband was examined by the PCR/single-strand conformation polymorphism technique followed by direct DNA sequencing, five novel mutations were detected. Each mutation was present in affected relatives of the respective proband. This work should increase the efficiency of screening mutations associated with HERG.

Amino Acid Sequence↗
PubMed1998

Detection of a new de novo mutation at codon 251 of exon 8 of thyroid hormone receptor beta gene in an Italian kindred with resistance to thyroid hormone.

Resistance to thyroid hormone (RTH) is almost invariably associated with mutations of the thyroid hormone (TH) receptor beta (hTR beta) gene and is inherited as an autosomal dominant disease. Mutations of hTR beta identified in patients affected by RTH cluster generally at two spots of the ligand binding domain. We investigated whether an Italian kindred with RTH had a mutation in the thyroid hormone (TH) receptor beta gene. Blood samples were obtained from the available family members for biochemical and genetic analyses. Thyroid function tests in basal conditions, and in the case of the propositus also following incremental doses of T3, were performed. Exon 4 to 10 of hTR beta gene were amplified using the polymerase chain reaction (PCR) and the mutation was identified by direct sequence analysis. The affinity constant of this mutated receptor for T3 was measured by in vitro transcription-translation and was then compared with that of wild type. We identified a heterozygous G to A transition at nucleotide 1037 of exon 8 at codon 251, resulting in a glycine (G) to glutamic acid (E) substitution (G251E) in the patient affected by RTH and in his affected offspring, but not in the normal family members. This novel mutation represents a de novo mutation since both parents of the index case were unaffected and did not have this genomic mutation. When expressed in vitro, the mutant protein (G251E) showed a marked decrease of the affinity for T3, suggesting an impaired ligand-dependent transactivation activity of this mutant receptor. In vivo studies with incremental doses of L-T3 demonstrated a reduced sensitivity to TH in the index case, in particular at the pituitary level where the thyrotrophs' activity was not completely inhibited even by 200 micrograms/day of L-T3. G251E mutation represents the fourth mutation described up to now in exon 8 of hTR beta among the subjects affected by RTH. A third cluster of mutations of the c-erbA beta gene located proximally with respect to the other two so far described begins to emerge in RTH patients.

Adult↗
PubMed1988

The effect of the reproductive system on mutation load.

J. B. S. Haldane (Amer. Nat. 71, 337-349, 1937) argued that, in equilibrium populations, the effect of deleterious mutation on average fitness depends primarily on the mutation rate and is independent of the severity of the mutations. Specifically, the equilibrium population fitness is e-microH, where microH is the haploid genomic mutation rate. Here we extend Haldane's result to a variety of reproductive systems. Using an analysis based on the frequency of classes of individuals with a specified number of mutations, we show that Haldane's principle holds exactly for haploid sex, haploid apomixis, and facultative haploid sex. In the cases of diploid automixis with terminal fusion, diploid automixis with central fusion, and diploid selfing, Haldane's principle holds exactly for recessive mutations and approximately for mutations with some heterozygous effect. In the cases of K-ploid apomixis, diploid endomitosis, and haplodiploidy, we show that Haldane's principle holds exactly for recessive lethal mutations. In addition we extend Haldane's result to various mixtures of the above-mentioned reproductive systems. In the case of diploid out-crossing sexuals, we do not obtain an exact analytic result, but present arguments and computer simulations which show that Haldane's result extends to this case as well in the limit as the number of loci becomes large. Although diverse reproductive systems are equally fit at equilibrium, different reproductive systems harbor vastly different numbers of recessive genes at equilibrium and we provide estimates of these numbers. These different numbers of mutations may create transient selective pressures on individuals with reproductive systems different from that of the equilibrium population.

Animals↗
PubMed2005

Estimation of the mtDNA mutation rate in aging mice by proteome analysis and mathematical modeling.

The accumulation of mitochondria containing mutated genomes was proposed to be an important factor involved in aging. Although the level of mutated mtDNA has shown to increase over time, it is currently not possible to directly measure the mtDNA mutation rate within living cells. The combination of mathematical modeling and controlled experiments is an alternative approach to obtain an estimate for the mutation rate in a well-defined system. In order to judge the relevance of mitochondrial mutations for the aging process, we used a mouse model to study age-related alterations of the mitochondrial proteins. Based on these experimental data we constructed a mathematical model of the mitochondrial population dynamics to estimate mtDNA mutation rates. Mitochondria were isolated from mouse brain and liver at six different ages (newborn to 24-months). A large-gel 2D-electrophoresis-based proteomics approach was used to analyze the mitochondrial proteins. The expression of two respiratory chain complex I subunits and one complex IV subunit decreased significantly with age. One subunit of complex III and one subunit of complex V increased in expression during aging. Together, these data indicate that complex I and IV deficiency in aged tissues might be accompanied by feedback regulation of other protein complexes in the respiratory chain. When we fitted our experimental data to the mathematical model, mtDNA mutation rate was estimated to be 2.7x10(-8) per mtDNA per day for brain and 3.2x10(-9) per mtDNA per day for liver. According to our model and in agreement with the mitochondrial theory of aging, mtDNA mutations could cause the detrimental changes seen in mitochondrial populations during the normal lifespan of mice, while at the same time ensure that the mitochondrial population remains functional during the developmental and reproductive period of mice.

Aging↗
PubMed2002

Molecular and immunohistochemical analysis of signaling adaptor protein Crk in human cancers.

Crk is a signaling adaptor protein which is mostly composed of SH2 and SH3 domains, and has been shown to play a pivotal role in cell proliferation, differentiation, and migration. Because Crk was originally isolated as an avian sarcoma virus CT10 encoding oncoprotein v-Crk, we examined a potential role for c-Crk in the carcinogenesis of human cancers. First, to analyze gene mutations of c-Crk, we isolated a human bacterial artificial chromosome clone containing Crk genome and exon/intron structures. However, polymerase chain reaction-single strand conformation polymorphism methods failed to show any genomic mutations in the Crk exon which could be related to carcinogenesis. Second, immunohistochemical analysis of c-Crk-II demonstrated that the levels of c-Crk-II were significantly elevated in most of the tumors, particularly in the colon and lung cancers. Furthermore, immunoblot analysis using human lung cancer cell lines revealed that the expression levels of c-Crk-II were correlated to growth rates of cells. The elevated expression levels of c-Crk-II might be related to the development of human cancers.

Adult↗
PubMed2000

Somatic mutation rates and specificities at TC/AG and GT/CA microsatellite sequences in nontumorigenic human lymphoblastoid cells.

We have examined mutational events at TC/AG microsatellites, the second most abundant dinucleotide repetitive motif in the human genome. Mutational targets were constructed containing TC/AG alleles up to 20 units in-frame within the coding region of the herpes simplex virus thymidine kinase (HSV-tk) gene. These targets were incorporated into oriP shuttle vectors, which replicate episomally in human lymphoblastoid cells. The overall HSV-tk mutant frequencies measured after 10 population doublings in cells derived from a clinically normal donor were slightly increased over the background of mutations recovered in Escherichia coli. DNA sequence analyses revealed that replication of TC/AG vectors in human cells increased the mutation frequencies at the microsatellite motif up to 3-fold, relative to background. Additionally, the median HSV-tk mutation rate of single-cell clones carrying the [TC/AG]17 vector was significantly different from that of clones harboring the control vector. The median rate of allele length alterations within the [TC/AG]11 tract was 2 x 10(-6) mutations/cell generation, with an equivalent rate of deletion and expansion mutations. In contrast, a [GT/CA]10 vector showed no increase in microsatellite mutation frequency after replication in human cells, and mutation rates of clones carrying a [GT/CA]16 vector were not significantly different from controls. Intriguingly, replication in human cells of all microsatellite-containing vectors resulted in elevated mutation frequencies at the downstream HSV-tk coding sequence of up to 20-fold, an effect not observed for the control vector. These results demonstrate that the frequency of mutational events at TC/AG motifs is slightly greater than at GT/CA motifs of similar allele length. This is the first report to our knowledge of the mutation rates at TC/AG microsatellite alleles in eukaryotic or prokaryotic cells.

Base Sequence↗
PubMed1992

DNA mutation analysis of Gaucher patients.

We evaluated 62 Gaucher patients to determine whether patients with similar phenotypes had the same DNA point mutations. Genomic DNA from these Gaucher patients was screened for the 3 most frequent single-point mutations, occurring in 69% of the 124 patient alleles, and resulting in changes in amino acids 370, 444, and 463. Many different genotypes were observed, at least one of which is present in all 3 types of Gaucher disease. No specific symptom complex could be correlated with a unique genotype. Even the more clinically homogeneous subgroups of Gaucher patients contained several genotypes. This study further emphasizes the need for caution in making clinical predictions on the basis of current genotype analysis, especially since one might not discern a fetus affected with type 2 disease by current DNA studies. The severity of involvement in type 1 disease could also not be predicted. Thus, even limiting our focus to 3 isolated common point mutations, a given genotype cannot be uniquely correlated with a specific prognosis.

Adolescent↗
PubMed2001

Alterations of the double-strand break repair gene MRE11 in cancer.

MRE11 plays a role in DNA double-strand break repair. Hypomorphic mutations of MRE11 have been demonstrated in ataxia-telangiectasia (AT)-like disorder. ATM mutations play a causal role in AT and have been demonstrated in lymphoid malignancies in patients without AT histories. By analogy with the relationship of ATM to lymphoid malignancies, it is probable that alterations of MRE11 are associated with tumor formation. We performed a mutation analysis of MRE11 in 159 unselected primary tumors. Three missense mutations at conserved positions were found in breast and lymphoid tumors. Additionally, an aberrant transcript without genomic mutation was found in a breast tumor. These findings suggest an occasional role for MRE11 alterations in the development of primary tumors.

Alternative Splicing↗
PubMed2002

Mutational analysis of HIV-1 long terminal repeats to explore the relative contribution of reverse transcriptase and RNA polymerase II to viral mutagenesis.

HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.

Base Sequence↗
PubMed1997

Polymerase chain reaction-based technique for the selective enrichment and analysis of mosaic arg201 mutations in G alpha s from patients with fibrous dysplasia of bone.

Mutations in the arg201 codon of the alpha s G protein-subunit have been associated with a variety of disorders, but analysis of such mutations has been complicated by their mosaic presentation. To overcome the problems associated with the analysis of genomic mutations that may be present in low and variable yield throughout the body, a polymerase chain reaction (PCR)-based technique has been developed that allows the selective amplification of products from the mutant allele. This technique uses site-directed mutagenesis to generate a PCR product from the normal allele that is susceptible to restriction endonuclease digestion, whereas that from the mutant allele is resistant to digestion. Consecutive and repeated cycles of amplification and digestion allow selective enrichment of the product from the mutant allele. The technique has been applied to the analysis of patients with fibrous dysplasia of bone, where the consequence of G alpha s mutations may vary from monostotic to polyostotic lesions, and has been performed with DNA isolated from either bone biopsy specimens or peripheral blood leukocytes. In addition to the previously described arg-->his and arg-->cys substitutions, the analyses have detected a novel arg-->ser substitution in one of the patients. This patient presented with a panostotic disease and may represent a unique subgroup of fibrous dysplasia.

Arginine↗
PubMed2002

Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii.

The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6 kDa) to an intermediate-sized product iSS (18.3 kDa) and to the mature SS (16.3 kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.

Animals↗
PubMed2001

P53 mutation and response to hepatic arterial floxuridine in patients with colorectal liver metastases.

PURPOSE: To establish the relationship between the number and site of p53 genomic mutations in metastatic colorectal cancer, and the response to hepatic arterial floxuridine. METHODS: Liver metastasis biopsies were collected, at the time of laparotomy for hepatic arterial cannulation. in 28 patients with metachronous colorectal liver metastases. p53 Gene mutations were assessed using reverse transcription, nested polymerase chain reaction, single strand conformational polymorphism and gene sequencing. Chemotherapy response was determined from computerised liver tomograms after 4 months of treatment. RESULTS: Liver metastasis p53 mutation was identified in 21 (75%), and p53 "hot spot" mutation in 11 (39%) patients. There was a significantly lower prevalence (Fisher's, P=0.001) of patients with p53 "hot spot"-mutated liver metastases in stable disease and partial response (5/22) than in progressive (6/6) disease groups. Significantly fewer (Mann-Whitney U, P=0.002) p53 "hot spot" mutations/biopsy were observed in liver metastases from stable disease and partial response (median 0, iqr. 0-0) than in progressive (median 1, iqr 1-2) disease patients. p53 "Hot spot"-mutated liver metastases were associated with significantly shorter survival times (logrank P=0.05) after hepatic arterial floxuridine. Significant response or survival-time differences by total or L2/L3 zinc-binding site p53 mutations were not detected. CONCLUSIONS: The results support a role for p53 "hot spot" mutations in colorectal liver metastasis resistance to fluorinated pyrimidines, and suggest that the presence of such mutations may be a contra-indication to treatment of colorectal liver metastases with hepatic arterial floxuridine.

Adult↗
PubMed1998

Inferences on genome-wide deleterious mutation rates in inbred populations of Drosophila and mice.

A theoretical analysis was carried out on the mutation load observed in long-maintained inbred lines from two experiments with Drosophila and mice. The rate of decline in fitness and its sampling distribution were predicted for both experiments using Monte Carlo simulation with a range of mutational parameters and models. The predicted rates of change in fitness were compared to the empirical observed rates, which were close to zero. The classical hypothesis of many deleterious mutations (about one event per genome per generation) of small effect (1-2%) resulting in a mutation pressure for fitness of about 1% per generation is incompatible with the data. Recent estimates suggesting an overall mutation pressure for fitness traits of about 0.1% are, however, compatible with the observed load.

Animals↗
PubMed2002

Allele separation facilitates interpretation of potential splicing alterations and genomic rearrangements.

Mutations that alter normal splice patterns and genomic rearrangements are common causes of hereditary diseases including hereditary nonpolyposis colorectal cancer. However, abnormal transcripts can be difficult to detect and interpret because splicing patterns are often heterogeneous even in normal cells. Standard techniques including sequencing and Southern hybridization fail to detect some genomic rearrangements. We show here that separation of alleles in somatic cell hybrids, through "conversion" technology, considerably facilitates the interpretation of abnormal splicing patterns and the detection of genomic rearrangements. We detected novel mutations in MLH1 in each of four hereditary nonpolyposis colorectal cancer patients. The genomic mutations were CAG>CAA predicting Q346Q; GAG>AAG predicting E102K; a>g at nucleotide 1559-2 at intron 13, and a tandem duplication involving exons 7-12. By separating the two alleles, we showed that one allele produced only abnormal transcript or no transcript whereas the other allele produced only normal transcript. These results allowed pathogenicity to be unambiguously assigned to the mutations and increased the sensitivity of genomic testing.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Screening for TP53 mutations in patients and tumours from 109 Swedish breast cancer families.

To estimate the prevalence of TP53 mutations in familial breast cancer, constant denaturant gel electrophoresis (CDGE) was used to screen exons 5-8 of the TP53 gene for germline mutations. Genomic DNA from 128 breast cancer patients belonging to 109 families with familial cancer were screened. No germline mutations were found in any of the patients. We also studied TP53 mutations in tumour DNA from 51 of the same individuals and found mutations in 14%. This is similar to what has been reported in sporadic breast cancer.

Breast Neoplasms↗
PubMed2025

MitoScribe single-cell molecular recorder logs graded signaling dynamics into mitochondrial DNA.

Genetically encoded DNA recorders convert transient biological events into stable genomic mutations, offering a means to reconstruct past cellular states. However, current approaches to log historical events by modifying genomic DNA have limited capacity to record the magnitude of biological signals within individual cells. Here, we introduce MitoScribe, a mitochondrial DNA (mtDNA)-based recording platform that uses mtDNA base editors (DdCBEs) to write graded biological signals into mtDNA as neutral, single-nucleotide substitutions at a defined site. Taking advantage of the hundreds to thousands of mitochondrial genome copies per cell, we demonstrate MitoScribe enables reproducible, highly sensitive, non-destructive, durable, and high-throughput measurements of molecular signals, including hypoxia, NF-κB activity, BMP and Wnt signaling. We show multiple modes of operation, including multiplexed recordings of two independent signals, and coincidence detection of temporally overlapping signals. Coupling MitoScribe with single-cell RNA sequencing and mitochondrial transcript enrichment, we further reconstruct signaling dynamics at the single-cell transcriptome level. Applying this approach during the directed differentiation of human induced pluripotent stem cells (iPSCs) toward mesoderm, we show that early heterogeneity in response to a differentiation cue predicts the later cell state. Together, MitoScribe provides a scalable platform for high-resolution molecular recording in complex cellular contexts.

Journal Article↗
PubMed1987

Saccharomyces cerevisiae mutants that tolerate centromere plasmids at high copy number.

Two yeast (Saccharomyces cerevisiae) mutants that tolerate centromere (CEN) plasmids at high copy number have been isolated. The mutations relieve the restraint normally imposed on plasmid copy number by a cloned CEN sequence. Our CEN plasmids specify resistance to G418 and are high copy plasmids only when the mutant host cells are grown on medium containing this antibiotic. The high copy number of the plasmids is independent of the specific cloned CEN sequence and recovered plasmids show no alteration in structure or function of the CEN DNA. The efficiency with which CEN plasmids go to high copy number is increased if the mutant cell is cotransformed by another CEN plasmid. The genomic mutation responsible for the high copy number (COP) is dominant and stable, and it segregates in a Mendelian manner. Homozygous COP/COP a/alpha diploids do not tolerate CEN plasmids at high copy number, suggesting that the mutation is regulated by mating type. The genomic DNA from both mutant cells contains an altered transposon (Ty) restriction fragment that cosegregates with the COP phenotype in crosses of mutant and wild-type strains. The mutations may be transposon-mediated events that identify a gene involved in centromere or mitotic spindle function.

Centromere↗