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Results for “Genome mutation”

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At least 127 records · Page 7Linked to original sources
PubMed1975

Colcemid-induced polyploidy and aneuploidy in normal and tumour cells in vitro.

The frequency of colcemid-induced genome mutations (aneuploidy and polyploidy) in normal and SV40-transformed cultures of Djungarian hamster embryonic cells was studied. Genome mutations were easily induced by the drug in transformed but not in normal cultures. Elevation of colcemid concentration and prolongation of the incubation period did not substantially increase the frequency of genome mutations in normal cells. An attempt was made to study the causes of the differences in sensitivity to colcemid-mutagenicity of normal and transformed cells. Transformed cells did not include more 3H-colchicine than normal cells, and binding of the drug to cell homogenates was similar in both kinds of cultures. According to these data the higher sensitivity of transformed cells to colcemid is not connected either with increased permeability of the cell to the drug or with changes in its binding to tubulin.

Aneuploidy↗
PubMed1999

The effect of overdominance on characterizing deleterious mutations in large natural populations.

Alternatives to the mutation-accumulation approach have been developed to characterize deleterious genomic mutations. However, they all depend on the assumption that the standing genetic variation in natural populations is solely due to mutation-selection (M-S) balance and therefore that overdominance does not contribute to heterosis. Despite tremendous efforts, the extent to which this assumption is valid is unknown. With different degrees of violation of the M-S balance assumption in large equilibrium populations, we investigated the statistical properties and the robustness of these alternative methods in the presence of overdominance. We found that for dominant mutations, estimates for U (genomic mutation rate) will be biased upward and those for h (mean dominance coefficient) and s (mean selection coefficient), biased downward when additional overdominant mutations are present. However, the degree of bias is generally moderate and depends largely on the magnitude of the contribution of overdominant mutations to heterosis or genetic variation. This renders the estimates of U and s not always biased under variable mutation effects that, when working alone, cause U and s to be underestimated. The contributions to heterosis and genetic variation from overdominant mutations are monotonic but not linearly proportional to each other. Our results not only provide a basis for the correct inference of deleterious mutation parameters from natural populations, but also alleviate the biggest concern in applying the new approaches, thus paving the way for reliably estimating properties of deleterious mutations.

Animals↗
PubMed2004

[The application of human mutation databases].

Researches on genome mutation are becoming more and more important with the finish of human genome DNA draft. This review is to classify the existing human mutation databases, including mutation database, SNP(single nucleotide polymorphisms) databases, mutation databases about disease, mutation databases about proteins, mutation databases about map and mutation information about specific gene. We also give advice on how to utilize these mutation databases, and discuss problems of existing databases.

Databases, Factual↗
PubMed2004

Directly fishing out subtle mutations in genomic DNA with histidine-tagged Thermus thermophilus MutS.

Tth MutS, a mismatch repair protein from Thermus thermophilus, was reported to effectively recognize all eight possible types of base pair mismatches and insertions or deletions up to three base pairs at a wide temperature range up to 60 degrees C. Here a procedure for directly fishing out subtle unknown mutations in bacterial genome with Tth MutS was described. Wild type genomic DNA and mutant one were mixed, digested with restriction enzymes, denatured and re-annealed. Hetero-duplex DNA carrying mispaired bases were bound to Tth MutS and recovered through Ni-NTA His-Bind((R)) Resin. The recovered DNA was cloned into plasmids, producing a mini-library with inserts of the mutated regions. Further DNA sequencing and genetic complementation demonstrated that the method was extremely efficient in fishing out the mutations from total genomic DNA. Using this method, the mutations existed in a Psedomonas aeruginosa mutant strain were screened, indicating that A/G transitions at nt 181 and nt 314 in chloramphenicol acetyltransferase (catB7) gene conferred this strain with a high chloramphenicol dosage resistant. Compared with those reported previously, this protocol can screen the mixed mutations more easily.

Adenosine Triphosphatases↗
PubMed2005

On the mutation rate of herpes simplex virus type 1.

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.

Animals↗
PubMed2000

A multi-site study for detection of the factor V (Leiden) mutation from genomic DNA using a homogeneous invader microtiter plate fluorescence resonance energy transfer (FRET) assay.

The goal of this multicenter study was to evaluate the second-generation Invader technology for detecting the factor V (Leiden) mutation directly from genomic DNA of different sample types. Invader assay results were compared with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) or allele-specific PCR (AS-PCR) analysis. The Invader assay is a PCR-independent methodology that uses a microtiter plate format. In the assay, a specific upstream Invader oligonucleotide and a downstream probe hybridize in tandem to a complementary DNA template and form a partially overlapping structure. The Cleavase VIII enzyme recognizes and cuts this structure to release the 5' flap of the probe. This flap then serves as an Invader oligonucleotide to direct cleavage of a fluorescence resonance energy transfer (FRET) probe in a second invasive cleavage reaction. Cleavage of this FRET probe results in the generation of a fluorescent signal. The results of the Invader assay were 99.5% concordant with the PCR-based methods. Of the 372 samples tested once, only two gave discordant results (one from operator error and one from unknown causes), but were concordant on retesting. These results indicate that a simple microtiter plate-based Invader assay can reliably genotype clinical patient samples for the factor V (Leiden) point mutation directly from genomic DNA without prior target amplification.

Base Sequence↗
PubMed2024

ERCC2 mutations alter the genomic distribution pattern of somatic mutations and are independently prognostic in bladder cancer.

Excision repair cross-complementation group 2 (ERCC2) encodes the DNA helicase xeroderma pigmentosum group D, which functions in transcription and nucleotide excision repair. Point mutations in ERCC2 are putative drivers in around 10% of bladder cancers (BLCAs) and a potential positive biomarker for cisplatin therapy response. Nevertheless, the prognostic significance directly attributed to ERCC2 mutations and its pathogenic role in genome instability remain poorly understood. We first demonstrated that mutant ERCC2 is an independent predictor of prognosis in BLCA. We then examined its impact on the somatic mutational landscape using a cohort of ERCC2 wild-type (n = 343) and mutant (n = 39) BLCA whole genomes. The genome-wide distribution of somatic mutations is significantly altered in ERCC2 mutants, including T[C>T]N enrichment, altered replication time correlations, and CTCF-cohesin binding site mutation hotspots. We leverage these alterations to develop a machine learning model for predicting pathogenic ERCC2 mutations, which may be useful to inform treatment of patients with BLCA.

Humans↗
PubMed1999

Hyperglycemia-induced embryonic dysmorphogenesis correlates with genomic DNA mutation frequency in vitro and in vivo.

Congenital malformations affecting multiple organ systems are at least three times more common in infants of mothers with IDDM than in infants born to nondiabetic mothers. Numerous studies have confirmed the teratogenic effect of hyperglycemia on the developing embryo, although no direct mechanism has been determined. In this study, we aimed to correlate the frequency of lacI mutations with degree of hyperglycemic exposure and severity of malformations in mouse embryos from in vitro cultures. Day 8 transgenic mouse embryos cultured in 30 or 50 mmol/l glucose for 48 h exhibited a higher incidence of morphological abnormalities, as well as an increase in lacI mutation frequency, compared with embryos cultured in 10 mmol/l glucose with no abnormalities and a lower frequency of lacI mutations. We also used a transgenic lacI rat system to evaluate the relationship between abnormal embryonic development and DNA mutation frequency in day 11 embryos of severely diabetic rats (serum glucose >20 mmol/l). Compared with control embryos, the embryos from diabetic rats displayed significantly more malformations, shorter crown-rump lengths, fewer somites, and more than six times greater genomic DNA mutation frequency. Genetic analysis of the mutated lacI gene from both in vitro cultured mouse embryos and in vivo developed rat embryos revealed that the majority of mutations were due to base substitutions (transitions and transversions), but that the rate of large DNA mutations tended to increase in embryos exposed to a diabetic environment. Our results support the interrelationship between increased rates of congenital malformations and DNA mutations in the offspring of diabetic pregnancy.

Animals↗
PubMed2004

[Construction of recombinant replication competent plasmids containing HBV genome with mutations of precore and basic core promoter].

OBJECTIVE: To construct a series of recombinant replication competent plasmids of hepatitis B virus (HBV) full-length genome with varying mutations of precore and basic core promoter. METHODS: The plasmid containing the entire HBV (1.2 copies) genome was used to construct objective plasmids. The objective competent vectors were constructed by molecular cloning and PCR-based site-directed mutagenesis in vitro and confirmed with sequence analysis. After introducing the plasmids into hepatocellular carcinoma cell line (Huh7) by calcium phosphate transfection, the culture supernatant was collected to detect HBsAg and HBV DNA to analyze viral replication and expression. RESULTS: Ten competent vectors with varying precore and basic core promoter mutations were constructed. After transfecting into Huh7, the vectors replicated and secreted viral particles. CONCLUSIONS: Ten full-length competent HBV recombinants were obtained, which were harbouring varying mutations within precore and basic core promoter.

Carcinoma, Hepatocellular↗
PubMed2006

Clinical profiles of four patients with Rett syndrome carrying a novel exon 1 mutation or genomic rearrangement in the MECP2 gene.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked MECP2 gene encoding methyl CpG binding protein 2 (MeCP2). Recently, a new isoform of MeCP2 including exon 1 was identified. This new isoform is more abundantly expressed in brain than the isoform including exons 2-4. Very little is known about the phenotypes associated with mutations in exon 1 of MECP2 since only a limited number of RTT patients carrying such mutations have been identified so far. In this study, we screened a cohort of 20 girls with RTT for exon 1 mutations by sequencing and multiplex ligation-dependent probe amplification (MLPA). We identified one girl with a novel exon 1 mutation (c.30delCinsGA) by sequencing and three with genomic rearrangements by MLPA. Comparison of the phenotypes showed that the girls carrying a mutation or rearrangement encompassing exon 1 were more severely affected than the girls with rearrangements not affecting exon 1.

Adolescent↗
PubMed1991

Direct molecular identification of the mouse pink-eyed unstable mutation by genome scanning.

DNA sequences associated with the mouse pink-eyed unstable mutation were identified in the absence of closely linked molecular markers and without prior knowledge of the encoded gene product. This was accomplished by "genome scanning," a technique in which high-resolution Southern blots of genomic DNAs were hybridized to a dispersed and moderately repetitive DNA sequence. In this assay, pink-eyed unstable DNA was distinguished from the DNA of wild-type and revertant mice by enhanced hybridization to one of several hundred resolved fragments. The fragment showing enhanced hybridization in pink-eyed unstable DNA was cloned and found to lie within a DNA duplication that is located close to, or within, the pink-eyed dilution locus. The duplication associated with the mouse pink-eyed unstable mutation may mediate the high reversion frequency characteristic of this mutation.

Alleles↗
PubMed1997

Lethal mutations and genomic instability.

The delayed expression of cell death in progeny of irradiated survivors was christened 'lethal mutations' by Tikvah Alper in 1984. The effect occurs when clones, or populations of cells grown up from irradiated progenitor cells, are replated and reassessed for cloning efficiency or population doubling time. The effect has been shown to be associated with the low dose shoulder region of the survival curve and is due to events occurring in the first two hours post irradiation, i.e. the 'fast repair' period. In this review the lethal mutation data accumulated over the past ten years is discussed in relation to our modern understanding of cellular and molecular events in radiation carcinogenesis and genomic instability research. It is suggested that lethal mutations are associated with a general epigenetic or field effect occurring in all irradiated cells, which makes them more prone to mutations, some of which are lethal. The implications of this for our current approach to risk estimates and therapeutic dose calculation, need to be addressed.

Animals↗
PubMed1990

Increased virulence of a mouse-adapted variant of influenza A/FM/1/47 virus is controlled by mutations in genome segments 4, 5, 7, and 8.

To cause disease, influenza virus must possess several genetically determined abilities that mediate stages in pathogenesis. The virulent mouse-adapted variant A/FM/1/47-MA (FM-MA), derived from the avirulent A/FM/1/47 (FM) strain, had acquired mutations in genes that control virulence. The purpose of this study was to identify those genes that had mutated to result in increased virulence and to obtain viruses that differed in virulence because of differences in individual genome segments. The genes that had mutated to increase virulence were initially identified by genetic analysis of reassortants obtained by crossing FM-MA with the avirulent strain A/HK/1/68 (HK). FM-MA genome segments 4, 5, 7, and 8 were significantly associated with virulence, as determined by using the Wilcoxon ranked sum analysis. The role of FM-MA segments 4, 7, and 8 was confirmed by reintroduction of these genes into the parental strain, which also provided virus strains that differed in virulence because of mutations in individual genome segments. Segments 4, 7, and 8 were responsible for a 10(3.6)-fold increase in virulence that was proportioned 10(2.2)-, 10(0.7)-, and 10(0.8)-fold, respectively. The role of segment 5 could not be confirmed on transfer back into the parental strain because of reversion during preparation of such reassortants. The incidence of reversion was shown to be significantly associated with culturing of FM-MA in chicken embryo cells but was not associated with growth in MDCK cells. The genetic analysis of FM-MA suggests that adaptation to increased virulence is an incremental process that involves the acquisition of mutations in multiple genes, each of which plays an individual role in pathogenesis. The structural and functional properties of segments 4, 7, and 8 that control the virulence of FM-MA can now be determined by using viruses that differ in virulence because of mutations in these individual genome segments.

Animals↗
PubMed1995

Function of a 5'-end genomic RNA mutation that evolves during persistent mouse hepatitis virus infection in vitro.

Persistently infected cultures of DBT cells were established with mouse hepatitis virus strain A59 (MHV-A59), and the evolution of the MHV leader RNA and 5' end of the genome was studied through 119 days postinfection. Sequence analysis of independent clones demonstrated an overall mutation frequency approaching 1.2 x 10(-3) to 6.7 x 10(-3). The rate of fixation of mutations was about 1.2 x 10(-5) to 7.6 x 10(-5) per nucleotide (nt) per day. In contrast to finding in bovine coronavirus, the MHV leader RNA sequences were extremely stable and did not evolve significantly during persistent infection. Rather, a 5' untranslated region (UTR) A-to-G mutation at nt 77 in the genomic RNA emerged by day 56 and accumulated until 50 to 80% of the genome-length molecules retained the mutation by 119 days postinfection. Although other 5'-end mutations were noted, only the nt 77 mutation was significantly associated with viral persistence in vitro. Mutations were also found in the 5' end of the p28 coding region, but no specific alterations accumulated in genome-length molecules through 119 days postinfection. The 5' UTR nt 77 mutation resulted in an 18-amino-acid open reading frame (ORF) upstream of the ORF 1a AUG start site. By in vitro translation assays, the small ORF was not translated into detectable product but the mutation significantly enhanced translation of the downstream p28 ORF about 2.5-fold. Variant viruses, containing either the nt 77 A-to-G mutation (V16-ATG+) or wild-type sequences at this locus (V1-ATG-), were isolated at 119 days postinfection. The variant viruses replicated more efficiently than wild-type virus and were extremely cytolytic in DBT cells, suggesting that the A-to-G mutation did not encode a nonlytic or attenuated phenotype. Consistent with the in vitro translation results, a significant increase (approximately 3.5-fold) in p28 expression was also observed with the mutant virus (V16-ATG+) in DBT cells compared with that in wild-type controls. These data indicate that MHV persistence was significantly associated with mutation and evolution in the 5'-end UTR which enhanced the translation of the ORF 1a and potentially ORF 1b polyproteins which function in virus transcription and replication.

Amino Acid Sequence↗
PubMed2000

[Concerning cell cultures for biocompatibility-testing: monitoring by DNA-fingerprinting].

Cell cultures are innovative tools for e.g. biocompatibility testing of biomaterials in vitro. In our studies we used fibroblast, endothelial cell and chondrocyte cultures of human origin and of the test animal species most common for this purpose in vivo. Verification of the identity of these cells is obligatory for reproducibility of the tests and valid interpretation of the results. Cultured cells have to be checked for identity, contaminations of various origins and also for genomic mutations occuring during prolonged cultivation in vitro or due to exposition to biomaterials. Furthermore, the risk of genetic cross-contamination with other cells increases with the number of cell cultures passaged parallel in the same laboratory. Therefore, we generated reference fingerprints of the cultures in varying passages for comparative monitoring of cells purposed for in vitro tests. Minisattelite DNA polymorhism resulting in reproducible individual DNA fingerprints is very discriminatory and can be used for cell culture monitoring. The patterns are stable over several passages, although sudden changes did happen in two cases, i.e. loss/gain of bands or changes in band-intensity, indicating massive genomic mutations of the cultures in vitro. Influences of biomaterials on the prints could not be detected. Several tasks can be followed at the same time: detection of contaminant cells, identification of these cells of primary culture origin used for in vitro testing and finally, monitoring for eventual genomic mutations due to prolonged cultivation or contact to biomaterials. Inconclusive results in just one of these aspects should lead to the disqualification of the monitored cultures from usage in vitro.

Animal Testing Alternatives↗
PubMed1984

[Capacity for differentiation and normalization of tumor cell populations transplanted into the anterior chamber. IV. Ehrlich ascites carcinoma].

The activity of LDH M- and H-forms, nuclear DNA contents and of genome mutation rates in the Ehrlich ascitic carcinoma cells were studied after transplantation to the intraperitoneal cavity, subcutaneous connective tissue (SCT) and the eye anterior chamber (EAC). SCT and EAC cultivations demonstrate the increase of M- to H-forms activity ratio due to a lesser H-form activity; so, LDH profiles appear to be associated with such a spontaneously highly differentiated adenocarcinoma of murine breast cancer. SCT and EAC cultivation leads also to changes in the karyotypic structure of cell population--there are no polyploid cells (DNA content is more than 4c). It may result from a sharp fall in genome mutation rates in the Ehrlich ascitic carcinoma clones following EAC proliferation which appears 47 times lower than those during lung proliferation. The data obtained favor a hypothesis that the increase in differentiation level and decrease in tumorigenety of cancer cells during EAC proliferation may be due to selection of the near-diploid cells and to the reduction in genome mutation rates, which in the whole results in decreasing genotypic and epigenotypic variability in tumor cell populations.

Animals↗
PubMed1981

Genome and chromosome mutations in porphyria cutanea tarda.

Spontaneous genome mutations in ten patients with porphyria cutanea tarda (PCT) amounted to 1.02% as compared to 0.36% in the control group of healthy individuals (P less than 0.01). Spontaneous structural chromosome aberrations in patients were seen in 2.39% of the examined cells versus 0.70% in the cells of controls (P less than 0.01). The increased percentage of cells with spontaneous genome and chromosome mutations in patients with PCT cannot be associated with alcohol only.

Adult↗