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D Beare

Publications and source records attributed to D Beare.

11 recordsLinked to original sources
PubMed2000

Molecular methods for the detection of mutations.

We report the results of a collaborative study aimed at developing reliable, direct assays for mutation in human cells. The project used common lymphoblastoid cell lines, both with and without mutagen treatment, as a shared resource to validate the development of new molecular methods for the detection of low-level mutations in the presence of a large excess of normal alleles. As the "gold standard, " hprt mutation frequencies were also measured on the same samples. The methods under development included i) the restriction site mutation (RSM) assay, in which mutations lead to the destruction of a restriction site; ii) minisatellite length-change mutation, in which mutations lead to alleles containing new numbers of tandem repeat units; iii) loss of heterozygosity for HLA epitopes, in which antibodies can be used to direct selection for mutant cells; iv) multiple fluorescence-based long linker arm nucleotides assay (mf-LLA) technology, for the detection of substitutional mutations; v) detection of alterations in the TP53 locus using a (CA) array as the target for the screening; and vi) PCR analysis of lymphocytes for the presence of the BCL2 t(14:18) translocation. The relative merits of these molecular methods are discussed, and a comparison made with more "traditional" methods.

Base Sequence↗
PubMed1999

Mapping and complex expression pattern of the human NPAP60L nucleoporin gene.

From a clone mapping to human chromosome 22q13.3, we have identified NPAP60L, the human homolog of the rat nuclear pore-associated protein gene, Npap60. The expression pattern of the human copy is much more complex that that of the rat, although conservation of the potential specific function of NPAP60L in male germ cells can be seen for one of the five transcripts. The exon-intron organization of the NPAP60L gene shows the presence of at least three alternate 3' ends, and Northern analysis indicates the possible presence of alternate 5' ends. Somatic cell hybrid mapping revealed additional related copies of NPAP60L on human chromosomes 5, 6, and 14, although it is not known if these are functional genes.

Alternative Splicing↗
PubMed1996

Development of new molecular procedures for the detection of genetic alterations in man.

The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10(-6) at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lacI gene of Escherichia coli was 10(-5), and we were able to detect DNA damage and repair after treatment with N-methyl-N-nitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasI and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.

Animals↗
PubMed1996

Characterisation and antiproliferative activity of an alpha-type murine interferon from embryonic fibroblasts.

Interferons play a part in the negative control of cell proliferation of mammalian cells. Here a natural interferon has been isolated from soluble proteins secreted by secondary murine embryonic fibroblasts using Blue Sepharose chromatography, immunoaffinity exclusion and Q Sepharose ion exchange fractionation. Partial amino acid sequencing assigns it to the interferon alpha family. Its biological and physico-chemical properties single it out from all other murine alpha interferons. The embryonic interferon has stronger antiproliferative activity, is acid labile, has stronger affinity for Blue Sepharose and weak affinity for antibodies which recognise other murine interferon alpha subtypes.

Amino Acid Sequence↗
PubMed1995

U.v.-hypermutability of xeroderma pigmentosum cells demonstrated with a DNA-based mutation system.

We have developed a DNA-based system, to detect mutations at restriction sites without any selection in culture. DNA is exhaustively digested with a restriction enzyme. Primers flanking a chosen site for this enzyme are used in the polymerase chain reaction (PCR). Only DNA molecules mutated at the chosen site are resistant to digestion and can serve as templates for the PCR. We have initially used this system to demonstrate the generation of mutations by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells, and by u.v.-C irradiation at a TaqI site in the hprt gene of human fibroblasts. In repair-deficient xeroderma pigmentosum (XP) cells the u.v.-induced mutant frequency was greatly enhanced. We have been able to detect and analyse mutations in XP cells at TaqI sites in three different genes, hprt, p53 and c-Ha-ras1. Both u.v.-C and u.v.-B irradiation have been used as mutagenic agents with both lymphoblastoid and fibroblast cells from XP patients from complementation group G. The mutant DNA molecules have been sequenced. Following u.v.-C-irradiation, the majority of mutations analysed were GC-->AT transitions, but several double and tandem mutations were also found.

Animals↗
PubMed1995

Determination of hprt mutant and mutation frequencies and the molecular characterization of human derived in vivo T-lymphocyte mutants.

Using a T-lymphocyte clonal assay, 73 6-thioguanine resistant T-lymphocytes were isolated from two blood samples obtained 4 months apart from a 50-year-old male subject. Sixty-six of these mutants were characterized at the DNA sequence level using cDNA. One particular single base substitution was recovered a total of 23 times. The majority of T-cell receptors (TCR) of these mutants all share a common gamma-TCR rearrangement, and thus likely represent a single mutational event that underwent clonal expansion in vivo. Siblings of this clone were recovered in both collections. Three other single base substitutions were also recovered more than once. In two of the three cases, the mutants were also found to be clonally related, while in one case they were not. A number of identical exon loss events were also recovered, yet none of these were clonally related. This probably reflects the multiple pathways by which these mutations can arise. The TCR data was used to correct the observed mutant frequency to produce an estimate of the actual mutation frequency. The two mutant frequencies, 18 x 10(-6) and 19 x 10(-6), obtained from the first and second sampling periods, respectively, can thus be corrected to yield true mutation frequency's of 12 x 10(-6) each.

Base Composition↗
PubMed1994

Sleepiness and ethanol effects on simulated driving.

Twelve healthy young men were assessed in each of four experimental conditions presented in a Latin Square design: 8-hr time in bed (TIB) and placebo, 4-hr TIB and placebo, 8-hr TIB and ethanol, and 4-hr TIB and ethanol. After consuming ethanol (0.6 g/kg) or placebo (0900-0930 hr) with 20% supplements at 1030 and 1100 hr, subjects were tested for sleepiness (Multiple Sleep Latency Test at 1000, 1200, 1400, and 1600 hr) and divided attention (1030 hr) performance on day 1, and for simulated driving and divided attention (1000-1200 and 1400-1600 hr) performance on day 2. In the morning testing, with breath ethanol concentrations (BECs) averaging 0.049%, sleepiness was increased, divided attention reaction times increased (on both days), and simulated driving performance was disturbed in the ethanol and 4-hr TIB relative to placebo. Similarly in the afternoon, with BECs averaging 0.013%, the ethanol and 4-hr TIB condition increased sleepiness and disrupted divided attention and simulated driving performance. The results show that sleepiness and low-dose ethanol combine to impair simulated automobile driving, an impairment that extends beyond the point at which BEC reaches zero. They provide a possible explanation for the incidence of alcohol-related automobile accidents at low BECs.

Adult↗
PubMed1993

Molecular analysis of mutations in the hprt gene in circulating lymphocytes from normal and DNA-repair-deficient donors.

Circulating lymphocytes from patients with the DNA-repair-deficient disorders, xeroderma pigmentosum (XP) and ataxia telangiectasia (A-T) have elevated frequencies of mutants at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus. We have analysed the DNA sequence of the hprt gene in mutants from normal donors, and compared them with mutants from XP and A-T individuals. In normal donors we found a range of mutations including principally transitions (40%), transversions (32%) and small deletions (20%). In an excision-deficient XP donor from complementation group C the mutation spectrum was similar to that from normal donors, whereas in an XP variant there was a significantly higher frequency (44%) of small deletions. In the two A-T donors, there was a high frequency of large deletions (22 and 75%) compared with only 4% in normal donors.

Adult↗
PubMed1991

Possible association between mutant frequency in peripheral lymphocytes and domestic radon concentrations.

To investigate whether previously found geographical correlations between leukaemia incidence and exposure to radon are reflected in a detectable mutagenic effect on individuals, the frequency of mutations in the hypoxanthine guanine phosphoribosyl transferase gene (hprt) in peripheral blood T lymphocytes was measured in subjects with known domestic radon concentrations. These concentrations were measured in December, 1989, in houses in Street, Somerset, UK, by passive alpha-track radon detectors. 20 non-smoking subjects aged 36-55 years were selected from the patient list at the local health centre on the basis of the radon concentrations in their homes--the range selected varied by a factor of ten. Blood samples for preparation of T lymphocytes were taken in July, 1990. There was a significant association between the log mutant frequency and radon concentration (t = 3.47, p less than 0.01). A second analysis of a further set of radon measurements (October, 1990, to January, 1991), in both living rooms and bedrooms, and repeated mutant frequency determinations also showed a significant relation, which remained significant even after exclusion of the highest frequency and adjustment for subject's age and cloning efficiency. These data must be regarded as preliminary and further more extensive studies should be done to determine whether the observed association is causal.

Acute Disease↗
PubMed1985

Cell cycle position and expression of encephalomyocarditis virus in mouse embryo fibroblasts.

Infection of mouse embryo fibroblasts in G1 or S phase with encephalomyocarditis virus gave different kinetics of viral RNA synthesis. In S phase cells, RNA synthesis was faster and reached higher levels than in G1 cells. Virus-specified proteins were fewer in G1 cells than in S cells during the early stage of the infection and c.p.e. in G1 cells appeared about 4 h later than in S cells. Addition of a cellular factor with ability to affect cell conformation had an inhibitory effect on viral RNA synthesis.

Animals↗