Cookie-cutter shark Isistius spp. predation upon different tuna species from the south-western Atlantic Ocean.

The predation of cookie-cutter sharks Isistius spp. upon the early life stages of yellowfin tuna Thunnus albacares, skipjack tuna Katsuwonus pelamis and little tunny Euthynnus alletteratus are described. New evidence suggesting a connection between commercial fishing and predation by Isistius sp. is presented, with these sharks biting tunas hooked in surface waters during daylight. The healing patterns of the wounds made by the sharks are described in detail and, although such damage is known to negatively influence market price elsewhere, it is not the case on the south-east Brazilian coast.

COSMIC: High-Resolution Cancer Genetics Using the Catalogue of Somatic Mutations in Cancer.

COSMIC (http://cancer.sanger.ac.uk) is an expert-curated database of somatic mutations in human cancer. Broad and comprehensive in scope, recent releases in 2016 describe over 4 million coding mutations across all human cancer disease types. Mutations are annotated across the entire genome, but expert curation is focused on over 400 key cancer genes. Now encompassing the majority of molecular mutation mechanisms in oncogenetics, COSMIC additionally describes 10 million non-coding mutations, 1 million copy-number aberrations, 9 million gene-expression variants, and almost 8 million differentially methylated CpGs. This information combines a consistent interpretation of the data from the major cancer genome consortia and cancer genome literature with exhaustive hand curation of over 22,000 gene-specific literature publications. This unit describes the graphical Web site in detail; alternative protocols overview other ways the entire database can be accessed, analyzed, and downloaded. © 2016 by John Wiley & Sons, Inc.

Piscivory and trophic position of Anguilla anguilla in two lakes: importance of macrozoobenthos density.

The feeding habits of the European eel Anguilla anguilla (>300 mm total length, L(T)) were compared in two lakes of different environmental state: Lake Grosser Vätersee (LGV), Germany (clear water, mesotrophic and submerged macrophytes), and Lake Vallum (LV), Denmark (turbid, eutrophic and no submerged macrophytes). The density of macrozoobenthos was higher in LV (3500 individuals m(-2)) than in LGV (1500 individuals m(-2)). The abundance of small prey fishes (40-99 mm L(T)) was highest in LV. In LV, A. anguilla fed on macrozoobenthos, in particular, chironomid larvae. In LGV, A. anguilla used fishes as the main food component. Stable isotope analyses confirmed the stomach contents dietary results. The estimated mean +/-s.d. trophic positions of A. anguilla in LGV (3.7 +/- 0.2) was one level higher than those of fish in LV (2.7 +/- 0.2). Based on these results, it is concluded that piscivory among A. anguilla was generally controlled by the density of macrozoobenthos. Stable isotope analysis further indicated that A. anguilla may act as integrators between benthic and pelagic food webs when density of insect larvae is low.

The DNA sequence and analysis of human chromosome 13.

Chromosome 13 is the largest acrocentric human chromosome. It carries genes involved in cancer including the breast cancer type 2 (BRCA2) and retinoblastoma (RB1) genes, is frequently rearranged in B-cell chronic lymphocytic leukaemia, and contains the DAOA locus associated with bipolar disorder and schizophrenia. We describe completion and analysis of 95.5 megabases (Mb) of sequence from chromosome 13, which contains 633 genes and 296 pseudogenes. We estimate that more than 95.4% of the protein-coding genes of this chromosome have been identified, on the basis of comparison with other vertebrate genome sequences. Additionally, 105 putative non-coding RNA genes were found. Chromosome 13 has one of the lowest gene densities (6.5 genes per Mb) among human chromosomes, and contains a central region of 38 Mb where the gene density drops to only 3.1 genes per Mb.

The DNA sequence and analysis of human chromosome 6.

Chromosome 6 is a metacentric chromosome that constitutes about 6% of the human genome. The finished sequence comprises 166,880,988 base pairs, representing the largest chromosome sequenced so far. The entire sequence has been subjected to high-quality manual annotation, resulting in the evidence-supported identification of 1,557 genes and 633 pseudogenes. Here we report that at least 96% of the protein-coding genes have been identified, as assessed by multi-species comparative sequence analysis, and provide evidence for the presence of further, otherwise unsupported exons/genes. Among these are genes directly implicated in cancer, schizophrenia, autoimmunity and many other diseases. Chromosome 6 harbours the largest transfer RNA gene cluster in the genome; we show that this cluster co-localizes with a region of high transcriptional activity. Within the essential immune loci of the major histocompatibility complex, we find HLA-B to be the most polymorphic gene on chromosome 6 and in the human genome.

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

The DNA sequence and comparative analysis of human chromosome 20.

The finished sequence of human chromosome 20 comprises 59,187,298 base pairs (bp) and represents 99.4% of the euchromatic DNA. A single contig of 26 megabases (Mb) spans the entire short arm, and five contigs separated by gaps totalling 320 kb span the long arm of this metacentric chromosome. An additional 234,339 bp of sequence has been determined within the pericentromeric region of the long arm. We annotated 727 genes and 168 pseudogenes in the sequence. About 64% of these genes have a 5' and a 3' untranslated region and a complete open reading frame. Comparative analysis of the sequence of chromosome 20 to whole-genome shotgun-sequence data of two other vertebrates, the mouse Mus musculus and the puffer fish Tetraodon nigroviridis, provides an independent measure of the efficiency of gene annotation, and indicates that this analysis may account for more than 95% of all coding exons and almost all genes.

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.

The DNA sequence of human chromosome 22.

Knowledge of the complete genomic DNA sequence of an organism allows a systematic approach to defining its genetic components. The genomic sequence provides access to the complete structures of all genes, including those without known function, their control elements, and, by inference, the proteins they encode, as well as all other biologically important sequences. Furthermore, the sequence is a rich and permanent source of information for the design of further biological studies of the organism and for the study of evolution through cross-species sequence comparison. The power of this approach has been amply demonstrated by the determination of the sequences of a number of microbial and model organisms. The next step is to obtain the complete sequence of the entire human genome. Here we report the sequence of the euchromatic part of human chromosome 22. The sequence obtained consists of 12 contiguous segments spanning 33.4 megabases, contains at least 545 genes and 134 pseudogenes, and provides the first view of the complex chromosomal landscapes that will be found in the rest of the genome.

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.

Correlated mutagenesis of bcl2 and hprt loci in blood lymphocytes.

In vivo measurement of human somatic mutations may be a valuable biodosimeter of exposure to carcinogens and of cancer risk. We have surveyed translocations at the bcl2 locus in B lymphocytes, and mutations at hprt in T lymphocytes, in 120 individuals with varying exposure to radon and cigarette smoke. bcl2 t(14:18) translocation is the commonest chromosomal alteration observed in non-Hodgkins lymphoma (NHL). We observed a significantly larger range of bcl2 translocation frequency (range: 0-372 x 10(-6), median: 1.9 x 10(-6)) than of hprt mutation frequency (range: 0-76.4 x 10(-6), median: 11.1 x 10(-6)), which is likely the result of clonal proliferation of deathless B cell mutants. We observed that the frequencies of these two distinct lymphocytic mutations are significantly correlated. Although some of the correlated variation is explained by age, a significant correlation of bcl2 mutagenesis persists after age adjustment. Correlated mutagenesis at distinct loci in distinct cell types could be explained by the existence of a mutator phenotype or by variation in exposure to environmental mutagens. NHL is commoner in men than in women, and our data indicate a trend toward higher bcl2 mutagenesis in males than females. There is mounting epidemiological evidence for a worldwide increase in NHL, which may have an environmental basis; molecular epidemiological analysis of bcl2 mutagenesis in exposed populations might be especially relevant to the identification of putative environmental causes. Given the relative ease of the bcl2 assay versus the hprt assay, and the consistency with which data are reproduced from laboratory to laboratory, it is likely that the bcl2 assay will be soon added to the array of assays used in human mutational surveillance.

Biomonitoring of possible human exposure to environmental genotoxic chemicals: lessons from a study following the wreck of the oil tanker Braer.

In January 1993 the oil tanker Braer ran aground in the Shetland Islands, Scotland. Approximately 80,000 tons of crude oil were released. Exceptionally high winds caused extensive pollution and exposure of the local population to crude oil. We describe the study which was immediately set in place to examine the exposed population for evidence of genotoxic exposure. Blood samples were taken and primary DNA damage was measured in the mononuclear cell fraction by the butanol modification of the 32P-postlabelling method. Mutation was measured at the hprt locus in T lymphocytes. No evidence of genotoxicity was obtained for either end point, but nevertheless, we believe that useful lessons were learnt, which should be incorporated into the design of future studies: (1) A rapid response is essential, and even if sufficient funds are not immediately available, it is still worth attempting to obtain samples quickly and use cryopreservation, also to attempt to estimate exposure. (2) Adequate numbers of volunteers must be sought, together with enough controls, not just to allow meaningful analysis but to overcome loss of samples and failure of things to go according to plan. (3) Points concerning laboratory practice include: (i) samples should be coded, (ii) clearly defined and proven protocols should be used, (iii) irreplaceable samples should not be used for method development, (iv) should a problem become apparent during the study, work on such samples should cease immediately until the problem is solved, (v) all critical experimental components should be pretested against a laboratory standard. (4) The study design should include replicate experiments to monitor experimental variability and reproducibility, as well as internal standards and cryopreserved "in house" samples. Care must be taken that samples from any one exposure group are spread between a number of independent experiments and that each experiment includes samples from a number of exposure groups. (5) A computerised data base should be maintained with full details of experimental variables, donor attributes, and raw data so that any contribution of experimental artefacts to "outlier" results can be monitored. (6) Because of the nature of the statistical variation for many environmental genotoxicity end points, only a large-scale study is likely to be capable of yielding useful information.

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.

Lack of evidence for an association between the frequency of mutants or translocations in circulating lymphocytes and exposure to radon gas in the home.

Radon measurements in the living room and main bedroom of 41 houses in the town of Street, Somerset, England have been made. Exposure levels, weighted using the formula of the UK National Radiological Protection Board, of 19-484 Bq m-3 (about half > 100 Bq m-3) were found. Blood samples were obtained from a total of 66 occupants in these homes, and the frequency of genetic alterations in lymphocytes was estimated using two different end points. Gene mutations at the hypoxanthine guanine phosphoribosyl transferase locus were determined in T lymphocytes for 65 subjects using a clonal assay, and the frequency of the BCL-2 t(14;18) translocation, a chromosomal event associated with leukemia/lymphoma, was estimated in lymphocytes using a polymerase chain reaction-based technique for 64 subjects. In neither case was a significant correlation with radon levels in the home found, in contrast to our earlier observation with a smaller series.

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.

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.

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.

An analysis of in vivo hprt mutant frequency in circulating T-lymphocytes in the normal human population: a comparison of four datasets.

In this paper, we have compared mutant frequency data at the hprt locus in circulating T-lymphocytes from four large datasets obtained in the UK (Sussex), the USA (Vermont), France (Paris) and The Netherlands (Leiden). In total, data from > 500 non-exposed individuals ranging in age from newborns (cord blood samples) to > 80 years old have been included in the analysis. Based on raw data provided by the four laboratories, a model is presented for the analysis of mutant frequency estimations for population monitoring. For three of the laboratories, a considerable body of data was provided on replicate estimates of mutant frequency from single blood samples, as well as estimates from repeat blood samples obtained over a period of time from many of the individual subjects. This enabled us to analyse the sources of variation in the estimation of mutant frequency. Although some variation was apparent in the results from the four laboratories, overall the data were in general agreement. Thus, in all laboratories, cellular cloning efficiency of T-cells was generally high (> 30%), although in each laboratory considerable variation between experiments and subjects was seen. Mutant frequency per clonable T-cell was in general found to be inversely related to cloning efficiency. With the exception of a few outliers (which are to be expected), mutant frequencies at this locus were in the same range in each dataset; no effect of subject gender was found, but an overall clear age effect was apparent. When log mutant frequency was analysed vs log (age + 0.5) a consistent trend from birth to old age was seen. In contrast, the effect of the smoking habit did differ between the laboratories, there being an association of smoking with a significant increase in mutant frequency in the Sussex and Leiden datasets, but not in those from the Vermont or Paris datasets. Possible reasons for this are discussed. One of the objectives of population monitoring is an ability to detect the effect of accidental or environmental exposure to mutagens and carcinogens among exposed persons. The large body of data from non-exposed subjects we have analysed in this paper has enabled us to estimate the size of an effect that could be detected, and the number of individuals required to detect a significant effect, taking known sources of variation into account.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

An improved procedure for the in vitro expansion of human T-lymphocyte clones for mutant analysis.

Assays detecting mutants present in human peripheral blood T-lymphocytes have been developed to monitor the population for somatic mutations. In order to investigate the nature of the mutations, colonies are further expanded in vitro by repeated lectin stimulation. To characterise fully each mutant clone, sufficient cells (approximately 10(7)) must be available for several molecular and biochemical techniques to be employed. These techniques, and their importance to the assay for population monitoring, are discussed briefly. We report here that the expansion of mutant colonies to approximately 10(7) cells by repeated lectin stimulation is not effective for all T-cell clones but that an alternative "lectin free" expansion method has enabled us to expand all the clones tested from a variety of normal donors and other individuals of interest.