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.

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.

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.

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.

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)

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.

Elevated hprt mutant frequency in circulating T-lymphocytes of xeroderma pigmentosum patients.

The mutant frequency to 6-thioguanine resistance in circulating T-lymphocytes from 10 xeroderma pigmentosum patients (including complementation groups D and G and XP variants) has been determined. A highly significantly elevated frequency was observed, compared to age-matched, non-smoking control donors (x 2.1-fold higher than the mutant frequency in normal control donors, adjusted for age and cloning efficiency, p less than 0.001). The mutant frequency of 5 XP heterozygotes was in the normal range, when age, smoking habit and log cloning efficiency were taken into account. A number of possible factors which may account for the elevated mutant frequency seen in the XP donors (including an elevated spontaneous mutation rate, UV mutagenesis of the T-cells as they pass through the skin, an effect of environmental mutagens such as tobacco smoke, or as a consequence of immune deficiency) are discussed.

Comparative human cellular radiosensitivity: III. Gamma-radiation survival of cultured skin fibroblasts and resting T-lymphocytes from the peripheral blood of the same individual.

Skin and blood samples were obtained from 34 donors, for whom there was no indication of abnormal radiosensitivity. From these, in 33 cases both fibroblast and T-lymphocyte cultures were obtained and in 26 cases at least three fibroblast and at least two G0 (resting) T-lymphocyte survival assays were possible. Within this set of results, differences in radiosensitivity between donors were significant for fibroblasts but not T-lymphocytes, although the range of radiosensitivity was similar for the two cell types (D 0.90-1.68 Gy for fibroblasts; 1.26-2.15 Gy for T-lymphocytes). Furthermore, there was little evidence for a correlation in radiosensitivity between the two cell types. These results suggest limitations in the predictive value of conventional measurement of cell survival.

Comparative human cellular radiosensitivity: IV. The increased sensitivity of human neonatal cord blood lymphocytes to gamma-irradiation compared with lymphocytes from children and adults.

We have compared the gamma-irradiation survival of G0 peripheral blood lymphocytes from 18 neonatal cord blood samples in a cloning assay with results from 21 controls (age range 1-65 years and consisting of 20 adults and one child). Using mean inactivation dose as the discriminating parameter, the cord blood cells showed a significantly greater radiosensitivity (mean inactivation dose for pooled data = 1.54 Gy) than the normal controls (mean inactivation dose for pooled data = 1.90 Gy, p less than 0.001). These results confirm and extend earlier work suggesting that T-lymphocytes in newborn children are more radiosensitive than normal controls, and this may have implications for the radiation protection of the unborn child.