Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA.
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Alternative Splicing↗
Biomedical subjects
K Gardiner
Publications and source records attributed to K Gardiner.
The sequence of human chromosome 21 and implications for research into Down syndrome.
The recent completion of the DNA sequence of human chromosome 21 has provided the first look at the 225 genes that are candidates for involvement in Down syndrome (trisomy 21). A broad functional classification of these genes, their expression data and evolutionary conservation, and comparison with the gene content of the major mouse models of Down syndrome, suggest how the chromosome sequence may help in understanding the complex Down syndrome phenotype.
Animals↗
Transcription factor GATA-2 gene is located near 3q21 breakpoints in myeloid leukemia.
Rearrangements affecting chromosome band 3q21 are observed in a subgroup of patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). However, little is known about the molecular consequences of such aberrations. We therefore established a PAC contig in the 3q21 breakpoint region and identified potential protein coding sequences by exon trapping. One of the exons isolated was from the human GATA-2 gene, which we showed to be transcribed from telomere to centromere. The majority of 3q21 breakpoints are located telomeric to the transcribed portion of this gene in a region that in mice appears to be necessary for proper promoter function. Results of GATA-2 expression analyses in leukemic cell lines as well as primary patient samples are compatible with the hypothesis that 3q21 aberrations contribute to leukemogenesis through deregulation of the hematopoietic transcription factor GATA-2.
Acute Disease↗
Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish.
One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.
Adenosine Deaminase↗
Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish.
The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation.
Adenosine Deaminase↗
The DNA sequence of human chromosome 21.
Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.
Base Sequence↗
Criteria for gene identification and features of genome organization: analysis of 6.5 Mb of DNA sequence from human chromosome 21.
To establish criteria for and the limitations of novel gene identification, to identify novel genes of potential relevance to Down Syndrome and to investigate features of genome organization, 6. 550kb. In total, 41 novel gene models were predicted, and for a subset of these, RT-PCR experiments helped to verify and refine the models, and were used to assess expression in early development and in adult brain regions of potential relevance to Down syndrome. Results suggest generally low and/or restricted patterns of expression, and also reveal examples of complex alternative processing, especially in brain, that may have important implications for regulation of protein function. Analysis of complete gene structures of the known genes identified a number of very large introns, a number of very short intergenic distances, and at least one potentially bi-directional promoter. At least 3/4 of known genes and 1/2 of predicted genes are associated with CpG islands. For novel genes, three cases of overlapping genes are predicted. Results of these analyses illustrate some of the complexities inherent in mammalian genome organization and some of the limitations of current sequence analysis technologies. They also doubled the number of potential genes within the region.
Adult↗
Risk factors for bronchial hyperresponsiveness in workers exposed to acid anhydrides.
Risk factors for bronchial hyperresponsiveness have previously been established in the general community. In settings where occupational asthma is a risk, it has not been established whether occupational sensitization or measures of exposure are important. Bronchial responsiveness to histamine was measured in a cohort of 506 workers exposed to acid anhydrides. Skin-prick tests were performed with conjugates of phthalic, maleic and trimellitic anhydride with human serum albumin and with common inhalant allergens. Employment and smoking histories were recorded. Occupational exposure was measured using personal air samplers and estimates of past exposure made by retrospective exposure assessment. Three hundred and seventy workers (73%) had bronchial responsiveness measured (median age 39 yrs, range 18-77) and 46 (12%) of these were hyperresponsive (provocative dose causing a 20% fall in forced expiratory volume in one second (FEV1; PD20) < or = 8 micromol). Twelve (3%) of these responsive workers had a skin-prick test reaction to an acid anhydride conjugate, 124 (34%) to a common inhalant allergen, and 148 (40%) were current smokers. Multivariate analysis showed that occupational sensitization, sensitization to a common inhalant allergen, age, and pack-years of smoking were independent risk factors for bronchial hyperresponsiveness. Of these only occupational sensitization was completely independent of baseline FEV1. It is concluded that sensitization to acid anhydrides is a significant risk factor for bronchial hyperresponsiveness. However, measures of personal acid anhydride exposure were not associated with bronchial hyperresponsiveness.
Adolescent↗
Report of the ninth international workshop on the identification of transcribed sequences.
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Animals↗
Trends in levels of inhalable dust exposure, exceedance and overexposure in the European carbon black manufacturing industry.
In an attempt to investigate the relationship between exposure to carbon black and respiratory morbidity, a study of the complete carbon black manufacturing industry in Western Europe was commissioned. As part of this study, a large number of personal inhalable (n=8015) dust exposure measurements was taken during three phases of data collection between 1987 and 1995. Repeated measurements on the same worker were taken in the last two phases, which enabled the estimation of the within- and between-worker components of variance. Simultaneously, the fixed effects of phase and factory were estimated using mixed-effects analysis of variance. The results show that the personal inhalable dust exposure has reduced significantly since the first phase of the study. In addition, the interaction term between phase and factory was significant in most job categories, confirming that the reduction of exposure was not equal across all factories. When all factories were considered together, the probability that the mean exposure of a randomly selected worker (overexposure) or the probability that the exposure on a randomly selected day for a randomly selected worker (exceedance) was higher than 3.5mg m(-3), was 10% or less for all job categories in the last phase. However, when the factories were considered separately, it appeared that the probability of overexposure or exceedance was in excess of 10% for the job categories 'Fitter/Welder', 'Warehouseman' and 'Site crew' for a number of factories. Therefore, even though exposure levels of inhalable dust have dropped considerably across the whole carbon black manufacturing industry in Western Europe, further reductions in exposure levels are required in these areas to make sure that the probability of overexposure and exceedance falls below a level of 10%.
Air Pollutants, Occupational↗
Human phosphoribosylformylglycineamide amidotransferase (FGARAT): regional mapping, complete coding sequence, isolation of a functional genomic clone, and DNA sequence analysis.
Purines play essential roles in many cellular functions, including DNA replication, transcription, intra- and extra-cellular signaling, energy metabolism, and as coenzymes for many biochemical reactions. The de-novo synthesis of purines requires 10 enzymatic steps for the production of inosine monophosphate (IMP). Defects in purine metabolism are associated with human diseases. Further, many anticancer agents function as inhibitors of the de-novo biosynthetic pathway. Genes or cDNAs for most of the enzymes comprising this pathway have been isolated from humans or other mammals. One notable exception is the phosphoribosylformylglycineamide amidotransferase (FGARAT) gene, which encodes the fourth step of this pathway. This gene has been cloned from numerous microorganisms and from Drosophila melanogaster and C. elegans. We report here the identification of a human cDNA containing the coding region of the FGARAT mRNA and the isolation of a P1 clone that contains an intact human FGARAT gene. The P1 clone corrects the purine auxotrophy and protein deficiency of Chinese hamster ovary (CHO) cell mutants (AdeB) deficient in both the activity and the protein for FGARAT. The P1 clone was used to regionally map the FGARAT gene to chromosome region 17p13, a location consistent with our prior assignment of this gene to chromosome 17. A comparison of the DNA sequence of the human FGARAT and FGARAT DNA sequence from 17 other organisms is reported. The isolation of this gene means that DNA clones for all the 10 steps of IMP synthesis have been isolated from humans or other mammals.
Amino Acid Sequence↗
Juxta-centromeric region of human chromosome 21 is enriched for pseudogenes and gene fragments.
A physical map including four pseudogenes and 10 gene fragments and spanning 500 kb in the juxta-centromeric region of the long arm of human chromosome 21 is presented. cDNA fragments isolated from a selected cDNA library were characterized and mapped to the 831B6 YAC and to two BAC contigs that cover 250 kb of the region. An 85 kb genomic sequence located in the proximal region of the map was analyzed for putative exons. Four pseudogenes were found, including psiIGSF3, psiEIF3, psiGCT-rel whose functional copies map to chromosome 1p13, chromosome 2 and chromosome 22q11, respectively. The TTLL1 pseudogene corresponds to a new gene whose functional copy maps to chromosome 22q13. Ten gene fragments represent novel sequences that have related sequences on different human chromosomes and show 97-100% nucleotide identity to chromosome 21. These may correspond to pseudogenes on chromosome 21 and to functional genes in other chromosomes. The 85 kb genomic sequence was analyzed also for GC content, CpG islands, and repetitive sequence distribution. A GC-poor L isochore spanning 40 kb from satellite 1 was observed in the most centromeric region, next to a GC-rich H isochore that is a candidate region for the presence of functional genes. The pericentric duplication of a 7.8 kb region that is derived from the 22q13 chromosome band is described. We showed that the juxta-centromeric region of human chromosome 21 is enriched for retrotransposed pseudogenes and gene fragments transferred by interchromosome duplications, but we do not rule out the possibility that the region harbors functional genes also.
Animals↗
Human chromosome 21. Why 40 Mb?
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Chromosome Mapping↗
Structures, sequence characteristics, and synteny relationships of the transcription factor E4TF1, the splicing factor U2AF35 and the cystathionine beta synthetase genes from Fugu rubripes.
A cosmid containing the beta-amyloid precursor protein (APP) from Fugu rubripes has been completely sequenced. In addition to APP, the cosmid contains the E4TF1-60 transcription factor, the U2AF35 pre-mRNA splicing factor, and the cystathionine beta synthetase (CBS) gene. The human homologues of all four genes map to human chromosome 21 but are not clustered; APP and E4TF1-60 map within 21q21, whereas U2AF35 and CBS map approximately 20Mb distal in 21q22. 3. The protein sequences of the Fugu genes vary in their overall level of similarity to their mammalian homologues, but several regions of functional importance are almost identical. As expected, the intron/exon structures of the homologous pairs of genes are highly conserved, but there are significant differences in the compaction ratios. The introns of APP and E4TF1-60 are 49- and 24-fold smaller in Fugu than in human, and the intergenic distance is compressed at least 100-fold. For U2AF35 and CBS, the introns are compressed only five- to eightfold. These size differences were compared with those for a number of previously reported Fugu genes; in general, levels of compaction of Fugu genes are consistent with the isochore locations of the human homologues.
Amino Acid Sequence↗
Maternal occupational exposure to electromagnetic fields before, during, and after pregnancy in relation to risks of childhood cancers: findings from the Oxford Survey of Childhood Cancers, 1953-1981 deaths.
BACKGROUND: The concern that maternal exposure to electromagnetic fields (EMF) might be related to childhood cancer risks, particularly leukemia risks. METHODS: Maternal occupational data already collected as part of the Oxford Survey of Childhood Cancers have been reviewed. Information on occupations held before, during, and after the pregnancy was sought for 15,041 children dying of cancer in Great Britain in the period 1953-1981, and for an equal number of matched controls. Each period of working was classified under one of five headings: (1) sewing machinist; (2) textile industry workers (other than sewing machinists) with likely exposures to EMF; (3) other machinists and other jobs with likely "higher" EMF exposure; (4) other jobs with likely exposure to some EMF, and (5) jobs with little potential for EMF exposure. RESULTS: Relative to risks in the children of mothers who held occupations with little potential for EMF exposure during pregnancy (a category that included housewives), risks of all childhood cancers were close to unity both for the children of sewing machinists (22 case and 31 control mothers, RR 0.72, 95% CI 0.42 to 1.25) and for the children of other machinists with likely "higher" EMF exposures (44 case and 47 control mothers, RR 0.93, 95% CI 0.61 to 1.41). Corresponding risks for all childhood leukemias and for all childhood brain cancers were similarly unexceptional. Simultaneous adjustment for social class, maternal age at birth of child, and sibship position had little effect. CONCLUSIONS: The study findings did not indicate that maternal occupational exposure to EMF during pregnancy is a risk factor for childhood leukemias, childhood brain cancers, or the generality of all childhood cancers.
Adolescent↗
Assessment of the sensitivity of the relation between current exposure to carbon black and lung function parameters when using different grouping schemes.
BACKGROUND: Equations using variance components in exposure data to predict attenuation and standard error of exposure--response slopes have been published recently. However, to date, no comparisons have been made between results of applying these equations to a real data set with the exposure-response relations estimated directly. METHODS: Data on lung function parameters and personal inhalable and respirable dust exposure levels from the European carbon black respiratory health study were used. The predicted attenuation and standard error of the relation between current inhalable and respirable dust levels and lung function parameters (FVC and FEV(1)) were calculated for various exposure grouping schemes. These results were compared with the observed exposure-response relations. Workers were grouped by Job Category, the combination of factory and Job Category and five a posteriori created Exposure Groups. In addition, the individual approach was also used, as exposure data were available for each worker. RESULTS: The rank orders of the coefficients from the regression analyses using the different grouping schemes were similar to those predicted by the equations, although the differences were larger than predicted. When using inhalable dust exposure, the predicted standard errors of the exposure-response slopes were slightly lower than those estimated directly; for respirable dust the predicted standard errors were about a factor two to three smaller than those from the regression analyses. When considering FVC, the predicted exposure-response relations were all statistically significant, whilst the observed relation was only significant when using the five a posteriori Exposure Groups. When reviewing the relations between dust exposure and level of FEV(1), all relations were statistically significant, with the exception of the (observed) relation between respirable dust and FEV(1), when the individual approach was used. CONCLUSIONS: Using different grouping schemes for estimating exposure can have large effects on the slope and standard error of the exposure-response relation. It is, therefore, important that the effect of the different grouping schemes on the level and precision of the exposure-response slope be estimated. Despite violation of most of the assumptions when applying the equations to predict attenuation and the standard error of the exposure-response slope, the similarities in predicted and observed exposure-response relations and standard errors are indicative of the robustness of these equations. Therefore, the equations appear to be a useful tool in establishing the most efficient way of utilizing exposure measurements.
Adult↗
Gene expression relevant to Down syndrome: problems and approaches.
The long arm of human chromosome 21 likely contains several hundred genes. To determine which of these are responsible for specific aspects of the Down Syndrome phenotype, protein functional analysis coupled to phenotypic analysis of transgenic mice will be required. Because such experiments are both time consuming and expensive, prioritizing 21q genes for further studies would be advantageous. Here, we discuss expression analysis, specifically the use of Northern analysis, cDNA array screening and RNA tissue in situ hybridization to assess place and time of expression of forty-two genes. For a subset of these, over expression in normal versus trisomy cell lines and mouse tissues is discussed. Lastly, several examples of alternative processing and their potential for generation of brain specific proteins are described. Together, these experiments give information on time, place and level of expression of a number of 21q genes and suggest some interesting candidates worth further investigation for relevance to Down Syndrome. These data also illustrate the complexities and ambiguities inherent in interpretation and use of expression information.
Adult↗
Saturation identification of coding sequences in genomic DNA.
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Base Sequence↗