Report and abstracts of the Sixth International Workshop on Human Chromosome 21 Mapping 1996. Cold Spring Harbor, New York, USA. May 6-8,1996.
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Animals↗
Biomedical subjects
K Gardiner
Publications and source records attributed to K Gardiner.
Molecular genetic characterization and comparative mapping of the human PCP4 gene.
The mouse Pcp4 gene is highly expressed in brain, primarily in cerebellar Purkinje cells. It maps to chromosome 16 (Chr 16), in a region of conserved synteny with human chromosome 21 (Chr 21). To further characterize PCP4 and its possible contribution to cerebellar hypoplasia in trisomy 21, or Down Syndrome (DS), we cloned and sequenced the full length human cDNA, isolated a YAC which carries the entire gene, determined the gene structure, and characterized its expression. The gene spans at least 55 kb and contains two introns, the placement of which is the same in mouse. Expression in the mouse brain during development was detected at embryonic day 10, and thereafter through development. The PCP4 YAC was placed on the human Chr 21 YAC contig by a link to a YAC carrying the markers D21S15 and D21S349. This placement distal to ETS2 was confirmed by mapping on a somatic cell hybrid panel of Chr 21 translocations. This position caused an apparent break in gene order with mouse Chr 16. However, mapping in the mouse was reassessed, and Pcp4 and a linked marker, D16Mit71, were both moved distal to Ets2, corresponding to the position of PCP4 on Chr 21.
Amino Acid Sequence↗
Occupational exposure to carbon black in its manufacture: data from 1987 to 1992.
Carbon black is a very pure form of very finely divided particulate carbon used mainly in the automotive tyre industry. Its carbonaceous nature and submicron size (unpelleted) have raised concerns with regard to its ability to affect respiratory morbidity. This paper describes the exposure to carbon black dust in the first and second phase of a large multi-national epidemiological study investigating the magnitude of these exposure-related effects. In Phase I, 1278 respirable dust samples were taken (SIMPEDS cyclone) which increased to 2941 in Phase II with a similar rise in the number of total inhalable dust samples (IOM head) from 1288 in Phase I to 3433 Phase II. Exposure dropped markedly between the two phases with total inhalable dust showing a bigger reduction (49.9%) than respirable dust (42%), although the mean exposure for certain factories and job categories dropped more than others. The data are presented by the 14 job titles/numbers (21-34). The highest mean exposure in both phases and for both dust fractions is experienced by the warehouse packers and they are also most likely to exceed the OES of 3.5 mg m-3 (35.1% of samples in Phase I and 12.0% in Phase II).
Air Pollutants, Occupational↗
Base composition and gene distribution: critical patterns in mammalian genome organization.
Recent success in developing transcriptional maps of large genomic regions provide excellent opportunities for the investigation of mammalian genome organization. Detailed definition of organizational features will, in the short term, aid in prioritizing genomic sequencing efforts and in interpreting sequencing results and, in the long term, will surely provide insights into the structural, functional and evolutionary basis for the mammalian chromosome and chromosomal banding patterns. For such efforts, human chromosome 21 provides an excellent model system because the physical and clone maps are detailed, and several transcriptional mapping projects have provided large numbers of novel genes. It is, therefore, valuable at this point to examine these transcriptional mapping data and to compare them with the isochore model of the mammalian genome, which describes patterns in base composition and predicts gene distributions. Not only do compelling organizational patterns appear, but new questions about additional possible patterns in gene size, structure, conservation and transcription can be asked.
Animals↗
Integration of 30 CA-repeat markers into the cytogenetic, genetic and YAC maps of human chromosome 21.
The number of polymorphic DNA markers developed for the whole human genome during the last 2 years has been vastly increased. For this reason, the genetic map is continuously improving, but the cytogenetic and physical maps are not progressing at the same speed. Therefore, there is a need to integrate genetic, cytogenetic and physical mapping data. We have developed and localized on the breakpoint map of human chromosome 21 thirty microsatellite markers. Twenty of them have been used in the construction of a genetic map of chromosome 21, which contains a total of 44 markers. This map has 39 uniquely placed loci at 23 anchor points, ordered with odds of at least 1,000:1. The sex average length of the map is 64.4 cM, with the male and female lengths being 49.4 and 79.2 cM, respectively. Twenty-six of these newly developed markers have been localised on the CEPH/Généthon and Joint YAC Screening Effort YACs. Although these microsatellites were found uniformly spread along chromosome 21, the detection of various markers in the same or adjacent YACs suggests that CA-repeat microsatellites are clustered in several regions. The localization of these markers on the cytogenetic, genetic and YAC maps has provided a refined location for them and is a step further towards the construction of an integrated map of HC21.
Chromosome Mapping↗
Localization of cDNAs to a region poorly represented in the CEPH chromosome 21 YAC contig: candidate genes for genetic diseases mapped to 21q22.3.
Fifty-three cDNA fragments previously obtained by hybridization selection from random clones in the chromosome 21 cosmid library LL21CNO2 failed to identify clones in the chromosome 21 YAC contig described by Chumakov et al. (1992, Nature 359: 380-387). Using an expanded panel of somatic cell hybrids, we have verified that the majority of these cDNAs map to chromosome 21 and that in particular a very high proportion, approximately 85%, localize to a 5-Mb region of distal 21q22.3. Pulsed-field analysis coupled with information from the NotI restriction map of the region further indicate that 17 cDNA fragments map within 650 kb of the PFKL gene and thus may be candidates for genetic diseases linked to this gene. This work helps to characterize a region poorly represented in the CEPH YAC contig and adds to the number of cDNAs useful in analysis of chromosome 21-associated diseases.
Animals↗
Mutant U5A cells are complemented by an interferon-alpha beta receptor subunit generated by alternative processing of a new member of a cytokine receptor gene cluster.
The cellular receptor for the alpha/beta interferons contains at least two components that interact with interferon. The ifnar1 component is well characterized and a putative ifnar2 cDNA has recently been identified. We have cloned the gene for ifnar2 and show that it produces four different transcripts encoding three different polypeptides that are generated by exon skipping, alternative splicing and differential use of polyadenylation sites. One polypeptide is likely to be secreted and two are transmembrane proteins with identical extracellular and transmembrane domains but divergent cytoplasmic tails of 67 and 251 amino acids. A mutant cell line U5A, completely defective in IFN-alpha beta binding and response, has been isolated and characterized. Expression in U5A cells of the polypeptide with the long cytoplasmic domain reconstitutes a functional receptor that restores normal interferon binding, activation of the JAK/STAT signal transduction pathway, interferon-inducible gene expression and antiviral response. The IFNAR2 gene maps at 0.5 kb from the CRFB4 gene, establishing that together IFNAR2, CRFB4, IFNAR1 and AF1 form a cluster of class II cytokine receptor genes on human chromosome 21.
Alternative Splicing↗
The human myosin light chain kinase (MLCK) from hippocampus: cloning, sequencing, expression, and localization to 3qcen-q21.
Myosin light chain kinase (MLCK), a key enzyme in muscle contraction, has been shown by immunohistology to be present in neurons and glia. We describe here the cloning of the cDNA for human MLCK from hippocampus, encoding a protein sequence 95% similar to smooth muscle MLCKs but less than 60% similar to skeletal muscle MLCKs. The cDNA clone detected two RNA transcripts in human frontal and entorhinal cortex, in hippocampus, and in jejunum, one corresponding to MLCK and the other probably to telokin, the carboxy-terminal 154 codons of MLCK expressed as an independent protein in smooth muscle. Levels of expression were lower in brain compared to smooth muscle. We show that within the protein sequence, a motif of 28 or 24 residues is repeated five times, the second repeat ending with the putative methionine start codon. These repeats overlap with a second previously reported module of 12 residues repeated five times in the human sequence. In addition, the acidic C-terminus of all MLCKs from both brain and smooth muscle resembles the C-terminus of tubulins. The chromosomal localization of the gene for human MLCK is shown to be at 3qcen-q21, as determined by PCR and Southern blotting using two somatic cell hybrid panels.
Aged↗
A second-generation YAC contig map of human chromosome 3.
A map of human chromosome 3 which integrates both physical and genetic data has been developed from the fusion of two large collections of markers and corresponding yeast artificial chromosome (YAC) clones. The map contains 972 megabase-sized YACs identified with 593 primary markers, of which 162 are highly polymorphic sequence-tagged sites (STSs) and form a closely spaced genetic linkage map; the remaining markers are hybridization-based. Chromosome 3 is now represented by 24 large YAC contigs whose order and orientation is largely known. The map generated by fusion of these hybridization- and STS-based datasets covers about 80% (over 160 megabases) of the chromosome and will provide the foundation necessary for rapid development of a detailed genetic understanding for this large autosome.
Chromosome Mapping↗
A search for genes from the dark band regions of human chromosome 21.
As part of an effort to isolate genes from the entire long arm of human chromosome 21, we performed cDNA selection with 15 YACs from the regions of the pericentromeric heterochromatin and the two Giemsa dark bands of this chromosome using cDNA libraries from six different tissues. Nine of these YACs mapped to the Giemsa dark band, 21q21. The 9 YACs cover approximately 6 Mb of DNA, representing 15% of 21q and a significant portion of the 12-15 Mb estimated to be within this band. Several lines of evidence from analysis of the selected cDNA libraries suggest that this region of 21q has very few single-copy transcribed sequences. An EcoRI library was constructed with DNA from 1 of the 9 YACs. Grail analysis of the sequences of both ends of 24 YAC-specific clones from this EcoRI library revealed no potential exons. In contrast to these results, the selected cDNA libraries of a control YAC from the human MHC region in 6p21.3 as well as those from most of the other 21q YACs consisted largely of YAC-specific single-copy cDNA clones. Given the success of the cDNA selection method for finding a large number of genes in YACs from other chromosomal regions, these results suggest that the 6 Mb of DNA in the dark band 21q21 contains few single-copy sequences expressed in this tissue set. In contrast, selected cDNA libraries from the pericentromeric region, the telomeric border of the dark band 21q21, and the dark band 21q22.2 yielded more than 30 new ESTs.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Bacterial translocation during peroperative colonic lavage of the obstructed rat colon.
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Animals↗
YAC analysis and minimal tiling path construction for chromosome 21q.
We have undertaken a detailed analysis of several hundred YACs from widely available YAC libraries which map to human chromosome 21 with the goal of improving the physical map of chromosome 21 and determining the feasibility of producing a minimal tiling path of well characterized, stable, non-chimeric YACs spanning the long arm of the chromosome (21q). We report information on over 500 YACs known to contain STS from 21q including information on size, stability, chimerism, marker content, and NotI restriction sites. YACs derive from the CEPH and St. Louis YAC libraries, and STSs include the set of 198 markers originally used do assemble a YAC contig of 21q, as well as additional anonymous probes and gene markers. This information has assisted in refinements of STS order, has defined a region of general instability in 2lq22.3, has identified an increased number of NotI restriction sites, and has defined cryptic gaps, particularly in 2lq2l, for which few or no markers are available. These results have allowed us to develop and assess a minimal tiling path of overlapping YACs consisting of 59 YACs (and two PI clones), largely non chimeric, stable, and of verified STS content. They total 30 mb of non-overlapping DNA, and contain all chromosome 21 specific STSs originally used to define the 810 YAC 21q YAC contig. When integrated with the analysis of a somatic cell hybrid mapping panel of chromosome 21 reported in the accompanying manuscript, a greatly enhanced understanding of the physical map of chromosome 21 is obtained.
Chromosome Mapping↗
Molecular analysis and breakpoint definition of a set of human chromosome 21 somatic cell hybrids.
Rodent-human somatic cell hybrids containing single human chromosomes or chromosome fragments are extremely valuable in physical mapping, marker analysis, and disease mapping. Chromosome 21 has been extensively studied in this fashion, and a single set of hybrids has been utilized in mapping the majority of chromosome 21 markers. The utility of a set of hybrids depends upon the definition of the human chromosome content. Recently, Chumakov and coworkers (1) utilized 198 chromosome 21 markers in the preliminary analysis of YACs spanning chromosome 21q. We have used these same markers to evaluate the STS content of a set of 27 chromosome 21 somatic cell hybrids, resulting in the description of the breakpoints at the molecular level, as well as the definition of 35 "bins. " The detailed molecular definition of chromosome 21 content of the hybrids, in combination with the further analysis of chromosome 21 YACs (2), has resulted in the most detailed picture of chromosome 21 to date.
Animals↗
Polycyclic aromatic hydrocarbon exposure in coal liquefaction workers: the value of urinary 1-hydroxypyrene excretion in the development of occupational hygiene control strategies.
Studies in coal liquefaction workers were conducted to examine relationships between work activities, exposure to polycyclic aromatic hydrocarbons (PAHs) and excretion of urinary 1-hydroxypyrene (1-OHP). In a single-week study in operators, the end of work period 1-OHP excretion was an order of magnitude higher than in a shadow control subject, who experienced exposure by inhalation only (shadowed operator = 15.8 mumol mol-1 creatinine; shadow control = 1.07 mumol mol-1 creatinine). Dermal exposure was estimated to account for over 70% of excreted 1-OHP in exposed workers. A second study over 4 consecutive weeks (multi-week study) showed consistency in 1-OHP excretion from week to week both in operators and laboratory technicians, indicating little variation in weekly exposures for these workers. Excretion levels in maintenance workers however, showed some week to week variation, consistent with the variable nature of exposures associated with this type of work. A further study involving an end of work period spot assessment for all personnel showed a clear distinction between exposed workers (operators and maintenance staff) and other workers not thought to be exposed (laboratory technicians and office staff). Findings in the course of these studies indicated that there is no loss of 1-OHP at 4 degrees C, compared with -20 degrees C. On the basis of work to date at the coal liquefaction facility, hygiene control strategies have been proposed involving periodic urinary 1-OHP monitoring.
Coal↗
Exposure to acid anhydrides in three resin and one cushioned flooring manufacturing plants.
Acid anhydrides are reactive organic chemicals of low molecular weight which cause occupational asthma. No previous research on the relationship between exposure to these chemicals and respiratory sensitization and development of occupational asthma has been reported. A retrospective cohort study was carried out in four factories (three alkyd resin factories and one cushioned flooring factory) to investigate the nature of exposure-response relationships for sensitization to phthalic anhydride (PA), trimellitic anhydride (TMA) and maleic anhydride (MA). This paper describes the results of full-shift and task-specific exposure measurements. Exposure to PA was low in relation to the Occupational Exposure Standard (OES). The highest full-shift PA exposures occurred among resin operators in the resin factory that used solid PA as compared to other resin factories where liquid PA was used. Arithmetic mean exposure levels to TMA and MA in the resin factories were well below their respective OESs. Short-term high exposures occurred during loading of acid anhydrides into the reactors and sampling and testing of the resin. Relatively high full-shift exposure to TMA occurred in the cushioned flooring factory, although no high peak exposures were detected.
Anhydrides↗
Cloning of breakpoints in 3q21 associated with hematologic malignancy.
Rearrangements of band 3q21 have been well documented in leukemia. To analyze the region involved, we have isolated a normal genomic P1 clone that spans the 3q21 breakpoints derived from two leukemia patients carrying t(3;3)(q21;q26). Both breakpoints are contained within a 50-kb NotI restriction fragment, but are not identical. They are separated by 9-11 kb and may disrupt genes associated with either the proximal or distal NotI sites. We further show that one patient carried a small insertion of material from chromosome 2 in the 3q- derivative. The P1 clone will allow isolation of candidate genes for these breakpoints and investigations into the clustering of other 3q21 leukemia breakpoints.
Chromosomes, Human, Pair 3↗
Human genome organization.
Recent advances have been made in addressing three intriguing aspects of human genome organization: the organization of protein-coding sequences within chromosomes, the structural basis of the metaphase chromosomal banding pattern, and the function of non protein coding DNA. At the cytogenetic level, R band heterogeneity has been examined by fluorescence in situ hybridization using complex fractions of genomic DNA as probes. DNA fractionated according to GC content and CpG is island density both generated patterns related G and R bands and directly demonstrated regional variations in gene densities. A structural basis for metaphase bands has been proposed that is based on the differential size and packing of DNA loops and matrix attachment sites in G versus R bands. The model presents interesting opportunities for structure/function and organization investigations. At the molecular level, the human genome initiative has resulted in extensive genomic clone coverage for many large chromosomal regions, permitting detailed documentation of CpG islands, base composition and repeat sequence context, as well as fueling comprehensive gene searches. Sequence and functional characteristics are being examined at the kilobase level and are prompting new suggestions of roles for 'junk' DNA. Because of these developments, opportunities are now emerging for direct assessment of the molecular characteristics of individual metaphase bands.
Chromosome Banding↗
Getting the message: identifying transcribed sequences.
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Animals↗