A microsatellite genetic linkage map of human chromosome 13

We have characterized 21 polymorphic (CA)n microsatellites for the development of a genetic map of chromosome 13. Fifteen markers were isolated from a flow-sorted chromosome 13 library, four CA repeats were derived from NotI-containing cosmid clones, and two polymorphic markers were described previously (J. L. Weber, A. E. Kwitek, and P. E. May, 1990, Nucleic Acids Res. 18: 4638; L. Warnich, I. Groenwald, L. Laubscher, and A. E. Retief, 1991, Am. J. Hum. Genet. 49(Suppl.): 372 (Abstract)). Regional localization for all of the markers was performed by amplification of DNA from five somatic cell hybrids containing different deletions of chromosome 13. Genetic markers were shown to be distributed throughout 6 of the 11 resolvable chromosomal subregions. Using data from nine families provided by the Centre d'Etude du Polymorphisme Humain (CEPH), a framework map of 12 of these 21 markers was developed. Six of the 12 markers form three pairs, with each two members of a pair being tightly linked, such that nine systems of markers can be distinguished. The average heterozygosity of these 12 markers is 0.75. The total length of the sex-averaged map is 65.4 cM (Kosambi), with an average distance of 8.2 cM between systems of markers (eight intervals). Seven remaining markers were placed provisionally into the framework map.     

Refined radiation hybrid map of mouse chromosome 17

We have made a radiation hybrid map of mouse Chromosome (Chr) 17 with 75 microsatellite markers, including those from McCarthy et al. (Genome Res 7, 1153-1161, 1997). Seventy-four of the markers are linked at LOD > 9, and all link at LOD > 5. A LOD 3 framework of 18 markers was used to construct a placement map. The order obtained is in good agreement with genetic maps, and distance estimates give an idea of how recombination rates vary across the chromosome. Recombination is remarkably low with respect to RH break frequency in the region from the centromere to the end of H2. This is similar in interspecific and intersubspecific crosses despite the inversion of a substantial part of this region in Mus spretus with respect to Mus musculus.     

Map integration at human chromosome 10: molecular and cytogenetic analysis of a chromosome-specific somatic cell hybrid panel and genomic clones, based on a well-supported genetic map

Well-characterized, chromosome-specific somatic cell hybrid panels are powerful tools for the analysis of the human genome. We have characterized a panel of human x hamster somatic cell hybrids retaining fragments of human chromosome 10 by fluorescence in situ hybridization and associated them to genetic markers. Most of the hybrids were generated by the radiation-reduction method, starting from a chromosome 10-specific monochromosomal hybrid, whereas some were collected from hybrids retaining chromosome 10-specific fragments as a result of spontaneous in vitro rearrangements. PCR was used to score the retention of 57 microsatellite markers evenly distributed along a well-supported framework genetic map containing 149 loci uniquely placed at 69 anchor points (odds exceeding 1,000:1), with an average spacing of 2.8 cM. As an additional resource for genomic studies involving human chromosome 10, we report the cytogenetic localization of a series of YAC and PAC clones recognized by at least one genetic marker. Somatic cell hybrids provide a powerful source of partial chromosome paints useful for detailed clinical cytogenetic and primate chromosome evolution investigations. Furthermore, correlation of the above physical, genetic, and cytogenetic data contribute to an emerging consensus map of human chromosome 10.     

A high-resolution metric HAPPY map of human chromosome 14

We have mapped 1001 novel sequence-tagged sites on human chromosome 14. The mean spacing between markers is approximately 90 kb, most markers are mapped with a resolution of better than 100 kb, and physical distances are determined. The map was produced using HAPPY mapping, a simple and widely applicable in vitro approach that is analogous to linkage or to radiation hybrid mapping, but that circumvents many of the difficulties and potential artifacts associated with these methods. We show also that the map serves as a robust scaffold for building physical maps using large-insert clones.     

Integrated radiation hybrid and yeast artificial chromosome map of chromosome 9p

OBJECTIVES: To determine concentrations of chondroitin sulphate (CS) and keratan sulphate (KS) epitopes, glycosaminoglycans (GAGs) and hyaluronan (HA) in knee synovial fluid (SF) from normal subjects and patients with osteoarthritis (OA) or rheumatoid arthritis (RA), to test whether these variables may be used as markers of the OA process. METHODS: OA was subdivided into large joint OA (LJOA), nodal generalised OA (NGOA), and OA with calcium pyrophosphate crystal deposition (CPA). Clinical assessment of inflammation (0-6) was undertaken on OA and RA knees. Knee SF was examined by enzyme linked immunosorbent assay for: CS epitopes, using monoclonal antibodies 3-B-3 and 7-D-4; KS epitope using monoclonal antibody 5-D-4; and HA, using biotinylated HA binding region of cartilage proteoglycan. Total sulphated GAGs were measured by dye binding with 1:9 dimethylmethylene blue. RESULTS: Increased SF 3-B-3 concentrations and 3-B-3/GAG ratio were found in OA, compared with RA or normal knees, with higher 3-B-3 and 3-B-3/GAG in LJOA and NGOA than in CPA. SF 7-D-4 and 7-D-4/GAG were reduced in RA, compared with normal and OA; SF 5-D-4 was reduced in OA compared with normal. GAG and HA concentrations were decreased in both OA and RA. No correlations with radiographic scores were observed, but SF 7-D-4 was lower in 'inflamed' compared with 'non-inflamed' RA and OA knees. In patients with bilateral samples there were strong correlations between right and left knees for all SF variables. CONCLUSIONS: Changed concentrations of SF CS and KS can be detected in OA with a profile that differs from that seen in RA. Clinical subgrouping and local joint inflammation may influence these measures, supporting different pathogenesis within OA subgroups and requirement for careful patient characterisation in SF studies.     

Variable X chromosome inactivation patterns in near-tetraploid murine EC x somatic cell hybrid cells differentiated in vitro

For the cytogenetic study of X chromosome inactivation as an X chromosome dosage compensation mechanism, we isolated a number of XXXX, XXX, and XXY near-tetraploid mouse hybrid cell clones by fusing XX or XO embryonal carcinoma cells with lymphocytes carrying a structurally altered X chromosome(s). The inactive X chromosome from the female lymphocyte was reactivated in these hybrid clones which retained embryonal carcinoma morphology so far as they were cultured on the collagen-coated plastic surface in the medium supplemented with leukemia inhibitory factor (LIF) and betamercaptoethanol (BME). Some of these clones developed balloon-like cystic embryoid bodies when they were allowed to form cell aggregates in medium without LIF and BME in bacteriological petri dishes to which they do not adhere. X chromosome inactivation occurring during this process detected by the incorporation of 5-bromodeoxyuridine did not conform to the expected pattern leaving two X chromosomes active in every tetraploid cells. This may suggest either that the X-inactivation mechanism evolved primarily, for the diploid cell is unable to deal with tetraploid conditions efficiently, or that the present system of in vitro differentiation represents an anomalous situation never encountered in vivo.     

A bacterial artificial chromosome-based framework contig map of human chromosome 22q

We have constructed a physical map of human chromosome 22q using bacterial artificial chromosome (BAC) clones. The map consists of 613 chromosome 22-specific BAC clones that have been localized and assembled into contigs using 452 landmarks, 346 of which were previously ordered and mapped to specific regions of the q arm of the chromosome by means of chromosome 22-specific yeast artificial chromosome clones. The BAC-based map provides immediate access to clones that are stable and convenient for direct genome analysis. The approach to rapidly developing marker-specific BAC contigs is relatively straightforward and can be extended to generate scaffold BAC contig maps of the rest of the chromosomes. These contigs will provide substrates for sequencing the entire human genome. We discuss how to efficiently close contig gaps using the end sequences of BAC clone inserts.     

A NotI restriction map of the entire long arm of human chromosome 21

A variety of maps of the human genome have been constructed, including cloned DNA maps. We have isolated 40 of the 42 NotI sites that exist on the long arm of human chromosome 21, as NotI linking clones and constructed a complete NotI restriction map spanning the entire region. This map, which provides the most reliable ordering and distance estimation in the region from a pericentromeric locus to the terminus, demonstrates the usefulness of linking clone mapping for analysing human chromosomes.     

Seven genes from human chromosome 18 map to chromosome 24 in the bovine

Bovine sequence tagged sites (STSs) were developed for seven genes and used for synteny mapping with a hybrid bovine x rodent cell line panel. The genes were thymidylate synthase (TYMS), pituitary adenylate cyclase activating peptide (ADCYAP1), and melanocortin-2 receptor (MC2R) from the short arm of human chromosome (HSA) 18 and N-cadherin (CDH2), transthyretin (TTR), gastrin-releasing peptide (GRP), and plasminogen activator inhibitor 2 (PAI2) from the long arm of HSA 18. Primers for these genes were designed with human, ovine, or bovine sequences aligned with a sequence from a second species. The bovine PCR product was cloned, and the fragment was sequenced to verify that the homologous gene was indeed amplified. A second set of bovine-specific PCR primers were developed for each gene from these sequences. These STSs were used for synteny mapping, and all seven genes were syntenic with markers of bovine chromosome (BTA) 24. The concordance with BTA 24 was at least 96.5% for all genes.     

Assignment of the dynamin-1 gene (DNM1) to human chromosome 9q34 by fluorescence in situ hybridization and somatic cell hybrid analysis

The dynamins are recently discovered GTP-binding proteins postulated to mediate the scission of clathrin-coated vesicles at the plasma membrane. Of the three known mammalian dynamins, dynamin-1 (DNM1) appears to be particularly important for the formation of synaptic vesicles at presynaptic nerve termini. To investigate the possibility that mutations in the DNM1 gene cause a human disease, we determined the chromosomal localization of human DNM1. We conclude from fluorescence in situ hybridization and from the analysis of somatic cell hybrids that the map position in 9q34. This region has syntenic homology with mouse chromosome 2p, in agreement with the map position of the mouse DNM1 gene [see accompanying article by Klocke et al. (1997, Genomics 41:290-292)]. We discuss the potential relevance of the human DNM1 localization to diseases that were mapped genetically to the same chromosomal region.     

A somatic cell hybrid panel for pig regional gene mapping characterized by molecular cytogenetics

A panel of 27 pig x rodent somatic cell hybrids was produced and characterized cytogenetically. The first step of this study consisted of hybridizing a SINE probe to GTG-banded metaphases of each hybrid clone in order to count and identify the normal pig chromosomes and to detect rearranged ones. The second step consisted of using the DNA of each clone as a probe after pIRS-PCR (porcine interspersed repetitive sequence-polymerase chain reaction) amplification to highly enrich it in pig sequences. These probes, hybridized to normal pig metaphase chromosomes, enabled the identification of the complete porcine complement in the hybrid lines. Whole chromosomes and fragments were characterized quickly and precisely, and results were compared. In addition to this cytogenetic characterization, molecular verification was also carried out by using primers specific to six microsatellites and to one gene previously mapped to pig chromosomes. The results obtained allow us to conclude that we have produced a panel that is informative for all porcine chromosomes. This panel constitutes a highly efficient tool to establish not only assignments of genes and markers but also regional localizations on pig chromosomes.     

A physical map of chromosome 7 of Candida albicans

As part of the ongoing Candida albicans Genome Project, we have constructed a complete sequence-tagged site contig map of chromosome 7, using a library of 3840 clones made in fosmids to promote the stability of repeated DNA. The map was constructed by hybridizing markers to the library, to a blot of the electrophoretic karyotype, and to a blot of the pulsed-field separation of the SfiI restriction fragments of the genome. The map includes 149 fosmids and was constructed using 79 markers, of which 34 were shown to be genes via determination of function or comparison of the DNA sequence to the public databases. Twenty-five of these genes were identified for the first time. The absolute position of several markers was determined using random breakage mapping. Each of the homologues of chromosome 7 is approximately 1 Mb long; the two differ by about 20 kb. Each contains two major repeat sequences, oriented so that they form an inverted repeat separated by 370 kb of unique DNA. The repeated sequence CARE2/Rel2 is a subtelomeric repeat on chromosome 7 and possibly on the other chromosomes as well. Genes located on chromosome 7 in Candida are found on 12 different chromosomes in Saccharomyces cerevisiae.     

Radiation hybrid mapping of the human MN/CA9 locus to chromosome band 9p12-p13

A previously healthy 12-year-old Japanese girl developed meningoencephalitis due to Cryptococcus neoformans. During the course of her illness she suffered persistent high fever, severe pancytopenia, hypercytokinemia and liver dysfunction. Laboratory findings, including results of a bone marrow examination, strongly indicated complication by haemophagocytic syndrome (HPS). The preceding cryptococcal infection was thought to be a cause of the HPS because no other viral or bacterial infection could be confirmed. The girl died of acute respiratory failure during the progressive course of HPS. This may be the first reported case of HPS due to cryptococcal infection in an otherwise healthy child.     

A transcript map of an 800-kb region on human chromosome 11q13, part of the candidate region for SCA5 and BBS1

OBJECTIVE: To evaluate whether small iodine supplements decrease the incidence of adolescent thyroid hypertrophy in an iodine-sufficient population or whether such thyroid enlargement should be considered an inevitable physiological phenomenon. DESIGN: Beginning in September 1991 (after an initial examination in September 1990), 54 11-year-old children in Bardejov, Slovakia were given small iodine supplements (Thyrojod depot tablets containing 1530 microg iodide) every 2 weeks for 2 years followed by once weekly for 2 years. A second group of 63 children served as controls. In June 1995, there were still 52 treated and 60 control children in the study and these were examined; 44 treated and 48 control children remained in the study until June 1997. METHODS: In 1990, 1993, 1995, 1996 and 1997 the thyroid volume (ThV) was measured by ultrasound. Serum levels of TSH, thyroglobulin, total and free thyroxine and tri-iodothyronine and anti-thyroid peroxidase (anti-TPO), anti-thyroglobulin (anti-TG) and anti-TSH receptor (TSR) antibodies were estimated in 1990 and 1994, while only TSH, and anti-TPO and anti-TSR antibodies were measured in 1997. RESULTS: There was no difference between the groups at any interval in the serum levels of the hormones measured. Marginally increased TSH was found in two treated and two control children. Anti-TSR antibodies were negative in all children, while anti-TPO and anti-TG antibodies were found in one treated and four control children. At the age of 10 years (1990), 84% of all ThVs were less than 4 ml, indicating a previous life-long sufficient iodine intake. After the treatment was completed (June 1995), a significant difference in ThV (P < 0.04) was found between the whole treated (5.78 +/- 0.19 ml) and the whole control group (6.56 +/- 0.30 ml). However, there was already a marked difference in the 75th percentile (6.4 ml in treated vs 8.5 ml in controls) due to more rapid thyroid growth in certain children of the control group (ThV > 7.0 ml in 6/52 treated children vs 24/60 controls; P < 0.01). Since such differences were much higher in 1997, the children in each group whose ThV was in the range of the upper 25% in 1997 were retrospectively evaluated as arbitrary separate subgroups in all the time intervals and compared with the remaining 75% of children who showed moderate thyroid growth rate. Two years after the termination of treatment (June 1997), excessive thyroid growth continued in the upper quarter of 12 controls with the highest ThV (13.60 +/- 0.40 ml or 7.60 +/- 0.29 ml/m2; 12/12 with ThV > 11.0 ml), and a similar subgroup now also appeared in 11 previously treated children (10.79 +/- 0.51 ml or 6.19 +/- 0.30 ml/m2; 5/11 with ThV > 11.0 ml). At the same time, ThV in the remaining 75% of both control (8.12 +/- 0.38 ml or 4.82 +/- 0.17 ml/m2; 3/36 with ThV > 11.0 ml) and treated (7.20 +/- 0.30 ml or 4.39 +/- 0.17 ml/m2; 0/33 with ThV > 11.0 ml) children was significantly less (P < 0.01 to P < 0.001) than that in the appropriate rapidly growing subgroups. During the whole observation period (1990-1997), no difference was found between treated and control subgroups with moderate thyroid growth. CONCLUSIONS: Since iodine intake in Slovakia has been adequate for decades and sporadic iodine deficiency is highly unlikely, the observed excessive thyroid growth in certain adolescents may result from causes other than simple iodine deficiency (e.g. hereditary), which are nevertheless ameliorated by small iodine supplements. The question remains whether such a subgroup with rapidly growing thyroids should be included in the range of normal thyroid volumes in adolescents.     

A linkage map of human chromosome 21:43 PCR markers at average intervals of 2.5 cM

A genetic linkage map of human chromosome 21q (HC21q) containing 43 markers genotyped by the polymerase chain reaction in the CEPH pedigrees is presented. The markers placed on this map are highly polymorphic with an average heterozygosity of 61%. The average interval size of the markers localized at 1000:1 odds is 2.5 cM. The map has a total length of 65.5 cM, with male and female lengths of 47.7 and 83.3 cM, respectively. The genotypes used in the construction of this map were subjected to rigorous error checking, which is reflected in the shorter map length compared to previous maps; the estimated error rate in genotyping is less than 0.04%. As noted in previous linkage maps there is increased recombination in females on proximal HC 21q and in the male in a region near the telomere. This map of HC 21 represents a highly informative and dense meiotic linkage map and will be useful in linking disease phenotypes to loci on this chromosome.