The effect of archistriatal lesions on ''open field'' and fear/avoidance behaviour in the domestic chick

DNA samples from about 100 human-hamster somatic cell hybrids, previously characterized by conventional banding techniques, were amplified with dual-Alu PCR. The products were then used as probes in FISH experiments on normal human metaphases for an accurate cytogenetic characterization of the human material retained in each hybrid. In addition to entire chromosomes, most hybrids were found to contain one or a few chromosome fragments, as a result of rearrangements that had occurred in vitro. Forty additional primary hybrids, in which conventional cytogenetic analysis failed to reveal any complete human chromosome, contained many human chromosome fragments. More than 300 chromosome fragments were scored and their precise chromosomal location recorded. We show data indicating that subchromosomal painting libraries generated from these hybrids can be favorably used in the fine characterization of chromosomal rearrangements encountered in clinical cytogenetics or in tumor cytogenetics, and in tracking chromosomal changes that occurred in primate evolution.     

Construction of a whole-genome radiation hybrid panel for high-resolution gene mapping in pigs

We have developed a panel of 152 whole-genome radiation hybrids by fusing irradiated diploid pig lymphocytes or fibroblasts with recipient hamster permanent cells. The number and size of the porcine chromosome fragments retained in each hybrid clone were checked by fluorescence in situ hybridization with a SINE probe or by primed in situ labeling (PRINS) with SINE-specific primers. A strategy based on the interspersed repetitive sequence polymerase chain reaction (IRS-PCR) was developed for selected clones to determine if the large fragments painted by the SINE probe corresponded to one pig chromosome or to different fragments of several chromosomes. This strategy was buttressed by a double PRINS approach using primers specific for alpha-satellite sequences of two different groups of swine chromosomes. Genome retention frequency was estimated for each clone by PCR with 32 markers localized on different porcine chromosomes. Of the 152 hybrids produced, 126 were selected on the basis of cytogenetic content and chromosome retention frequency to construct a radiation hybrid map of swine chromosome 8. Our initial results for this chromosome indicate that the resolution of the radiation hybrid map is 18 times higher than that obtained by linkage analysis.     

A somatic cell hybrid map of human chromosome 13

We have constructed a chromosome 13 somatic cell hybrid map using seven cell lines: PGMEA6, a hybrid containing the entire chromosome 13, and six hybrids containing various deletions of chromosome 13 (BARF7, PPF22, KBF11, KSF39, CF25, and CF27). We have mapped 80 markers that define 10 regions of chromosome 13 with respect to 10 breakpoints in the mapping panel; these regions range in size from 4 to 24 Mb, with an average size of 8 Mb. The 80 markers sublocalized on our mapping panel include 10 Alu-PCR clones, 6 of which were converted to sequence-tagged sites; 40 (CA)n repeat-containing clones, 27 of which are microsatellite PCR markers; 8 (AAAG)n repeat-containing PCR markers, 1 two-allele PCR marker, 4 genes or expressed sequences, and 17 anonymous DNA probes. This low-resolution physical map can be used as a backbone map for more refined physical mapping using radiation hybrids or yeast artificial chromosomes.     

Integration of 101 DNA markers across human Xp11 using a panel of somatic cell hybrids

One hundred and one DNA markers previously assigned to the short arm of the human X chromosome were localized on a hybrid mapping panel consisting of ten radiation-reduced, and four classical somatic cell hybrids. Of the 101 DNA markers, 16 are genes, two are pseudogenes, 13 are expressed sequence tags, 32 are simple tandem repeats (STRs), four are restriction fragment length polymorphisms, one is a variable number of tandem repeats, and 33 are sequence tagged sites (STSs). Three of these markers, two STSs and one STR, were generated from the products of an inter-Alu PCR library of a radiation-reduced hybrid containing Xp11.4-->p11.22 as its only human DNA content. A second STR was isolated from a region-specific cosmid containing the gene ZNF21. The 101 DNA markers fell into 22 bins based on their retention on the hybrids of this panel, which, in combination with YAC contig data, could be further resolved into 24 bins. This hybrid map of Xp11 has an average resolution of approximately 0.8 Mb.     

Porcine linkage and cytogenetic maps integrated by regional mapping of 100 microsatellites on somatic cell hybrid panel

Recently two main genetic maps [Rohrer et al. Genetics 136, 231 (1994); Archibald et al. Mamm. Genome 6, 157 (1995)] and a cytogenetic map [Yerle et al. Mamm. Genome 6, 175 (1995)] for the porcine genome were reported. As only a very few micro-satellites are located on the cytogenetic map, it appears to be important to increase the relationships between the genetic and cytogenetic maps. This document describes the regional mapping of 100 genetic markers with a somatic cell hybrid panel. Among the markers, 91 correspond to new localizations. Our study enabled the localization of 14 new markers found on both maps, of 54 found on the USDA map, and of 23 found on the PiGMaP map. Now 21% and 43% of the markers on the USDA and PiGMaP linkage maps respectively are physically mapped. This new cytogenetic information was then integrated within the framework of each genetic map. The cytogenetic orientation of the USDA linkage maps for Chromosomes (Chrs) 3, 8, 9, and 16 and of PiGMaP for Chr 8 was determined. USDA and PiGMaP linkage maps are now oriented for all chromosomes, except for Chrs 17 and 18. Moreover, the linkage group "R" from the USDA linkage map was assigned to Chr 6.     

Identification of a yeast artificial chromosome clone encoding an accessory factor for the human interferon gamma receptor: evidence for multiple accessory factors

Human chromosomes 6 and 21 are both necessary to confer sensitivity to human interferon gamma (Hu-IFN-gamma), as measured by the induction of human HLA class I antigen. Human chromosome 6 encodes the receptor for Hu-IFN-gamma, and human chromosome 21 encodes accessory factors for generating biological activity through the Hu-IFN-gamma receptor. A small region of human chromosome 21 that is responsible for encoding such factors was localized with hamster-human somatic cell hybrids carrying an irradiation-reduced fragment of human chromosome 21. The cell line with the minimum chromosome 21-specific DNA is Chinese hamster ovary 3x1S. To localize the genes further, 10 different yeast artificial chromosome clones from six different loci in the vicinity of the 3x1S region were fused to a human-hamster hybrid cell line (designated 16-9) that contains human chromosome 6q (supplying the Hu-IFN-gamma receptor) and the human HLA-B7 gene. These transformed 16-9 cells were assayed for induction of class I HLA antigens upon treatment with Hu-IFN-gamma. Here we report that a 540-kb yeast artificial chromosome encodes the necessary species-specific factor(s) and can substitute for human chromosome 21 to reconstitute the Hu-IFN-gamma-receptor-mediated induction of class I HLA antigens. However, the factor encoded on the yeast artificial chromosome does not confer antiviral protection against encephalomyocarditis virus, demonstrating that an additional factor encoded on human chromosome 21 is required for the antiviral activity.     

Visuospatial judgment: a cross-national comparison

To define the position of a 13q12 breakpoint from a patient with ganglioneuroblastoma, a series of somatic cell hybrids carrying human chromosome translocations with breakpoints in the proximal part of chromosome 13 has been compiled. Sequence-tagged sites (STS) have been generated from a series of Alu-PCR probes previously shown to be in the 13q12 region. Together with an STS for the oncogene FLT1, these have been used to define the relative positions of the translocation breakpoints in the hybrids. In this way, four markers have been ordered in three subregions of 13q12 and reference breakpoints established. The refined physical map of 13q12 provides a series of reference markers with known locations and will be invaluable in the further characterization of breakpoints in this region.     

Impaired interaction of naturally occurring mutant NF2 protein with actin-based cytoskeleton and membrane

The loss of large segments or an entire copy of chromosome 10 is the most common genetic alteration in human glioblastomas. To address the biological and molecular consequences of this chromosomal alteration, we transferred a human chromosome 10 into a glioma cell clone devoid of an intact copy. The hybrid cells exhibited an altered cellular morphology, a decreased saturation density, and a suppression of both anchorage-independent growth and tumor formation in nude mice. The hybrids also expressed the recently identified candidate tumor suppressor gene MMAC1/PTEN. To further identify gene products that may be involved in glioma progression, a subtractive hybridization was performed between the human glioblastoma cells and the phenotypically suppressed hybrid cells to identify differentially expressed gene products. Sixty-one clones were identified, with nine clones being preferentially expressed in the hybrid cells. Four cDNA clones represented markers of differentiation in glial cells. Two cDNA clones shared homology with platelet derived growth factor-alpha and the insulin receptor, respectively, both genes previously implicated in glioma progression. A novel gene product that was expressed predominantly in the brain, but which did not map to chromosome 10, was also identified. This clone contained an element that was also present in three additional clones, two of which also exhibited differential expression. Consequently, the presence of a functional copy of chromosome 10 in the glioma cells results in differential expression of a number of gene products, including novel genes as well as those associated with glial cell differentiation.     

Randomly amplified polymorphic DNAs assess recombination following an induced parasexual cycle in Penicillium roqueforti

Random amplified polymorphic DNAs (RAPDs) were used as a genetic marker system to characterize recombinant strains following the parasexual cycle of Penicillium roqueforti. After protoplast fusion and haploidization of diploid hybrids, segregants characterized by a reassortment of the parental genetic markers displayed specific RAPD fingerprints. The appearance or the loss of RAPD fragments demonstrate that these markers provide an efficient method to analyze recombination and to characterize somatic hybrids.     

Contribution to the physically anchored linkage map of the pig

Thirty-three microsatellites have been mapped on the PiGMaP porcine genetic map. By comparison with the previously published PiGMaP maps, the maps of chromosome 2 (140 cM/70 cM) and chromosome 3 (180 cM/110 cM) were extended and new markers were mapped on the p-arm extremity of chromosome 7 and on the centromeric extremity of chromosome 15. New orders are proposed for markers on chromosomes 3 and 17. Six microsatellites isolated from cosmids were also localized on the cytogenetic map by fluorescent in situ hybridization. We tested the subcloning ligation mixture-polymerase chain reaction (SLiM-PCR) method for isolating microsatellites from cosmids. Subcloning is more effective when the cosmid harbours several microsatellites whereas SLiM-PCR is more straightforward when the cosmid contains a single microsatellite. Fifteen anonymous microsatellites were regionally assigned by using a hybrid cell panel. For map integration, the determination of a regional assignment of anonymous microsatellites by using a hybrid cell panel offers an alternative to microsatellite isolation from cosmids and their localizations by in situ hybridization.     

Integrated mapping analysis of the Werner syndrome region of chromosome 8

The Werner syndrome locus (WRN) is located at 8p11-p12. To facilitate eventual cloning of the WRN gene, a 10,000-rad radiation-reduced hybrid (RH) cell panel was generated to map genetic markers, sequence-tagged sites (STSs), and genes in this region. A hamster cell line carrying an intact human chromosome 8 was fused with another hamster cell line. Two sets of hybrid cell panels from 2 separate fusions were generated; each panel consisted of 50 independent clones; 33 and 34 cell lines from the 2 fusions retained human chromsome material as determined by inter-Alu PCR. The combined panel was genotyped for 52 markers spanning the entire chromosome, including 10 genes, 29 anonymous polymorphic loci, and 13 STSs. Seventeen of these markers have not been previously described. Markers near the centromere were retained at a higher frequency than more distal markers. Fluorescence in situ hybridization was also used to localize and order a subset of the markers. A RH map of the WRN region was constructed using a maximum likelihood method, giving the following most likely order: D8S131-D8S339 (GSR)-D8S124-D8S278-D8S259-(D8S71)-D8S283- D8S87-D8S105-D8S135 (FGFR1)-D8S135PB-D8S255-ANK1. A genetic map of 15 short tandem repeat polymorphic loci in the WRN region was also constructed. The marker orders from the genetic and RH maps were consistent. In addition, an integrated map of 24 loci in the WRN region was generated using information from both genetic and RH mapping methods. A 1000:1 framework map for 6 loci (LPL-D8S136-D8S137-D8S87-FGFR1-ANK1) was determined by genetic mapping, and the resulting locus order was fixed during analysis of the RH genotype data. The resulting integrated map contained more markers than could confidently be ordered by either genetic or RH mapping alone.     

Allocation of random amplified polymorphic DNA markers and enzyme activities to Aspergillus nidulans and Aspergillus tetrazonus chromosomes

Chromosome-substituted haploid segregants of an A. nidulans x A. tetrazonus somatic hybrid were used to allocate several random amplified polymorphic DNA and isoenzyme markers to parental chromosomes. Twenty-six amplified DNA fragments, and nine isoenzyme activities, including lactate dehydrogenase, superoxide dismutase, and arylesterase isoenzymes were assigned to chromosomes. Chromosomes-specific markers were found for each A. nidulans and A. tetrazonus chromosome. These markers could be used to saturate the genetic map of A. nidulans. The formation of two secondary metabolites was also assigned to chromosomes III and VIII. Attempts were made to allocate extracellular enzyme activities to parental chromosomes, mostly without success, possibly because multiple enzyme forms located on different chromosomes could be responsible for the production of an enzyme activity.     

Binding of thyroid hormone to the goat testicular Leydig cell induces the generation of a proteinaceous factor which stimulates androgen release

Chromosome 3 comprises 7% of the genome and contains at least 210 Mb of DNA. To expedite the analysis of this chromosome, we have assembled a somatic cell hybrid mapping panel that subdivides human chromosome 3 into 23 intervals using a total of 19 hybrids. Hybrids were constructed from 16 patients' cells containing chromosome 3 translocations. All of these hybrids selectively retained the derivative 3 chromosome. In addition, we utilized 2 radiation-reduced hybrids and 3 hybrids carrying spontaneous translocations between human chromosome 3 and rodent chromosomes. The entire panel has 9 short arm breakpoints that involve bands p24.2, p22, p21, p14, and p12 plus a total of 11 long arm breakpoints that involve bands q13, q21, q25, q26, and q27. In addition, two cell lines appear to have breakpoints at or near the centromere. To date, we have used this panel to localize 92 sequences regionally on the short arm, 89 sequences on the long arm, and 7 sequences near the centromere. These hybrids are useful tools that allow the rapid localization of markers on chromosome 3 and greatly assist other mapping efforts.     

The development of sequence-tagged sites for human chromosome 4

As part of our efforts to construct a high-resolution physical map of human chromosome 4, we developed a systematic approach for efficiently generating large numbers of chromosome-specific sequence-tagged sites (STSs). In this paper, we describe how rate-limiting steps in our STS development were identified and overcome, and detail our current development strategy. We present information for 822 new human chromosome 4-specific STSs, including PCR amplification conditions and subchromosomal localization data, obtained by analysis of the STS with somatic cell hybrids containing different portions of human chromosome 4. Although most STSs presented here were developed from anonymous clones whose sequences were determined in this laboratory, several STSs were developed for genes and other DNA sequences that were previously mapped to chromosome 4. Our data indicate that the availability of DNA sequence for an STS locus, in addition to the sequences of the two PCR oligonucleotides, significantly increases the transfer of that STS by allowing investigators to select new oligonucleotides best suited to the standard conditions used in their laboratories.     

High resolution mapping of the renal sodium-phosphate cotransporter gene (NPT2) confirms its localization to human chromosome 5q35

The precise chromosomal localization of the type II renal-specific Na+-phosphate (Pi) cotransporter (NPT2) gene (gene symbol SLC17A2) is necessary for the identification of closely linked polymorphic markers to determine whether NPT2 is a candidate gene for inherited disorders of renal Pi reabsorption. Recent studies by two different groups localized NPT2 to human chromosome 5q35 and 5q13, respectively. To resolve this discrepancy, we used three independent methods. The results using a human chromosome 5/rodent somatic cell hybrid deletion panel, fluorescence in situ hybridization with a PAC clone containing the NPT2 locus, and analysis of a chromosome 5-specific radiation hybrid panel were all consistent with the 5q35 assignment of the NPT2 gene. The radiation hybrid results placed NPT2 between polymorphic microsatellite markers D5S498 and D5S469. These findings will allow the initiation of linkage analysis to determine if NPT2 has a causative role in Mendelian disorders of renal Pi wasting.     

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.     

Fatty acid profiles in subcutaneous and mesenteric adipose tissues from Zucker rats after energy restriction. Influence of dietary fat

We have constructed a collection of canine-rodent microcell hybrid cell lines by fusion of canine fibroblast microcell donors with immortalized rodent recipient cells. Characterization of the hybrid cell lines using a combination of fluorescence in situ hybridization and PCR analysis of canine microsatellite repeat sequences allowed selection of a panel of hybrids in which most canine chromosomes are represented. Approximately 90% of genetic markers and genes that were tested could be assigned to 1 of 31 anonymous canine chromosome groups, based on common patterns of retention in the hybrid set. Many of these putative chromosome groups have now been validated by linkage analysis. This panel of cell lines provides a tool for development of genetic, physical, and comparative maps of the canine genome.