Mapping the Down syndrome chromosome region. Establishment of a YAC contig spanning 1.2 megabases

The ocular artifacts that contaminate the EEG derive from the potential difference between the cornea and the fundus of the eye. This corneofundal or corneoretinal potential can be considered as an equivalent dipole with its positive pole directed toward the cornea. The cornea shows a steady DC potential of approximately +13 mV relative to the forehead. Blink potentials are caused by the eyelids sliding down over the positively charged cornea. The artifacts from eye-movements result from changes in orientation of the corneo-fundal potential. The scalp-distribution of the ocular artifacts can be described in terms of propagation factors--the fraction of the EOG signal at periocular electrodes that is recorded at a particular scalp location. These factors vary with the location of the scalp electrode. Propagation factors for blinks and upward eye-movements are significantly different.     

Construction of a yeast artificial chromosome contig spanning the spinal muscular atrophy disease gene region

The childhood spinal muscular atrophies (SMAs) are the most common, serious neuromuscular disorders of childhood second to Duchenne muscular dystrophy. A single locus for these disorders has been mapped by recombination events to a region of 0.7 centimorgan (range, 0.1-2.1 centimorgans) between loci D5S435 and MAP1B on chromosome 5q11.2-13.3. By using PCR amplification to screen yeast artificial chromosome (YAC) DNA pools and the PCR-vectorette method to amplify YAC ends, a YAC contig was constructed across the disease gene region. Nine walk steps identified 32 YACs, including a minimum of seven overlapping YAC clones (average size, 460 kb) that span the SMA region. The contig is characterized by a collection of 30 YAC-end sequence tag sites together with seven genetic markers. The entire YAC contig spans a minimum of 3.2 Mb; the SMA locus is confined to roughly half of this region. Microsatellite markers generated along the YAC contig segregate with the SMA locus in all families where the flanking markers (D5S435 and MAP1B) recombine. Construction of a YAC contig across the disease gene region is an essential step in isolation of the SMA-encoding gene.     

A cosmid and yeast artificial chromosome contig containing the complete ryanodine receptor (RYR1) gene

The ryanodine receptor (RYR1) gene is responsible for some forms of malignant hyperthermia and has been localized to 19q13.1. Central core disease is a genetic myopathy that is genetically linked to RYR1. We have identified an overlapping set of cosmid and YAC clones that spans more than 800 kb and includes the RYR1 gene (approximately 205 kb). Cosmids from this region were identified by screening three chromosome 19 cosmid libraries (11-fold coverage) with six subclones representing the entire RYR1 cDNA. Genomic sequences from positive cosmids were then used as probes to identify additional cosmids. A minimally overlapping set of 23 cosmids was assembled into two contigs on the basis of restriction fragment analysis and hybridization data. Three YAC clones were isolated by screening a human YAC library with selected cosmid inserts. Overlaps among these YACs and the cosmid contigs were determined by hybridizing YAC Alu-PCR products to cosmid DNAs. The YACs bridged the gap between the cosmid contigs and extended the contig on both sides. Fluorescence in situ hybridization experiments positioned the RYR1 contig between GPI, MAG, and D19S191 on the proximal side and D19S190, CYP2A, CYP2F, SNRPA, BCKDHA, and other markers on the distal side. The 800-kb contig of cloned reagents will facilitate the detailed characterization of the RYR1 gene and other loci that may be closely related to central core disease.     

A yeast artificial chromosome (YAC) library containing 10 haploid chicken genome equivalents

We report the construction of a YAC library that provides 10-fold redundant coverage of the chicken genome. The library was made by transforming S. cerevisiae AB1380 with YAC constructs consisting of partially digested and size fractionated (>465 kb) EcoRI genomic fragments ligated to pCGS966 YAC vector arms. The primary library provides 8.5-fold redundant coverage and consists of 16,000 clones arrayed in duplicate 96-well microtiter plates and gridded on nylon membranes at high density (18,000 clones/484cm2). The average insert size, 634 kb, was derived from size fractionation of a random sample of 218 YACs. Hybridization of five unlinked chicken genes to colony blots revealed six or more positive clones. This is consistent with the theoretical expectation from average insert sizes and number of clones. A second collection of clones consists of a further 20,000 colonies, of which 20% contain inserts larger than 450 kb and 80% contain only coligated vector arms. We estimate that these clones provide a further 1.5-fold redundant coverage of the chicken genome; thus, the total collection of 36,000 clones provides 10-fold redundant coverage of the chicken genome. The library is intended as a resource for fine-scale analysis of the organization of the chicken genome and is presently being used to construct a contig map of chicken Chromosome (Chr) 16, which contains the MHC and nucleolar organizer.     

Expression screening of a yeast artificial chromosome contig refines the location of the mouse H3a minor histocompatibility antigen gene

The H3 complex, on mouse Chromosome 2, is an important model locus for understanding mechanisms underlying non-self Ag recognition during tissue transplantation rejection between MHC-matched mouse strains. H3a is a minor histocompatibility Ag gene, located within H3, that encodes a polymorphic peptide alloantigen recognized by cytolytic T cells. Other genes within the complex include beta2-microglobulin and H3b. A yeast artificial chromosome (YAC) contig is described that spans the interval between D2Mit444 and D2Mit17, a region known to contain H3a. This contig refines the position of many genes and anonymous loci. In addition, 23 new sequence-tagged sites are described that further increase the genetic resolution surrounding H3a. A novel assay was developed to determine the location of H3a within the contig. Representative YACs were modified by retrofitting with a mammalian selectable marker, and then introduced by spheroplast fusion into mouse L cells. YAC-containing L cells were screened for the expression of the YAC-encoded H3a(a) Ag by using them as targets in a cell-mediated lympholysis assay with H3a(a)-specific CTLs. A single YAC carrying H3a was identified. Based on the location of this YAC within the contig, many candidate genes can be eliminated. The data position H3a between Tyro3 and Epb4.2, in close proximity to Capn3. These studies illustrate how genetic and genomic information can be exploited toward identifying genes encoding not only histocompatibility Ags, but also any autoantigen recognized by T cells.     

A YAC contig encompassing the XRCC5 (Ku80) DNA repair gene and complementation of defective cells by YAC protoplast fusion

The Chinese hamster ovary xrs mutants are sensitive to ionizing radiation, defective in DNA double-strand break rejoining, and unable to carry out V(D)J recombination effectively. Recently, the gene defective in these mutants, XRCC5, has been shown to encode Ku80, a component of the Ku protein and DNA-dependent protein kinase. We present here a YAC contig involving 25 YACs mapping to the region 2q33-q34, which encompasses the XRCC5 gene. Eight new markers for this region of chromosome 2 are identified. YACs encoding the Ku80 gene were transferred to xrs cells by protoplast fusion, and complementation of all the defective phenotypes has been obtained with two YACs. We discuss the advantages and disadvantages of this approach as a strategy for cloning human genes complementing defective rodent cell lines.     

Loss of heterozygosity of the von Hippel Lindau gene locus in polypoid dysplasia but not flat dysplasia in ulcerative colitis or sporadic adenomas

Carcinoma in ulcerative colitis (UC) develops from dysplastic precursor lesions, which include flat dysplasia (FD) and polypoid dysplasias (PD). PD may present as single or multiple polypoid structures or as plaque-like lesions that, independent of histological grade, are an indication for colectomy. PDs are histologically similar to adenomas and may not be readily distinguished by light microscopy. It is not known whether FD and PD are different entities, or whether they represent etiologically similar lesions with different morphological expression. We microdissected 25 cases of UC with PD and 19 samples of FD with surrounding chronic colitis (CC) in UC. Loss of heterozygosity (LOH) at the von Hippel Lindau (vHL) gene locus and the putative tumor suppressor genes APC, INK4A (9p16), and p53 was studied. LOH of the vHL gene, INK4A (9p16), and APC was also studied in 11 sporadic adenomas of the colon. LOH at the vHL locus was present in 50% of the samples of PD and in 12% of the samples of FD. LOH was seen in CC close to PD and FD in 26% and 12% of cases, respectively. No adenoma showed LOH of the vHL gene markers studied. LOH in p53 was seen in PD in 16% cases and in FD in 42% cases and in CC close to PD and FD in 0% and 14% cases, respectively. LOH patterns between PD and FD of the markers for APC and 9p16 were not different. LOH in APC was seen in two of five cases of adenoma. We conclude that PD and FD share genetic alterations in APC and 9p16 genes. More frequent involvement of the VHL gene in PD and surrounding CC and involvement of p53 in HGD and CC in FD may represent genetic differences between the development of PD and FD and may be the cause of the different morphology. The infrequency of LOH at the vHL locus in adenomas versus PD may serve as a discriminator between adenomas and PD in diagnostically problematic cases.     

A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice

Expression of transgenes in mice often fails to follow the normal temporal and spatial pattern and to reach the same level as the endogenous copies. Only in exceptional cases has position-independent and copy number-dependent expression been reproduced. The size constraint of standard constructs may prevent the inclusion of important remote regulatory elements. Yeast artificial chromosomes (YACs) provide a means of cloning large DNA fragments and the transfer of YAC DNA into somatic cells has been reported. We have previously produced transgenic mice carrying a 35 kilobase YAC construct. Here we report the transfer of a 250 kilobase YAC covering the mouse tyrosinase gene into mice by pronuclear injection of gel-purified YAC DNA. The YAC was inserted into the mouse genome without major rearrangements and expression of the YAC-borne tyrosinase gene resulted in complete rescue of the albino phenotype of the recipient mice. Expression from the transgene reached levels comparable to that of the endogenous gene and showed copy number dependence and position independence.     

Position-independent expression of a human nerve growth factor-luciferase reporter gene cloned on a yeast artificial chromosome vector

Two yeast artificial chromosomes containing the entire human nerve growth factor gene were isolated and mapped. By homologous recombination a luciferase gene was precisely engineered into the coding portion of the NGF gene and a neomycin selection marker was placed adjacent to one of the YAC telomeres. Expression of the YAC-based NGF reporter gene and a plasmid-based NGF reporter gene were compared with the regulation of endogenous mouse NGF protein in mouse L929 fibroblasts. In contrast to the plasmid-based reporter gene, expression and regulation of the YAC-based reporter gene was independent of the site of integration of the transgene. Basic fibroblast growth factor and okadaic acid stimulated expression of the YAC transgene, whereas transforming growth factor-beta and dexamethasone inhibited it. Although cyclic AMP strongly stimulated production of the endogenous mouse NGF, no effect was seen on the human NGF reporter genes. Downregulation of the secretion of endogenous mouse NGF already occurred at an EC50 of 1-2 nM dexamethasone, but downregulation of the expression of NGF reporter genes occurred only at EC50 of 10 nM. This higher concentration was also required for upregulation of luciferase genes driven by the dexamethasone-inducible promoter of the mouse mammary tumor virus in L929 fibroblasts.     

Identification of complex chromosome rearrangements in the gibbon by fluorescent in situ hybridization (FISH) of a human chromosome 2q specific microlibrary, yeast artificial chromosomes, and reciprocal chromosome painting

Chromosome painting has revealed that the human chromosome homologs in lesser apes are often fragmented and translocated to a number of different hylobatid chromosomes. We investigated the fragmented human chromosome 2 homologs in gibbons to illustrate a new strategy in mapping regional and band-specific chromosomal homologies between species. Previous research showed that the DNA library specific to human chromosome 2 paints parts of four gibbon (lar species group) chromosomes (viz., 1, 10, 12, and 16) and yields five distinct hybridization signals (including two on gibbon chromosome 16). However, the exact segments of human chromosome 2 that were translocated to the various gibbon chromosomes could not be distinguished. To determine the origin of the human chromosome 2 signals, we hybridized a microlibrary for the long arm of human chromosome 2, as well as YACs specific for most of the major bands on this chromosome, to metaphases of the gibbon. For reciprocal chromosome painting, we hybridized flow-sorted gibbon chromosome probes to human chromosome 2. Each method added additional insights that helped clarify the shuffling of human chromosome 2 material in the highly reorganized gibbon genome. There was an excellent correspondence between these complementary techniques. YAC 958d2 identified the breakpoint between human chromosome 2 material present on gibbon chromosomes 10 and 16. The reciprocal chromosome painting permitted a more complete and regional assignment of homology between segments on various gibbon chromosomes to human chromosome 2. The results show that a combination of reciprocal chromosome painting, subregional microlibraries, and band-specific probes (such as YACs) can be used to identify homologies between species and to rapidly construct detailed comparative chromosome maps, especially when the karyotypes are highly rearranged.     

YAC contigs of the Rab1 and wobbler (wr) spinal muscular atrophy gene region on proximal mouse chromosome 11 and of the homologous region on human chromosome 2p

Despite rapid progress in the physical characterization of murine and human genomes, little molecular information is available on certain regions, e.g., proximal mouse chromosome 11 (Chr 11) and human chromosome 2p (Chr 2p). We have localized the wobbler spinal atrophy gene wr to proximal mouse Chr 11, tightly linked to Rab1, a gene coding for a small GTP-binding protein, and Glnsps1, an intronless pseudogene of the glutamine synthetase gene. We have now used these markers to construct a 1.3-Mb yeast artificial chromosome (YAC) contig of the Rab1 region on mouse Chr 11. Four YAC clones isolated from two independent YAC libraries were characterized by rare-cutting analysis, fluorescence in situ hybridization (FISH), and sequence-tagged site (STS) isolation and mapping. Rab1 and Glns-ps1 were found to be only 200 kb apart. A potential CpG island near a methylated NarI site and a trapped exon, ETG1.1, were found between these loci, and a new STS, AHY1.1, was found over 250 kb from Rab1. Two overlapping YACs were identified that contained a 150-kb region of human Chr 2p, comprising the RAB1 locus, AHY1.1, and the human homologue of ETG1.1, indicating a high degree of conservation of this region in the two species. We mapped AHY1.1 and thus human RAB1 on Chr 2p13.4-p14 using somatic cell hybrids and a radiation hybrid panel, thus extending a known region of conserved synteny between mouse Chr 11 and human Chr 2p. Recently, the gene LMGMD2B for a human recessive neuromuscular disease, limb girdle muscular dystrophy type 2B, has been mapped to 2p13-p16. The conservation between the mouse Rab1 and human RAB1 regions will be helpful in identifying candidate genes for the wobbler spinal muscular atrophy and in clarifying a possible relationship between wr and LMGMD2B.     

Direct isolation of human BRCA2 gene by transformation-associated recombination in yeast

Mutant forms of the BRCA2 gene contribute significantly to hereditary breast cancer. Isolation of the normal and mutant forms of the BRCA2 gene with its natural promoter would greatly facilitate analysis of the gene and its contribution to breast cancer. We have accomplished the direct isolation of the 90-kb gene from total human DNA by transformation-associated recombination in yeast using a small amount of 5' and 3' BRCA2 sequence information. Because the entire isolation procedure of a single chromosomal gene could be accomplished in approximately 2 weeks, the transformation-associated recombination cloning approach is readily applicable to studies of chromosome alterations and human genetic diseases.     

A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34

The Gene Expression Database (GXD) is a community resource that stores and integrates expression information for the laboratory mouse, with a particular emphasis on mouse development, and makes these data freely available in formats appropriate for comprehensive analysis. GXD is implemented as a relational database and integrated with the Mouse Genome Database (MGD) to enable global analysis of genotype, expression and phenotype information. Interconnections with sequence databases and with databases from other species further extend GXD's utility for the analysis of gene expression data. GXD is available through the Mouse Genome Informatics Web Site at http://www.informatics.jax.org/     

The proopiocortin (adrenocorticotropin/beta-lipoprotein) gene is located on chromosome 2 in humans

The proopiocortin gene is located on chromosome 2 in humans. A 13-kb DNA fragment containing proopiocortin gene sequences was identified in human cells while proopiocortin-related genes sequences of 9.8 and 6.2 kb were present in mouse cells. In human-mouse cell hybrids which contained reduced numbers of human chromosomes and a complete set of mouse chromosomes, the 9.8- and 6.2-kb fragments were always present while the 13-kb fragment segregated with human chromosome 2 and the chromosome 2 enzyme markers acid phosphatase-1 (ACP1), malate dehydrogenase-1 (MDHI), and isocitrate dehydrogenase-1 (IDH1). Analysis of a single cell hybrid with a broken chromosome 2 indicates that the proopiocortin and ACP1 genes are closely linked and in the distal region of the short arm of chromosome 2.