RESUMO
One of the major goals of the human genome project is to establish a physical map of each human chromosome with a density of sequence-tagged site (STS) markers exceeding one every 100 kb. We report here the generation of a human chromosome 5-specific radiation hybrid (RH) map that includes 556 markers. Of these markers, 132 loci are ordered with a maximum likelihood ratio of >1000:1 compared with the next most likely order. An additional 113 loci were ordered relative to these backbone markers with a maximum likelihood ratio of >10:1 but <1000:1. Together, these 245 loci form an ordered framework map for the chromosome. Using this framework, >300 more markers were localized based on two-point analysis with the ordered set. On average, there are 50 markers in common with the RH map presented here and other chromosome 5 maps included in the current whole genome cytogenetic, genetic, and physical maps. The accuracy of all the maps is evident in that there are no more than two discrepancies between any one of them and these data. All of the maps encompassing chromosome 5 complement each other providing excellent STS coverage with >2200 loci combined. The chromosome 5-specific RH map contains 20% of these independent loci. In addition, our RH map contains STSs derived from clones suitable for fluorescent in situ hybridization, allowing alignment to the cytogenetic map. Together, these maps will assist in the assembly of sequence-ready contigs and will aid in the identification of disease loci on chromosome 5 by positional cloning and positional candidate approaches.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 5/ultraestrutura , Sitios de Sequências Rotuladas , Centrômero , Cosmídeos , Marcadores Genéticos , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Funções Verossimilhança , Escore Lod , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
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.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 5 , Córtex Renal/metabolismo , Simportadores , Animais , Proteínas de Transporte/biossíntese , Mapeamento Cromossômico , Primers do DNA , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Fosfatos/metabolismo , Reação em Cadeia da Polimerase , Roedores , Deleção de Sequência , Proteínas Cotransportadoras de Sódio-Fosfato , Proteínas Cotransportadoras de Sódio-Fosfato Tipo I , Proteínas Cotransportadoras de Sódio-Fosfato Tipo II , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIIRESUMO
A cDNA library derived from human cerebral cortex was screened for the presence of sodium channel alpha subunit-specific clones. Ligation of three overlapping clones generated a full-length cDNA clone, HBA, that provided the complete nucleotide sequence coding for a protein of 2005 amino acids. The predicted structure suggests four homologous repeats and exhibits greatest homology and structural similarity to the rat brain sodium channel II. A second cDNA clone, HBB, that encodes a different subtype of sodium channel was isolated. Hybridization of DNA fragments from the 3' untranslated region of HBA and PCR with primers derived from HBB with human-hamster somatic cell hybrids localized these clones to human chromosome 2. In situ hybridization to human metaphase chromosomes mapped the structural genes for both HBA and HBB sodium channels to chromosome 2q23-24.3. The sodium channel HBA gene product was expressed by transfection in CHO cells. Expressed HBA currents were voltage-dependent, sodium-selective, and tetrodotoxin-sensitive and, thus, exhibit the biophysical and pharmacological properties characteristic of sodium channels.
Assuntos
Canais de Sódio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Cromossomos Humanos Par 2 , Clonagem Molecular , Cricetinae , DNA/genética , Humanos , Ativação do Canal Iônico , Potenciais da Membrana , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Mapeamento por Restrição , Alinhamento de Sequência , Canais de Sódio/fisiologia , TransfecçãoRESUMO
We report the isolation and sequencing of cDNAs encoding two human glutamate decarboxylases (GADs; L-glutamate 1-carboxy-lyase, EC 4.1.1.15), GAD65 and GAD67. Human GAD65 cDNA encodes a Mr 65,000 polypeptide, with 585 amino acid residues, whereas human GAD67 encodes a Mr 67,000 polypeptide, with 594 amino acid residues. Both cDNAs direct the synthesis of enzymatically active GADs in bacterial expression systems. Each cDNA hybridizes to a single species of brain mRNA and to a specific set of restriction fragments in human genomic DNA. In situ hybridization of fluorescently labeled GAD probes to human chromosomes localizes the human GAD65 gene to chromosome 10p11.23 and the human GAD67 gene to chromosome 2q31. We conclude that GAD65 and GAD67 each derive from a single separate gene. The cDNAs we describe should allow the bacterial production of test antigens for the diagnosis and prediction of insulin-dependent diabetes mellitus.
