RESUMO
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.
Assuntos
Cromossomos Humanos Par 13 , DNA Satélite , Ligação Genética , Alelos , Sequência de Bases , Feminino , Frequência do Gene , Humanos , Células Híbridas , Masculino , Dados de Sequência Molecular , Linhagem , Recombinação Genética , Caracteres SexuaisAssuntos
Cromossomos Humanos Par 4 , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Alelos , Sequência de Bases , DNA/genética , Frequência do Gene , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da PolimeraseRESUMO
The multigene family of human Na,K-ATPase is composed of 5 alpha-subunit genes, 3 of which were shown to encode the functionally active alpha 1, alpha 2 and alpha 3 isoforms of the catalytic subunits. This report describes the isolation, mapping and partial sequencing of the fourth gene (ATP1AL1) that was demonstrated here to be functionally active and expressed in human brain and kidney. Limited DNA sequencing of the ATP1AL1 exons allowed one to suggest that the gene probably encodes a new ion transport ATPase rather than an isoform of the Na,K-ATPase or the closely related H,K-ATPase.
Assuntos
Adenosina Trifosfatases/genética , Família Multigênica , ATPase Trocadora de Sódio-Potássio/genética , Transcrição Gênica , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico , Encéfalo/enzimologia , Eletroforese em Gel de Ágar , Éxons , Humanos , Íntrons , Rim/enzimologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase , Homologia de Sequência do Ácido NucleicoRESUMO
The sequences of a 1.5 kb long stretch of the 5' flanking region of the gene for the alpha 3 isoform of the catalytic subunit of human Na(+)-K+ ATPase (located on chromosome 19) and of more than a 2 kb stretch of the 5' flanking region of the gene for the alpha 2 isoform (located on chromosome 1) have been determined. Transcription start sites for the gene for the alpha 3 isoform have been mapped at positions -152 and -155 relative to the translation initiation codon by primer extension analysis and S1-nuclease mapping of mRNA from human brain. The 5' flanking region of the gene for isoform alpha 3 contains a CCAAT box on the noncoding chain and six putative Sp1 binding sites. Absence of a conventional TATA box and a high GC content are other features of the region. The 5' upstream region of the gene for the alpha 2 isoform contains potential TATA and CCAAT boxes and one potential Sp1 binding site. Upstream of the putative TATA box there is an octanucleotide repeat, GGGGGAGA, which is also found in several eukaryotic genes in analogous positions. Pairwise comparison of the putative 5' regulatory regions of the genes coding for the different isoforms of the Na(+)-K(+)-ATPase catalytic subunit shows the existence of conserved elements, as well as of oligonucleotide blocks with very different structures. It is suggested that the differences in the primary structure of the 5' upstream regions may provide the basis for tissue-specific expression of the Na(+)-K(+)-ATPase isoforms.
Assuntos
Sequências Reguladoras de Ácido Nucleico , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Mapeamento Cromossômico , Humanos , Isoenzimas , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Biossíntese de Proteínas , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The existence of a chromosome gene family containing at least one gene and one pseudogene was shown for the Na+,K+-ATPase beta-subunit. A partial structure of the beta 1-gene was determined, the coding part of which was completely homologous to cDNA of the Na+,K+-ATPase beta I-subunit from HeLa cells. The region encoding the putative protein transmembrane domain was shown to be bordered by two introns. The structure of a pseudogene (beta psi) was determined. This pseudogene is processed and contains multiple stop codons. Its homology to the beta I-subunit cDNA from HeLa cells is about 88%.
Assuntos
ATPase Trocadora de Sódio-Potássio/genética , Animais , Sequência de Bases , Células HeLa , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Família Multigênica , Pseudogenes , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , SuínosRESUMO
The primary structure of the putative regulatory region of a gene of the Na+,K+-ATPase multigene family in the human genome has been determined. This region includes the first exon with all of the untranslatable sequence of mRNA and a dozen nucleotides, coding for the first four amino acids of the hypothetic precursor of the alpha+-subunit. The entire region comprises over 1400 bp. The possible role of specific nucleotide blocks within this region in comparison with other genes is discussed.
Assuntos
Genes Reguladores , Genes , Família Multigênica , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cosmídeos , Humanos , Camundongos , Dados de Sequência Molecular , Coelhos , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
cDNA clones coding for either full-length or truncated forms of the beta-subunit of the Na+,K+-ATPase from pig kidney were engineered into a transcription vector based on a T7 promotor. In vitro transcription and subsequent translation of the mRNA in the presence of rough microsomes (RM) yielded beta-subunit molecules that were N-glycosylated and correctly inserted into the membrane. The signal peptide was not cleaved off. This membrane integration was found to be dependent on the function of the signal recognition particle (SRP). Several lines of evidence suggest that the hydrophilic aminoterminal domain of 34 amino acid residues preceding the postulated signal sequence is located on the cytoplasmic side whereas the carboxyterminal glycosylated domain is located on the exoplasmic side of the ER (endoplasmic reticulum)-membrane (type II membrane protein).
Assuntos
Rim/enzimologia , Ribonucleoproteínas/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Transcrição Gênica , Animais , Membrana Celular/enzimologia , Clonagem Molecular , DNA/genética , Vetores Genéticos , Substâncias Macromoleculares , Microssomos/enzimologia , Plasmídeos , Biossíntese de Proteínas , Mapeamento por Restrição , Partícula de Reconhecimento de Sinal , SuínosRESUMO
The expression of genes coding for alpha and alpha III isoforms of Na+,K+-ATPase alpha-subunit has been studied in human kidney, brain, thyroid and liver cells. The expression was shown to be subjected to a tissue-specific control and also depended on the developmental stage. The tissue-specific expression of genes coding for different isoforms of the catalytic subunit of Na+,K+-ATPase perhaps may be attributed to various functions of proteins belonging to this family.
Assuntos
Regulação da Expressão Gênica , Genes , ATPase Trocadora de Sódio-Potássio/genética , Adulto , Envelhecimento , Sequência de Aminoácidos , Sequência de Bases , Criança , Códon , Embrião de Mamíferos , Humanos , Isoenzimas/genética , Substâncias Macromoleculares , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Especificidade de Órgãos , RNA Mensageiro/genéticaRESUMO
Intra-individual tissue-specific restriction fragment length polymorphism (RFLP) has been demonstrated in DNA isolated from different mammalian tissues using cDNAs of alpha- and beta-subunits of Na+,K+-ATPase as hybridization probes. We propose that the RFLPs could result from gene rearrangements in the gene loci for the alpha- and beta-subunits of Na+,K+-ATPase. The changes in restriction patterns have been shown to occur during embryonic development and tumor formation. In addition, the tissue specificity of the expression of different genes of the family of Na+,K+-ATPase genes and their low expression in tumor cells have been demonstrated.
Assuntos
Polimorfismo Genético , Polimorfismo de Fragmento de Restrição , ATPase Trocadora de Sódio-Potássio/genética , Animais , DNA/genética , Feto/enzimologia , Regulação da Expressão Gênica , Humanos , Camundongos , Neoplasias/enzimologia , Hibridização de Ácido Nucleico , CoelhosRESUMO
Five different nucleotide sequences have been found in the human genome homologous to the gene of the alpha-subunit of Na+,K+-ATPase. A comparative analysis of the primary structure of these genes in the region 749-1328 (in coordinates of cDNA from the pig alpha-subunit) is presented.
Assuntos
Genes , Família Multigênica , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Sequência de Bases , DNA Recombinante , Humanos , Homologia de Sequência do Ácido NucleicoAssuntos
ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/análise , Enzimas de Restrição do DNA/metabolismo , Células HeLa/enzimologia , Humanos , Rim/enzimologia , Substâncias Macromoleculares , Hibridização de Ácido Nucleico , Ratos , Ovinos , SuínosRESUMO
The nucleotide sequence of the cDNA, containing coding region of the alpha-subunit of the pig kidney Na+, K+-ATPase, was determined. The region contains 3063 b.p. coding for 1021 amino acid residues. In the course of processing, five amino acid residues are cleaved to yield the mature Na+, K+-ATPase alpha-subunit containing 1016 amino acid residues.
Assuntos
Genes , Rim/enzimologia , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , SuínosRESUMO
mRNA coding for beta-subunit of Na+, K+-ATPase from pig kidney is about 24-25S as deduced from the hybridization pattern of poly (A)+-RNA with two synthetic oligonucleotides structurally corresponding to two peptides isolated from the tryptic hydrolyzate of beta-subunit. Cloning of cDNA allowed to determine the complete structure of the gene and to deduce the amino acid sequence of beta-subunit of Na+, K+-ATPase. The beta-subunit contains 302 amino acid residues and the protein is not processed at the N-nor at the C-terminus.
Assuntos
Genes , Rim/enzimologia , RNA Mensageiro/genética , DNA Polimerase Dirigida por RNA/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA/genética , Hidrólise , Hibridização de Ácido Nucleico , Suínos , Moldes Genéticos , TripsinaRESUMO
cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.