RESUMEN
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.
Asunto(s)
Regulación de la Expresión Génica , Genes , ATPasa Intercambiadora de Sodio-Potasio/genética , Adulto , Envejecimiento , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Codón , Embrión de Mamíferos , Humanos , Isoenzimas/genética , Sustancias Macromoleculares , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Especificidad de Órganos , ARN Mensajero/genéticaRESUMEN
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.
Asunto(s)
Médula Renal/enzimología , ATPasa Intercambiadora de Sodio-Potasio , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Membrana Celular/enzimología , Fenómenos Químicos , Química Física , ADN/genética , Membrana Dobles de Lípidos , Proteínas de la Membrana , Hibridación de Ácido Nucleico , Fragmentos de Péptidos , Poli A/genética , ARN/genética , ARN Mensajero/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , PorcinosRESUMEN
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.
Asunto(s)
Genes , Familia de Multigenes , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN Recombinante , Humanos , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , ADN/genética , Feto/enzimología , Regulación de la Expresión Génica , Humanos , Ratones , Neoplasias/enzimología , Hibridación de Ácido Nucleico , ConejosRESUMEN
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.
Asunto(s)
Genes Reguladores , Genes , Familia de Multigenes , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cósmidos , Humanos , Ratones , Datos de Secuencia Molecular , Conejos , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
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%.
Asunto(s)
ATPasa Intercambiadora de Sodio-Potasio/genética , Animales , Secuencia de Bases , Células HeLa , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Familia de Multigenes , Seudogenes , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , PorcinosRESUMEN
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.
Asunto(s)
Adenosina Trifosfatasas/genética , Familia de Multigenes , ATPasa Intercambiadora de Sodio-Potasio/genética , Transcripción Genética , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Transporte Biológico , Encéfalo/enzimología , Electroforesis en Gel de Agar , Exones , Humanos , Intrones , Riñón/enzimología , Datos de Secuencia Molecular , Sondas de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Ácido NucleicoRESUMEN
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.
Asunto(s)
Genes , Riñón/enzimología , ARN Mensajero/genética , ADN Polimerasa Dirigida por ARN/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Hidrólisis , Hibridación de Ácido Nucleico , Porcinos , Moldes Genéticos , TripsinaRESUMEN
Oligonucleotides deduced from the amino acid sequence of a hexapeptide Lys-Asp-Phe-Ala-Glu-Asn were synthesized and used as probes to screen a pig kidney cDNA library for a specific DNA sequence coding for the alpha-subunit of Na+, K+-ATPase. It was shown that the mixed oligoprobe, consisting of 64 heptadecamers, could be only suitable for mRNA blot analysis. To identify the clones with specific cDNA inserts, mixed oligoprobes were fractionated by HPLC technique. For the same purpose a new set of oligonucleotides, synthesized as four groups of 16 different heptadecamers each, was used.
Asunto(s)
ADN/genética , Genes , Oligonucleótidos/genética , ARN Mensajero/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Médula Renal/enzimología , Hibridación de Ácido Nucleico , Péptidos/genética , ATPasa Intercambiadora de Sodio-Potasio/análisis , PorcinosRESUMEN
Messenger RNA, coding for the alpha-subunit of the Na+, K+-ATPase, was isolated from outer medulla of pig kidney. Within 25S-26S region the mRNA yields a band of specific hybridization with three oligonucleotide probes synthesized according to data on structures of three peptides isolated from the tryptic hydrolysate of the protein. Translation of the enriched poly(A+)-fraction of RNA in Xenopus laevis oocytes followed by the immunochemical identification of the products confirmed the presence of RNA coding for the desired protein. This RNA preparation was used for synthesis and cloning of double stranded cDNA.
Asunto(s)
Clonación Molecular , Biosíntesis de Proteínas , ARN Mensajero/aislamiento & purificación , ADN Polimerasa Dirigida por ARN/genética , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , ADN/biosíntesis , ADN/genética , Médula Renal/enzimología , Hibridación de Ácido Nucleico , ARN Mensajero/genética , ATPasa Intercambiadora de Sodio-Potasio/análisis , Porcinos , Xenopus laevisRESUMEN
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.
Asunto(s)
Genes , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , PorcinosAsunto(s)
ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/análisis , Enzimas de Restricción del ADN/metabolismo , Células HeLa/enzimología , Humanos , Riñón/enzimología , Sustancias Macromoleculares , Hibridación de Ácido Nucleico , Ratas , Ovinos , PorcinosRESUMEN
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).
Asunto(s)
Riñón/enzimología , Ribonucleoproteínas/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , Transcripción Genética , Animales , Membrana Celular/enzimología , Clonación Molecular , ADN/genética , Vectores Genéticos , Sustancias Macromoleculares , Microsomas/enzimología , Plásmidos , Biosíntesis de Proteínas , Mapeo Restrictivo , Partícula de Reconocimiento de Señal , PorcinosRESUMEN
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.
Asunto(s)
Secuencias Reguladoras de Ácidos Nucleicos , ATPasa Intercambiadora de Sodio-Potasio/genética , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Mapeo Cromosómico , Humanos , Isoenzimas , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genética , Biosíntesis de Proteínas , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
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.