RESUMEN
Aquaporins (AQPs) belong to a transmembrane protein family of water channels that are permeable to water by the osmotic gradient. There are two isoforms of mouse AQP4 - M1 and M23. Their balance in the cell determines water permeability of the plasma membrane. These two isoforms are encoded by three mRNAs: M1 isoform is encoded by M1 mRNA and M23 isoform is encoded by M23 and M23X mRNAs. Here we found a new fourth mRNA of mouse AQP4 - M23A mRNA. The start of transcription is different for M23A mRNA from all the known AQP4 mRNAs. The 5'-untranslated region (5'-UTR) of M23A mRNA is encoded by four new exons (A, B, C, and D), which are located in the 5' region from exon-0 of the AQP4 gene. Alternative splicing between the exons-A, -B, -C, and -D leads to formation of multiple variants of M23A mRNA. We cloned six of these variants, all of which code full length M23 isoform of AQP4. Using RT-PCR we detected tissue-specific expression of the new M23A and already known M23, M23X, and M1 mRNAs. The M23A mRNA is expressed mostly in kidney, liver, and brain. Analysis of mRNA 5'-UTR structure showed low translation efficacy for M1 mRNA in comparison with high translation efficacy for M23A, M23X, and M23 mRNAs. We propose that AQP4 expression is controlled tissue-specifically by independent promoters. Thus multiple AQP4 mRNAs may allow long-term regulation of the balance between M1 and M23 AQP4 isoforms in the cell and thus water permeability of the plasma membrane.
Asunto(s)
Acuaporina 4/metabolismo , Encéfalo/metabolismo , Riñón/metabolismo , Hígado/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 5' , Empalme Alternativo , Animales , Acuaporina 4/química , Acuaporina 4/genética , Secuencia de Bases , Clonación Molecular , Exones , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Ratones , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de ADNAsunto(s)
Aldehído Deshidrogenasa/metabolismo , Fabaceae/enzimología , Plantas Medicinales , Aldehído Deshidrogenasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Fabaceae/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de AminoácidoRESUMEN
Two degenerate oligonucleotide sequence primers and polymerase chain reactions on total DNA have been utilized to clone on 651--bp gene fragment coding the central part of amino acid sequence of an earlier unknown aldehyde dehydrogenase (ALDH) from mung bean. The deduced partial amino acid sequence for this aldehyde dehydrogenase shows about 65% sequence identity to ALDHs of Vibrio cholerae Rhodococcus sp., Alcaligenes eutrophus and about 45% sequence identity to mammalian ALDHs 1 and 2, ALDHs of Aspergillus niger and A, nidulans, the betain aldehyde dehydrogenase from spinach. Alignment of the mung bean aldehyde dehydrogenase partial amino acid sequence with the sequence of 16 NAD(P)(+)-dependent aldehyde dehydrogenases has demonstrated that all strictly conserved amino acid residues and all three conservative regions are identical.
Asunto(s)
Aldehído Deshidrogenasa/genética , Fabaceae/genética , Genes de Plantas , Plantas Medicinales , Secuencia de Aminoácidos , Clonación Molecular , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Vasopressin plays an essential role for the regulation of water balance by activating the collecting duct-specific water channel, aquaporin-2 (AQP2). Here we present evidence that vasopressin may also act as a long-term, transcriptional regulator of AQP2. The studies were performed on LLC-PK1 cells, which normally express V2 receptor (V2R) and which were transfected with a fragment of the human AQP2 promoter. Activation of the adenylate cyclase-coupled V2R in LLC-PK1 cells induced phosphorylation of adenosine 3',5'-cyclic monophosphate (cAMP) responsive element binding protein (CREB) and expression of c-Fos. Binding of these factors to the CRE and AP1 site did, in combination, lead to AQP2 promoter activation. These results establish the role of vasopressin as a regulator of transcription and are the first example of how a message from a highly specific receptor is, via a dual effect of the cAMP signal on CREB and immediate early gene expression, transduced to the transcription of a final target protein with known biological effects.
Asunto(s)
Adenilil Ciclasas/metabolismo , Acuaporinas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Canales Iónicos/genética , Regiones Promotoras Genéticas , Receptores de Vasopresinas/fisiología , Factor de Transcripción AP-1/metabolismo , Vasopresinas/farmacología , Animales , Acuaporina 2 , Acuaporina 6 , Sitios de Unión , Línea Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , AMP Cíclico/metabolismo , Desamino Arginina Vasopresina/farmacología , Regulación de la Expresión Génica , Humanos , Canales Iónicos/biosíntesis , Cinética , Modelos Biológicos , Forbol 12,13-Dibutirato/farmacología , Fosforilación , Regiones Promotoras Genéticas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-fos/biosíntesis , Proteínas Proto-Oncogénicas c-jun/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Transducción de Señal , TransfecciónRESUMEN
Three exon regions of the myoglobin gene of Baikal seal Phoca sibirica have been amplified and sequenced. The sequences have been found identical to those for the myoglobin gene of the North Atlantic seal Halichoerus grypus. Thus, these seal species have separated not earlier than 2.2 billion years before present.
Asunto(s)
Exones/genética , Mioglobina/genética , Phocidae/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , SiberiaRESUMEN
Characteristics of G-proteins were studied in renal medulla of rats and mice of different ages. It was established that G-protein alpha-subunit gene expression has a specific dynamics with maximum on the 5-8-th day of life and at the end of weaning. It was shown that vasopressin does not stimulate G-protein GTPase activity in membrane preparations from immature kidneys. The differences of G-protein chromatographic characteristics were revealed. It is suggested that the development of vasopressin sensitivity of the kidney is related with the changes in G-protein system.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Médula Renal/crecimiento & desarrollo , Envejecimiento/metabolismo , Animales , Autorradiografía , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/análisis , Regulación del Desarrollo de la Expresión Génica/fisiología , Médula Renal/metabolismo , Masculino , Ratones , Ratones Endogámicos CBA , Ratas , Ratas Wistar , Vasopresinas/fisiología , DesteteRESUMEN
Introns of human tyrosine aminotransferase (TAT) gene were sequenced. Combined with the literature data about the exon-intron structure of the gene and the sequence of the TAT mRNA, the obtained nucleotide sequences yielded on uninterrupted segment 10989 b. p. long of the human TAT gene.
Asunto(s)
Tirosina Transaminasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN , Exones , Humanos , Intrones , Datos de Secuencia MolecularRESUMEN
EcoRI-fragment No. 5 of the rat tyrosine aminotransferase gene containing exons K, L, intron 11, and a part of the 3'-nontranslatable region was digested with several restriction endonucleases (BspRI, Sau3A, BamHI, Ecl136II, AluI), the subfragments obtained were cloned into M13mp19 and sequenced using the Sanger technique with dye-labelled primers on the automated sequencer "Applied Biosystems", model 370A. The sequences were combined by means of a PC-GENE package and original programs to yield the primary structure (1064 b. p.) of the above fragment No. 5, adjacent to the previously sequenced EcoRI-fragment No. 4 (3677 b.p.).
Asunto(s)
Desoxirribonucleasa EcoRI/metabolismo , Tirosina Transaminasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Datos de Secuencia Molecular , RatasRESUMEN
cDNA for proopiomelanocortin (POMC) of mink has been cloned and sequenced. A comparative analysis of the primary structures of mRNAs coding for proopiomelanocortins of eight animal species and man has been performed. The analysis has revealed conserved and variable POMC mRNA regions. High variability of some of the regions is suggested to be due to the peculiarities of their structural organization. A putative mechanism responsible for the mutations in variable regions is proposed. A tree of the evolutionary relations of POMC has been developed.
Asunto(s)
ADN/química , Visón/genética , Hipófisis/química , Proopiomelanocortina/genética , ARN Mensajero/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Haplorrinos , Humanos , Ratones , Datos de Secuencia Molecular , Proopiomelanocortina/biosíntesis , ARN Mensajero/aislamiento & purificación , Ratas , Salmón , Alineación de Secuencia , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Porcinos , XenopusRESUMEN
A probe was constructed containing a fragment of DNA replica of hepatitis A virus (HAV) RNA within bacteriophage M13 single-stranded DNA which allowed 10(-12) g of viral RNA to be tested. Hybridization of 32P-labeled probe with total RNA from 559 samples of blood, saliva, and urine from patients with viral hepatitis A revealed the presence of HAV RNA in 14% of the samples. In the 1st week of the jaundice period HAV RNA was detected in 40% (15 positive samples out of the 39 tested), in the 2nd week 26% (14 out of 54). HAV RNA was demonstrated in 20 out of 236 blood samples from subjects in a focus of HA outbreak. Six out of 9 subjects subsequently developing the disease were detected 7-10 days before the onset of the clinical symptoms. The proposed method may be useful for detection of HAV carriers in the latent period for their isolation in order to prevent further development of epidemic.
Asunto(s)
Bacteriófagos/genética , Sondas de ADN , ADN Viral , ADN , Hepatovirus/aislamiento & purificación , Portador Sano/diagnóstico , Portador Sano/microbiología , Hepatitis A/diagnóstico , Hepatitis A/microbiología , Hepatovirus/genética , Humanos , Hibridación de Ácido Nucleico , ARN Viral/genética , Saliva/microbiología , Orina/microbiología , Viremia/diagnóstico , Viremia/microbiologíaAsunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Hormonas/fisiología , Mamíferos/genética , Tirosina Transaminasa/genética , Animales , Secuencia de Bases , Clonación Molecular , Regulación Enzimológica de la Expresión Génica/genética , Genes tat/genética , Genes tat/fisiología , Hígado/enzimología , Datos de Secuencia Molecular , Especificidad de Órganos/genética , Mapeo RestrictivoRESUMEN
The sequence of a 3677 nucleotide EcoRI fragment was determined that codes for part of the rat liver tyrosine aminotransferase gene. The sequence was compared with the previously determined cDNA sequence and the intron and exon boundaries were deduced.
Asunto(s)
Hígado/enzimología , Tirosina Transaminasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN , Datos de Secuencia Molecular , Ratas , Mapeo RestrictivoRESUMEN
Tyrosine aminotransferase (TAT) displays a higher activity in rat liver than in cardiac, renal and cerebral tissues. In the latter three tissues the enzyme is not activated by hydrocortisone. In order to investigate the nature of TAT tissue specificity and to understand how its activity is regulated by hormones in different tissues, the content of TAT mRNA in rat liver, kidney, heart and brain was measured by dot-hybridization using TAT cRNA. TAT mRNA was found in all tissues under study, although in the liver its amount was 10 times as high as in kidney, heart and brain, which is consistent with different levels of TAT enzymatic activity. Upon hydrocortisone administration the amount of TAT mRNA in the liver showed a constant increase reaching a maximum (7-fold) thereafter. The TAT mRNA levels in cardiac and cerebral tissues remained unchanged. These data suggest that the tissue-specific expression and regulation of TAT activity are effectuated by changes in the mRNA contents which may be due to the level of TAT gene transcription.
Asunto(s)
Hidrocortisona/farmacología , ARN Mensajero/metabolismo , Tirosina Transaminasa/genética , Animales , ADN/genética , Inducción Enzimática , Regulación Enzimológica de la Expresión Génica , Hígado/enzimología , Hígado/metabolismo , Masculino , Hibridación de Ácido Nucleico , Especificidad de Órganos , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Transcripción Genética , Tirosina Transaminasa/biosíntesis , Tirosina Transaminasa/metabolismoRESUMEN
Treatment of Wistar rats with a single dose of hydrocortisone acetate resulted in a transient induction of tyrosine aminotransferase (TAT) in the liver. TAT activity and the level of TAT mRNA increased 4-6 h after addition of hydrocortisone acetate (induction phase) and declined thereafter (deinduction phase). TAT activity and TAT mRNA failed to respond to readdition of fresh hydrocortisone acetate during the deinduction period, but cycloheximide treatment at this period increased the level of TAT mRNA. Hydrocortisone acetate alone and cycloheximide alone or after hydrocortisone acetate treatment stimulated the rate of TAT gene transcription. TAT displayed a low activity in the rat heart, brain and kidney, and was not induced by hydrocortisone acetate in these tissues. In addition to liver, TAT mRNA was found in heart, brain and kidney, but in the latter three tissues its amount was about one tenth only as in the liver, which was consistent with different levels of TAT activity. Upon hydrocortisone acetate treatment the TAT mRNA levels in brain and heart remained unchanged.
Asunto(s)
Regulación Enzimológica de la Expresión Génica , Hidrocortisona/fisiología , Tirosina Transaminasa/genética , Animales , Cicloheximida/farmacología , Inducción Enzimática , Hígado/metabolismo , Masculino , Hibridación de Ácido Nucleico , ARN Mensajero/efectos de los fármacos , Ratas , Ratas Endogámicas , Distribución Tisular , Transcripción Genética , Tirosina Transaminasa/biosíntesisRESUMEN
Three fragments of a DNA copy complementary to tre hepatitis A virus RNA have been isolated from the pHAV-VPI-22 plasmid and cloned in M 13 mp 8 vector. The 140, 250 and 390 b. p. fragments corresponded to the viral genome fragment coding for VP1 protein. Single-stranded DNAs of the hybrid phages with 250 b. p. and 390 b. p. inserts have been used for radioactive probe synthesis. The specificity of the probes for viral RNA was confirmed in hybridization with hepatitis A virus RNA purified from the virus-infected cell culture. The hybridization was shown to detect up to 10(-12)-10(-13) g of the virus. A principal possibility to use the probes in diagnostic purposes was demonstrated by the virus detection in blood samples obtained from patients with hepatitis A infection.
Asunto(s)
Colifagos/genética , Sondas de ADN , Hepatovirus/genética , Hibridación de Ácido Nucleico , ARN Viral/genética , ADN Viral/genética , PlásmidosRESUMEN
A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.
Asunto(s)
Mapeo Cromosómico , ADN/genética , Visón/genética , Proopiomelanocortina/genética , Secuencia de Aminoácidos , Animales , Bovinos , Clonación Molecular , Cricetinae , Cricetulus , Humanos , Células Híbridas , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Homología de Secuencia de Ácido NucleicoRESUMEN
Total RNA was extracted from Mustella vison pituitary gland, and cDNA for proopiomelanocortin mRNA was synthesised and cloned. A 600 b. p. insert encoding for total ACTH, beta LPH and 3'-nontranslated end of the mRNA was sequenced using the Maxam-Gilbert technique.
Asunto(s)
Clonación Molecular , ADN/biosíntesis , Hipófisis/análisis , Proopiomelanocortina/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN/genética , Visón , Datos de Secuencia Molecular , Plásmidos , ARN Mensajero/genéticaRESUMEN
The poly(A)-containing mRNA from human pituitary and prolactinoma have been purified and translated in the cell-free system from rabbit reticulocytes. mRNA from prolactinoma was shown to be enriched with specific prolactin mRNA. DNA complementary to the prolactin mRNA from human pituitary was obtained and cloned. Sequencing of the 900 bp insert by the Maxam-Gilbert technique suggested the cDNA cloned to cole for the previously published amino acid sequence, mismatches with mRNA from prolactinoma occurring at the third positions of codons and thus not causing amino acid substitutions.
Asunto(s)
Clonación Molecular , ADN/genética , Hipófisis/análisis , Prolactina/genética , ARN Mensajero/genética , Secuencia de Bases , ADN/análisis , ADN/síntesis química , Electroforesis en Gel de Poliacrilamida , Humanos , Datos de Secuencia Molecular , Prolactina/análisis , ARN Mensajero/análisisRESUMEN
A simple and rapid method is proposed for the localization of antigenic determinants in proteins of known primary structure exemplified by human myoglobin. The polypeptide chain of myoglobin was cleaved with BrCN (at Met residues) or with bromosuccinimide (at Trp and Tyr residues) under conditions which on average gave less than one scission per myoglobin molecule. The "single-hit" cleavage products were separated by gel electrophoresis and transferred to nitrocellulose by electroblotting. The peptides containing intact antigenic determinants were vizualized by immuno-peroxidase staining with four monoclonal anti-myoglobin antibodies. Comparison of the lengths of the immuno-reactive peptides with the known positions of methionine, tryptophan and tyrosine residues suggested that the four monoclonal antibodies were bound by myoglobin over the region Trp-14 to Met-55. As compared with other methods of localization, the method proposed is much faster and takes much lesser amount of protein.