RESUMEN
Progressive ventricular dilatation commonly accompanies the transition to overt failure in chronically overloaded hearts; however, only recently have studies begun to elucidate underlying molecular alterations. In particular, the potential role of altered myocardial expression of the procollagenase gene in this process has not previously been examined. Biventricular hypertrophy and dilatation were produced in rats by creating an abdominal aortocaval fistula. The time courses of changes in expression of collagen I and III genes and of the procollagenase gene (matrix metalloproteinase-1, MMP-1) were assessed by Northern blot hybridization. Expression of all three genes increased promptly; however, collagenase gene expression peaked much earlier (8 h) than did expression of either of the collagen genes (7 days), and all returned to baseline levels by 45 days. These data corroborate earlier reports of increased collagen gene expression in this model, but more importantly, they provide the first evidence of concurrent activation of collagenase gene expression, suggesting that enhancement of collagen degradation may be a prerequisite for structural cardiac dilatation.
Asunto(s)
Aorta , Fístula Arteriovenosa/genética , Colágeno/genética , Expresión Génica/fisiología , Metaloproteinasa 1 de la Matriz/genética , Venas Cavas , Animales , Fístula Arteriovenosa/complicaciones , Fístula Arteriovenosa/diagnóstico por imagen , Fístula Arteriovenosa/patología , Gasto Cardíaco Bajo/etiología , Colágeno/metabolismo , Ecocardiografía , Atrios Cardíacos , Hipertrofia Ventricular Izquierda/etiología , Hipertrofia Ventricular Izquierda/patología , Hipertrofia Ventricular Derecha/etiología , Hipertrofia Ventricular Derecha/patología , Pulmón/patología , Masculino , Mortalidad , Miocardio/patología , Tamaño de los Órganos , ARN Mensajero/metabolismo , RatasRESUMEN
During lens regeneration in Pleurodeles waltl, the dorsal iris zone is the cell source of the lens regeneration, while the ventral iris zone can serve as the cells' source of lens regeneration only under certain experimental conditions. The method of subtractive hybridization was used for the identification of genes responsible for the different proliferative potential of these zones. Differential screening of the enriched cDNA libraries, which were obtained as a result of subtractive hybridization of the cDNA samples of the ventral and dorsal iris zones 14 days after lens removal, revealed four clones specific to the dorsal iris and six clones specific to the ventral iris. Two of these, LeR-1 and VeR-1, were structurally characterized. Comparison of their primary structure with data from the Gene Bank showed no essential homology with the known sequences. Time-related changes in LeR-1 and VeR-1 expression were shown during lens regeneration. LeR-1 and VeR-1 expression was activated at the early stages of lens regeneration. The peaks of LeR-1 and VeR-1 expression were observed on the 14th day of lens regeneration in the dorsal and ventral iris zones, respectively. Furthermore, LeR-1 is activated during retina regeneration. The results of Southern hybridization suggest the presence of sequences complementary to LeR-1 in the genomes of Pleurodeles waltl and Rana temporaria. We propose that the activation of LeR-1 expression is related to the triggering of lens regeneration, while the activation of VeR-1 expression accompanies the inhibition of proliferative activity in the ventral iris zone.
Asunto(s)
Expresión Génica , Cristalino/fisiología , Regeneración , Retina/fisiología , Salamandridae/fisiología , Animales , Secuencia de Bases , Southern Blotting , Cartilla de ADN , ADN Complementario , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Salamandridae/genéticaRESUMEN
This paper constitutes a review of the methodical approaches allowing analysis of the mechanisms underlying development and differentiation. Progress in investigation of the mechanisms underlying embryogenesis is related to the discovery of genic families in the Drosophila genome, which are responsible for different periods of embryogenesis. The true revolution in studies of developmental mechanisms began with the application of molecular-genetic methods for analysis of Drosophila mutant lines. The clarification and analysis of the genes controlling regeneration is one of the most effective paths toward an understanding of the mechanisms underlying regeneration. No mutations affecting regeneration are, and the development of alternative (i.e., not based on mutation analysis) methods of discovery of the genes controlling regeneration is necessary for investigation of the genetic mechanisms of regeneration. The advantages and drawbacks of the two main approaches for discovery of the genes controlling regeneration are considered. The first approach is based on the production of a bank of sequences expressed in the regenerating structures and subsequent screening of the bank by the known probes. This approach also involves analysis of the structure, function, and expression pattern of the obtained homologs. The second approach is based on subtractive hybridization, which allows identification of the genes specifically expressed in the regenerating structures. This approach was made it possible to identify, for the first time, new genes specifically expressed during lens and retina regeneration in amphibians.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Regeneración/genética , Animales , Drosophila/embriología , Drosophila/genética , Drosophila/fisiología , MutaciónRESUMEN
Physicochemical and molecular-biological properties of the proteins from the family of NADP-dependent reductases are reviewed in the article. Physicochemical properties of aldehyde/aldosoreductases are well studied. Information on the genes, coding for these proteins has appeared recently as well. Comparison of the protein structures has revealed, that taxon-specific protein of the frog lens-rho-crystallin--is structurally related to the superfamily of NADP-dependent reductases, though it does not have an enzymatic activity. Sequence alignment reveals a set of clusters, conserved in all members of superfamily. Among them there are two highly conserved regions, providing for binding of NADP coenzyme. Secondary structure of the proteins is similar as well. All the members of superfamily predominantly have beta-sheets. Comparison of structural data for proteins, isolated from various organisms from bacterium to human, suggests phylogenetic relatedness of all the members of superfamily, including rho-crystallin. All the data presented enable to suppose, that rho-crystallin and other members of superfamily have a common ancestor gene. A set of successive duplications and mutations of the ancestor gene resulted in the appearance of rho-crystallin gene, that has lost the enzymatic activity and acquired the ability for tissue specific superexpression in the lens cells.
Asunto(s)
Cristalinas/química , NADH NADPH Oxidorreductasas/química , Filogenia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Fenómenos Químicos , Química Física , Cristalinas/genética , Datos de Secuencia Molecular , NADH NADPH Oxidorreductasas/genética , Rana temporaria , Especificidad de la EspecieRESUMEN
Four recombinant cDNA clones coding for a 23 kDa beta-crystallin polypeptide of the frog (Rana temporaria) were identified in a collection of cloned cDNA and two of them were sequenced. The cDNA present in these clones codes for a polypeptide 198 amino-acid residues in length, which appears to be the frog beta A1-crystallin because of its high homology with the sequences of beta A1-crystallins from other species. Furthermore, the nucleotide sequence coding for the compact folded region of the protein is highly conserved. Virtually no homology was found in the 3' nontranslated regions of the mRNA. The amino-acid sequence of the Rana beta A1-crystallin was used to build a three-dimensional model based on the coordinates of the homologous bovine gamma II. An analysis of the model shows that the surface residues of the beta A1-crystallin (amphibian, mammalian and bird) are more highly conserved than the buried residues. It is suggested that this is related to the oligomeric nature of the lens beta-crystallins.
Asunto(s)
Gráficos por Computador , Cristalinas/genética , ADN/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Pollos , ADN Recombinante , Humanos , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Rana temporaria , Ratas , Homología de Secuencia de Ácido NucleicoAsunto(s)
Cristalinas/genética , ADN Recombinante/aislamiento & purificación , ADN/aislamiento & purificación , Código Genético , Cristalino/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular/métodos , ADN/genética , Escherichia coli/genética , Ratones , Hibridación de Ácido Nucleico , Rana temporariaRESUMEN
The nucleotide sequence of a cloned DNA coding for the 35-kDa polypeptide of the eye lens of the frog (Rana temporaria) has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins: alpha-, beta-, gamma- or delta-crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3'-non-translated region with the polyadenylation signal sequence and a poly(A) tract; the 5'-non-translated region is lost along with part of the coding region; this accounts for about 1/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin - Finkelstein method shows the presence of predominantly beta-strands with only a few alpha-helical regions. We conclude that the 35-kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name epsilon-crystallin.
Asunto(s)
Cristalinas/análisis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cristalinas/genética , ADN/análisis , ADN Recombinante/aislamiento & purificación , Peso Molecular , Plásmidos , Rana temporariaRESUMEN
The nucleotide sequence of a cloned cDNA (clone pRt(1)297; GENE (1982) 17, 131) coding for a 18 kDa polypeptide of the frog eye lens has been determined. The sequence, 791 nucleotide in length has only one long open reading frame (447 nucleotides). The derived amino acid sequence in this frame has greater than 90% homology with the region 25-173 of alpha A2-crystallin amino acid sequence from a related frog species Rana pipiens. The 5'-terminal part of mRNA corresponding to the first 24 amino acids of alpha A2-crystallin has been lost in cloning and substituted by an artefactual sequence. The 3'-terminal part appears to be intact as follows from the presence of the universal poly(A) addition site and poly(A) tract. The 3'-nontranslated region present in frog alpha A2-crystallin mRNA (130 nucleotides) is about 4-times shorter than in mammalian alpha A2-crystallin mRNA. Intact alpha A2-crystallin mRNA with a size of about 700 nucleotides as determined by Northern blot hybridization is about twice smaller than corresponding mammalian mRNAs.
Asunto(s)
Cristalinas/genética , ARN Mensajero , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Evolución Biológica , Bovinos , ADN , ADN Recombinante , Ratones , Hibridación de Ácido Nucleico , Rana pipiens , Rana temporaria , RatasRESUMEN
Poly(A)+ RNA from the lens of the frog Rana temporaria contains three components (1200 +/- 50, 1000 +/- 50, and 900 +/- 50 bp in size) and a more heterogeneous RNA species with a length of 650-750 nucleotides. This RNA was used as a template for the AMV reverse transcriptase and Escherichia coli DNA-polymerase I and the total cDNA obtained was cloned in the PstI site of the pBR322 plasmid vector. Recombinant plasmids corresponding to abundant poly(A)+ RNA classes contain cDNA inserts from less than or equal to 200 to 1200 nucleotides in length. Part of the library (clonotheque) was divided into classes differing in the presence of absence of the restriction sites for BamHI, EcoRI and HindIII restriction endonucleases. The clones belonging to each of the five classes were characterized by the hybridization-translation test. The translation product of mRNA hybridizing with the clone pRT(1)294 has an M4 of about 22 000 and is specifically precipitated by the antiserum to lambda-crystallins of Rana temporaria. The size of the cDNA present in pRT(1)294, equal to 580 +/- 20 bp, is sufficient for coding the greater part of the lambda-crystallin amino acid sequence. On the basis of these data, we conclude that the clone pRT(1)294 codes for one of the frog lambda-crystallins.
Asunto(s)
Cristalinas/genética , ADN Recombinante/análisis , ADN/genética , Genes , Rana temporaria/genética , Animales , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Escherichia coli/genética , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , ARN Mensajero/genéticaAsunto(s)
ADN Mitocondrial , Peces/metabolismo , Animales , Fenómenos Químicos , Química , Enzimas de Restricción del ADNAsunto(s)
Acridinas/farmacología , Acriflavina/farmacología , Antibacterianos/farmacología , ADN/biosíntesis , Etidio/farmacología , Hígado/efectos de los fármacos , Animales , Antibióticos Antineoplásicos/farmacología , Dactinomicina/farmacología , Equinomicina/farmacología , Técnicas In Vitro , Hígado/metabolismo , Olivomicinas/farmacología , RatasRESUMEN
Sibiromycin, an antitumour antibiotic forming a stable complex with the double-stranded DNA selectively inhibits the incorporation of [3H]thymidine into the H-strand of mtDNA during the incubation of isolated rat liver mitochondria in vitro. A model accounting for this result is presented. It is concluded that the H-strand is a leading strand throughout mtDNA replication adn consequently the replication of rat liver monomer mtDNA is unidirectional.