RESUMEN
We analyzed associations of single nucleotide polymorphisms rsl13004520 (R247T), rs11562975 (L250L), rs7593557 (S419N), rs11563208 (I1016I), and rs11563071 (V1058V) of the cold receptor TRPM8 (2q37.1) gene with blood plasma lipids and anthropometric parameters in Russian population (randomly chosen residents of Novosibirsk: 507 women and 459 men, mean age 57 years). The studied polymorphisms are localized in regions encoding NH2-terminal (R247T, L250L, S419N) and COOH-terminal (I1016I, V1058V) cytoplasmic domains of the channel. We showed association of single nucleotide polymorphism V1058V with the levels of total cholesterol and LDL and HDL cholesterol, and association of I1016I polymorphism with triglyceride content. Polymorphisms L250L and S419N correlated with anthropometric parameters (body mass index and waist and hip circumferences).
Asunto(s)
Lípidos/sangre , Polimorfismo de Nucleótido Simple/genética , Canales Catiónicos TRPM/genética , Antropometría , Índice de Masa Corporal , Colesterol/sangre , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Federación de Rusia , Triglicéridos/sangre , Relación Cintura-CaderaRESUMEN
The region of intron 2 of the SDF1 gene encoding a chemokine of the CXC subfamily has been resequenced in Galloway, Hereford, and Black Pied cattle. Five of the single-nucleotide polymorphisms (SNPs) that were earlier detected by other authors in various breeds of cattle in North America (99C/G, 128T/C, 206C/T, 267C/G and 313C/T) have been found. The 270insC polymorphic marker has proved to be monomorphic in Russian cattle breeds. Hereford cattle significantly differ from Galloway and Black Pied cattle in the frequencies of some SNP variants and their combinations. The number of SNP combinations in Hereford and Galloway cattle exceeds that in Black Pied cattle.
Asunto(s)
Bovinos/genética , Quimiocina CXCL12/genética , Polimorfismo de Nucleótido Simple/genética , Animales , Marcadores Genéticos , Especificidad de la EspecieRESUMEN
Human HFE gene haplotype analysis with reference to IVS2(+4)t/c, IVS4(-44)t/c, IVS5(-47)a/g polymorphic sites was performed in different North Asian ethnic groups. Of the eight possible intronic haplotypes, TTG, TTA, CTA and CCA were identified. High frequency of the CCA haplotype appears to be a characteristic feature of all Asian native populations. Potential functional importance of IVS4(-44)t/c polymorphism is demonstrated. Patients presenting with iron overload syndrome are shown to have low frequency of IVS4(-44)c.
Asunto(s)
Proyecto Mapa de Haplotipos , Antígenos de Histocompatibilidad Clase I/genética , Sobrecarga de Hierro/etnología , Sobrecarga de Hierro/genética , Proteínas de la Membrana/genética , Polimorfismo Genético , Pueblo Asiatico/genética , Proteína de la Hemocromatosis , Humanos , Intrones , Hierro/metabolismo , Sobrecarga de Hierro/metabolismo , Redes y Vías Metabólicas/genética , SiberiaRESUMEN
2'-5'-oligoadenylate synthetases are a family of interferon-induced enzymes which play an important role in the antiviral defense in mammals. In human genome three genes encoding functional synthetases (OAS1, OAS2 and OAS3) form a cluster. Previously we found that particular genotypes and/or alleles of five single nucleotide polymorphisms (SNPs) located within OAS2 and OAS3 genes are associated with predisposition to severe forms of tick-borne encephalitis (TBE) in Russian population. In current study we investigated the distribution of three of that SNPs (OAS3rs2285932 (C/T Ile438Ile), OAS3rs2072136 (G/A, Ser567Ser) and OAS2 rs15895 (G/A, Trp720Ter relative to p71 isoform)) in seven populations from North Eurasia: Caucasians (Russians and Germans (from Altai region)), Central Asian Mongoloids (Altaians, Khakasses, Tuvinians and Shorians) and Arctic Mongoloids (Chukchi). Differences between populations in genotype, allele and haplotype frequencies and in linkage disequilibrium structure for these SNPs were detected. We found that these frequencies correlate with the ethnicity of the populations and with their supposed differential exposure to TBE virus. Particularly, the lowest frequencies of G/G genotype for OAS3 gene rs2072136 SNP (that according to our previously obtained data is associated with predisposition to severe forms of TBE) were found in Altaians, Khakasses, Tuvinians and Shorians who may highly contact with TBE virus in places of their habitation. Thus, data obtained allow to suppose that TBE virus might act as a selection factor for particular OAS genes variants in Central Asian Mongoloids.
Asunto(s)
2',5'-Oligoadenilato Sintetasa/genética , Encefalitis Transmitida por Garrapatas/genética , Predisposición Genética a la Enfermedad , Polimorfismo de Nucleótido Simple , Asia Central , Europa (Continente) , Humanos , Desequilibrio de Ligamiento , Federación de RusiaRESUMEN
The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.
Asunto(s)
Anisoles/toxicidad , Carcinógenos/toxicidad , Glucocorticoides/farmacología , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas/metabolismo , Hígado/enzimología , Proteínas de Neoplasias/metabolismo , Derivados de Alilbenceno , Animales , Inducción Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Femenino , Neoplasias Hepáticas/inducido químicamente , Masculino , Ratones , Ratones Endogámicos ICR , Especificidad de Órganos/efectos de los fármacos , Pentaclorofenol/farmacología , Ratas , Ratas Wistar , Caracteres SexualesRESUMEN
It is known that the carcinogenic effect of estragole, a component of essential oils of many spicy plants, is characterized by species, tissue, and sex specificity. It causes mainly liver tumors in female mice but is not carcinogenic for male mice and for rats. In this work, the estragole hepatocarcinogenicity was shown for female mice of previously not studied ICR line. The strict correlation between estragole hepatocarcinogenicity and its ability to decrease the level of glucocorticoid induction of liver-specific enzymes tyrosine aminotransferase (TAT) and tryptophan oxygenase (TO) was found. Inhibition of TAT and TO inducibility by estragole takes place only in female mice but not in male mice and in rats. Studying the estragole effect on DNA-binding activity of transcription factors, present mainly in liver and regulating expression of genes encoding liver-specific proteins, has shown that estragole decreases FOXA and HNF4 activities but not activities of C/EBP and HNF1, and this happens only in female mice, for which this substance is hepatocarcinogen, but not in male mice and in rats. Pentachlorophenol, preventing hepatocarcinogenic effect of estragole, abolishes inhibitory influence of the latter on the TAT and TO glucocorticoid induction and restores DNA-binding activity of FOXA and HNF4. Thus, a correlation was revealed between the estragole hepatocarcinogenic effect and decrease in DNA-binding activity of transcription factors FOXA and HNF4, which might be indicative of the role of these factors in tumor suppression mechanisms in liver.
Asunto(s)
Anisoles/farmacología , Carcinógenos/farmacología , Factores de Transcripción Forkhead/metabolismo , Glucocorticoides/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Derivados de Alilbenceno , Animales , Femenino , Factores de Transcripción Forkhead/genética , Factor Nuclear 4 del Hepatocito/genética , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos ICR , Ratas , Ratas Wistar , Caracteres Sexuales , Especificidad de la Especie , Triptófano Oxigenasa/metabolismo , Tirosina Transaminasa/metabolismoRESUMEN
We conducted a comparative study of the properties of topoisomerase I isolated from maize nuclei and mitochondria. We found that nuclear and mitochondrial enzymes possess different ability to bind single stranded DNA. Study of the enzyme activity dependence on Mg2+ demonstrated an absolute dependence of the mitochondrial topoisomerase activity. Contrary, nuclear enzyme activity was not absolutely dependent but stimulated by the magnesium cation. Mitochondrial topoisomerase formed covalent bond with the 5'-end of the cleaved DNA what is unique property of prokaryotic topoisomerase I. Nuclear enzyme bound covalently to the 3'-end like all eukaryotic topoisomerases I. The search through databases revealed genes which could encode mitochondrial topoisomerase I in the genomes of higher plants. Using both cDNA sequencing and in silico methods we demonstrated an existence of the ortholog gene in the maize genome. This gene shares significant homology with prokaryotic topoisomerase I genes that may explain differences in the properties of the mitochondrial and nuclear enzyme. Data obtained is of a significant interest both from the point of view of plant organelle evolution and mitochondrial genome expression mechanisms study.
Asunto(s)
Núcleo Celular/enzimología , ADN-Topoisomerasas de Tipo I/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Plantas/metabolismo , Zea mays/enzimología , Núcleo Celular/química , Núcleo Celular/genética , Clonación Molecular , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , ADN Complementario/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Evolución Molecular , Genoma Mitocondrial/fisiología , Magnesio/química , Magnesio/metabolismo , Mitocondrias/química , Mitocondrias/genética , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Zea mays/genéticaRESUMEN
Statistical analysis of the frequency of chromosomes carrying three types (A, B, and C) of porcine endogenous retroviruses (PERV) was based on molecular-genetic testing of populations of domestic pigs and wild boars. Domestic pigs were shown to have higher frequency of PERV and haplotypes containing two and three types of PERV than wild boars.
Asunto(s)
Cromosomas de los Mamíferos/genética , Retroviridae/genética , Porcinos/genética , Animales , Genética de Población/métodos , Haplotipos , Especificidad de la EspecieRESUMEN
The spectrum of mutations in the low-density lipoprotein (LDL) receptor gene was studied in a sample of hypercholesterolemia patients of Caucasoid origin from the population of Russia. The examined patients were 45 to 49-years-old and had the highest level of total serum cholesterol in this age group. Seven previously non-described mutations have been revealed in exon 9 (R410G; M412V) and in exon 12 (Y/Y576; N/N591; L605V; L605R; A612G). Twelve previously described mutations have been identified in exons 2 (C/C27), 5 (C261F; E240X), 6 (E288K), 8 (A391T), 9 (E418G; L432R; D433E), 11 (G/G549; E558K; L/L568), and 12 (G592E). Only one of these mutations was previously described in Russia in a clinical sample of patients with familial hypercholesterolemia. The spectrum of LDL receptor gene mutations in the population sample of patients with hypercholesterolemia significantly differs from the mutation spectrum in patients with familial hypercholesterolemia (clinical samples). Sequencing of the LDL receptor gene is a highly efficient method for identifying the markers of hypercholesterolemia predisposition in a population.
Asunto(s)
Sustitución de Aminoácidos , Predisposición Genética a la Enfermedad , Hipercolesterolemia/genética , Mutación Missense , Receptores de LDL/genética , Población Blanca/genética , Exones/genética , Femenino , Marcadores Genéticos/genética , Genética de Población/métodos , Humanos , Masculino , Federación de RusiaRESUMEN
The SF-1 (Steroidogenic Factor-1) is a transcription factor known as a key regulator of the steroidogenic gene expression. SF-1 is required for the development and functioning at all levels of the hypothalamic-pituitary-gonadal and adrenal axis. Also it plays an essential role in sex determination. SF-1 is a member of the nuclear receptor superfamily and it activates gene expression by binding to DNA in a monomeric form. Here, we report the results of potential SF-1 binding sites identification by using the SiteGA recognition method. The SiteGA method was implemented using a genetic algorithm (GA) involving a iterative discriminant analyses of local dinucleotide context characteristics. These characteristics were compiled not only over the core binding sites region but over its flanks as well. Developed SiteGA method is characterized by considerably better recognition accuracy when compared to that for the weight matrix method. The experimental tests demonstrated that 83% of the sites recognized by the SiteGA method in the regulatory regions of steroidogenic genes, indeed, interact with the SF-1 factor. We also estimated the density of predicted sites in regulatory region of genes, the members of different functional groups and developed the criterion to search for new SF-1 target genes in genome sequences.
Asunto(s)
Algoritmos , Regulación de la Expresión Génica/fisiología , Genoma Humano/fisiología , Proteínas de Homeodominio/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Elementos de Respuesta/fisiología , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismo , Animales , Femenino , Humanos , Masculino , Ratones , Valor Predictivo de las Pruebas , Unión Proteica/fisiología , Ovinos , Factor Esteroidogénico 1 , PorcinosRESUMEN
We purified DNA binding proteins which interact with the promoter region of cox1 gene from maize mitochondria. Presence of poly[dIdC-dIdC] and KCl in concentrations up to 500 mM had no influence on binding efficiency demonstrating high specificity of complexes formed. Surprisingly, we did not detect DNA binding when probes containing promoter regions of other mitochondrial genes (cox3, rrn26) were used. Mobility shift competition studies also suggest that the protein posseses binding specificity towards cox1 promoter. The core motif AAGTA proved to be necessary for DNA binding. Using combination of EMSA and elution of proteins from PAG we showed that DNA-protein complex formed contains three polypeptides with molecular mass 60, 44 and 22 kD. We suggest that the isolated proteins may play an important role in the regulation of plant cox1 gene transcription.
Asunto(s)
Proteínas de Unión al ADN/química , Mitocondrias/química , Proteínas de Plantas/química , Regiones Promotoras Genéticas , Prostaglandina-Endoperóxido Sintasas/genética , Zea mays/química , Ciclooxigenasa 1 , Proteínas de Unión al ADN/aislamiento & purificación , Mitocondrias/genética , Proteínas de Plantas/aislamiento & purificación , Zea mays/genéticaRESUMEN
We have analyzed an interaction of the general transcription complex RNA polymerase II proteins (RNA polymerase II, factors TBP, TFIIB, TFIIF, TFIIE and TFIIH) S. cerevisiae with the oligoribonucleotides. With the help of method EMSA was shown that labeled 32P labeled oligoribonucleotide 5'-ACUCUCUUCCGCAUCGC-3' (r-17) binds with the proteins and generates three species of the complexes with the three major shifts. All the three species of the complexes are RNA specific because a total RNA S. cerevisiae was a competitor for all three species but the TATA-containing oligodeoxyribonucleotide (500-fold molar excess) was not a competitor for its. Complexes 32P-r-17 with the proteins belonging to the middle shift are the sequence specific because unlabeled r-17 was a competitor for its binding (100-fold molar excess) but unlabeled UA-rich oligoribonucleotide (5'-AUAUUAUGUUCAAAA-3) was not a competitor for this shift (500-fold molar excess). Complexes belonging to the upper shift are RNA specific probably. We think 32P-r-17 interaction with the proteins belonging to the under shift is nonspecific corresponding to a sorbtion of 32P-r-17 on a protein. The data presented demonstrate that oligoribonucleotide and oligodeoxyribonucleotide don't compete for the binding sites on a basal transcription complex proteins.
Asunto(s)
Oligorribonucleótidos/química , ARN Polimerasa II/química , Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/química , Factores de Transcripción TFII/química , Ensayo de Cambio de Movilidad Electroforética , Complejos Multiproteicos/química , Radioisótopos de FósforoRESUMEN
Single base mutations G-->A at position 663 and G-->T at position 666 of intron 6 of the human tryptophan oxygenase gene (TDO2) are associated with a variety of psychiatric disorders [Comings, D.E. et al. (1996) Pharmacogenetics 6, 307-318]. Binding of rat liver nuclear extract proteins to synthetic double-strand oligonucleotides corresponding to three allelic states of the region between 651 bp and 680 bp of human TDO2 intron 6 has been studied by gel shift assay. It has been demonstrated that to each allelic state of the region there corresponds a specific set of proteins that interacts with it. With the aid of computer analysis and using specific anti-YY-1 antibodies it has been shown that both mutations damage the YY-1 transcription factor binding site.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Trastornos Mentales/genética , Factores de Transcripción/metabolismo , Triptófano Oxigenasa/genética , Animales , Sitios de Unión , Daño del ADN , Factores de Unión al ADN Específico de las Células Eritroides , Humanos , Intrones , Trastornos Mentales/enzimología , Mutación Puntual , Ratas , Triptófano Oxigenasa/metabolismoRESUMEN
Antisense strategy was used to investigate the role of alpha2A-adrenoceptor (alpha2A-AR) subtype in anxiety-related behavior. A 18-mer phosphorothioate oligodeoxynucleotide (AS-ODN) complementary to the alpha2A-AR mRNA was administered to the adult male rats for 3 days (1 nmol/5 microl/day) into the region of locus coeruleus (LC). Control groups received infusions of either oligodeoxynucleotide of a random sequence (RS-ODN) or saline. Treatment with AS-ODN significantly reduced the levels of alpha2A-AR mRNA in the brain stem. At the same time, AS-ODN treatment caused only a small reduction in [(3)H]clonidine binding (by 26-32%) in the brain stem which was not significant. Compared to both RS-ODN and saline controls, treatment with AS-ODN significantly increased the percentage of open arm entries in the elevated plus-maze while the total number of arm entries was unaltered. Also, AS-ODN treatment elevated basal levels of plasma corticosterone by 217% and 96% compared to both RS-ODN and saline controls. These changes in the hormone concentrations were at a level of marginal significance (p<0.1 versus random group). Taken together, the data indicate that administration of AS-ODN against alpha2A-ARs in the LC significantly reduced expression of alpha2A-AR mRNA in brain stem, moderately increased plasma corticosterone and had anxiolytic-like effect in the elevated plus-maze.
Asunto(s)
Ansiedad/psicología , Conducta Animal/efectos de los fármacos , Corticosterona/sangre , Oligonucleótidos Antisentido/farmacología , Receptores Adrenérgicos alfa 2/genética , Agonistas alfa-Adrenérgicos/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/fisiología , Clonidina/metabolismo , Locus Coeruleus , Masculino , Microinyecciones , Oligonucleótidos Antisentido/administración & dosificación , Ratas , Ratas Wistar , Receptores Adrenérgicos alfa 2/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The paper deals with the energetics of the transition to left-handed Z form in DNA with an arbitrary base sequence. There is a brief outline of the statistical-mechanical model of the B-Z transition allowing for three possible states of each base pair. The parameters of the model can be determined by comparing the theory with experimental data for the B-Z transition in inserts with given sequences in circular DNA. The model contains six energy parameters, most of which have been determined before. In order to find the remaining parameters of the model and test its adequacy, a number of oligonucleotide sequences were synthesized and inserted into the pUC 19 plasmid. Two-dimensional gel electrophoresis was used to determine the superhelical density at which the inserts adopt the Z form. A statistical-mechanical treatment of these data yielded a complete set of six energy parameters for the B-Z transition. The theoretical assumption that the free energy of Z-form pairs does not depend on the type of adjacent pairs proved to be in agreement with the experimental data.
Asunto(s)
ADN/ultraestructura , Conformación de Ácido Nucleico , Oligodesoxirribonucleótidos , Fenómenos Químicos , Química Física , Metabolismo Energético , Modelos QuímicosRESUMEN
The levels of polymorphism of genes of angiotensin converting enzyme (ACE) and apolipoprotein E (Apo E) were studied in elderly and long-living people in Novosibirsk. The results of the study in the investigated group (97 subjects) were compared with polymorphism of these genes in Novosibirsk population group aged 25-64 who were investigated in MONICA Project survey and had DNA data base formed. Frequency of D/D genotype among senile and long-living men was 5.9%. It is 5 times lower than in men 55-64 years of age (p = 0.04). Similar decrease of this gene frequency was also found in women of the same age. In men older than 83 years of age 4 times lowering of 3/4 genotype of Apo E gene and 2 times increasing of frequency of 2/3 genotype were revealed when comprising frequency of these genotypes in people of middle age. In subjects of senile age and long-livers of both sexes genotype 4/4 was not revealed. Lipid levels were more favorable in women with genotype 2/3 of Apo E gene (comparatively lower mean level of total cholesterol and higher level of HDL cholesterol) if compared with genotypes 3/3 and 3/4.
Asunto(s)
Apolipoproteínas E/genética , Longevidad/genética , Peptidil-Dipeptidasa A/genética , Polimorfismo Genético , Adulto , Anciano , Anciano de 80 o más Años , Envejecimiento/sangre , Envejecimiento/genética , Colesterol/sangre , Femenino , Genotipo , Humanos , Lipoproteínas HDL/sangre , Masculino , Persona de Mediana Edad , Siberia , Triglicéridos/sangreRESUMEN
Sequences homologous to the rat frain specific identifier sequences were found in the mouse genome. These sequences were defined by dot- and blotting-hybridisation in different DNA and RNA preparations. It was shown that there are (1-2) X 10(3) copies of these sequences per genome. They are not transcribed in mouse brain and possibly are not connected with mouse brain specific genes expression.
Asunto(s)
Secuencia de Bases , ADN/análisis , Genes Reguladores , Homología de Secuencia de Ácido Nucleico , Animales , ADN/genética , Intrones , Ratones , Hibridación de Ácido Nucleico , ARN/análisis , Ratas , Especificidad de la EspecieRESUMEN
The sequence of K-ras intron 2, which has been associated with lung tumor susceptibility in inbred mouse strains, was analyzed in susceptible strain GR and in resistant strains PT and UT. In the latter case, the intron had a tandem repeat of a 37-bp sequence with variant GC of its two single-nucleotide polymorphisms (SNPs), as earlier reported for resistant strains AKR, C57BL/c, and C3H/A. Strain GR did not differ in intron structure from susceptible strains A/He and ICR, having one copy of the 37-bp sequence with SNP variant CA. By gel retardation assay, a DNA probe corresponding to "susceptible" allele CA of K-ras region 278-307 formed an additional complex with nuclear proteins extracted from the lungs, as compared with probes corresponding to the "resistant" GC and "intermediate" CC alleles. With specific antibodies, the protein binding to the susceptible allele was identified as transcription factor GATA-6. Reverse transcription with subsequent multiplex PCR did not reveal a significant difference in K-ras expression for susceptible and resistant strains. The results suggest that SNPs of K-ras intron 2 do not affect the level of K-ras expression but do control the binding of GATA-6, which plays an important role in lung differentiation.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes ras , Predisposición Genética a la Enfermedad , Neoplasias Pulmonares/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Factor de Transcripción GATA6 , Regulación de la Expresión Génica , Intrones , Pulmón/citología , Pulmón/fisiología , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple , Factores de Transcripción/genéticaRESUMEN
Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/enzimología , Proteína de Unión a TATA-Box/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de AminoácidoRESUMEN
With PCR, RT-PCR, and direct sequencing, complete nucleotide sequences were established for the Elymus sibiricus mitochondrial cytochrome c oxidase subunit 3 gene (cox3) and its cDNA. The cox3 transcript was shown to have 12 editing sites with changes affecting the amino acid sequence of the protein product. The editing of the primary cox3 transcript was found to change the position of a site of protein-protein interactions. The results demonstrate again the important role of mRNA editing in posttranscriptional regulation of the expression of plant mitochondrial genes.