RESUMEN
The possibility of using oligonucleotides from MER1 family repeats as PCR primers for the amplification of human genome DNA fragments was studied. The recommended oligonucleotide primers were chosen by computer analysis of the data base of the EMBL (release 37.0) nucleotide sequences. Use of one of the oligonucleotides was shown to allow specific human DNA amplification to be carried out.
Asunto(s)
Fragmentación del ADN , Bases de Datos Factuales , Genoma Humano , Familia de Multigenes , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Bases , Cartilla de ADN , Frecuencia de los Genes , Humanos , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Programas InformáticosRESUMEN
A fragment of cDNA and an intron-containing fragment of the human L19 ribosomal protein (RPL19) gene, and introns of the human ribosomal proteins S26 (RPS26) and L32 (RPL32) genes were cloned and sequenced. The intron-containing genes of these ribosomal proteins were mapped to human chromosomes by means of polymerase chain reaction (PCR) using a human/rodent hybrid DNA panel.
Asunto(s)
Cromosomas Humanos , Proteínas Ribosómicas/genética , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Humanos , Células Híbridas , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Roedores , Homología de Secuencia de Ácido NucleicoRESUMEN
The 5'-terminal region of the mink S26 ribosomal protein cDNA was cloned using polymerase chain reaction. The nucleotide sequences of the 5'-UTR (24 bp) and a 120-bp fragment of the coding region of RPS26 mRNA were determined. The homology between the coding regions of the human and mink RPS26 mRNAs proved to be 90.8%. The nucleotide sequences of the 5'-UTRs of mink, human, and rat RPS26 mRNAs, as well as mRNAs of the Drosophila S31 protein and Neurospora crp-5 protein, which are homologous to mammalian RPS26, were compared. A highly conserved 9-bp sequence located immediately upstream of the AUG codon was revealed in the 5'-UTR of the RPS26 mRNAs from different species.
Asunto(s)
Proteínas de Drosophila , Proteínas Ribosómicas/genética , Animales , Secuencia de Bases , ADN Complementario , Humanos , Visón , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Several detergents and chemical compounds--Tweens 40, 60, 80, Triton X-100, Triton WR-1340, polyvinylpyrrolidone, dimethylsulfoxide, urea, n-butyl, MESK, and combinations thereof were used for the isolation of surface proteins of vaccinia virus. Optimal conditions for the treatment of the virus with detergents were selected, permitting isolation of vaccinia virus surface proteins p35 and p61. Mouse experiments yielded data on the protective properties of the isolated proteins. Protein p35 may turn to be one of the major proteins responsible for the formation of protective immunity in vaccination with vaccinia virus.
Asunto(s)
Detergentes , Inmunidad/fisiología , Virus Vaccinia/fisiología , Proteínas del Envoltorio Viral/fisiología , Animales , Electroforesis en Gel de Poliacrilamida , Masculino , Ratones , Microscopía Electrónica , Virus Vaccinia/ultraestructura , Proteínas del Envoltorio Viral/aislamiento & purificaciónRESUMEN
A new method of mapping transcriptionally active genes of ribosomal proteins onto human chromosomes is proposed. The method is based on the detection of the expression of human ribosomal protein mRNA in rodent-human hybrid cells carrying different human chromosomes. Using this method, the functional gene of the human ribosomal protein S17 was mapped and the location of the S14 ribosomal protein gene on chromosome 5 was confirmed.
Asunto(s)
Cromosomas Humanos Par 5 , Proteínas Ribosómicas/genética , Animales , Secuencia de Bases , Mapeo Cromosómico/métodos , ADN Complementario , Humanos , Células Híbridas , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genéticaRESUMEN
A polymerase chain reaction strategy was employed to isolate cDNA encoding L11 human ribosomal protein. Based on the known nucleotide sequence of 5'-region of the ribosomal protein L11 mRNA, we have designed primers and used them in amplification of corresponding sequence of human cDNA from total placenta cDNA. The fragment of RPS26 cDNA was cloned in plasmid vector and sequenced. Sequence analysis showed that there is high homology (88%) between coding regions of RPS26 mRNAs in rat liver and human placenta. The amino acid exchanges were observed at positions: 91 (Asp-->Glu), 217 (Thr-->Ala), 352 (Lys-->Glu).
Asunto(s)
ARN Mensajero/genética , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Datos de Secuencia Molecular , Plásmidos , Reacción en Cadena de la Polimerasa , RatasRESUMEN
The polymerase chain reaction technique was employed to isolate cDNA encoding S26 human ribosomal protein. Based on the known sequence of rat S26 ribosomal protein we have designed primers and amplified the corresponding sequence of human cDNA from total placenta cDNA. The fragment of S26 cDNA was cloned in a plasmid vector and sequenced by the Sanger method. Extremely high homology (87.7%) between coding regions of S26 mRNAs in rat liver and human placenta was revealed. The only amino acid substitution (Ser-->Val) at position 38 was observed. Results of blot hybridization with partially digested human genomic DNA suggest the presence of more than 6 copies of the S26 gene.
Asunto(s)
Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Placenta/metabolismo , Plásmidos , Ratas , Proteínas Ribosómicas/química , Homología de Secuencia de AminoácidoRESUMEN
Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of beta-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.
Asunto(s)
Secuencia Conservada , ADN Viral/genética , Genoma Viral , Orthopoxvirus/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Peso Molecular , Orthopoxvirus/ultraestructura , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Virales/químicaRESUMEN
Genes I3 and A2 of the vaccinia virus strain L-IVP DNA were cloned into bacterial expressing vectors. The monospecific antisera to the expression products of these genes in E. coli were obtained. By means of immunochemical cross-analysis two polypeptides of equal electrophoretic mobility were found in the virion preparations in the band of the major envelope protein p35. The major of them is the product of gene I3, and the minor is encoded by gene A2.
Asunto(s)
Escherichia coli , Genes Virales , Virus Vaccinia/genética , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Inmunoquímica , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
The HindIII--J HindIII-F fragments of the vaccinia virus DNA strain Lister have been analysed by the technique of mRNA hybridization selection with the subsequent translation in cell-free protein synthesizing system from the rabbit reticulocytes. The viral mRNA hybridizable with the HindIII--J fragment was shown to direct the synthesis of 30 kDa polypeptide in the cell-free system. This polypeptide was demonstrated to react specifically with antiserum to plasma membrane protein p34. The viral mRNA hybridizable with the HindIII-F fragment was shown to direct the synthesis of 37 kDa polypeptide in the cell-free system. This polypeptide reacts specifically with antiserum to major membrane protein p40.
Asunto(s)
Genes Virales , Proteínas de la Membrana/genética , ARN Mensajero/genética , ARN Viral/genética , Virus Vaccinia/genética , Sistema Libre de Células , Clonación Molecular , Hibridación de Ácido Nucleico , Biosíntesis de Proteínas , Mapeo RestrictivoRESUMEN
The I5 gene from the HindIII-I-fragment of the vaccinia virus strain L-IVP DNA was cloned into bacterial vector pUC19. The monospecific antiserum to the expression product of this gene in E. coli was obtained. This antiserum was demonstrated to co-precipitate the virion protein p90. The vaccinia virus strain L-LVP DNA was shown to have only one ORF coding the p90 protein instead of two ORF H5 and H4 as known for vaccinia virus strain WR. This protein is associated with the core of vaccinia virion, but some of its antigenic determinants are exposed on the surface of the viral particles.
Asunto(s)
Virus Vaccinia/genética , Western Blotting , Clonación Molecular , ADN Viral/genética , Desoxirribonucleasa HindIII , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Genes Virales , Inmunoquímica , Sistemas de Lectura Abierta , Plásmidos , Biosíntesis de Proteínas , Mapeo Restrictivo , Relación Estructura-Actividad , ViriónRESUMEN
The I6 gene from the HindIII-I-fragment of the vaccinia virus strain L-IVP DNA was cloned into bacterial vector pUC19. The monospecific antiserum to the expression product of this gene in E. coli was obtained. Using this antiserum the I6 gene was shown to code the viral protein of 34 kDa molecular weight estimated from SDS-PAGE. This protein is not included into the mature virion, but can be detected in the cytoplasm of the vaccinia virus infected cells.
Asunto(s)
Genes Virales , Virus Vaccinia/genética , Proteínas Virales de Fusión/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Viral/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Datos de Secuencia Molecular , Plásmidos , Mapeo Restrictivo , Relación Estructura-ActividadRESUMEN
The sequence of vaccinia virus (VV) LIVP HindIII N DNA fragment has been determined to be 2215 b.p. Three open reading frames designated LN1, N1 and NO1 and coding polypeptides with the calculated molecular masses (32.5 kDa, 21.8 kDa and 47.8 kDa) were located. mRNAs selected by hybridization with the VV HindIII N were translated in rabbit reticulocyte lysate. Proteins of genes for host range and resistance to alpha-amanitin corresponding to 29 and 47 kDa were detected. Two forms of polypeptides of the ORF LN1 (32 and 29K), and anomalous electrophoresis mobility of the ORF LN1 are discussed.
Asunto(s)
ADN Viral/análisis , Genoma Viral , Biosíntesis de Proteínas/genética , Virus de la Viruela/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Peso Molecular , Plásmidos/genética , Conejos , Proteínas Virales/análisisRESUMEN
Genes encoding virus-specific late proteins with molecular mass 36 kDa and 12 kDa were mapped in HindIII-P DNA fragment of vaccinia virus strain L-IVP by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. RNA origin site of the 36K protein was detected in HindIII-J fragment. Nucleotide sequences of these genes were determined. Amino acid sequences of the 36K and 12K polypeptides were compared with the protein bank PIR.
Asunto(s)
ADN Viral/genética , Genes Virales , Virus Vaccinia/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Desoxirribonucleasa HindIII , Datos de Secuencia Molecular , Peso Molecular , ARN Viral/genética , Mapeo Restrictivo , Proteínas Virales/químicaRESUMEN
Vaccinia virus gene encoding 36K protein was cloned in pUR290 bacterial expressing vector and resulted in the synthesis of a chimeric protein in E. coli. The chimeric protein consists of beta-galactosidase and virus protein in C-termini. It has virus antigen specificity. By monospecific antibody 36K protein of vaccinia virus was determined to be non-virion. It is localized in the cytoplasm of infected cells.
Asunto(s)
Desoxirribonucleasa HindIII , Genes Virales , Virus Vaccinia/genética , Proteínas Virales/genética , ADN Viral/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Vectores Genéticos , Plásmidos , Radioinmunoensayo , Proteínas Virales de Fusión/química , Proteínas Virales de Fusión/genética , Proteínas Virales/químicaRESUMEN
The left HindIII-A-Sal fragment of the vaccinia virus DNA has been analyzed by the technique of mRNA hybridizational selection with the subsequent translation in cell-free protein-synthesizing system from the rabbit reticulocytes. The viral mRNA hybridizable with the fragment was shown to direct the synthesis of 12, 17, 27, 42, 70 kD polypeptides in the cell-free protein-synthesizing system. Each of 12 and 42 kD polypeptides was demonstrated to react specifically with antisera to structural p12 and p42 coat proteins. The structural coat proteins p12, p20, p42 of the vaccinia virus are concluded to be the products of the same viral gene.
Asunto(s)
ADN Viral/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Virus Vaccinia/genética , Proteínas del Envoltorio Viral/análisis , Proteínas Estructurales Virales/análisis , Animales , Línea Celular , ADN Viral/análisis , Desoxirribonucleasa HindIII , Desoxirribonucleasas de Localización Especificada Tipo II , Inmunoquímica , Hibridación de Ácido Nucleico , ARN Mensajero/análisis , Mapeo RestrictivoRESUMEN
The monospecific antiserums to the major proteins p12, p19, p20, p42, p61 of the vaccinia virus coat were obtained and analyzed. The dynamics of the proteins accumulation in the infected cells has been studied. Products of the cell-free translation of the total viral mRNA precipitated by the obtained antiserums were identified. The p20 protein has been found to be a result of p12 protein reversible oligomerization under the conditions of electrophoresis. The antigenic relation of p12 and p20 proteins to a p42 protein, also a p12 oligomer, has been demonstrated. The possibility is discussed to use the obtained antiserums for mapping of the genes corresponding to structural proteins in the genome of the vaccinia virus.
Asunto(s)
Cápside/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , Virus Vaccinia/genética , Animales , Northern Blotting , Sistema Libre de Células , Genes Virales , Sueros Inmunes , Inmunohistoquímica , Conejos , Radioinmunoensayo , Proteínas Estructurales Virales/genéticaRESUMEN
Three monospecific antisera to the major 35 kD (p35) surface protein of vaccinia and ectromelia viruses have been obtained. Two of them are obtained to p35 protein isolated by electrophoresis in the presence of sodium dodecylsulfate from the protein fractions of vaccinia virus, soluble in NP40 and NP40 with dithiothreitol (NP40 and DTT-fractions). The third serum is obtained to NP40-fraction of ectromelia virus, containing practically only p35 protein. The obtained antisera were compared in the reactions with the different fractions of viral proteins in two versions of solid phase radioimmunoassay. The effect of such reagents as sodium dodecylsulfate, NP40, 2-mercaptoethanol, ethanol on the antigenic properties of p35 protein from vaccinia virus is discussed.