RESUMEN
Libraries of hybrid plasmids carrying DNA fragments of complete genomes of 8 variola virus strain from the Russian Collection belonging to 2 epidemical types and isolated in various geographic regions of the world were obtained. Genomic sequences of variola virus can be thus preserved for a long time in a biologically safe form and provide the research work on studying the genetic organization of this unique virus and on developing modern methods for rapid detection of variola virus and other orthopoxviruses.
Asunto(s)
Genoma Viral , Virus de la Viruela/genética , ADN Viral/análisis , ADN Viral/genética , Salud Global , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyltransferases from the BstF5I restriction-modification system.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Geobacillus stearothermophilus/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Bacteriófago lambda/genética , Secuencia de Bases , Metilación de ADN , ADN Viral/genética , ADN Viral/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Sales (Química)/farmacología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
The nucleotide sequence was established for the full-length Flavobacterium aquatile operon coding for the FauI restriction-modification system. The operon is unusual in structure and has the gene order control protein gene-DNA methyltransferase A gene-restriction endonuclease gene-DNA methyltransferase B gene, other than in the known analogs. The genes are similarly oriented and overlap. On evidence of sequence analysis, both methyltransferases are C5 enzymes, the control protein is similar to that of other restriction-modification systems, and restriction endonuclease is low-homologous to other enzymes cleaving the DNA upper strand in position 4 or 5 relative to the recognition site.
Asunto(s)
Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Clonación Molecular , Flavobacterium/enzimología , Flavobacterium/genética , Regulación Bacteriana de la Expresión Génica , Orden Génico , Datos de Secuencia Molecular , Operón , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
A fragment containing the SfeI restriction-modification system (RMS) operon was cloned from a Streptococcus faecalis SE72 plasmid. Nucleotide sequence analysis revealed its high (99.2%) homology to the operon for Lactococcus lactis subsp. cremoris W56 LlaBI RMS recognizing the same site, 5'-CTRYAG-3'. A substantial difference was that SfeI RMS operon had an additional 198-bp fragment and a larger gene for the putative control protein. No homology was observed between operon-flanking sequences of the two closely related species, suggesting horizontal transfer of the operon.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Secuencia de Aminoácidos , Clonación Molecular , ADN Intergénico , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Enterococcus faecalis/genética , Transferencia de Gen Horizontal , Lactococcus lactis/genética , Datos de Secuencia Molecular , Operón , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
The fourth DNA-methyltransferase of the BstF5I restriction-modification (RM) system from Bacillus stearothermophilus F5 (M.BstF5I-4) was discovered, which modifies the adenine residue within the upper strand of the recognition site 5'-GGATG-3'/5'-CATCC-3'. Thus, unlike other known RM systems, the BstF5I RM system comprises four genes encoding DNA-methyltransferases, three of which possess the same substrate specificity and methylate adenine within the 5'-GGATG sequence. The English version of the paper.
Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Geobacillus stearothermophilus/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Secuencia de Aminoácidos , Geobacillus stearothermophilus/genética , Datos de Secuencia Molecular , Familia de MultigenesRESUMEN
The BstF5I restriction-modification system from Bacillus stearothermophilus F5, unlike all known restriction-modification systems, contains three genes encoding DNA methyltransferases. In addition to revealing two DNA methylases responsible for modification of adenine in different DNA strands, it has been first shown that one bacterial cell has two DNA methylases, M.BstF5I-1 and M.BstF5I-3, with similar substrate specificity. The boundaries of the gene for DNA methyltransferase M.BstF5I-1 have been verified. The bstF5IM-1 gene was cloned in pJW and expressed in Escherichia coli. Homogeneous samples of M.BstF5I-1 and M.BstF5I-3 were obtained by chromatography with different sorbents. The main kinetic parameters have been determined for M.BstF5I-1 and M.BstF5I-3, both modifying adenine in the recognition site 5'-GGATG-3'.
Asunto(s)
Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Geobacillus stearothermophilus/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Secuencia de Bases , Clonación Molecular , Geobacillus stearothermophilus/genética , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Especificidad por SustratoAsunto(s)
Metilasas de Modificación del ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Geobacillus stearothermophilus/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoAsunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/genética , Geobacillus stearothermophilus/genética , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Metilasas de Modificación del ADN/genética , Genes Bacterianos , Datos de Secuencia Molecular , Alineación de Secuencia , Homología de SecuenciaRESUMEN
The T7 RNA polymerase-dependent transcription was studied as a function of nucleotide sequence structures positioned upstream of the T7 promoter. Model double-stranded DNA templates were constructed for this purpose. They contained a target sequence of 485 base pairs (cDNA fragment of Venesuelian encephalomyelitis equine virus genome), T7 promoter consensus and different extra base sequences upstream of the T7 promoter. The level of the target sequence transcription was clearly determined by the extra base sequence. The presence of one extra base pair G.C ensured the most pronounced effect, transcription was increased one order of magnitude in comparison with template which has only a canonical T7 promoter sequence at the 5'-end.