RESUMO
The site-specific endonuclease R.BspKT6I and the cognate site-specific methylase M.BspKT6I have been isolated from the thermophilic strain of Bacillus species KT6 using gel-filtration on Sephadex G100 followed by chromatography on heparin-Sepharose and hydroxyapatite. Endonuclease BspKT6I is an isomer but not an isoschizomer of Sau3AI and MboI. It recognized on the DNA molecule the GAT decreases C sequence and cleaves it; however, unlike Sau3AI and MboI it produces 3'-protruding dinucleotides. The site cleavage is inhibited by dam-methylation. The sticky ends resulting from the BspKT6I cleavage are identical and complementary to the ends formed after the PvuI cleavage. The isolated from the B. species KT6 methylase protects the DNA from subsequent cleavage by BspKT6I. Adenine is a methylated base.
Assuntos
Bacillus/enzimologia , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Cromatografia em Gel , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Eletroforese em Gel de Ágar , DNA Metiltransferases Sítio Específica (Adenina-Específica)/isolamento & purificação , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Especificidade por SubstratoRESUMO
The new site-specific endonuclease BspKT5I free from interfering impurities has been isolated from thermophilic soil bacteria Bacillus species KT5 by successive chromatography on blue agarose, hydroxyapatite and heparin-Sepharose. BspKT5I on double-stranded DNA recognizes the sequence 5'-CTGAAG16N decreases 3'-GACTTC14N increases and cleaves the DNA at the recognition site as indicated by the arrows to form dinucleotide 3'-protruding termini. The isolated endonuclease is an isoschisomer of Eco57I. However, unlike Eco57I, it is not stimulated by S-adenosylmethionine (SAM) and can therefore be related to subclass IIs but not to IV, as Eco57I. In addition, endonuclease BspKT5I, unlike Eco57I, has no methylase activity.
Assuntos
Bacillus/enzimologia , Enzimas de Restrição do DNA/metabolismo , Sequência de Bases , Cromatografia em Gel , DNA/metabolismo , Enzimas de Restrição do DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Hidrólise , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
A site-specific endonuclease activity was found in extract of Bacillus stearothermophilus M6 isolated from molasses. The functionally pure enzyme designated as BstM6I was obtained by consecutive chromatographies on blue sepharose, hydroxyapatite, and heparin-sepharose. The endonuclease recognizes the nucleotide sequence CC decreases WGG in double-stranded DNA and cleaves it as indicated by the arrow to give one-nucleotide 5'-protruding ends. Consequently, the site-specific endonuclease BstM6I is an isoshizomer of BstNI.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Geobacillus stearothermophilus/enzimologia , Sequência de Bases , DNA Bacteriano , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Eletroforese em Gel de Ágar , Dados de Sequência Molecular , Especificidade por SubstratoRESUMO
The relationship between the type of agar used to prepare solid nutrient media and the growth of Bl. trispora strains (+)8A and (-)8A was under study. Bl. trispora culture virtually failed to grow in the media prepared with powder agar, GOST 16280-70: it died after 2-month storage with 2 reinoculations. Bl. trispora grew fairly well in culture media with white frozen lamellar agar (GOST 16280-70) or with white frozen microbiologic agar, GOST 17206-71; the characteristic features of the culture were preserved after 10 monthly reinoculations.
