bacteriophage λ dna  (New England Biolabs)

Journal: Nucleic Acids Research

Article Title: Cleavage of host tRNAs by mycoplasma membrane-associated nuclease

doi: 10.1093/nar/gkag055

Figure Lengend Snippet: Recombinant M. hyorhinis MnuA2 recapitulates host tRNA cleavage pattern. ( A ) Purification of recombinant M. hyorhinis MnuA2. ( B ) M. hyorhinis MnuA2 shows strong RNase activity. SYBR Gold staining is shown. ( C, D ) The recombinant MnuA2 treatment (3 nM) shows the same cleavage pattern as that by live M. hyorhinis , and the MnuA2-induced tRNA cleavage is inhibited by EGTA treatment (5 mM) of the lysate. ( C ) SYBR Gold staining and ( D ) northern blotting for human tRNA Gly-GCC and tRNA iMet-CAT . Blue arrowheads indicate mature tRNAs, and red arrowheads indicate tDRs. ( E ) MnuA2 possesses both DNase and RNase activity. Linear dsDNA (1 µg λ DNA), circular dsDNA (1 µg pcDNA3.1(+)), or 5 µg total RNA was digested by 5 ng (roughly 1.05 nM) MnuA2. ( F ) M. hyorhinis lysate shows DNase activity at the same molecular weight range as the purified MnuA2 (indicated by a red arrowhead) in the presence of 3 mM CaCl 2 . Fifteen nanograms of MnuA2 corresponds to 0.32 pmol.

Article Snippet: Five nanograms of the MnuA2 were incubated at 37°C for the indicated time periods in 10 mM Tris-HCl (pH 8.0), 5 mM CaCl 2 , containing 1 μg λ phage DNA (TOYOBO), 1 μg pcDNA3.1(+) (Thermo Fisher Scientific), or 5 μg U2OS total RNA as a substrate.

Techniques: Recombinant, Purification, Activity Assay, Staining, Northern Blot, Molecular Weight

Journal: Nucleic Acids Research

Article Title: Cleavage of host tRNAs by mycoplasma membrane-associated nuclease

doi: 10.1093/nar/gkag055

Figure Lengend Snippet: H262Q + H428Q double mutation completely abolishes the nuclease activity of MnuA2. ( A ) Conserved domains and putative catalytic sites of MnuA2, M. pulmonis MnuA ( WP_010925495.1 ) and mature ( N -terminal signal peptide-removed) human DNASE1 ( NP_005214.2 ). Amino acids comprising catalytic sites are indicated by red arrowheads, and two putative catalytic histidines are highlighted in red. ( B ) Purification of H262Q + H428Q mutant MnuA2 (mut-MnuA2). ( C, D ) Total RNA digestion by wild type (WT) or mutant (mut-) MnuA2 (3 nM) in the U2OS lysate. ( C ) SYBR Gold staining and ( D ) northern blotting for tRNA Gly-GCC . ( E ) in vitro DNA digestion by MnuA2. λ DNA and pcDNA3.1(+) were digested by 5 ng (roughly 1.05 nM) MnuA2.

Article Snippet: Five nanograms of the MnuA2 were incubated at 37°C for the indicated time periods in 10 mM Tris-HCl (pH 8.0), 5 mM CaCl 2 , containing 1 μg λ phage DNA (TOYOBO), 1 μg pcDNA3.1(+) (Thermo Fisher Scientific), or 5 μg U2OS total RNA as a substrate.

Techniques: Mutagenesis, Activity Assay, Purification, Staining, Northern Blot, In Vitro