Journal: bioRxiv

Article Title: OTTER, a new method quantifying absolute amounts of tRNAs

doi: 10.1101/2020.05.18.101501

Figure Lengend Snippet: tRNA quantification using OTTER ( A ) Schematic drawing of tRNA fluorescence labeling reaction in OTTER. A target tRNA is first hybridized with a specifically-designed antisense oligo DNA with the 5’-extension of dAdTdTdTdT (Type 1 oligo DNA). The overhang in the tRNA/DNA hybrid was filled by Klenow fragment (3’-5’ exo − ) with dATP and fluorescence-labeled dUTP (such as TMR-dUTP; marked by star) as substrates. ( B ) An example for tRNA-Arg UCU quantification. Typical reactions of OTTER for tRNA-Arg UCU were analyzed by urea-PAGE and scanned with a fluorescence scanner. The fluorescence-labeled tRNA-Arg UCU (closed triangle) as well as the fluorescence-labeled template oligo DNA by a weak reverse transcription activity of the Klenow fragment (asterisk) were detected. Since the 3’ end of the oligo DNA falls on the TΨC region rather conserved even among different tRNA species, unrelated tRNAs also acted as templates to produce the strong signal. Three replicates of the reaction were analyzed. The amounts of the standard TMR-oligo DNA on the gel (open triangle) were 0.500, 0.250, 0.100, 0.050, and 0.020 pmol/lane. ( C ) The three OTTER reaction products for tRNA-Arg UCU shown in ( B ) (“+” lanes) were subjected to Northern blotting with a reaction without the template oligo DNA (“−” lane).

Article Snippet: After adding final concentration of 10 μM TMR-dUTP (Roche Diagnostics, Basel, Switzerland), 250 μM dATP, 10 mM MgCl2 , and 2.0 units of Klenow Fragment (3’-5’ exo− )(New England Biolabs, Ipswich, Massachusetts, USA), total 10 μl of the mixture was incubate at 37°C for 90 min.

Techniques: Fluorescence, Labeling, Polyacrylamide Gel Electrophoresis, Activity Assay, Northern Blot

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

doi: 10.1093/nar/gkv545

Figure Lengend Snippet: Effect of RecO on RecA nucleation and polymerization. Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with the indicated concentration of RecO for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results. The amount of ATP or dATP hydrolyzed was calculated as described in ‘Materials and Methods’ section.

Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma.

Techniques: Incubation, Concentration Assay, Activity Assay

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

doi: 10.1093/nar/gkv545

Figure Lengend Snippet: RecO facilitates RecA assembly on SsbA- or SsbB-coated ssDNA. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) was pre-incubated with SsbA (0.3 μM) or SsbA and RecO for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecA (0.8 μM) was added and the ATPase activity measured for 25 min. ( B ) Circular ssDNA was pre-incubated with SsbB (0.3 μM) (or both SsbA [0.3 μM] and SsbB [0.3 μM]) and when indicated with RecO (0.1 and 0.2 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. Then RecA (0.8 μM) was added and the (d)ATPase activity measured for 25 min. ( C ) The circular ssDNA was pre-incubated with a fixed amount of SsbB (0.6 μM), SsbA (0.3 μM) plus SsbB (0.3 μM) or a fixed amount of SsbB (0.3 μM) and increasing SsbA (0.03 to 0.3 μM) concentrations for 5 min at 37°C in buffer A containing 5 mM ATP. Then RecO (0.1 μM) was added to the preformed SSB·ssDNA complexes and incubated for 5 min at 37°C. Finally RecA (0.8 μM) was added and the ATPase activity measured for 25 min. All reactions were repeated three or more times with similar results.

Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma.

Techniques: Incubation, Activity Assay

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RecO and SsbA are crucial for RecA-mediated recombinational DNA repair

doi: 10.1093/nar/gkv545

Figure Lengend Snippet: SsbA and RecO contribute to RecA-promoted DNA strand exchange. ( A ) Circular pGEM3 Zf (+) ssDNA (10 μM in nt) and homologous linear dsDNA (20 μM in nt) were pre-incubated with increasing concentrations of SsbA or SsbB (0.15 and 0.3 μM) or RecO (0.2 and 0.4 μM) for 5 min at 37°C in buffer A containing 5 mM ATP or dATP. In the indicated reactions, RecO (0.2 and 0.4 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA (SSB 0.3 μM) complexes and incubated for 5 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. ( B ) The circular ssDNA and homologous linear dsDNA were pre-incubated with SsbA or SsbB (0.3 μM) for 5 min at 37°C in buffer A containing 5 mM dATP, ATP or ATPγS, and RecO (0.2 μM) was added to the preformed SsbA·ssDNA or SsbB·ssDNA complexes and incubated for 10 min at 37°C. Finally, RecA (1.2 μM) was added and the reaction was incubated for 60 min at 37°C. The products of the reactions were deproteinized, separated on a 0.8% AGE with ethidium bromide and quantified as described in ‘Materials and Methods’ section. The positions of the bands corresponding to css, cds, lds, jm, nc and ATPγS-generated prd products are indicated. Symbols + and − denote the presence or absence, respectively, of the indicated protein. In A (lane 14) and in B (lane 17) nicked circular pGEM3 Zf (+) plasmid DNA was added as a mobility control ( C ). The amounts of jm s and products ( nc and prd ) are indicated and expressed as the percentage of total substrate added. The results are the average value obtained from more than three independent experiments (the results given stand within a 5% standard error).

Article Snippet: IPTG was from Calbiochem; DNA restriction enzymes, DNA ligase, etc. were supplied by Roche; and polyethyleneimine, DTT, ATP, dATP, ATPγS and AMP–PNP were from Sigma.

Techniques: Incubation, Generated, Plasmid Preparation

Journal:

Article Title: Differential Regulation of Smac/DIABLO and Hsp-70 during Brain Maturation

doi: 10.1007/s12017-007-8007-9

Figure Lengend Snippet: Model of the intrinsic pathway of apoptosis during brain maturation. In the intrinsic pathway of apoptosis, cyt c is released to the cytosol, where in the presence of dATP it assembles with Apaf-1 and procaspase-9 (pC9). Caspase 9 (C9) activates procaspase-3

Article Snippet: Briefly, 50 µg of cell-free extracts of mouse brain at different stages of maturation were activated with 10 µM horse heart cyt c (Sigma, #C7752) and 1 mM dATP (Invitrogen, #10216-018) at 37°C for 60 min. Control samples in the absence of cyt c and dATP were run under the same conditions.

Techniques: