Journal: Journal of Human Immunity

Article Title: A heterozygous USB1 variant linked to immunodeficiency

doi: 10.70962/jhi.20250110

Figure Lengend Snippet: The USB1 de novo variant is catalytically active and correctly processes U6 snRNA. (A) Total RNA extracted from the indicated BEBV cell lines were treated with T4 PNK or with buffer only (PNKBuff) in mild acidic conditions. RNA was subsequently treated with poly(A) polymerase (PAP). Nontreated RNA was loaded as a control, ( n = 2). (B) 3′ RACE analysis of U6 oligo(U) tails in the indicated cell lines. At least 24 clones per sample in each experiment ( n = 2) were sequenced. Bars and error bars are averages of the number of U's within U6 oligo(U) tails and SEM from two independent experiments. (C and D) Indicated cell lines were treated with actinomycin D for 0, 4, and 8 h. RNA samples were processed by northern blotting for detection of U6 and 5S ( n = 2). L: marker of known length (67 nucleotides). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. (E and F) U6 relative abundance quantification by qPCR analysis on patients and control cell lines (ctr1 n = 2, ctr2 n = 3, ctr3 n = 1, USB1 −/− n = 3, P1 n = 3) (E), and USB1 −/− cells transduced with the indicated lentiviral constructs ( n = 2) (F). U6 signals were normalized through the corresponding 5S signals and successively expressed as fold decrease over U6 signal at time 0. Error bars are averages of SEM from two independent experiments. EV, empty vector. Source data are available for this figure: .

Article Snippet: RNA was then treated with Escherichia coli Poly(A) Polymerase (#M0276; New England Biolabs) and incubated at 37°C for 30 min.

Techniques: Variant Assay, Control, Clone Assay, Northern Blot, Marker, Transduction, Construct, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Fibrillarin/Nop1 perturbs RNA folding and assembly independently of liquid–liquid phase separation

doi: 10.1093/nar/gkag065

Figure Lengend Snippet: Nop1–rRNA interactions are displaced by assembly with ribosomal proteins. ( A ) Cy3-Nop1 (10 nM) binding to the 16S 5WJ rRNA was monitored by single-molecule colocalization. Ten nanomolar E. coli ribosomal proteins (RP) uS4 or uS7 were added to challenge Nop1. ( B ) Nop1 occupancy, defined as the fraction of time 5WJ rRNA is bound by Nop1. Orange symbols, average value over one trial (∼100 molecules); gray bars, average of independent trials. * P < .05 by one-way ANOVA. ( C ) Occupancy of Cy3-S4 on 5WJ rRNA with or without 10 nM unlabeled Nop1. The P -values were calculated by a one-tailed t -test. See for rastergrams.

Article Snippet: Proteins were overexpressed from plasmids and purified from E. coli BL21(DE3) (NEB; cat# C2527H) and Rosetta(DE3) (Novagen; cat# 71397-3) cells as described below.

Techniques: Binding Assay, One-tailed Test

Journal: Nucleic Acids Research

Article Title: Fibrillarin/Nop1 perturbs RNA folding and assembly independently of liquid–liquid phase separation

doi: 10.1093/nar/gkag065

Figure Lengend Snippet: Nonspecific Nop1 binding limits unwinding by CsdA. ( A ) Escherichia coli 5WJ rRNA tagged with ATTO 532 and ATTO 647N at its 3′ and 5′ ends has high FRET efficiency when folded . Unfolding by DEAD-box protein CsdA in 4 mM MgCl 2 and 1 mM ATP results in low FRET. Phase separation was initiated as in Fig. to investigate its effect on CsdA unwinding. ( B ) Change in average FRET efficiency for all 5WJ molecules. –Nop control: 200 nM CsdA was added at time 0. Inside and outside droplets, 200 nM CsdA was pre-mixed with Nop1 before phase separation. See for 2D histograms. ( C ) The change in rRNA FRET efficiency with or without 0.2 mg/ml tRNA. ( D – G ) 2 µM Nop1 was added without initiating phase separation, with or without 200 nM CsdA. ( H ) 200 nM Nop1 was pre-mixed with CsdA and added to the rRNA. ( I ) The rRNA was pre-incubated with CsdA, then 200 nM Nop1 plus CsdA was added at time 0. See for fitted parameters and confidence intervals.

Article Snippet: Proteins were overexpressed from plasmids and purified from E. coli BL21(DE3) (NEB; cat# C2527H) and Rosetta(DE3) (Novagen; cat# 71397-3) cells as described below.

Techniques: Binding Assay, Control, Incubation

Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Constitutive expression of rpfG restores motility and EPS production of the mutant Δ tfmR . And Xoc TfmR binds directly to the promoter of RpfG and activates its transcription. ( A ) Constitutive expression of rpfG restores motility of the mutant Δ tfmR . ( i ) Example photo of a bacterial strain. ( ii ) Mean measurements of colony diameter for each strain on “swarming” plates. ( iii ) Mean measurements of colony diameter for each strain on “swimming” plates. Data shown are the mean ± SD ( n = 10). Significance was determined by ANOVA and Dunnett’s post hoc test for comparison with to the wild type. ** p < 0.01; n.s., not significant. ( B ) Constitutive expression of rpfG restores the yield of the mutant Δ tfmR EPS. ( i ) Xoc strains were grown on NA plates supplemented with 2% sucrose for 3 days. ( ii ) Xoc strains were cultured in NB medium supplemented with 2% sucrose for 3 days and EPS was precipitated from the culture supernatant. Values given are the means ± SD of triplicate measurements from a representative experiment, and significance was determined by analysis of variance (ANOVA) and Dunnett’s post hoc test for comparison with the wild type. * p < 0.05; n.s., not significant. Similar results were obtained in two other independent experiments. ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of rpfG ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) ß–Glucuronidase (GUS) activity of the gusA reporter of the rpfG gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) Detection of Δ tfmR mutant and wild–type expression of rpf genes in NB medium ( i ), or XOM2 medium ( ii ), revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( F ) Fold enrichment of the promoter region of rpfG in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t -test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( G ) In vitro transcription experiments showing TfmR activates the transcription of rpfG . RNA was produced from a 323 bp template DNA fragment containing the rpfG promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 150 bp template DNA fragment of the rpfG coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Expressing, Mutagenesis, Comparison, Cell Culture, Electrophoretic Mobility Shift Assay, Negative Control, Activity Assay, Standard Deviation, Quantitative RT-PCR, In Vitro, Produced, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: The Transcriptional Regulator TfmR Directly Regulates Two Pathogenic Pathways in Xanthomonas oryzae pv. oryzicola

doi: 10.3390/ijms25115887

Figure Lengend Snippet: Xoc TfmR binds directly to the promoter of HrpX and activates its transcription. ( A ) ß–Glucuronidase (GUS) activity of the gusA reporter of the hrpG and hrpX gene promoter in the Δ tfmR mutant and the wild type in NB medium ( i ), or in XOM2 medium ( ii ). The data shown are the mean and standard deviation of three measurements. The experiment was repeated three times and similar results were obtained. Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( B ) Detection of Δ tfmR –mutant and wild-type expression of T3SS genes in NB medium ( i ), or XOM2 medium ( ii ) revealed by RT–qPCR analysis. Values are the means ± SD ( n = 3 biological replicates). Differences were evaluated by Student’s t -test (** p < 0.01; * p < 0.05; n.s., no significance at p ≤ 0.05). ( C ) Electrophoretic mobility shift and competition assays of TfmR with the promoter region of hrpX ( i ) and hutG ( ii ) (negative control); the bound– and free–DNA fragments are marked with the words Bound probe and Free probe, respectively, and the concentrations are indicated at the top of each lane. ( D ) Fold enrichment of the promoter region of hrpX in the GX01/TfmR::3 × Flag–ChIP samples compared with the Mock–ChIP samples (with anti–HA antibody), as measured by ChIP–qPCR using hutG as the negative control. Data are presented as means ± SD ( n = 3). Differences were evaluated using Student’s t –test (* p < 0.05; n.s., no significance at p ≤ 0.05). ( E ) In vitro transcription experiments showing TfmR activates the transcription of hrpX . RNA was produced from a 371 bp template DNA fragment containing the hrpX promoter using E. coli RNA polymerase (RNAP) holoenzyme. A 334 bp template DNA fragment containing the hutG promoter and a 161 bp template DNA fragment of the hrpX coding sequence were used as controls. Lane 1, template DNA alone; lane 2, template DNA with RANP; lanes 3–4, template DNA with RANP and 5 and 10 nM TrxA–TfmR.

Article Snippet: The TrxA-TfmR protein and DNA fragments were incubated in transcription buffer at 28 °C for 30 min. Then, the NTP mixture (250 μM each of ATP, CTP, and GTP; 250 μM biotin 16-UTP) and 0.5 U of E. coli RNA polymerase holoenzyme (New England BioLabs, Ipswich, MA, USA) were added and the reaction was carried out for 1 h at 37 °C.

Techniques: Activity Assay, Mutagenesis, Standard Deviation, Expressing, Quantitative RT-PCR, Electrophoretic Mobility Shift Assay, Negative Control, In Vitro, Produced, Sequencing

Journal: Microbial Cell Factories

Article Title: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

doi: 10.1186/s12934-019-1078-0

Figure Lengend Snippet: GFP-specific scFv purification and antibody-antigen binding assay. a SDS-Page representing purification steps for the GFP-specific scFv antibody fragment from culture supernatants of B. subtilis strain KO7A/pJHS11, including immobilized metal ion affinity (IMAC) purification via the polyhistidine-tag and NiNTA, affinity purification via the StrepII-tag and StrepTactin agarose, and removal of the StrepII-SUMO part via SenP protease treatment. Cells were induced with IPTG at an OD 600 of 0.8 and incubated for further 14 h at 30 °C. StrepII-SUMO-scFv fusion protein is marked with one star (*), GFP-specific scFv is marked with two stars (**), and StrepII-SUMO is marked with three stars (***). Lane 1 and 2: culture supernatant concentrated by TCA precipitation before (B) and after (A) NiNTA-agarose treatment. Lane 3: sample of wash fraction from first IMAC (W); lane 4: sample of elution fraction from first IMAC (E); lane 5: sample of elution fraction from StrepII-tag affinity purification; lane 6: sample of elution fraction from StrepII-tag affinity purification incubated with SenP-protease for 1 h at 37 °C; lane 7: sample of flow-through fraction of second IMAC (F); lane 8: sample of elution fraction from second IMAC (E). For details, see “ ” section. b SDS-Page analysis of pull down assay using NiNTA magnetic agarose beads. GFP-specific scFv antibody fragments were bound to magnetic beads via the C-terminal polyhistidine tag (lanes are marked with a plus: +). Unloaded magnetic beads served as a control (marked with a minus: −). GFP was then added to the magnetic beads in excess as a purified native protein (lanes 3–6) or as a cleared lysate from E. coli cells overproducing GFP (lanes 7–10). As a negative control, no GFP was added (lanes 1 and 2). After 1 h incubation at 4 °C allowing interaction complex formation, the magnetic beads were removed, and a sample of each remaining supernatant (S) submitted to SDS-Page (lanes 1, 3, 5, 7 and 9). The beads were then washed and protein was eluted using 250 mM imidazole. The elution fractions (E) were submitted to SDS-Page (lanes 2, 4, 6, 8 and 10). GFP-specific scFv is marked with two stars (**)

Article Snippet: Blots were developed with polyclonal antibodies against E. coli alkaline phosphatase PhoA (OriGene Technologies, Inc.) at a dilution of 1:5000 and polyclonal secondary anti Rabbit-HRP antibodies (Carl Roth, #4750.1) according to the manufacturer’s specifications.

Techniques: Purification, Binding Assay, SDS Page, Affinity Purification, Incubation, TCA Precipitation, Pull Down Assay, Magnetic Beads, Negative Control

Journal: Microbial Cell Factories

Article Title: The YoaW signal peptide directs efficient secretion of different heterologous proteins fused to a StrepII-SUMO tag in Bacillus subtilis

doi: 10.1186/s12934-019-1078-0

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Blots were developed with polyclonal antibodies against E. coli alkaline phosphatase PhoA (OriGene Technologies, Inc.) at a dilution of 1:5000 and polyclonal secondary anti Rabbit-HRP antibodies (Carl Roth, #4750.1) according to the manufacturer’s specifications.

Techniques: Plasmid Preparation, Expressing, Purification, Clone Assay