Structured Review

Thermo Fisher cry21aa2
Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or <t>Cry21Aa2</t> (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .
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Images

1) Product Images from "Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes"

Article Title: Host–Pathogen Coevolution: The Selective Advantage of Bacillus thuringiensis Virulence and Its Cry Toxin Genes

Journal: PLoS Biology

doi: 10.1371/journal.pbio.1002169

Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .
Figure Legend Snippet: Virulence of BT-679 pathogens with or without nematocidal toxin genes. Mean virulence of plasmid-lacking BT-679 (Cry-) with reintroduced Cry14Aa1 (+14) or Cry21Aa2 (+21; left panel) or two concentrations of Cry21Aa2-expressing E . coli (+EC21_low, +EC21_high; right panel). Cry+, toxin gene plasmid-bearing BT-679; Cry-_0, empty vector control for BT-679; EC0, empty vector control for E . coli . The data is provided in S6 Data .

Techniques Used: Plasmid Preparation, Expressing

Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and
Figure Legend Snippet: Frequency of BT-679 toxin genes cry21Aa2 and cry35Aa4 among the evolved replicate populations. The different shades of blue indicate alternative combinations of toxin genes present, as indicated. The toxin genes were all restricted to evolved clones of the BT-679 background (i.e., horizontal transfer was not detected). The top two rows refer to the coevolved, the middle two rows to one-sided adapted, and the bottom two rows to the control evolved replicate populations. Replicate populations are given along the horizontal axis. Data is shown for both transfer 12 and 20 and a total of 55 replicate populations. Crosses indicate extinction of replicates and "miss" that genetic material for the population was unavailable. The original data is shown in S4 Data .

Techniques Used: Clone Assay

2) Product Images from "Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae"

Article Title: Cloning and characterization of filamentous temperature-sensitive protein Z from Xanthomonas oryzae pv. Oryzae

Journal: SpringerPlus

doi: 10.1186/s40064-016-1876-3

Analysis of the process for FtsZ expression and purification by SDS-PAGE ( a ) and western blotting analysis of the purified FtsZ ( b ). The FtsZ was expressed in E. coli BL21 (DE3)/pET22b- ftsZ induced by 0.6 mM IPTG at 37 °C for 3 h. Lane M 1 and M 2 protein Marker; lane 1 purified FtsZ via Ni-NTA agarose column; lane 2 supernatant after ultrasonication under induced conditions; lane 3 total protein under induced conditions; lane 4 supernatant of broth under induced conditions; lane 5 supernatant of lysed E. coli BL21 cells containing pET-22b- ftsZ ; lane 6 purified FtsZ via Ni-NTA agarose column. The migration difference of the 44.3 kDa protein between ( a ) and ( b ) is due to gel electrophoresis with different pulse time
Figure Legend Snippet: Analysis of the process for FtsZ expression and purification by SDS-PAGE ( a ) and western blotting analysis of the purified FtsZ ( b ). The FtsZ was expressed in E. coli BL21 (DE3)/pET22b- ftsZ induced by 0.6 mM IPTG at 37 °C for 3 h. Lane M 1 and M 2 protein Marker; lane 1 purified FtsZ via Ni-NTA agarose column; lane 2 supernatant after ultrasonication under induced conditions; lane 3 total protein under induced conditions; lane 4 supernatant of broth under induced conditions; lane 5 supernatant of lysed E. coli BL21 cells containing pET-22b- ftsZ ; lane 6 purified FtsZ via Ni-NTA agarose column. The migration difference of the 44.3 kDa protein between ( a ) and ( b ) is due to gel electrophoresis with different pulse time

Techniques Used: Expressing, Purification, SDS Page, Western Blot, Marker, Positron Emission Tomography, Migration, Nucleic Acid Electrophoresis

3) Product Images from "Ligand-directed profiling of organelles with internalizing phage libraries"

Article Title: Ligand-directed profiling of organelles with internalizing phage libraries

Journal: Current protocols in protein science / editorial board, John E. Coligan ... [et al.]

doi: 10.1002/0471140864.ps3004s79

Cloning strategy to generate the iPhage library. The f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII) and a recombinant protein VIII (rpVIII). The recombinant gene VIII contains a foreign DNA insert with a Hin
Figure Legend Snippet: Cloning strategy to generate the iPhage library. The f88-4 phage vector contains two capsid genes encoding a wild-type (wt) protein VIII (pVIII) and a recombinant protein VIII (rpVIII). The recombinant gene VIII contains a foreign DNA insert with a Hin

Techniques Used: Clone Assay, Plasmid Preparation, Recombinant

4) Product Images from "Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4"

Article Title: Influenza M1 Virus-Like Particles Consisting of Toxoplasma gondii Rhoptry Protein 4

Journal: The Korean Journal of Parasitology

doi: 10.3347/kjp.2017.55.2.143

PCR amplification of T. gondii ROP4 (A) and influenza M1 genes (B). T. gondii ROP4 (1,728 bp) gene was RCR-amplified from cDNA synthesized using a Prime Script 1st Strain cDNA Synthesis Kit using total RNA extracted from T. gondii RH. Influenza M1 gene was PCR amplified from total RNA extract from influenza virus (A/PR/8/34). M, DNA marker; TgROP4, T. gondii ROP4; M1, influenza M1.
Figure Legend Snippet: PCR amplification of T. gondii ROP4 (A) and influenza M1 genes (B). T. gondii ROP4 (1,728 bp) gene was RCR-amplified from cDNA synthesized using a Prime Script 1st Strain cDNA Synthesis Kit using total RNA extracted from T. gondii RH. Influenza M1 gene was PCR amplified from total RNA extract from influenza virus (A/PR/8/34). M, DNA marker; TgROP4, T. gondii ROP4; M1, influenza M1.

Techniques Used: Polymerase Chain Reaction, Amplification, Synthesized, Marker

5) Product Images from "DNA vaccine constructs against enterovirus 71 elicit immune response in mice"

Article Title: DNA vaccine constructs against enterovirus 71 elicit immune response in mice

Journal: Genetic Vaccines and Therapy

doi: 10.1186/1479-0556-5-6

Amplification of VP1 genes of EV71 isolate S2/86/1 and isolate 410/4 by PCR . Lane M shows a 1 kb molecular marker (Fermentas). Lane 1 and lane 3 each shows a band of size 923 bp, which represent the PCR product of VP1 gene of EV71 isolate S2/86/1 and isolate 410/4, respectively. Lane 2 and lane 4 which show no band of PCR product are PCR negative controls that used water as template.
Figure Legend Snippet: Amplification of VP1 genes of EV71 isolate S2/86/1 and isolate 410/4 by PCR . Lane M shows a 1 kb molecular marker (Fermentas). Lane 1 and lane 3 each shows a band of size 923 bp, which represent the PCR product of VP1 gene of EV71 isolate S2/86/1 and isolate 410/4, respectively. Lane 2 and lane 4 which show no band of PCR product are PCR negative controls that used water as template.

Techniques Used: Amplification, Polymerase Chain Reaction, Marker

6) Product Images from "R1, a Novel Repressor of the Human Monoamine Oxidase A"

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A

Journal: The Journal of biological chemistry

doi: 10.1074/jbc.M410033200

R1 interacted with the endogenous MAO A promoter directly in vivo The occupation of R1 on the MAO A core promoter or 5′-irrelevant region was determined by ChIP assay combined with quantitative real time PCR using SK-N-BE (2) C cells. A , a schematic representation of the MAO A promoter. Real time PCR-targeted regions containing core promoter and Sp1 sites (from −360 to −17 bp) and 5′-irrelevant loci (from −1377 to −1024 bp for negative control) were indicated. B , representative R1 ChIP/quantitative real time PCR amplification plots (triplicates). The ChIP/quantitative real time PCR amplification was performed for cross-linked inputs, R1-associated MAO A core promoter, or 5′-irrelevant loci in SK-N-BE (2) C cells. The nuclear protein-DNA complex was immunoprecipitated by anti-R1 antibody and was quantitatively analyzed by real time PCR. C , analysis of association of R1 with MAO A core promoter or 5′-irrelevant locus. The relative differences between input sample and R1 or negative control were determined using the Δ C T method (see “Materials and Methods”). These values were presented as percent input in which the DNA cross-linked input sample was taken as 100%. Data were the mean ± S.D. from triplicate samples of three independent experiments.
Figure Legend Snippet: R1 interacted with the endogenous MAO A promoter directly in vivo The occupation of R1 on the MAO A core promoter or 5′-irrelevant region was determined by ChIP assay combined with quantitative real time PCR using SK-N-BE (2) C cells. A , a schematic representation of the MAO A promoter. Real time PCR-targeted regions containing core promoter and Sp1 sites (from −360 to −17 bp) and 5′-irrelevant loci (from −1377 to −1024 bp for negative control) were indicated. B , representative R1 ChIP/quantitative real time PCR amplification plots (triplicates). The ChIP/quantitative real time PCR amplification was performed for cross-linked inputs, R1-associated MAO A core promoter, or 5′-irrelevant loci in SK-N-BE (2) C cells. The nuclear protein-DNA complex was immunoprecipitated by anti-R1 antibody and was quantitatively analyzed by real time PCR. C , analysis of association of R1 with MAO A core promoter or 5′-irrelevant locus. The relative differences between input sample and R1 or negative control were determined using the Δ C T method (see “Materials and Methods”). These values were presented as percent input in which the DNA cross-linked input sample was taken as 100%. Data were the mean ± S.D. from triplicate samples of three independent experiments.

Techniques Used: In Vivo, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Amplification, Immunoprecipitation

7) Product Images from "The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster"

Article Title: The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster

Journal: The FEBS journal

doi: 10.1111/febs.14677

Generation of the period -AID-EGFP allele by CRISPR/Cas9-induced homologous recombination. (A) A schematic of AID in the circadian rhythm system. The Gal4-UAS system is used to introduce TIR1 from rice (OsTIR1), which includes an F box that allows integration into an SCF ubiquitin ligase complex together with the endogenous Drosophila proteins. The endogenous PER protein is fused with the AID (PER-AID). In the presence of auxin, PER-AID is recruited to OsTIR1, resulting in its polyubiquitinylation and proteasomal degradation. (B) Strategy for tagging the period gene with AID-EGFP. The two target sites located in the last exon and downstream of the 3’UTR are shown. The circle donor vector containing two homology arms was designed to insert the AID-EGFP tag before the stop codon of period . (C) PCR analysis of 5’ and 3’ integration of AID-EGFP and representative sequencing results of the period -AID-EGFP allele. P, positive ( y,sc,v,period -AID-EGFP), N, negative ( y,sc,v ), M, maker.
Figure Legend Snippet: Generation of the period -AID-EGFP allele by CRISPR/Cas9-induced homologous recombination. (A) A schematic of AID in the circadian rhythm system. The Gal4-UAS system is used to introduce TIR1 from rice (OsTIR1), which includes an F box that allows integration into an SCF ubiquitin ligase complex together with the endogenous Drosophila proteins. The endogenous PER protein is fused with the AID (PER-AID). In the presence of auxin, PER-AID is recruited to OsTIR1, resulting in its polyubiquitinylation and proteasomal degradation. (B) Strategy for tagging the period gene with AID-EGFP. The two target sites located in the last exon and downstream of the 3’UTR are shown. The circle donor vector containing two homology arms was designed to insert the AID-EGFP tag before the stop codon of period . (C) PCR analysis of 5’ and 3’ integration of AID-EGFP and representative sequencing results of the period -AID-EGFP allele. P, positive ( y,sc,v,period -AID-EGFP), N, negative ( y,sc,v ), M, maker.

Techniques Used: CRISPR, Homologous Recombination, Introduce, Plasmid Preparation, Polymerase Chain Reaction, Sequencing

8) Product Images from "Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life"

Article Title: Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2019.00212

Overview of the functional metagenomic approach, samples collection and patient history with antibiotic therapy. (A) Schematic representation of the experimental workflow. (B) We used 130 nasal swabs from 26 infants with CF enrolled in a cohort study. We chose samples containing a decent amount bacterial DNA (≥40 ng/μL 16S rRNA PCR product) for functional metagenomic analysis. Samples are represented according to patient antibiotic history at the time of sampling; circles: antibiotic naïve, squares with red border: during an antibiotic treatment and squares: after one or more antibiotic therapy.
Figure Legend Snippet: Overview of the functional metagenomic approach, samples collection and patient history with antibiotic therapy. (A) Schematic representation of the experimental workflow. (B) We used 130 nasal swabs from 26 infants with CF enrolled in a cohort study. We chose samples containing a decent amount bacterial DNA (≥40 ng/μL 16S rRNA PCR product) for functional metagenomic analysis. Samples are represented according to patient antibiotic history at the time of sampling; circles: antibiotic naïve, squares with red border: during an antibiotic treatment and squares: after one or more antibiotic therapy.

Techniques Used: Functional Assay, Polymerase Chain Reaction, Sampling

9) Product Images from "The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription"

Article Title: The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20082678

pKMP2 -derived transcripts and mutations of the Amp r gene in plasmids selected in ampicillin. (A) RT-PCR of RNA of E. coli transformed with unmutated (WT) or two mutated (Mut1 and Mut2) pKMP2 plasmids that confer ampicillin resistance ( Fig. 5 F and Fig. 6 , clones 1 and 7). Kan r and Amp r are transcripts of the respective genes in fourfold sequential dilutions. Equal amounts of cDNA were used for the respective Kan r and Amp r reactions. RT-PCR products without reverse transcription with RNA samples corresponded to the highest concentrations of WT ( 1 ), Mut1 ( 2 ), and Mut2 ( 3 ) RNAs, respectively. (B–D) Mutation patterns in pKMP2 after exposure to low amounts of AID for a short time. Treatments of the plasmids are indicated, with numbers of colonies obtained in E. coli selected in ampicillin shown in parenthesis. The y axes represent numbers of mutations. Relative to Fig. 5 , the scales were expanded by factors of 7.25, 4.8, and 6.9 for B, C, and D, respectively, for easier comparison with the data in Fig. 5 F (for B) and Fig. 5 D (for C and D), where larger numbers of colonies were obtained. The sequences are shown in Figs. S9–S11 . (E) AID acts processively/cooperatively. Selection of AID-treated plasmids in ampicillin. 2: no ampicillin resistance was seen with nucleosomal DNA and 5, 10, or 20 min of exposure to AID, T7 RNA polymerase, and BamHI. 5: no ampicillin resistance was seen with naked DNA and 5 min of exposure to AID and T7 RNA polymerase. The size markers are 100, 200, and 300 bp. The experiments were done four (E4), three (E1 and 2), two (A, C, D, and E5-7), and one (B and E3) times with similar results.
Figure Legend Snippet: pKMP2 -derived transcripts and mutations of the Amp r gene in plasmids selected in ampicillin. (A) RT-PCR of RNA of E. coli transformed with unmutated (WT) or two mutated (Mut1 and Mut2) pKMP2 plasmids that confer ampicillin resistance ( Fig. 5 F and Fig. 6 , clones 1 and 7). Kan r and Amp r are transcripts of the respective genes in fourfold sequential dilutions. Equal amounts of cDNA were used for the respective Kan r and Amp r reactions. RT-PCR products without reverse transcription with RNA samples corresponded to the highest concentrations of WT ( 1 ), Mut1 ( 2 ), and Mut2 ( 3 ) RNAs, respectively. (B–D) Mutation patterns in pKMP2 after exposure to low amounts of AID for a short time. Treatments of the plasmids are indicated, with numbers of colonies obtained in E. coli selected in ampicillin shown in parenthesis. The y axes represent numbers of mutations. Relative to Fig. 5 , the scales were expanded by factors of 7.25, 4.8, and 6.9 for B, C, and D, respectively, for easier comparison with the data in Fig. 5 F (for B) and Fig. 5 D (for C and D), where larger numbers of colonies were obtained. The sequences are shown in Figs. S9–S11 . (E) AID acts processively/cooperatively. Selection of AID-treated plasmids in ampicillin. 2: no ampicillin resistance was seen with nucleosomal DNA and 5, 10, or 20 min of exposure to AID, T7 RNA polymerase, and BamHI. 5: no ampicillin resistance was seen with naked DNA and 5 min of exposure to AID and T7 RNA polymerase. The size markers are 100, 200, and 300 bp. The experiments were done four (E4), three (E1 and 2), two (A, C, D, and E5-7), and one (B and E3) times with similar results.

Techniques Used: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Clone Assay, Mutagenesis, Selection

10) Product Images from "HexaPrime: A novel method for detection of coronaviruses"

Article Title: HexaPrime: A novel method for detection of coronaviruses

Journal: Journal of Virological Methods

doi: 10.1016/j.jviromet.2012.11.039

Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
Figure Legend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

Techniques Used: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

11) Product Images from "A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication"

Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

Journal: Journal of Virology

doi:

Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.
Figure Legend Snippet: Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.

Techniques Used: In Vitro, Purification, Incubation, Generated, Agarose Gel Electrophoresis, Produced, Acrylamide Gel Assay

12) Product Images from "Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase ?"

Article Title: Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase ?

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.23.7995-8006.2001

Assays for distributive DNA synthesis and proofreading exonuclease of human Polμ. (A) DNA polymerase assays were performed with 1.5 ng (27 fmol) of human Polμ at 30°C for various times as indicated, using a 40-mer DNA template containing a 16-mer 5′ 32 P-labeled (asterisk) primer as shown on the right. (B) DNA substrates (50 fmol) containing a T-A (template-primer) pair (lanes 1 to 3) or a T-T mismatch (lanes 4 to 6) (sequences shown on the right) at the primer 3′ end were incubated with purified human Polμ (5 ng; 90 fmol) for 10 min at 37°C in the DNA polymerase assay buffer without dNTPs. Similar assays were performed with the purified Klenow fragment (1 U) of E. coli DNA polymerase I, except that the incubation time was reduced to 2 min. The reaction products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Lanes 1 and 4, no DNA polymerase. DNA size markers in nucleotides are indicated on the sides.
Figure Legend Snippet: Assays for distributive DNA synthesis and proofreading exonuclease of human Polμ. (A) DNA polymerase assays were performed with 1.5 ng (27 fmol) of human Polμ at 30°C for various times as indicated, using a 40-mer DNA template containing a 16-mer 5′ 32 P-labeled (asterisk) primer as shown on the right. (B) DNA substrates (50 fmol) containing a T-A (template-primer) pair (lanes 1 to 3) or a T-T mismatch (lanes 4 to 6) (sequences shown on the right) at the primer 3′ end were incubated with purified human Polμ (5 ng; 90 fmol) for 10 min at 37°C in the DNA polymerase assay buffer without dNTPs. Similar assays were performed with the purified Klenow fragment (1 U) of E. coli DNA polymerase I, except that the incubation time was reduced to 2 min. The reaction products were separated by electrophoresis on a 20% denaturing polyacrylamide gel. Lanes 1 and 4, no DNA polymerase. DNA size markers in nucleotides are indicated on the sides.

Techniques Used: DNA Synthesis, Labeling, Incubation, Purification, Electrophoresis

13) Product Images from "Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells"

Article Title: Inactivation of class II transactivator by DNA methylation and histone deacetylation associated with absence of HLA-DR induction by interferon-γ in haematopoietic tumour cells

Journal: British Journal of Cancer

doi: 10.1038/sj.bjc.6601602

Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.
Figure Legend Snippet: Bisulphite sequencing of CIITA. Amplified PCR products were cloned into pCR4 vector using a TOPO-TA cloning Kit (Invitrogen) and plasmid DNA was purified. Sequencing reaction was performed using a Big-Dye terminator Kit (Applied Biosystems) and electrophoresed using an ABI3100 system (Applied Biosystems). CpG sites are shown above. Methylated alleles are shown as solid circles; unmethylated alleles as shown as open circles.

Techniques Used: Bisulfite Sequencing, Amplification, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, TA Cloning, Purification, Sequencing, Methylation

14) Product Images from "Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders"

Article Title: Duplication and concerted evolution of MiSp-encoding genes underlie the material properties of minor ampullate silks of cobweb weaving spiders

Journal: BMC Evolutionary Biology

doi: 10.1186/s12862-017-0927-x

Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)
Figure Legend Snippet: Neighbor joining tree for N-terminal MiSp encoding sequences demonstrates variation within and among species. Variants are shown for L. hesperus ( Lh ) and TOPO clones from L. tredecimguttatus ( Lt ), L. geometricus ( Lg ), and S. grossa ( Sg ). Units are number of substitutions. Latrodectus clones are arbitrarily numbered, except that Lt M1-7 and Lg M1-5 resulted from amplification with primers designed from L. hesperus , while higher clone numbers resulted from amplification with species-specific primers. S. grossa TOPO clones resulted from two separate PCR reactions, which are indicated here as “A#” and “B#”. Completely sequenced clones are indicated by bolded names. See Table 1 and Additional file 1 : Table S4 for accession numbers. Boxes indicate distinct clusters identified in neighbor joining trees based on N-terminal sequences including adjacent repetitive sequences and in C-terminal and adjacent repetitive region encoding sequences (Additional file 1 : Figures S2 and S3)

Techniques Used: Clone Assay, Amplification, Polymerase Chain Reaction

Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap
Figure Legend Snippet: Arrangement of amino acid motifs in MiSp sequences of cobweb and orb-web weaving spiders. Latrodectus and S. grossa accession numbers are in Table 1 . Lh , L. hesperus ; Lt , L. tredecimguttatus ; Lg , L. geometricus ; Sg , S. grossa ; Pt , P. tepidariorum based on combining the incompletely assembled MiSp-encoding region from Scaffold 853 of the i5K genome with an ~2.3 kb TOPO-cloned PCR product, KX584004; Av , A. ventricosus, JX513956.1 [ 30 ]. Lengths of missing amino acid sequences in Pt MiSp_v1 are based on the length of gaps in Scaffold 853 of the i5K genome, assuming no introns are present within those gaps. The last gap was predicted to be 1782 bp long (594 aa if no intron) in Scaffold 853, but our TOPO clone added 2236 bp, including a 1175 bp intron, which was longer than the predicted gap. The repetitive encoding region at the 3’ end of our TOPO clone did not overlap with the encoding region present in the i5K genome prior to the last gap

Techniques Used: Clone Assay, Polymerase Chain Reaction

15) Product Images from "Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens"

Article Title: Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens, et al. Generation of cattle knockout for galactose‐α1,3‐galactose and N‐glycolylneuraminic acid antigens

Journal: Xenotransplantation

doi: 10.1111/xen.12524

Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1
Figure Legend Snippet: Editing of GGTA1 and CMAH genes in male and female fibroblasts. A, Target sequences for selected sgRNAs and ss CMAH ‐STOP oligo sequence. For each bovine gene ( GGTA1 and CMAH ), target sequences are indicated on the respective exons recognized by the selected sgRNAs. PAM sequences are highlighted in blue. In the ss CMAH ‐STOP oligo sequence, the TAA (STOP) codon is highlighted in bold character; the Afl II restriction site is underlined. B, PCR analyses of female colonies. The results of the PCR analyses performed for the genomic characterization of the female colonies (A1, A2, A3, A4, A5 and A6) selected after Dynabeads sorting are reported as an example. Each colony was analysed for the GGTA1 gene (739 bp) and for the CMAH gene (225 bp). Resulting electrophoretic patterns determined directly that some colonies were characterized by visible Indels , creating bands different from the WT controls. This situation is clear for colonies A1 (double band), A2 (deletion) and A6 (deletion) in PCR analyses for the GGTA1 gene (°) and for colonies A1 (double band) and A5 (deletion) in PCR analyses for the CMAH gene (#). Resulting CMAH ‐PCR products were also digested with the Afl II restriction enzyme, detecting the alleles interested by the targeting event. Due to the introduction of a STOP codon (TAA) in the START position (ATG) of the CMAH gene, only the HDR‐ CMAH alleles will be cut by the restriction enzyme producing two lower bands (152 + 73 bp). A simple agarose electrophoresis enabled us to identify possible additional edited colonies detecting the STOP codon insertion (**) for colonies A2 and A6 and the single insertion (*) for colonies A3 and A4. In these last ones, the not targeted allele resulted uncut (225 bp) as the WT sample. For this reason, the final determination of the exact Indels , occurred in all the edited colonies, was determined by Sanger sequencing of the resulting TOPO TA E coli clones. 100 = 100 bp ladder (Thermo Fisher Scientific); A1, A2, A3, A4, A5 and A6 = transfected females colonies; WT = wild‐type female line; H 2 0 = Nucleases‐free water. C, Sequences alignments of colonies used for the SCNT. Sanger sequencing outlining the mutations affecting the GGTA1 and the CMAH genes of colonies selected for the SCNT step. For the GGTA1 gene, the exon 9 was used as reference for the male colonies and a PCR product including the exon 4 was used for the female ones. In both cases, deletions of different lengths were obtained (Table S1 ). For the CMAH gene, all edited alleles of the edited colonies were aligned using as reference a PCR product including the exon 2 sequence. In this case, in both lines, we were able to determine the TAA substitution, as result of the targeting event mediated by the site‐specific cut, produced by the CRISPR/Cas9 system driven by the sgRNA bt CMAH cr1

Techniques Used: Sequencing, Polymerase Chain Reaction, Electrophoresis, Clone Assay, Transfection, Produced, CRISPR

16) Product Images from "In vivo dissection of the chromosome condensation machinery"

Article Title: In vivo dissection of the chromosome condensation machinery

Journal: The Journal of Cell Biology

doi: 10.1083/jcb.200109056

Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.
Figure Legend Snippet: Topo II and phospho-H3 are not essential for rDNA condensation. CH325 ( top2-4 ), JHY90 ( WT ), JHY91 ( H3-S10A ), and JHY93 ( H3-S10,28A ) cultures were synchronized in G1, and released into a Nz block either at 37°C (top2-4) or 23°C. After rearrest in M phase, cells were fixed and processed for rDNA FISH. rDNA loops were scored as condensed. Greater than 100 nuclei/sample were scored. Data for the condensins is from Fig. 2 and is shown for comparison.

Techniques Used: Blocking Assay, Fluorescence In Situ Hybridization

17) Product Images from "Accurate quantification of supercoiled DNA by digital PCR"

Article Title: Accurate quantification of supercoiled DNA by digital PCR

Journal: Scientific Reports

doi: 10.1038/srep24230

Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).
Figure Legend Snippet: Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).

Techniques Used: Digital PCR, Plasmid Preparation

PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).
Figure Legend Snippet: PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).

Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Expressing

Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).
Figure Legend Snippet: Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).

Techniques Used: Plasmid Preparation, Concentration Assay, Digital PCR, Labeling

Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).
Figure Legend Snippet: Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).

Techniques Used: Digital PCR, Serial Dilution, Labeling

18) Product Images from "poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance"

Article Title: poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance

Journal: bioRxiv

doi: 10.1101/2019.12.31.891960

pUG RNA shortening may act as a brake on TEI. a , The gel shown is the same as in Fig. 5a , except that oma-1 pUG RNAs from the P 0 generation are included. b, oma-1 pUG RNA reads from MiSeq were mapped to oma-1 and the length of the oma-1 mRNA portion of each pUG RNA was determined (y-axis). Shown is a Box and Whisker plot representing the interquartile range (IQR, box) and median (line in the box) of lengths at the indicated generations after dsRNA-treatment. The y-axis starts at the aug of the oma-1 mRNA. The whiskers extend to values below and above 1.5*IQR from the first and third quartiles, respectively. Data beyond the end of the whiskers are outliers and plotted as points. The data support the gel in a , showing that pUG RNAs get shorter in each generation during RNAi-directed TEI. c, A “ratchet” model to explain pUG RNA shortening. pUG RNA shortening may be due to the 3’→5’ directionality of RdRPs, which causes each turn of the pUG/siRNA cycle (see model in Fig. 5g ) to trigger cleavage and pUGylation of target mRNAs at sites more 5’ than in the previous cycle until, eventually, pUG RNAs are too short to act as RdRP templates, thereby ending the cycle. Additional support for the ratchet model comes from Fig. 3g and 5c , which show that RNAi-triggered pUG RNAs are longer in mut-16 and MAGO12 mutant animals than in wild-type animals. Our data indicates that loss of MUT-16 or the WAGOs blocks pUG/siRNA cycling, suggesting that MUT-16 and the WAGOs act during the pUG/siRNA cycling phase of pUG RNA-mediated gene silencing (see model in Fig. 5g ). In the absence of cycling, pUG shortening does not occur and pUG RNAs are longer in mut-16 and MAGO12 animals. Finally, a number of recent studies report transgenerational inheritance of acquired traits in C. elegans , which last 3-4 generations 32 – 37 . oma-1 RNAi-directed pUG RNAs also perdure for 3-4 generations ( Fig. 5a ). These shared generational timescales of inheritance suggest that the inheritance of acquired traits in C. elegans may be mediated by pUG RNAs whose generational “half-life” is limited to 3-4 generations due to the built-in brake on TEI provided by pUG RNA shortening.
Figure Legend Snippet: pUG RNA shortening may act as a brake on TEI. a , The gel shown is the same as in Fig. 5a , except that oma-1 pUG RNAs from the P 0 generation are included. b, oma-1 pUG RNA reads from MiSeq were mapped to oma-1 and the length of the oma-1 mRNA portion of each pUG RNA was determined (y-axis). Shown is a Box and Whisker plot representing the interquartile range (IQR, box) and median (line in the box) of lengths at the indicated generations after dsRNA-treatment. The y-axis starts at the aug of the oma-1 mRNA. The whiskers extend to values below and above 1.5*IQR from the first and third quartiles, respectively. Data beyond the end of the whiskers are outliers and plotted as points. The data support the gel in a , showing that pUG RNAs get shorter in each generation during RNAi-directed TEI. c, A “ratchet” model to explain pUG RNA shortening. pUG RNA shortening may be due to the 3’→5’ directionality of RdRPs, which causes each turn of the pUG/siRNA cycle (see model in Fig. 5g ) to trigger cleavage and pUGylation of target mRNAs at sites more 5’ than in the previous cycle until, eventually, pUG RNAs are too short to act as RdRP templates, thereby ending the cycle. Additional support for the ratchet model comes from Fig. 3g and 5c , which show that RNAi-triggered pUG RNAs are longer in mut-16 and MAGO12 mutant animals than in wild-type animals. Our data indicates that loss of MUT-16 or the WAGOs blocks pUG/siRNA cycling, suggesting that MUT-16 and the WAGOs act during the pUG/siRNA cycling phase of pUG RNA-mediated gene silencing (see model in Fig. 5g ). In the absence of cycling, pUG shortening does not occur and pUG RNAs are longer in mut-16 and MAGO12 animals. Finally, a number of recent studies report transgenerational inheritance of acquired traits in C. elegans , which last 3-4 generations 32 – 37 . oma-1 RNAi-directed pUG RNAs also perdure for 3-4 generations ( Fig. 5a ). These shared generational timescales of inheritance suggest that the inheritance of acquired traits in C. elegans may be mediated by pUG RNAs whose generational “half-life” is limited to 3-4 generations due to the built-in brake on TEI provided by pUG RNA shortening.

Techniques Used: Whisker Assay, Mutagenesis

pUG tails convert otherwise inert RNAs into agents of gene silencing. a , Fluorescent micrographs showing -1 to -3 oocytes of adult rde-1(ne219); gfp::h2b animals injected in the germline with RNAs consisting of the indicated 3’ terminal repeats appended to the first 369nt of gfp mRNA. % of progeny with gfp silenced was counted. b, oma-1(zu405ts) animals lay arrested embryos at 20°C unless oma-1(zu405ts) is silenced 14 . Adult rde-1(ne219); oma-1(zu405ts) animals were injected with RNAs consisting of the indicated 3’ terminal repeats appended to the first 541nt of oma-1 mRNA. c, Adult rde-1(ne219); oma-1(zu405ts) animals were injected with the same oma-1 mRNA fragment as in b with varying 3’ pUG tail length, different UG repeat sequences or with the pUG sequence appended to the 3’ end, 5’ end or in the middle of the oma-1 mRNA. b-c, 5 progeny per injected animal were pooled and the % hatched embryos (# of hatched embryos/total embryos laid) was counted. Insets show injected RNAs run on a 2% agarose gel to assess RNA integrity. n=5-15 injected animals. a-c, Repeats were 36nt in length unless otherwise indicated. Error bars are standard deviations (s.d.) of the mean.
Figure Legend Snippet: pUG tails convert otherwise inert RNAs into agents of gene silencing. a , Fluorescent micrographs showing -1 to -3 oocytes of adult rde-1(ne219); gfp::h2b animals injected in the germline with RNAs consisting of the indicated 3’ terminal repeats appended to the first 369nt of gfp mRNA. % of progeny with gfp silenced was counted. b, oma-1(zu405ts) animals lay arrested embryos at 20°C unless oma-1(zu405ts) is silenced 14 . Adult rde-1(ne219); oma-1(zu405ts) animals were injected with RNAs consisting of the indicated 3’ terminal repeats appended to the first 541nt of oma-1 mRNA. c, Adult rde-1(ne219); oma-1(zu405ts) animals were injected with the same oma-1 mRNA fragment as in b with varying 3’ pUG tail length, different UG repeat sequences or with the pUG sequence appended to the 3’ end, 5’ end or in the middle of the oma-1 mRNA. b-c, 5 progeny per injected animal were pooled and the % hatched embryos (# of hatched embryos/total embryos laid) was counted. Insets show injected RNAs run on a 2% agarose gel to assess RNA integrity. n=5-15 injected animals. a-c, Repeats were 36nt in length unless otherwise indicated. Error bars are standard deviations (s.d.) of the mean.

Techniques Used: Injection, Sequencing, Agarose Gel Electrophoresis

Endogenous RNAs are pUGylated and localize to germline Mutator foci. a , Total RNA isolated from adult wild-type or rde-3 mutant animals was subjected to Tc1 pUG PCR analysis ( Fig. 1a ). Rescue strategies are described in the Main text and Methods. b, A 36nt pUG tail was appended to a 338nt Tc1 RNA fragment and this Tc1 pUG RNA was injected into germlines of rde-3(-); unc-22::tc1 animals with a co-injection marker. 25 co-injection marker expressing progeny were pooled per injected animal. Each data point represents the # of mobile progeny (indicating Tc1 mobilized from unc-22 ) per pool. Error bars represent s.d. c-e, Fluorescent micrographs of adult pachytene germ cell nuclei. c, Wild-type or rde-3(-) animals expressing a marker of chromatin (mCherry:HIS-58, magenta) and C38D9.2::GFP (green), which is expressed diffusely in the germline syncytium, wherein germ cell nuclei share a common cytoplasm. d, RNA FISH to detect pUG RNAs (pUG RNA FISH) was performed on germlines dissected from wild-type or rde-3(-) animals using an 18nt long poly(AC) oligo conjugated to Alexa 647 (magenta). RNA FISH to detect ama-1 mRNA (green) was performed simultaneously as a positive control. DNA was stained with DAPI (blue). e, pUG RNA FISH (magenta) and immunofluorescence to detect a GFP- and degron-tagged RDE-3 (green). DNA was stained with DAPI (blue). f, Tc1, dpy-11 , and oma-1 pUG PCR was performed on total RNA isolated from glp-1(q224 or ts) animals grown at 15°C (permissive temperature, germ cells present) or 25°C (non-permissive temperature,
Figure Legend Snippet: Endogenous RNAs are pUGylated and localize to germline Mutator foci. a , Total RNA isolated from adult wild-type or rde-3 mutant animals was subjected to Tc1 pUG PCR analysis ( Fig. 1a ). Rescue strategies are described in the Main text and Methods. b, A 36nt pUG tail was appended to a 338nt Tc1 RNA fragment and this Tc1 pUG RNA was injected into germlines of rde-3(-); unc-22::tc1 animals with a co-injection marker. 25 co-injection marker expressing progeny were pooled per injected animal. Each data point represents the # of mobile progeny (indicating Tc1 mobilized from unc-22 ) per pool. Error bars represent s.d. c-e, Fluorescent micrographs of adult pachytene germ cell nuclei. c, Wild-type or rde-3(-) animals expressing a marker of chromatin (mCherry:HIS-58, magenta) and C38D9.2::GFP (green), which is expressed diffusely in the germline syncytium, wherein germ cell nuclei share a common cytoplasm. d, RNA FISH to detect pUG RNAs (pUG RNA FISH) was performed on germlines dissected from wild-type or rde-3(-) animals using an 18nt long poly(AC) oligo conjugated to Alexa 647 (magenta). RNA FISH to detect ama-1 mRNA (green) was performed simultaneously as a positive control. DNA was stained with DAPI (blue). e, pUG RNA FISH (magenta) and immunofluorescence to detect a GFP- and degron-tagged RDE-3 (green). DNA was stained with DAPI (blue). f, Tc1, dpy-11 , and oma-1 pUG PCR was performed on total RNA isolated from glp-1(q224 or ts) animals grown at 15°C (permissive temperature, germ cells present) or 25°C (non-permissive temperature,

Techniques Used: Isolation, Mutagenesis, Polymerase Chain Reaction, Injection, Marker, Expressing, Fluorescence In Situ Hybridization, Positive Control, Staining, Immunofluorescence

pUG tails must be appended to sense RNAs of > 50 nts for functionality. rde-1(ne219); oma-1(zu405ts) animals were injected with: a, an oma-1 pUG RNA consisting of the sense or antisense strand of the same 541nt long oma-1 mRNA fragment (beginning at the atg) and a 36nt 3’ pUG tail. b, oma-1 pUG RNAs consisting of oma-1 mRNA fragments of varying lengths (with position 1 starting at the aug of the oma-1 mRNA sequence) all appended to a 36nt pUG tail. For a and b , % embryonic arrest was scored at the non-permissive temperature for oma-1(zu405ts) . n=8-17 injected animals.
Figure Legend Snippet: pUG tails must be appended to sense RNAs of > 50 nts for functionality. rde-1(ne219); oma-1(zu405ts) animals were injected with: a, an oma-1 pUG RNA consisting of the sense or antisense strand of the same 541nt long oma-1 mRNA fragment (beginning at the atg) and a 36nt 3’ pUG tail. b, oma-1 pUG RNAs consisting of oma-1 mRNA fragments of varying lengths (with position 1 starting at the aug of the oma-1 mRNA sequence) all appended to a 36nt pUG tail. For a and b , % embryonic arrest was scored at the non-permissive temperature for oma-1(zu405ts) . n=8-17 injected animals.

Techniques Used: Injection, Sequencing

pUG RNAs and siRNAs cooperate to drive heritable gene silencing. a , oma-1 pUG PCR was performed on RNA isolated from four generations of descendants (F 1 -F 4 ) derived from oma-1 dsRNA-treated animals. b, rde-1(ne219); gfp::h2b animals were injected with a gfp pUG RNA and gfp expression was monitored for six generations. c, MAGO12 animals, which harbor deletions in all twelve wago genes, were treated with oma-1 dsRNA. oma-1 pUG PCR was performed on total RNA from dsRNA-treated animals (P 0 ) and their progeny (F 1 ). Note: pUG RNAs appear longer in MAGO12 animals (see Extended Data Fig. 9 legend). d, c38d9.2 and Tc1 pUG RNA expression levels were quantified in embryos harvested from wild-type, MAGO12, or rde-3(-) animals. Shown is the fold change normalized to rde-3(-). e, rde-1(ne219); oma-1(zu405ts) animals were injected with an oma-1(SNP) pUG RNA. pUG RNAs were Sanger sequenced from F 2 progeny to determine the presence or absence of the SNP. f, Wild-type and rde-3(ne298) animals subjected to oma-1 RNAi were crossed and F 2 progeny were genotyped (not shown). RNA isolated from populations of rde-3(+) or rde-3(ne298) F 3 animals (3 biological replicates) was subjected to oma-1 pUG PCR. g, Model. Two major phases of the pUG RNA pathway, initiation and maintenance, are shown. Initiation : exogenous and constitutive (i.e. genomically-encoded such as dsRNA, piRNAs) triggers direct RDE-3 to pUGylate RNAs previously fragmented by RNAi, and possibly other, systems. Maintenance : pUG RNA are templates for RdRPs to make 2° siRNAs. Argonaute proteins (termed WAGOs) bind these 2° siRNAs and: 1) target homologous RNAs for transcriptional and translational silencing (previous work 25 , 30 , 38 , 39 ), and 2) direct the cleavage and de novo RDE-3-mediated pUGylation of additional mRNAs (this work). In this way, cycles of pUG RNA-based siRNA production and siRNA-directed mRNA pUGylation form a silencing loop, which is maintained over time and across generations to mediate stable gene silencing. pUG/siRNA cycling likely occurs in germline perinuclear condensates called Mutator foci.
Figure Legend Snippet: pUG RNAs and siRNAs cooperate to drive heritable gene silencing. a , oma-1 pUG PCR was performed on RNA isolated from four generations of descendants (F 1 -F 4 ) derived from oma-1 dsRNA-treated animals. b, rde-1(ne219); gfp::h2b animals were injected with a gfp pUG RNA and gfp expression was monitored for six generations. c, MAGO12 animals, which harbor deletions in all twelve wago genes, were treated with oma-1 dsRNA. oma-1 pUG PCR was performed on total RNA from dsRNA-treated animals (P 0 ) and their progeny (F 1 ). Note: pUG RNAs appear longer in MAGO12 animals (see Extended Data Fig. 9 legend). d, c38d9.2 and Tc1 pUG RNA expression levels were quantified in embryos harvested from wild-type, MAGO12, or rde-3(-) animals. Shown is the fold change normalized to rde-3(-). e, rde-1(ne219); oma-1(zu405ts) animals were injected with an oma-1(SNP) pUG RNA. pUG RNAs were Sanger sequenced from F 2 progeny to determine the presence or absence of the SNP. f, Wild-type and rde-3(ne298) animals subjected to oma-1 RNAi were crossed and F 2 progeny were genotyped (not shown). RNA isolated from populations of rde-3(+) or rde-3(ne298) F 3 animals (3 biological replicates) was subjected to oma-1 pUG PCR. g, Model. Two major phases of the pUG RNA pathway, initiation and maintenance, are shown. Initiation : exogenous and constitutive (i.e. genomically-encoded such as dsRNA, piRNAs) triggers direct RDE-3 to pUGylate RNAs previously fragmented by RNAi, and possibly other, systems. Maintenance : pUG RNA are templates for RdRPs to make 2° siRNAs. Argonaute proteins (termed WAGOs) bind these 2° siRNAs and: 1) target homologous RNAs for transcriptional and translational silencing (previous work 25 , 30 , 38 , 39 ), and 2) direct the cleavage and de novo RDE-3-mediated pUGylation of additional mRNAs (this work). In this way, cycles of pUG RNA-based siRNA production and siRNA-directed mRNA pUGylation form a silencing loop, which is maintained over time and across generations to mediate stable gene silencing. pUG/siRNA cycling likely occurs in germline perinuclear condensates called Mutator foci.

Techniques Used: Polymerase Chain Reaction, Isolation, Derivative Assay, Injection, Expressing, RNA Expression

pUG tails are added to mRNA fragments in vivo . a , PCR-based assay to detect gene-specific pUG RNAs. Total RNA was reverse transcribed (RT) using an (AC) 9 oligo modified with two PCR adapters and then degraded using RNase H. Two rounds of PCR were performed using gene-specific and adapter-specific primers. Note: the (AC) 9 RT oligo can complementary base-pair anywhere along the length of a pUG tail. b , oma-1 pUG PCR was performed on total RNA isolated from wild-type animals and two different rde-3 mutant strains fed E. coli expressing empty vector control or oma-1 dsRNA (RNAi). RDE-3 function was rescued in ne3370 and ne298 animals (see Main text and Methods for details). gsa-1 , which has an 18nt long genomically-encoded pUG repeat in its 3’UTR, is a loading control. c, oma-1 pUG PCR on RNA isolated from animals of the indicated genotypes, +/- oma-1 dsRNA. d, Sanger sequencing chromatogram of an oma-1 pUG PCR product showing that a pUG tail consists of perfect UG repeats and is longer than the RT oligo. e, Illumina MiSeq was performed on oma-1 pUG PCR products derived from wild-type and rde-3(-) animals +/- oma-1 dsRNA. # of sequenced pUG RNAs (y-axis) mapping to each pUGylation site (x-axis) is shown. Inset: total number of sequenced and spliced oma-1 pUG RNAs from indicated samples. f, % of oma-1 pUG RNAs (MiSeq reads) having each nucleotide (nt) at the last templated position (−1) is indicated. Logo analysis was used to determine the probability of finding each nt at both the first position of a pUG tail (+1), as well as at the second-to-last templated nt of oma-1 (−2).
Figure Legend Snippet: pUG tails are added to mRNA fragments in vivo . a , PCR-based assay to detect gene-specific pUG RNAs. Total RNA was reverse transcribed (RT) using an (AC) 9 oligo modified with two PCR adapters and then degraded using RNase H. Two rounds of PCR were performed using gene-specific and adapter-specific primers. Note: the (AC) 9 RT oligo can complementary base-pair anywhere along the length of a pUG tail. b , oma-1 pUG PCR was performed on total RNA isolated from wild-type animals and two different rde-3 mutant strains fed E. coli expressing empty vector control or oma-1 dsRNA (RNAi). RDE-3 function was rescued in ne3370 and ne298 animals (see Main text and Methods for details). gsa-1 , which has an 18nt long genomically-encoded pUG repeat in its 3’UTR, is a loading control. c, oma-1 pUG PCR on RNA isolated from animals of the indicated genotypes, +/- oma-1 dsRNA. d, Sanger sequencing chromatogram of an oma-1 pUG PCR product showing that a pUG tail consists of perfect UG repeats and is longer than the RT oligo. e, Illumina MiSeq was performed on oma-1 pUG PCR products derived from wild-type and rde-3(-) animals +/- oma-1 dsRNA. # of sequenced pUG RNAs (y-axis) mapping to each pUGylation site (x-axis) is shown. Inset: total number of sequenced and spliced oma-1 pUG RNAs from indicated samples. f, % of oma-1 pUG RNAs (MiSeq reads) having each nucleotide (nt) at the last templated position (−1) is indicated. Logo analysis was used to determine the probability of finding each nt at both the first position of a pUG tail (+1), as well as at the second-to-last templated nt of oma-1 (−2).

Techniques Used: In Vivo, Polymerase Chain Reaction, Modification, Isolation, Mutagenesis, Expressing, Plasmid Preparation, Sequencing, Derivative Assay

19) Product Images from "The full-length isoform of the mouse pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth"

Article Title: The full-length isoform of the mouse pleckstrin homology domain-interacting protein (PHIP) is required for postnatal growth

Journal: FEBS letters

doi: 10.1016/j.febslet.2010.08.042

Generation and characterization of Phip gene-trap mice ( A ) Schematic representation of wild-type ( Phip + ) and mutant ( Phip - ) Phip alleles. Filled boxes represent exons; the open box in Phip - denotes the gene trap cassette. Dashed lines indicate RNA splicing events. ( B ) Schematic representation of wild-type and mutant PHIP peptides generated from Phip + and Phip - , respectively. The number of amino acids for each peptide is indicated. The mutant peptide is a fusion protein containing the N-terminal 63 amino acids of PHIP1 and full-length βgeo. ( C and E ) RT-PCR analysis of liver RNAs from Phip +/+ , Phip +/- and Phip -/- mice. PCR primers (arrows) and their locations in corresponding cDNAs are indicated on the top. For each genotype, PCR products from the indicated two primer pairs were pooled and resolved using a 2% agarose gel. In C , upper and lower bands represent products amplified by F1/R2 and F1/R1, respectively. In E , upper and lower bands represent products amplified by F2/R3 and F3/R4, respectively. ( D and F ) Quantification of transcripts in C and E by quantitative RT-PCR. Genotypes of the RNA are indicated as +/+, +/- and -/-, respectively. qPCR primers are shown on top of the bar graph. N = 3 mice per genotype, *, p
Figure Legend Snippet: Generation and characterization of Phip gene-trap mice ( A ) Schematic representation of wild-type ( Phip + ) and mutant ( Phip - ) Phip alleles. Filled boxes represent exons; the open box in Phip - denotes the gene trap cassette. Dashed lines indicate RNA splicing events. ( B ) Schematic representation of wild-type and mutant PHIP peptides generated from Phip + and Phip - , respectively. The number of amino acids for each peptide is indicated. The mutant peptide is a fusion protein containing the N-terminal 63 amino acids of PHIP1 and full-length βgeo. ( C and E ) RT-PCR analysis of liver RNAs from Phip +/+ , Phip +/- and Phip -/- mice. PCR primers (arrows) and their locations in corresponding cDNAs are indicated on the top. For each genotype, PCR products from the indicated two primer pairs were pooled and resolved using a 2% agarose gel. In C , upper and lower bands represent products amplified by F1/R2 and F1/R1, respectively. In E , upper and lower bands represent products amplified by F2/R3 and F3/R4, respectively. ( D and F ) Quantification of transcripts in C and E by quantitative RT-PCR. Genotypes of the RNA are indicated as +/+, +/- and -/-, respectively. qPCR primers are shown on top of the bar graph. N = 3 mice per genotype, *, p

Techniques Used: Mouse Assay, Mutagenesis, Generated, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

20) Product Images from "Repression of FLOWERING LOCUS T Chromatin by Functionally Redundant Histone H3 Lysine 4 Demethylases in Arabidopsis"

Article Title: Repression of FLOWERING LOCUS T Chromatin by Functionally Redundant Histone H3 Lysine 4 Demethylases in Arabidopsis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0008033

Increased trimethylation of H3K4 at FT locus by elf6 and atjmj4 mutations. A) Schematic of FT locus showing regions (F, G, I, EX1, and N) amplified by the primers used for ChIP analysis. The front and the rear black boxes indicate 5′ and 3′ UTRs, respectively. White boxes indicate exons, while lines indicate introns and intergenic regions. B and C) ChIP assay of FT chromatin with antibody against H3K4me3 or H3K27me3. Plants of each genotype were grown in SD for 57 d and harvested for ChIP assay. ‘Input’ indicates chromatins before immunoprecipitation. ‘Mock’ refers to control samples lacking antibody. Actin1 was used as an internal control. D) qPCR analysis of the ChIP assay for H3K4me3 described in (B and C). The wt Col levels were set to 1 after normalization by input. Error bars represent sd. E) Coomassie-blue stained 6His-AtJmj4 protein purified from sf9 cells (left), and in vitro histone demethylation activity assay using the purified protein (right). Assays were performed without (−) or with either two (+) or four (++) µg of purified 6His-AtJmj4 protein. Mr (K), molecular mass in kilo-daltons.
Figure Legend Snippet: Increased trimethylation of H3K4 at FT locus by elf6 and atjmj4 mutations. A) Schematic of FT locus showing regions (F, G, I, EX1, and N) amplified by the primers used for ChIP analysis. The front and the rear black boxes indicate 5′ and 3′ UTRs, respectively. White boxes indicate exons, while lines indicate introns and intergenic regions. B and C) ChIP assay of FT chromatin with antibody against H3K4me3 or H3K27me3. Plants of each genotype were grown in SD for 57 d and harvested for ChIP assay. ‘Input’ indicates chromatins before immunoprecipitation. ‘Mock’ refers to control samples lacking antibody. Actin1 was used as an internal control. D) qPCR analysis of the ChIP assay for H3K4me3 described in (B and C). The wt Col levels were set to 1 after normalization by input. Error bars represent sd. E) Coomassie-blue stained 6His-AtJmj4 protein purified from sf9 cells (left), and in vitro histone demethylation activity assay using the purified protein (right). Assays were performed without (−) or with either two (+) or four (++) µg of purified 6His-AtJmj4 protein. Mr (K), molecular mass in kilo-daltons.

Techniques Used: Amplification, Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Staining, Purification, In Vitro, Demethylation Activity Assay

21) Product Images from "Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins"

Article Title: Detection of a G-Quadruplex as a Regulatory Element in Thymidylate synthase for Gene Silencing Using Polypurine Reverse Hoogsteen Hairpins

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms21145028

Identification of a G4FS in the TYMS gene and design of a PPRH targeting this site. ( A ) Putative G4-forming sequences detected in the TYMS gene using the QGRS mapper. The positions of the identified G4FS are referred to as the transcription start site considering the TYMS mRNA sequence NM_001071.3. The blue underlined Guanines (Gs) represent the ones implicated in G4 formation. ( B ) Localization of the G4FS with the higher G-score in the TYMS gene sequence (NM_001071.3). The orange highlighted sequence corresponds to G4FS. ( C ) The G4FS is located in the 5’UTR of the TYMS gene. PCR products (184 bp) obtained after amplification with primers 5’UTR-TYMS-Fw and 5’UTR-TYMS-Rv of both gDNA and cDNA/RNA samples. PCR products were resolved in a 6% polyacrylamide gel electrophoresis. ( D ) The first two polypurine sequences found in TYMS mRNA using the TFO searching tool. ( E ) Sequence of the specific PPRH targeting the G4FS (HpTYMS-G4-T), its corresponding Watson–Crick negative control (HpTYMS-G4-T-WC) and a scramble negative control (HpSC4). ( F ) Scheme showing the potential strand displacement produced when the PPRH (HpTYMS-G4-T) is mixed with dsDNA-G4FS. The formation of the antiparallel triplex by binding of the PPRH to the polypyrimidine oligonucleotide dissociates the polypurine oligonucleotide.
Figure Legend Snippet: Identification of a G4FS in the TYMS gene and design of a PPRH targeting this site. ( A ) Putative G4-forming sequences detected in the TYMS gene using the QGRS mapper. The positions of the identified G4FS are referred to as the transcription start site considering the TYMS mRNA sequence NM_001071.3. The blue underlined Guanines (Gs) represent the ones implicated in G4 formation. ( B ) Localization of the G4FS with the higher G-score in the TYMS gene sequence (NM_001071.3). The orange highlighted sequence corresponds to G4FS. ( C ) The G4FS is located in the 5’UTR of the TYMS gene. PCR products (184 bp) obtained after amplification with primers 5’UTR-TYMS-Fw and 5’UTR-TYMS-Rv of both gDNA and cDNA/RNA samples. PCR products were resolved in a 6% polyacrylamide gel electrophoresis. ( D ) The first two polypurine sequences found in TYMS mRNA using the TFO searching tool. ( E ) Sequence of the specific PPRH targeting the G4FS (HpTYMS-G4-T), its corresponding Watson–Crick negative control (HpTYMS-G4-T-WC) and a scramble negative control (HpSC4). ( F ) Scheme showing the potential strand displacement produced when the PPRH (HpTYMS-G4-T) is mixed with dsDNA-G4FS. The formation of the antiparallel triplex by binding of the PPRH to the polypyrimidine oligonucleotide dissociates the polypurine oligonucleotide.

Techniques Used: Sequencing, Polymerase Chain Reaction, Amplification, Polyacrylamide Gel Electrophoresis, Negative Control, Produced, Binding Assay

22) Product Images from "Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline"

Article Title: Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline

Journal: bioRxiv

doi: 10.1101/2020.08.26.268920

H3K9me3 and RNA level changes in piwi embryonic knockdown ovaries compared to control. (A) Log2 fold change of H3K9me3 ChIP/input enrichment shows satDNA H3K9me3 levels decrease. P-values are estimated by one sample t-test (mu=0) with FDR correction (Benjamini 1995). (B) Log2 fold change of small RNA abundance shows satDNA small RNA levels decrease with variation observed for replicate2. Small RNA aundance is normalized to the number of reads mapped to miRNAs. (C) Log2 fold change of total RNA abundance shows satDNA long RNA levels increase. Adjusted p-values for total RNA changes are reported by DESeq2. 20A and flamenco are uni-strand piRNA clusters, 42AB, 80F and 38C1 / 2 are dual-strand piRNA clusters. * adjusted p-value
Figure Legend Snippet: H3K9me3 and RNA level changes in piwi embryonic knockdown ovaries compared to control. (A) Log2 fold change of H3K9me3 ChIP/input enrichment shows satDNA H3K9me3 levels decrease. P-values are estimated by one sample t-test (mu=0) with FDR correction (Benjamini 1995). (B) Log2 fold change of small RNA abundance shows satDNA small RNA levels decrease with variation observed for replicate2. Small RNA aundance is normalized to the number of reads mapped to miRNAs. (C) Log2 fold change of total RNA abundance shows satDNA long RNA levels increase. Adjusted p-values for total RNA changes are reported by DESeq2. 20A and flamenco are uni-strand piRNA clusters, 42AB, 80F and 38C1 / 2 are dual-strand piRNA clusters. * adjusted p-value

Techniques Used: Chromatin Immunoprecipitation

SatDNA loci are regulated by the heterochromatin-dependent transcription machinery in Drosophila ovaries. (A) Heatmap showing the quantification of transcript abundance in small RNA-seq data from mutants of rhino, cutoff, deadlock , and moonshiner for satDNAs ( Rsp, 260-bp and 359-bp satellite) and piRNA clusters, normalized by miRNA level. GLKD: germline knockdown. (B) qPCR estimate of Rsp copy number in wild types and mutants. (C) qRT-PCR estimate of Rsp transcript level in mutants compared to wild types. ΔΔCt = ΔCt(wild type) - ΔCt(mutant), a negative value indicates lower expression in mutant. Student’s t-test, p-value=0.077, 0.048.
Figure Legend Snippet: SatDNA loci are regulated by the heterochromatin-dependent transcription machinery in Drosophila ovaries. (A) Heatmap showing the quantification of transcript abundance in small RNA-seq data from mutants of rhino, cutoff, deadlock , and moonshiner for satDNAs ( Rsp, 260-bp and 359-bp satellite) and piRNA clusters, normalized by miRNA level. GLKD: germline knockdown. (B) qPCR estimate of Rsp copy number in wild types and mutants. (C) qRT-PCR estimate of Rsp transcript level in mutants compared to wild types. ΔΔCt = ΔCt(wild type) - ΔCt(mutant), a negative value indicates lower expression in mutant. Student’s t-test, p-value=0.077, 0.048.

Techniques Used: RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Mutagenesis

23) Product Images from "Human norovirus hyper-mutation revealed by ultra-deep sequencing"

Article Title: Human norovirus hyper-mutation revealed by ultra-deep sequencing

Journal: Infection, Genetics and Evolution

doi: 10.1016/j.meegid.2016.04.017

NoV genetic map, regions sequenced, and setup of transfection assays. A. In the NoV genetic map, the VP1 capsid gene is shown in red. Molecular clones encompassing the entire VP1 gene were sequenced by the Sanger method. Illumina sequencing was used to analyze smaller regions mapping to the S domain of VP1 and the hyper-variable domain P2 (dark red bars). B. An infectious cDNA clone was transfected in HEK293 cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase, allowing for transcription of plus-strand NoV genomic RNA. A primer annealing to minus-strand copies was used for RT-PCR amplification and sequencing. Colored circles represent mutations/variants.
Figure Legend Snippet: NoV genetic map, regions sequenced, and setup of transfection assays. A. In the NoV genetic map, the VP1 capsid gene is shown in red. Molecular clones encompassing the entire VP1 gene were sequenced by the Sanger method. Illumina sequencing was used to analyze smaller regions mapping to the S domain of VP1 and the hyper-variable domain P2 (dark red bars). B. An infectious cDNA clone was transfected in HEK293 cells previously infected with a recombinant vaccinia virus expressing T7 RNA polymerase, allowing for transcription of plus-strand NoV genomic RNA. A primer annealing to minus-strand copies was used for RT-PCR amplification and sequencing. Colored circles represent mutations/variants.

Techniques Used: Transfection, Clone Assay, Sequencing, Infection, Recombinant, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification

24) Product Images from "Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera"

Article Title: Bridging the Synaptic Gap: Neuroligins and Neurexin I in Apis mellifera

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003542

Developmental Expression Profiles of the Neuroligins and Neurexin I in Honeybee Brain. Honeybee neuroligin and neurexin I expression was assessed by quantitative real time PCR amplification. The ribosomal gene RPL8 was used as the housekeeping gene. Methodology for data analysis and the presentation of results was taken from Collins et al [104] ; where by expression levels were normalised by subtraction against the threshold cycle of the RPL8 . Collins et al [104] found RPL8 to be the best correlate with RNA concentration across varying developmental life stages and varying tissues of the honeybee. Expression levels were examined from whole larvae (5 day old); and brain tissue from pupae (stage P8 as outlined by Ganeshina et al [101] ) 24 hour adult, 7 day adult and forager honeybees. Standards errors were negligible and less than +/−1.18 for all experimental results. The coloured lines illustrate the developmental expression profile of a single gene through development. Data points in columns illustrate the relative levels of neurexin I and neuroligin expression to one another at a particular stage of development. The developmental stage/gene with lowest expression relative to the control gene (neuroligin 1 at 7 days of age) was given an arbitrary expression level of 1. The data values are shown in Supplementary Data Table 3 .
Figure Legend Snippet: Developmental Expression Profiles of the Neuroligins and Neurexin I in Honeybee Brain. Honeybee neuroligin and neurexin I expression was assessed by quantitative real time PCR amplification. The ribosomal gene RPL8 was used as the housekeeping gene. Methodology for data analysis and the presentation of results was taken from Collins et al [104] ; where by expression levels were normalised by subtraction against the threshold cycle of the RPL8 . Collins et al [104] found RPL8 to be the best correlate with RNA concentration across varying developmental life stages and varying tissues of the honeybee. Expression levels were examined from whole larvae (5 day old); and brain tissue from pupae (stage P8 as outlined by Ganeshina et al [101] ) 24 hour adult, 7 day adult and forager honeybees. Standards errors were negligible and less than +/−1.18 for all experimental results. The coloured lines illustrate the developmental expression profile of a single gene through development. Data points in columns illustrate the relative levels of neurexin I and neuroligin expression to one another at a particular stage of development. The developmental stage/gene with lowest expression relative to the control gene (neuroligin 1 at 7 days of age) was given an arbitrary expression level of 1. The data values are shown in Supplementary Data Table 3 .

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Amplification, Concentration Assay

25) Product Images from "Salicylic acid treatment and expression of an RNA-dependent RNA polymerase 1 transgene inhibit lethal symptoms and meristem invasion during tobacco mosaic virus infection in Nicotiana benthamiana"

Article Title: Salicylic acid treatment and expression of an RNA-dependent RNA polymerase 1 transgene inhibit lethal symptoms and meristem invasion during tobacco mosaic virus infection in Nicotiana benthamiana

Journal: BMC Plant Biology

doi: 10.1186/s12870-016-0705-8

RDR activity, but not MtRDR1 transcript accumulation, is induced by SA treatment in MtRDR1 -transgenic N. benthamiana plants. Semi-quantitative RT-PCR analysis of PR1 transcript accumulation in leaves of ( a ) transgenic (empty vector) control and ( b ) MtRDR1 -transgenic plants infiltrated with a control solution of 0.05 % ( v/v ) ethanol or a solution of 1 mM SA in 0.05 % ( v/v ) ethanol. Infiltrated leaf tissue samples were harvested for RNA extraction at 72 h post-infiltration. PR1 transcript accumulation levels were determined by RT-PCR after 40 cycles of PCR and compared relative to the accumulation levels of the elongation factor 1 alpha ( EF1α ) transcript. Increased PR1 accumulation confirmed that SA was taken up by the tissues and was effective in inducing transcriptional changes. c Semi-quantitative RT-PCR analysis showed that there was little difference in MtRDR1 transcript accumulation in MtRDR1 -transgenic plants infiltrated with control solution or 1 mM SA. NbRDR1m transcript accumulation was up-regulated in both transgenic control and MtRDR1 -transgenic N. benthamiana plants after SA treatment, although the NbRDR1m protein itself is non-functional. MtRDR1 and NbRDR1m transcript accumulation levels after 27 and 35 cycles, respectively, were compared relative to the accumulation levels of EF1α . Infiltrated tissue samples were harvested at 72 h post-infiltration. d RT-qPCR analysis of MtRDR1 transcript levels in leaves of empty vector control and MtRDR1 -transgenic plants infiltrated with water control or 1 mM SA solution. MtRDR1 was not detected in empty vector control plants. Mean values for relative MtRDR1 levels (based on duplicate technical replicate values; 100 = mean value for the transcript level in untreated MtRDR1 plants) obtained from three plants (one plant = one independent sample) have been given for each treatment group. Error bars represent standard errors of the mean for the three samples. Relative transcript levels of MtRDR1 were calculated using the 2 -ΔΔC(t) method [ 59 ] using EF1α as an internal reference. e Enhancement of RDR activity by SA in MtRDR1 -transgenic plants. Leaves from tobacco ( N. tabacum , included as a positive control) and transgenic empty vector control and MtRDR1 -transgenic N. benthamiana plants were infiltrated with water (-) containing 0.05 % ( v/v ) ethanol or 2.5 mM SA in 0.05 % ( v/v ) ethanol (+) and harvested after 48 h for preparation of RDR1-enriched extracts. The proxy for RDR1 activity was the incorporation of α-[ 32 P] CTP into nascent RNA analysed by liquid scintillation counting of radioactivity incorporated (counts per minute) into trichloroacetic acid-precipitable material. Error bars are standard errors for the mean for three technical replicates (RDR assays) per sample
Figure Legend Snippet: RDR activity, but not MtRDR1 transcript accumulation, is induced by SA treatment in MtRDR1 -transgenic N. benthamiana plants. Semi-quantitative RT-PCR analysis of PR1 transcript accumulation in leaves of ( a ) transgenic (empty vector) control and ( b ) MtRDR1 -transgenic plants infiltrated with a control solution of 0.05 % ( v/v ) ethanol or a solution of 1 mM SA in 0.05 % ( v/v ) ethanol. Infiltrated leaf tissue samples were harvested for RNA extraction at 72 h post-infiltration. PR1 transcript accumulation levels were determined by RT-PCR after 40 cycles of PCR and compared relative to the accumulation levels of the elongation factor 1 alpha ( EF1α ) transcript. Increased PR1 accumulation confirmed that SA was taken up by the tissues and was effective in inducing transcriptional changes. c Semi-quantitative RT-PCR analysis showed that there was little difference in MtRDR1 transcript accumulation in MtRDR1 -transgenic plants infiltrated with control solution or 1 mM SA. NbRDR1m transcript accumulation was up-regulated in both transgenic control and MtRDR1 -transgenic N. benthamiana plants after SA treatment, although the NbRDR1m protein itself is non-functional. MtRDR1 and NbRDR1m transcript accumulation levels after 27 and 35 cycles, respectively, were compared relative to the accumulation levels of EF1α . Infiltrated tissue samples were harvested at 72 h post-infiltration. d RT-qPCR analysis of MtRDR1 transcript levels in leaves of empty vector control and MtRDR1 -transgenic plants infiltrated with water control or 1 mM SA solution. MtRDR1 was not detected in empty vector control plants. Mean values for relative MtRDR1 levels (based on duplicate technical replicate values; 100 = mean value for the transcript level in untreated MtRDR1 plants) obtained from three plants (one plant = one independent sample) have been given for each treatment group. Error bars represent standard errors of the mean for the three samples. Relative transcript levels of MtRDR1 were calculated using the 2 -ΔΔC(t) method [ 59 ] using EF1α as an internal reference. e Enhancement of RDR activity by SA in MtRDR1 -transgenic plants. Leaves from tobacco ( N. tabacum , included as a positive control) and transgenic empty vector control and MtRDR1 -transgenic N. benthamiana plants were infiltrated with water (-) containing 0.05 % ( v/v ) ethanol or 2.5 mM SA in 0.05 % ( v/v ) ethanol (+) and harvested after 48 h for preparation of RDR1-enriched extracts. The proxy for RDR1 activity was the incorporation of α-[ 32 P] CTP into nascent RNA analysed by liquid scintillation counting of radioactivity incorporated (counts per minute) into trichloroacetic acid-precipitable material. Error bars are standard errors for the mean for three technical replicates (RDR assays) per sample

Techniques Used: Activity Assay, Transgenic Assay, Quantitative RT-PCR, Plasmid Preparation, RNA Extraction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Functional Assay, Positive Control, Radioactivity

26) Product Images from "Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress"

Article Title: Drosophila melanogaster Activating Transcription Factor 4 Regulates Glycolysis During Endoplasmic Reticulum Stress

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.115.017269

Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. * P
Figure Legend Snippet: Atf4 binding sites within the promoters of Ldh and Pfk mediate Atf4-dependent transcriptional up-regulation. (A) We stably transfected S2 cells with the GFP reporter constructs diagrammed on the left. We then mock-treated or depleted cells of Atf4 by RNAi, incubated with and without DTT (2 mM, 5 hr), and measured relative GFP RNA levels by qPCR. (B) We transiently transfected S2 cells with reporter constructs containing wild-type or mutated promoter sequences as shown on the left. We then incubated cells with or without DTT (2 mM, 5 hr) and measured relative GFP RNA levels by qPCR. For all panels: shown are the means ± SDs of at least three independent experiments. * P

Techniques Used: Binding Assay, Stable Transfection, Transfection, Construct, Incubation, Real-time Polymerase Chain Reaction

Metabolic gene expression is regulated by Tm in S2 cells. We treated S2 cells with Tm (5 μg/mL) and measured relative RNA levels by quantitative polymerase chain reaction. We normalized all RNA measurements to the RNA levels of Ribosomal protein L19 (RpL19) . Data are presented as means ± SEs of three technical replicates, and are representative of two independent experiments. Pfk , phosphofructokinase ; TCA, tricarboxylic acid; Tm, tunicamycin; Tpi , triosephosphate isomerase .
Figure Legend Snippet: Metabolic gene expression is regulated by Tm in S2 cells. We treated S2 cells with Tm (5 μg/mL) and measured relative RNA levels by quantitative polymerase chain reaction. We normalized all RNA measurements to the RNA levels of Ribosomal protein L19 (RpL19) . Data are presented as means ± SEs of three technical replicates, and are representative of two independent experiments. Pfk , phosphofructokinase ; TCA, tricarboxylic acid; Tm, tunicamycin; Tpi , triosephosphate isomerase .

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Flies display metabolic changes during ER stress in vivo . (A) We fed male D. melanogaster w1118 with Tm (10 μg/mL, 23 hr) to induce ER stress and measured Ldh mRNA levels by qPCR. (B−C) We crossed UAS-Atf4 RNAi to hs-GAL4 to obtain Atf4 knockdown flies. We stressed each strain of flies as in (A) and compared the RNA levels of Atf4 (B), Ldh , and BiP (C) by qPCR. (D) We measured lactate levels in extracts from D. melanogaster fed with or without Tm as in A. Lactate concentrations were normalized using total protein concentrations. For all panels: data are presented as means ± SDs of 3 independent experiments. * P
Figure Legend Snippet: Flies display metabolic changes during ER stress in vivo . (A) We fed male D. melanogaster w1118 with Tm (10 μg/mL, 23 hr) to induce ER stress and measured Ldh mRNA levels by qPCR. (B−C) We crossed UAS-Atf4 RNAi to hs-GAL4 to obtain Atf4 knockdown flies. We stressed each strain of flies as in (A) and compared the RNA levels of Atf4 (B), Ldh , and BiP (C) by qPCR. (D) We measured lactate levels in extracts from D. melanogaster fed with or without Tm as in A. Lactate concentrations were normalized using total protein concentrations. For all panels: data are presented as means ± SDs of 3 independent experiments. * P

Techniques Used: In Vivo, Real-time Polymerase Chain Reaction

Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. * P
Figure Legend Snippet: Atf4 is necessary and sufficient for up-regulation of glycolytic genes and Ldh. (A−B) We incubated S2 cells with dsRNA targeting either GFP (as a negative control) or Atf4, allowed cells to recover, and incubated with and without either DTT (2 mM, 6 hr, A) or Tm (5 μg/mL, 16 hr, B). We then measured the relative RNA levels for the indicated genes by qPCR. * P

Techniques Used: Incubation, Negative Control, Real-time Polymerase Chain Reaction

27) Product Images from "Open Reading Frame 8a of the Human Severe Acute Respiratory Syndrome Coronavirus Not Only Promotes Viral Replication but Also Induces Apoptosis"

Article Title: Open Reading Frame 8a of the Human Severe Acute Respiratory Syndrome Coronavirus Not Only Promotes Viral Replication but Also Induces Apoptosis

Journal: The Journal of Infectious Diseases

doi: 10.1086/519166

The effect of orf8a small interfering RNA (siRNA) on viral replication and cytopathic effects of severe acute respiratory syndrome coronavirus (SARS-CoV) HKU39849. A, Verification of the effects of 2 sets of orf8a siRNAs. Top, Results of reverse-transcription polymerase chain reaction (RTPCR) of orf8a mRNA using total RNA extracted from VeroE6 cells cotransfected with pHA-ORF8a and different siRNAs. Lane 1, pcDNA3-HA; lanes 2-9, pHA-ORF8a. The siRNAs used in different assays were as follows: lanes 2 and 3, siRNA-GFP; lanes 4-6, orf8a siRNA set 1; and lanes 7-9, orf8a siRNA set 2. The cells were harvested at different time points after transfection (18 h for lanes 2, 4, and 7; 36 h for lanes 5 and 8; and 56 h for lanes 1, 3, 6, and 9). The relative intensity (ratio) of the orf8a, compared with b-actin, in the top panel was calculated and normalized using the ratio of lane 3 (56 h of siRNA-GFP) as the 100% value. B, Measurements of the viral loads in the cultural supernatants from VeroE6 cells that had been transfected with orf8a siRNA for 36 h before they were inoculated with SARS-CoV HKU39849. After infection, the cultural supernatants were collected at various time points for 5 days. Nos. of replicase open reading frame (ORF)-1b mRNA copies were determined using real-time PCR. Error bars indicate SDs. * P
Figure Legend Snippet: The effect of orf8a small interfering RNA (siRNA) on viral replication and cytopathic effects of severe acute respiratory syndrome coronavirus (SARS-CoV) HKU39849. A, Verification of the effects of 2 sets of orf8a siRNAs. Top, Results of reverse-transcription polymerase chain reaction (RTPCR) of orf8a mRNA using total RNA extracted from VeroE6 cells cotransfected with pHA-ORF8a and different siRNAs. Lane 1, pcDNA3-HA; lanes 2-9, pHA-ORF8a. The siRNAs used in different assays were as follows: lanes 2 and 3, siRNA-GFP; lanes 4-6, orf8a siRNA set 1; and lanes 7-9, orf8a siRNA set 2. The cells were harvested at different time points after transfection (18 h for lanes 2, 4, and 7; 36 h for lanes 5 and 8; and 56 h for lanes 1, 3, 6, and 9). The relative intensity (ratio) of the orf8a, compared with b-actin, in the top panel was calculated and normalized using the ratio of lane 3 (56 h of siRNA-GFP) as the 100% value. B, Measurements of the viral loads in the cultural supernatants from VeroE6 cells that had been transfected with orf8a siRNA for 36 h before they were inoculated with SARS-CoV HKU39849. After infection, the cultural supernatants were collected at various time points for 5 days. Nos. of replicase open reading frame (ORF)-1b mRNA copies were determined using real-time PCR. Error bars indicate SDs. * P

Techniques Used: Small Interfering RNA, Reverse Transcription Polymerase Chain Reaction, Transfection, Infection, Real-time Polymerase Chain Reaction

28) Product Images from "RCC1L (WBSCR16) isoforms coordinate mitochondrial ribosome assembly through their interaction with GTPases"

Article Title: RCC1L (WBSCR16) isoforms coordinate mitochondrial ribosome assembly through their interaction with GTPases

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1008923

RCC1L strongest interactors are mitoribosome biogenesis factors. ( A ) Co-immunoprecipitation of STREP2-FLAG-tagged RCC1L isoforms from purified mitochondria after induction of HEK cells with 3–10 ng/ml doxycycline for 3–4 days. In the immunoblots of endogenous RCC1L, the endogenous isoforms (37 or 50 kDa band) are marked (empty arrowhead) in the elution fractions. Immunoblots for mitochondrial ribosomal proteins and biogenesis factors are presented. ACO2 was used as mitochondrial negative control for unspecific binding. Additional immunoblots and longer exposures are shown in S5A and S5B Fig , respectively. ( B ) Quantification of pull-down enrichment of mtLSU and mtSSU proteins from panel (A) based on densitometric analysis of the elution signal and normalised to its input loading. Data represent mean ± SD from two independent experiments. ( C ) Co-immunoprecipitation of STREP2-FLAG-tagged and HA-tagged RCC1L isoforms from HEK purified mitochondria. Cells overexpressing STREP2-FLAG-tagged RCC1L V1 were transiently overexpressing HA-tagged version of each of the three isoforms: RCC1L V1 , RCC1L V2 and RCC1L V3 . Alternatively, cells overexpressing STREP2-FLAG-tagged RCC1L V2 or RCC1L V3 were transiently overexpressing HA-tagged RCC1L V1 . Parental HEK cells not expressing any STREP2-FLAG-tagged protein but transiently overexpressing HA-tagged RCC1L V1 were used as negative control for unspecific pull down. ( D ) Interaction between RCC1L isoforms is independent of nucleic acids. Cells overexpressing STREP2-FLAG-tagged RCC1L V2 or RCC1L V3 were transiently overexpressing HA-tagged RCC1L V1 . Mitochondrial lysates were split into four identical columns and before elution they were left untreated or treated with DNase I (20U/ml), RNase A (40 μg/ml) or Benzonase (12.5 U/ μl) for 1 h at 4°C. The effectiveness of the treatments was verified by measuring the amount of DNA or RNA present in the elution by qPCR ( S5C Fig ). Input (I) and flow through (FT) 3%, washes (W1-5-10) and elution (E) 8%. See S3 Table for quantitative data in this figure.
Figure Legend Snippet: RCC1L strongest interactors are mitoribosome biogenesis factors. ( A ) Co-immunoprecipitation of STREP2-FLAG-tagged RCC1L isoforms from purified mitochondria after induction of HEK cells with 3–10 ng/ml doxycycline for 3–4 days. In the immunoblots of endogenous RCC1L, the endogenous isoforms (37 or 50 kDa band) are marked (empty arrowhead) in the elution fractions. Immunoblots for mitochondrial ribosomal proteins and biogenesis factors are presented. ACO2 was used as mitochondrial negative control for unspecific binding. Additional immunoblots and longer exposures are shown in S5A and S5B Fig , respectively. ( B ) Quantification of pull-down enrichment of mtLSU and mtSSU proteins from panel (A) based on densitometric analysis of the elution signal and normalised to its input loading. Data represent mean ± SD from two independent experiments. ( C ) Co-immunoprecipitation of STREP2-FLAG-tagged and HA-tagged RCC1L isoforms from HEK purified mitochondria. Cells overexpressing STREP2-FLAG-tagged RCC1L V1 were transiently overexpressing HA-tagged version of each of the three isoforms: RCC1L V1 , RCC1L V2 and RCC1L V3 . Alternatively, cells overexpressing STREP2-FLAG-tagged RCC1L V2 or RCC1L V3 were transiently overexpressing HA-tagged RCC1L V1 . Parental HEK cells not expressing any STREP2-FLAG-tagged protein but transiently overexpressing HA-tagged RCC1L V1 were used as negative control for unspecific pull down. ( D ) Interaction between RCC1L isoforms is independent of nucleic acids. Cells overexpressing STREP2-FLAG-tagged RCC1L V2 or RCC1L V3 were transiently overexpressing HA-tagged RCC1L V1 . Mitochondrial lysates were split into four identical columns and before elution they were left untreated or treated with DNase I (20U/ml), RNase A (40 μg/ml) or Benzonase (12.5 U/ μl) for 1 h at 4°C. The effectiveness of the treatments was verified by measuring the amount of DNA or RNA present in the elution by qPCR ( S5C Fig ). Input (I) and flow through (FT) 3%, washes (W1-5-10) and elution (E) 8%. See S3 Table for quantitative data in this figure.

Techniques Used: Immunoprecipitation, Purification, Western Blot, Negative Control, Binding Assay, Expressing, Real-time Polymerase Chain Reaction

Nucleic acid binding properties of RCC1L isoforms. ( A ) Electrophoretic mobility shift assay (EMSA) using 10 nM [ 32 P]-end-labelled RNA 25-mer polyuridine (rU 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the s ignal in the region marked with a bracket and using the no-protein lane as reference (below). ( B ) Electrophoretic mobility shift assay (EMSA) using 10 nM [γ 32 P]-end-labelled RNA 25-mer polyadenosine (rA 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the signal in the region marked with a bracket and using the no-protein lane as reference (below). ( C ) Electrophoretic mobility shift assay (EMSA) using 10 nM [γ 32 P]-end-labelled DNA 25-mer polydeoxythymidine (dT 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the signal in the region marked with a filled arrowhead and using the no-protein lane as reference (below). In panels (A-C), increasing concentrations of dialysed recombinant RCC1L proteins (0–4 μM) were used. Equivalent volumes of HEK parental mitochondrial dialysed elution were used as negative control in each experiment. Data represent mean ± SD from two independent experiments. See S5 Table for quantitative data in this figure.
Figure Legend Snippet: Nucleic acid binding properties of RCC1L isoforms. ( A ) Electrophoretic mobility shift assay (EMSA) using 10 nM [ 32 P]-end-labelled RNA 25-mer polyuridine (rU 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the s ignal in the region marked with a bracket and using the no-protein lane as reference (below). ( B ) Electrophoretic mobility shift assay (EMSA) using 10 nM [γ 32 P]-end-labelled RNA 25-mer polyadenosine (rA 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the signal in the region marked with a bracket and using the no-protein lane as reference (below). ( C ) Electrophoretic mobility shift assay (EMSA) using 10 nM [γ 32 P]-end-labelled DNA 25-mer polydeoxythymidine (dT 25 ) oligo for 30 min at 37°C (above). Densitometric analysis was based on quantification of the signal in the region marked with a filled arrowhead and using the no-protein lane as reference (below). In panels (A-C), increasing concentrations of dialysed recombinant RCC1L proteins (0–4 μM) were used. Equivalent volumes of HEK parental mitochondrial dialysed elution were used as negative control in each experiment. Data represent mean ± SD from two independent experiments. See S5 Table for quantitative data in this figure.

Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Recombinant, Negative Control

29) Product Images from "Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells †"

Article Title: Reduced Expression of the Immediate-Early Protein IE0 Enables Efficient Replication of Autographa californica Multiple Nucleopolyhedrovirus in Poorly Permissive Spodoptera littoralis Cells †

Journal: Journal of Virology

doi: 10.1128/JVI.77.1.535-545.2003

Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (10 5 ) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35 S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with Ac M NPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of Ac M NPV polyhedrin.
Figure Legend Snippet: Metabolic labeling of polypeptides synthesized in virus-infected SF9 and SL2 cells. Cells (10 5 ) were infected at an MOI of 20. At 45 h postinfection, metabolic labeling was performed with a 35 S-labeled methionine-cysteine mixture. Analysis was done with extracts from cells infected with Ac M NPV (lanes 2 and 7), vAcSL2 (lanes 3 and 8), vBgl3 (lanes 4 and 9), and vHsp-1 (lanes 5 and 10) and with mock-infected SF9 cells (lane 1) and SL2 cells (lane 6). Ph, location of Ac M NPV polyhedrin.

Techniques Used: Labeling, Synthesized, Infection

30) Product Images from "Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein"

Article Title: Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein

Journal: Nature Communications

doi: 10.1038/s41467-017-01413-7

Assessment of the cyclization status of artificial lanthipeptides in cell lysates and displayed on phage. a Precursor peptides (full sequences in Supplementary Table 1 ) containing the NisA leader and indicated core sequences (residues involved in thioether formation colored) flanked by affinity tags were expressed with or without NisBC, captured from cell lysates, and subjected to FXa digestion and ELISA detection. The protease resistance relative to untreated (no FXa) samples was calculated and data representing mean ± s.d. of three independent cultures analyzed in duplicate is shown (unpaired, two-tailed t -test). b As in a , but core sequences were fused to the ProcA leader (full sequences in Supplementary Table 2 ) and expressed with or without ProcM enzyme. c Peptides containing the NisA leader sequence were translationally fused to the N- (left panel) or C-terminus (right panel) of phage pIII (full sequences in Supplementary Table 3 ), phage produced with or without NisBC co-expression, and the peptide cyclization status assessed on phage particles as described in a . d As in c , but phage displayed peptides containing ProcA leader sequences (full sequences in Supplementary Table 4 ) were tested after phage production with or without ProcM co-expression. Experiments shown in a – d were repeated three times
Figure Legend Snippet: Assessment of the cyclization status of artificial lanthipeptides in cell lysates and displayed on phage. a Precursor peptides (full sequences in Supplementary Table 1 ) containing the NisA leader and indicated core sequences (residues involved in thioether formation colored) flanked by affinity tags were expressed with or without NisBC, captured from cell lysates, and subjected to FXa digestion and ELISA detection. The protease resistance relative to untreated (no FXa) samples was calculated and data representing mean ± s.d. of three independent cultures analyzed in duplicate is shown (unpaired, two-tailed t -test). b As in a , but core sequences were fused to the ProcA leader (full sequences in Supplementary Table 2 ) and expressed with or without ProcM enzyme. c Peptides containing the NisA leader sequence were translationally fused to the N- (left panel) or C-terminus (right panel) of phage pIII (full sequences in Supplementary Table 3 ), phage produced with or without NisBC co-expression, and the peptide cyclization status assessed on phage particles as described in a . d As in c , but phage displayed peptides containing ProcA leader sequences (full sequences in Supplementary Table 4 ) were tested after phage production with or without ProcM co-expression. Experiments shown in a – d were repeated three times

Techniques Used: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Sequencing, Produced, Expressing

Phage selection outcome and characterization of identified lanthipeptides. a Amino-acid sequences of specific clones selected on uPA. The library in which the clones were found, the number of clones with identical sequences, and a unique clone ID are indicated. Fixed cysteine positions are boxed (gray), putative sites of dehydration (serines and threonines) are underlined, and a conserved three residue motif is highlighted (colored). b Sequence and disulfide pattern of bicyclic and monocyclic uPA-specific peptides described in the literature. c ELISA for uPA binding of purified His 6 -tagged leader-core peptides produced with (+ProcM) or without (unmodified) ProcM co-expression. Data representing mean ± s.d. of three replicates is shown (curve fit with nonlinear regression). d As in a , but amino-acid sequences of specific clones selected on streptavidin are shown. e ELISA for streptavidin binding of purified His 6 -tagged leader-core peptide PEP330 produced with or without ProcM co-expression and analyzed under reducing (red) and non-reducing (non-red.) conditions. f As in e , but binding curves of PEP331 are shown. Experiments shown in c , e , and f were repeated three times
Figure Legend Snippet: Phage selection outcome and characterization of identified lanthipeptides. a Amino-acid sequences of specific clones selected on uPA. The library in which the clones were found, the number of clones with identical sequences, and a unique clone ID are indicated. Fixed cysteine positions are boxed (gray), putative sites of dehydration (serines and threonines) are underlined, and a conserved three residue motif is highlighted (colored). b Sequence and disulfide pattern of bicyclic and monocyclic uPA-specific peptides described in the literature. c ELISA for uPA binding of purified His 6 -tagged leader-core peptides produced with (+ProcM) or without (unmodified) ProcM co-expression. Data representing mean ± s.d. of three replicates is shown (curve fit with nonlinear regression). d As in a , but amino-acid sequences of specific clones selected on streptavidin are shown. e ELISA for streptavidin binding of purified His 6 -tagged leader-core peptide PEP330 produced with or without ProcM co-expression and analyzed under reducing (red) and non-reducing (non-red.) conditions. f As in e , but binding curves of PEP331 are shown. Experiments shown in c , e , and f were repeated three times

Techniques Used: Selection, Clone Assay, Sequencing, Enzyme-linked Immunosorbent Assay, Binding Assay, Purification, Produced, Expressing

An ELISA-based reporter assay monitors the cyclization status of artificial lanthipeptides. a Peptides containing an FXa recognition site (red bar) flanked by affinity tags and residues involved in thioether bridge formation are captured via anti-tag1 antibodies, treated with FXa, and detected via anti-tag2 antibodies. Tag2 is proteolytically removed (left panel) in linear peptides, but remains connected via a covalent thioether (indicated by “S”, right panel) in cyclic peptides, resulting in low and high signals, respectively. b Sequences of synthetic linear or thioether-bridged peptides with FLAG- and His 6 -epitopes (boxed) flanking the FXa site (underlined) used for assay validation. c Synthetic peptides were captured via the His 6 -tag, incubated with or without FXa, and detected using anti-FLAG IgG. The protease resistance relative to untreated (no FXa) samples was calculated and data representing mean ± s.d. of three replicates is shown (unpaired, two-tailed t -test). The experiment was repeated three times
Figure Legend Snippet: An ELISA-based reporter assay monitors the cyclization status of artificial lanthipeptides. a Peptides containing an FXa recognition site (red bar) flanked by affinity tags and residues involved in thioether bridge formation are captured via anti-tag1 antibodies, treated with FXa, and detected via anti-tag2 antibodies. Tag2 is proteolytically removed (left panel) in linear peptides, but remains connected via a covalent thioether (indicated by “S”, right panel) in cyclic peptides, resulting in low and high signals, respectively. b Sequences of synthetic linear or thioether-bridged peptides with FLAG- and His 6 -epitopes (boxed) flanking the FXa site (underlined) used for assay validation. c Synthetic peptides were captured via the His 6 -tag, incubated with or without FXa, and detected using anti-FLAG IgG. The protease resistance relative to untreated (no FXa) samples was calculated and data representing mean ± s.d. of three replicates is shown (unpaired, two-tailed t -test). The experiment was repeated three times

Techniques Used: Enzyme-linked Immunosorbent Assay, Reporter Assay, Incubation, Two Tailed Test

Display of lanthipeptide precursors on the N- and C-termini of phage pIII. N-terminal pIII display of largely unmodified lanthipeptide precursors (left panel). Translocation of unstructured peptide–pIII fusions from the cytoplasm (CP) via the Sec pathway (Sec) is fast allowing little or no lanthionine introduction by ProcM, whereas modified lanthipeptides are poorly transported via the narrow Sec pore. Prior to phage assembly, the precursor peptide (ocher) is exposed to the periplasm (PP) and no longer accessible to ProcM. C-terminal display of lanthipeptides (right panel). After translocation of the pIII–peptide fusion, the C-terminal exposed peptide remains in the cytoplasm allowing ProcM-catalyzed lanthionine introduction (indicated by a blue cycle). Phage display of modified peptide is accomplished after incorporation of pIII into the phage coat and subsequent extrusion of the phage particle into the medium. Black arrows indicate movement of capsid proteins to the phage assembly site. OmpA signal sequence in pIII fusions (black box), leader peptide (yellow box), linker sequences (black line), relevant phage coat proteins (as numbered), outer membrane (OM), and inner membrane (IM) are highlighted
Figure Legend Snippet: Display of lanthipeptide precursors on the N- and C-termini of phage pIII. N-terminal pIII display of largely unmodified lanthipeptide precursors (left panel). Translocation of unstructured peptide–pIII fusions from the cytoplasm (CP) via the Sec pathway (Sec) is fast allowing little or no lanthionine introduction by ProcM, whereas modified lanthipeptides are poorly transported via the narrow Sec pore. Prior to phage assembly, the precursor peptide (ocher) is exposed to the periplasm (PP) and no longer accessible to ProcM. C-terminal display of lanthipeptides (right panel). After translocation of the pIII–peptide fusion, the C-terminal exposed peptide remains in the cytoplasm allowing ProcM-catalyzed lanthionine introduction (indicated by a blue cycle). Phage display of modified peptide is accomplished after incorporation of pIII into the phage coat and subsequent extrusion of the phage particle into the medium. Black arrows indicate movement of capsid proteins to the phage assembly site. OmpA signal sequence in pIII fusions (black box), leader peptide (yellow box), linker sequences (black line), relevant phage coat proteins (as numbered), outer membrane (OM), and inner membrane (IM) are highlighted

Techniques Used: Translocation Assay, Size-exclusion Chromatography, Modification, Sequencing

31) Product Images from "The Role of UbiX in Escherichia coli Coenzyme Q Biosynthesis"

Article Title: The Role of UbiX in Escherichia coli Coenzyme Q Biosynthesis

Journal: Archives of biochemistry and biophysics

doi: 10.1016/j.abb.2007.08.009

Disruption of the ubiX gene in E. coli leads to an accumulation of radioactive lipid co-eluting with 4-hydroxy-3-octaprenylbenzoate (HP 8 B) Lipid extracts from E. coli strains labeled with p -[U- 14 C]hydroxybenzoic acid were separated by normal-phase HPLC. Absorbance was monitored at 274 nm (to detect Q 8 ) and 250 nm (to detect HP 8 B); only one wavelength is depicted and is designated for each of the panels on the left side. The 14 C radioactivity (dpm) present in each fraction is depicted in (B, D, F, H, and J). Unless noted otherwise, the amount of lipid extract injected was 15μl of 500 μl total; chromatograms shown in panels (I and J) represent 15 μl of 50 μl total lipid extract. The paired chromatograms of absorbance and radioactivity (amount of dpm injected is indicated for each panel) correspond to separated lipid extracts from E. coli strains, MC4100 (A and B [6.67 × 10 3 dpm]); AN66, ubiD mutant (C and D [2.87 × 10 4 dpm]); LL1, ubiX mutant with pCH empty vector (E and F [1.87 × 10 3 dpm]); LL1 harboring ubiX on a high copy plasmid (G and H [5.02 × 10 3 dpm]); and LL1 harboring yeast PAD1 (I and J [4.42 × 10 3 dpm]). There was a 0.4 min time delay between the UV and in-line radiochromatography detector.
Figure Legend Snippet: Disruption of the ubiX gene in E. coli leads to an accumulation of radioactive lipid co-eluting with 4-hydroxy-3-octaprenylbenzoate (HP 8 B) Lipid extracts from E. coli strains labeled with p -[U- 14 C]hydroxybenzoic acid were separated by normal-phase HPLC. Absorbance was monitored at 274 nm (to detect Q 8 ) and 250 nm (to detect HP 8 B); only one wavelength is depicted and is designated for each of the panels on the left side. The 14 C radioactivity (dpm) present in each fraction is depicted in (B, D, F, H, and J). Unless noted otherwise, the amount of lipid extract injected was 15μl of 500 μl total; chromatograms shown in panels (I and J) represent 15 μl of 50 μl total lipid extract. The paired chromatograms of absorbance and radioactivity (amount of dpm injected is indicated for each panel) correspond to separated lipid extracts from E. coli strains, MC4100 (A and B [6.67 × 10 3 dpm]); AN66, ubiD mutant (C and D [2.87 × 10 4 dpm]); LL1, ubiX mutant with pCH empty vector (E and F [1.87 × 10 3 dpm]); LL1 harboring ubiX on a high copy plasmid (G and H [5.02 × 10 3 dpm]); and LL1 harboring yeast PAD1 (I and J [4.42 × 10 3 dpm]). There was a 0.4 min time delay between the UV and in-line radiochromatography detector.

Techniques Used: Labeling, High Performance Liquid Chromatography, Radioactivity, Injection, Mutagenesis, Plasmid Preparation

32) Product Images from "The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression"

Article Title: The Anopheles gambiae cE5, a tight- and fast-binding thrombin inhibitor with post-transcriptionally regulated salivary-restricted expression

Journal: Insect biochemistry and molecular biology

doi: 10.1016/j.ibmb.2012.04.008

Expression and purification of the recombinant cE5 protein
Figure Legend Snippet: Expression and purification of the recombinant cE5 protein

Techniques Used: Expressing, Purification, Recombinant

Effect of salt on thrombin inhibition by the An. gambiae cE5 and the An. albimanus anophelin
Figure Legend Snippet: Effect of salt on thrombin inhibition by the An. gambiae cE5 and the An. albimanus anophelin

Techniques Used: Inhibition

Effect of pre-incubation on thrombin inhibition by the An. gambiae cE5 and the An. albimanus anophelin
Figure Legend Snippet: Effect of pre-incubation on thrombin inhibition by the An. gambiae cE5 and the An. albimanus anophelin

Techniques Used: Incubation, Inhibition

Effects of cE5 and salivary extracts from An. gambiae and Ae. albopictus on thrombin activity
Figure Legend Snippet: Effects of cE5 and salivary extracts from An. gambiae and Ae. albopictus on thrombin activity

Techniques Used: Activity Assay

Tissue-specific expression of the cE5 protein in adult female salivary glands
Figure Legend Snippet: Tissue-specific expression of the cE5 protein in adult female salivary glands

Techniques Used: Expressing

IC 50 of cE5 for thrombin
Figure Legend Snippet: IC 50 of cE5 for thrombin

Techniques Used:

Schematic representation of the genomic region encoding the An. gambiae cE5
Figure Legend Snippet: Schematic representation of the genomic region encoding the An. gambiae cE5

Techniques Used:

33) Product Images from "The Cystathionine-β-synthase Domains on the Guanosine 5′-Monophosphate Reductase and Inosine 5′-Monophosphate Dehydrogenase Enzymes from Leishmania Regulate Enzymatic Activity in Response to Guanylate and Adenylate Nucleotide Levels"

Article Title: The Cystathionine-β-synthase Domains on the Guanosine 5′-Monophosphate Reductase and Inosine 5′-Monophosphate Dehydrogenase Enzymes from Leishmania Regulate Enzymatic Activity in Response to Guanylate and Adenylate Nucleotide Levels

Journal: Molecular microbiology

doi: 10.1111/mmi.13352

Analysis of nucleotide binding to LdIMPDH. (A) Full length LdIMPDH or (B) the LdIMPDH CBS domain (His 6 -ldimpdh112-223) were titrated with hypoxanthine (Hyp), XMP, IMP, GTP, GMP, or ATP and ligand binding was monitored by changes in intrinsic fluorescence
Figure Legend Snippet: Analysis of nucleotide binding to LdIMPDH. (A) Full length LdIMPDH or (B) the LdIMPDH CBS domain (His 6 -ldimpdh112-223) were titrated with hypoxanthine (Hyp), XMP, IMP, GTP, GMP, or ATP and ligand binding was monitored by changes in intrinsic fluorescence

Techniques Used: Binding Assay, Ligand Binding Assay, Fluorescence

34) Product Images from "CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer"

Article Title: CTCF Expression is Essential for Somatic Cell Viability and Protection Against Cancer

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19123832

CRISPR/Cas9-directed editing of Ctcf in hemizygous MEFs. ( A ) Ctcf +/pgkneo MEFs transduced with Cas9 and sgRNA-containing lentivectors (mouse Ctcf exon 3 sgRNAs #1, #2, #3, and #4; Rosa26 sgRNA) were FACS-enriched and subjected to T7EI digestion of Ctcf exon 3 amplicons amplified from isolated gDNA. Approximate expected sizes (in bp) for digested products #1 (427, 214), #2 (476, 165), #3 (428, 213), and #4 (399, 242). Clonogenicity assay ( B ); Western blot analysis of individual clones (from sgRNA#4, arrowheads indicate lower molecular weight Ctcf variants) ( C ); and molecular genetic analysis of individual clones; n=number of clones sequenced (in brackets) ( D ). ( E ) Examples of frequently occurring in-frame deletions in Ctcf +/pgkneo MEFs (from sgRNA#4). Quantitative data represent the mean ± SEM for three experiments each performed in triplicate. Statistical analysis was performed using Mann-Whitney U-test (ns = not significant, **** p
Figure Legend Snippet: CRISPR/Cas9-directed editing of Ctcf in hemizygous MEFs. ( A ) Ctcf +/pgkneo MEFs transduced with Cas9 and sgRNA-containing lentivectors (mouse Ctcf exon 3 sgRNAs #1, #2, #3, and #4; Rosa26 sgRNA) were FACS-enriched and subjected to T7EI digestion of Ctcf exon 3 amplicons amplified from isolated gDNA. Approximate expected sizes (in bp) for digested products #1 (427, 214), #2 (476, 165), #3 (428, 213), and #4 (399, 242). Clonogenicity assay ( B ); Western blot analysis of individual clones (from sgRNA#4, arrowheads indicate lower molecular weight Ctcf variants) ( C ); and molecular genetic analysis of individual clones; n=number of clones sequenced (in brackets) ( D ). ( E ) Examples of frequently occurring in-frame deletions in Ctcf +/pgkneo MEFs (from sgRNA#4). Quantitative data represent the mean ± SEM for three experiments each performed in triplicate. Statistical analysis was performed using Mann-Whitney U-test (ns = not significant, **** p

Techniques Used: CRISPR, Transduction, FACS, Amplification, Isolation, Western Blot, Clone Assay, Molecular Weight, MANN-WHITNEY

Inhibition of cell proliferation and clonogenicity following CRISPR/Cas9 targeting of CTCF in K562 cells. K562 cells were transduced with Cas9 and sgRNA-containing lentivectors (AAVS1 sgRNA = control; human CTCF exon 3 sgRNAs #2, #3, #5) and enriched for eGFP + mCherry + cells using FACS; Neg = untransduced K562 cells. ( A ) CTCF exon 3 PCR amplification and T7 endonuclease I (T7EI) digestion: approximate expected sizes (in bp) for digested products #2 (310, 345), #3 (296, 359) and #5 (323, 332). Analysis of CTCF protein levels in K562 clones: ( B ) immunoblot; and ( C ) densitometric analysis of upper 130 kDa band. CTCF protein expression normalised to GAPDH expression in each sample is shown relative to untransduced K562 cells. ( D ) Summary of results after sequencing of CTCF exon 3 PCR amplicons from individual clones; n = number of clones sequences (in brackets). Functional assays performed were MTT cell proliferation ( E ); and clonogenicity assays ( F ). Quantitative data represent the mean ± SEM for 3–4 experiments each performed in triplicate. Statistical analysis was performed using a Mann-Whitney U-test (ns = not significant, * p
Figure Legend Snippet: Inhibition of cell proliferation and clonogenicity following CRISPR/Cas9 targeting of CTCF in K562 cells. K562 cells were transduced with Cas9 and sgRNA-containing lentivectors (AAVS1 sgRNA = control; human CTCF exon 3 sgRNAs #2, #3, #5) and enriched for eGFP + mCherry + cells using FACS; Neg = untransduced K562 cells. ( A ) CTCF exon 3 PCR amplification and T7 endonuclease I (T7EI) digestion: approximate expected sizes (in bp) for digested products #2 (310, 345), #3 (296, 359) and #5 (323, 332). Analysis of CTCF protein levels in K562 clones: ( B ) immunoblot; and ( C ) densitometric analysis of upper 130 kDa band. CTCF protein expression normalised to GAPDH expression in each sample is shown relative to untransduced K562 cells. ( D ) Summary of results after sequencing of CTCF exon 3 PCR amplicons from individual clones; n = number of clones sequences (in brackets). Functional assays performed were MTT cell proliferation ( E ); and clonogenicity assays ( F ). Quantitative data represent the mean ± SEM for 3–4 experiments each performed in triplicate. Statistical analysis was performed using a Mann-Whitney U-test (ns = not significant, * p

Techniques Used: Inhibition, CRISPR, Transduction, FACS, Polymerase Chain Reaction, Amplification, Clone Assay, Expressing, Sequencing, Functional Assay, MTT Assay, MANN-WHITNEY

35) Product Images from "Functional genomic exploration reveals that Ss-RIOK-1 is essential for the development and survival of Strongyloides stercoralis larvae"

Article Title: Functional genomic exploration reveals that Ss-RIOK-1 is essential for the development and survival of Strongyloides stercoralis larvae

Journal: International journal for parasitology

doi: 10.1016/j.ijpara.2017.06.005

Subcellular localization of GFP-fused wild-type and mutant Strongyloides stercoralis ( Ss )-RIOK-1. Differential Interference Contrast (DIC) (A, C, E) and fluorescence images (B, D, F) of wild-type GFP fused Ss -RIOK-1 transgenic post free-living L2s. DIC (G) and fluorescence images (H) of GFP-fused mutant Ss -RIOK-1 transgenic PFL L2s. GFP is expressed in the hypodermis (h), head neurons (HN), body motor neurons (BN), tail neurons (TN), ventral ganglion (VG) and longitudinal nerve tracts (L). Scale bar = 100 μm.
Figure Legend Snippet: Subcellular localization of GFP-fused wild-type and mutant Strongyloides stercoralis ( Ss )-RIOK-1. Differential Interference Contrast (DIC) (A, C, E) and fluorescence images (B, D, F) of wild-type GFP fused Ss -RIOK-1 transgenic post free-living L2s. DIC (G) and fluorescence images (H) of GFP-fused mutant Ss -RIOK-1 transgenic PFL L2s. GFP is expressed in the hypodermis (h), head neurons (HN), body motor neurons (BN), tail neurons (TN), ventral ganglion (VG) and longitudinal nerve tracts (L). Scale bar = 100 μm.

Techniques Used: Mutagenesis, Fluorescence, Transgenic Assay

In vitro kinase assay of GFP-fused wild-type Strongyloides stercoralis ( Ss )-RIOK-1 and Ss -RIOK-1 D282A mutant. (A) Purified, bacterially expressed GFP-fused wild-type Ss -RIOK-1 and purified GFP-fused D282A mutant Ss -RIOK-1 as compared with molecular weight standards (M). (B) Autoradiograph comparing autophosphorylation activity of GFP-tag only (GFP), GFP-fused mutant (GFP- Ss -RIOK-1 D282A) and wild-type (GFP- Ss -RIOK-1) Ss -RIOK-1s after incubation with ATP[γ 32 P].
Figure Legend Snippet: In vitro kinase assay of GFP-fused wild-type Strongyloides stercoralis ( Ss )-RIOK-1 and Ss -RIOK-1 D282A mutant. (A) Purified, bacterially expressed GFP-fused wild-type Ss -RIOK-1 and purified GFP-fused D282A mutant Ss -RIOK-1 as compared with molecular weight standards (M). (B) Autoradiograph comparing autophosphorylation activity of GFP-tag only (GFP), GFP-fused mutant (GFP- Ss -RIOK-1 D282A) and wild-type (GFP- Ss -RIOK-1) Ss -RIOK-1s after incubation with ATP[γ 32 P].

Techniques Used: In Vitro, Kinase Assay, Mutagenesis, Purification, Molecular Weight, Autoradiography, Activity Assay, Incubation

The arrest/lethal phenotype resulting from GFP-fused D282A mutant Strongyloides stercoralis ( Ss )-RIOK-1 expression was rescued by co-expression of GFP-fused wild-type Ss -RIOK-1. Percentages of larvae classified into three developmental classes, young larvae, moulting larvae and infective L3s (iL3), from groups transformed with plasmid mixtures containing varying ratios of the mutant construct (pRP8) to the wild-type construct (pRP7), with the mutant or wild-type construct alone and the non-injection group. All the transgenic larvae were collected and classified into the three groups, including dead worms. Counts were done at 96 h after transformation. Error bars indicate S.D. Asterisks denote the proportion of iL3s in these groups were statistically much higher than that in pRP8 group, which was revealed by one-way ANOVA analysis with Bonferroni’s Multiple Comparison Test. * P
Figure Legend Snippet: The arrest/lethal phenotype resulting from GFP-fused D282A mutant Strongyloides stercoralis ( Ss )-RIOK-1 expression was rescued by co-expression of GFP-fused wild-type Ss -RIOK-1. Percentages of larvae classified into three developmental classes, young larvae, moulting larvae and infective L3s (iL3), from groups transformed with plasmid mixtures containing varying ratios of the mutant construct (pRP8) to the wild-type construct (pRP7), with the mutant or wild-type construct alone and the non-injection group. All the transgenic larvae were collected and classified into the three groups, including dead worms. Counts were done at 96 h after transformation. Error bars indicate S.D. Asterisks denote the proportion of iL3s in these groups were statistically much higher than that in pRP8 group, which was revealed by one-way ANOVA analysis with Bonferroni’s Multiple Comparison Test. * P

Techniques Used: Mutagenesis, Expressing, Transformation Assay, Plasmid Preparation, Construct, Injection, Transgenic Assay

Expression of GFP-fused mutant Strongyloides stercoralis ( Ss )-RIOK-1 in vivo caused arrest/lethal phenotype in post free-living larval stages. Length of transgenic larvae at different time points following transformation (A). Larval lengths measured from live parasites in the mutant Ss -RIOK-1 expression group (pRP8), wild-type Ss -RIOK-1 expression group (pRP7) and non-transformed control group at 48 h, 72 h and 96 h after transformation. Survival rates of transgenic larvae expressing different constructs at different time points following transformation (B). Pharynx length as a percentage of total body length in transgenic and wild-type larvae sampled 96 h after transformation (C). Data are means from three or more than three biological replicates of the experiment. Error bars indicate S.D. Asterisks denote statistically significant differences of body length and pharynx to body length ratio were revealed by one-way ANOVA with Bonferroni’s Multiple Comparison Test. * P
Figure Legend Snippet: Expression of GFP-fused mutant Strongyloides stercoralis ( Ss )-RIOK-1 in vivo caused arrest/lethal phenotype in post free-living larval stages. Length of transgenic larvae at different time points following transformation (A). Larval lengths measured from live parasites in the mutant Ss -RIOK-1 expression group (pRP8), wild-type Ss -RIOK-1 expression group (pRP7) and non-transformed control group at 48 h, 72 h and 96 h after transformation. Survival rates of transgenic larvae expressing different constructs at different time points following transformation (B). Pharynx length as a percentage of total body length in transgenic and wild-type larvae sampled 96 h after transformation (C). Data are means from three or more than three biological replicates of the experiment. Error bars indicate S.D. Asterisks denote statistically significant differences of body length and pharynx to body length ratio were revealed by one-way ANOVA with Bonferroni’s Multiple Comparison Test. * P

Techniques Used: Expressing, Mutagenesis, In Vivo, Transgenic Assay, Transformation Assay, Construct

36) Product Images from "An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs"

Article Title: An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Journal: PLoS ONE

doi: 10.1371/journal.pone.0027288

Optimisation and reproducibility of ribo-depleted RNA-Seq. ( A ) Distribution of different sequence tag types from RNA prepared from CCE differentiated ES cells subject to ribosomal RNA depletion using either the RiboMinus or the Ribo-Zero Kit and fragmented either by RNA-hydrolysis or by cDNA-shearing. Sequencing was performed in two different sequencing locations (Vienna-IMP, Nijmegen, RiboMinus) or in one sequencing location (Vienna-CeMM, Ribo-Zero). The percentage of tags in each category is shown for two technical sequencing replicates (CCE1, CCE2) of material prepared by RiboMinus and cDNA-shearing (sheared, lanes nr. 1,2,5,6) or RiboMinus and RNA-hydrolysis (hydrolysed, lanes nr. 3,4,7,8), for the combination of three technical sequencing replicates of RiboMinus and RNA-hydrolysis (lane nr. 9) and for one sequencing of Ribo-Zero and RNA-hydrolysis (lane nr. 10). green: unique tags matching only once in the genome; blue: rRNA+mitoRNA tags matching to ribosomal (RiboMinus and Ribo-Zero) or mitochondrial (RiboMinus) RNAs; red: repeat tags matching more than once in the genome; purple: nomatch tags do not match to the genome. ( B ) Scatter plots comparing the RPKM ( R eads P er K ilobase of exon model per M illion of reads) transcription levels of RefSeq protein-coding genes between combined tags from RiboMinus and RNA-hydrolysis (H) and RiboMinus and cDNA-shearing (S) from CCE within the same location: Vienna-IMP (left) and Nijmegen (right). ( C ) Scatter plots as in B comparing RPKM transcript levels of all combined tags from the two sequencing locations (Vienna-IMP and Nijmegen, left) or between the combined RiboMinus data and the Ribo-Zero data (right). R: Pearson's correlation, note that a perfect correlation is R = 1.
Figure Legend Snippet: Optimisation and reproducibility of ribo-depleted RNA-Seq. ( A ) Distribution of different sequence tag types from RNA prepared from CCE differentiated ES cells subject to ribosomal RNA depletion using either the RiboMinus or the Ribo-Zero Kit and fragmented either by RNA-hydrolysis or by cDNA-shearing. Sequencing was performed in two different sequencing locations (Vienna-IMP, Nijmegen, RiboMinus) or in one sequencing location (Vienna-CeMM, Ribo-Zero). The percentage of tags in each category is shown for two technical sequencing replicates (CCE1, CCE2) of material prepared by RiboMinus and cDNA-shearing (sheared, lanes nr. 1,2,5,6) or RiboMinus and RNA-hydrolysis (hydrolysed, lanes nr. 3,4,7,8), for the combination of three technical sequencing replicates of RiboMinus and RNA-hydrolysis (lane nr. 9) and for one sequencing of Ribo-Zero and RNA-hydrolysis (lane nr. 10). green: unique tags matching only once in the genome; blue: rRNA+mitoRNA tags matching to ribosomal (RiboMinus and Ribo-Zero) or mitochondrial (RiboMinus) RNAs; red: repeat tags matching more than once in the genome; purple: nomatch tags do not match to the genome. ( B ) Scatter plots comparing the RPKM ( R eads P er K ilobase of exon model per M illion of reads) transcription levels of RefSeq protein-coding genes between combined tags from RiboMinus and RNA-hydrolysis (H) and RiboMinus and cDNA-shearing (S) from CCE within the same location: Vienna-IMP (left) and Nijmegen (right). ( C ) Scatter plots as in B comparing RPKM transcript levels of all combined tags from the two sequencing locations (Vienna-IMP and Nijmegen, left) or between the combined RiboMinus data and the Ribo-Zero data (right). R: Pearson's correlation, note that a perfect correlation is R = 1.

Techniques Used: RNA Sequencing Assay, Sequencing

The template preparation protocol determines the comparability of ribo-depleted RNA-Seq to polyA RNA-Seq. The cDNA size distribution of genes showing more than 8× expression difference ( Figure S2 ), in the comparison of ( A ) FH RiboMinus - FH-RiboZero (left) and CCE RiboMinus - CCE Ribo-Zero (right). ( B ) as in A for the comparisons of CCE RiboMinus-Cloonan et al. EB (left), FH RiboMinus-Cui et al. adult mouse brain polyA (middle) and FH RiboMinus-Mortazavi et al. adult mouse brain polyA (right). ( C ) as in A for the comparisons of CCE Ribo-Zero-Cloonan et al. EB (left), FH Ribo-Zero-Cui et al. adult mouse brain polyA (middle) and FH Ribo-Zero-Mortazavi et al. adult mouse brain polyA (right). For Cloonan et al. EB both the gene expression data from the published alignment (shown in B, C, see Materials and methods ) and from an alignment done with the pipeline used here (data not shown) were used and produced the same highly significant differences. Two different size classes are shown with different bin sizes (0–2 kb, 100 bp bins and > 2 kb, 500 bp bins). Genes bigger than 11.5 kb are grouped in the last bin (arrow).
Figure Legend Snippet: The template preparation protocol determines the comparability of ribo-depleted RNA-Seq to polyA RNA-Seq. The cDNA size distribution of genes showing more than 8× expression difference ( Figure S2 ), in the comparison of ( A ) FH RiboMinus - FH-RiboZero (left) and CCE RiboMinus - CCE Ribo-Zero (right). ( B ) as in A for the comparisons of CCE RiboMinus-Cloonan et al. EB (left), FH RiboMinus-Cui et al. adult mouse brain polyA (middle) and FH RiboMinus-Mortazavi et al. adult mouse brain polyA (right). ( C ) as in A for the comparisons of CCE Ribo-Zero-Cloonan et al. EB (left), FH Ribo-Zero-Cui et al. adult mouse brain polyA (middle) and FH Ribo-Zero-Mortazavi et al. adult mouse brain polyA (right). For Cloonan et al. EB both the gene expression data from the published alignment (shown in B, C, see Materials and methods ) and from an alignment done with the pipeline used here (data not shown) were used and produced the same highly significant differences. Two different size classes are shown with different bin sizes (0–2 kb, 100 bp bins and > 2 kb, 500 bp bins). Genes bigger than 11.5 kb are grouped in the last bin (arrow).

Techniques Used: RNA Sequencing Assay, Expressing, Produced

Tag coverage of genes differs between fragmentation methods and ribosomal RNA depletion methods. The coverage of genes with sequence tags is shown as the normalized number of tags at relative positions throughout the gene length. UTRs and coding exons were analysed separately and are plotted as 10 bins for 5′UTRs and 3′UTRs and 100bins for the coding exons (separated by vertical dotted line). ( A ) Comparison of the coverage in the RiboMinus dataset for the combined tags of CCE and FH from RNA-hydrolysis (black) and cDNA-shearing (grey). ( B ) Comparison of the coverage in the RNA-hydrolysis RiboMinus dataset (dotted line, same as in A) and in Ribo-Zero dataset plotted separately for CCE (black) and FH (grey). For all analyses the genes were separated into three groups according to their cDNA length (coding exons and 5′ and 3′ UTRs) as indicated.
Figure Legend Snippet: Tag coverage of genes differs between fragmentation methods and ribosomal RNA depletion methods. The coverage of genes with sequence tags is shown as the normalized number of tags at relative positions throughout the gene length. UTRs and coding exons were analysed separately and are plotted as 10 bins for 5′UTRs and 3′UTRs and 100bins for the coding exons (separated by vertical dotted line). ( A ) Comparison of the coverage in the RiboMinus dataset for the combined tags of CCE and FH from RNA-hydrolysis (black) and cDNA-shearing (grey). ( B ) Comparison of the coverage in the RNA-hydrolysis RiboMinus dataset (dotted line, same as in A) and in Ribo-Zero dataset plotted separately for CCE (black) and FH (grey). For all analyses the genes were separated into three groups according to their cDNA length (coding exons and 5′ and 3′ UTRs) as indicated.

Techniques Used: Sequencing

37) Product Images from "The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair"

Article Title: The unstructured linker arms of MutL enable GATC site incision beyond roadblocks during initiation of DNA mismatch repair

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkz834

Single molecule DNA nanomanipulation assay for MMR strand incision. ( A ) The heteroduplex Rb-pREP4 DNA substrate is tethered and supercoiled in the magnetic trap in the presence of MMR components. Incision of DNA is observed as an abrupt increase in DNA extension. Drawing is not to scale. ( B ) Time-trace showing repeated cycles of supercoiling and subsequent incision of the substrate by 2.5 nM MutS, 10 nM MutL, 10 nM MutH. In between cycles incised DNA is religated by T4 DNA ligase which is present in the reaction. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events. T wait represents the time elapsed before a supercoiled DNA is incised. ( C ) Distribution of T wait is well-described by a single-exponential fit (red line) with mean
Figure Legend Snippet: Single molecule DNA nanomanipulation assay for MMR strand incision. ( A ) The heteroduplex Rb-pREP4 DNA substrate is tethered and supercoiled in the magnetic trap in the presence of MMR components. Incision of DNA is observed as an abrupt increase in DNA extension. Drawing is not to scale. ( B ) Time-trace showing repeated cycles of supercoiling and subsequent incision of the substrate by 2.5 nM MutS, 10 nM MutL, 10 nM MutH. In between cycles incised DNA is religated by T4 DNA ligase which is present in the reaction. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events. T wait represents the time elapsed before a supercoiled DNA is incised. ( C ) Distribution of T wait is well-described by a single-exponential fit (red line) with mean

Techniques Used:

Effect of RNAP roadblock on single molecule strand incision. Time-trace of incision assay carried out on the Rb-pREP4 construct using 2.5 nM MutS, 10 nM MutL, 10 nM MutH and RNA polymerase (RNAP) stalled between the bulge and the GATC site. RNAP addition ( 1 ) is followed by formation of a stalled elongation complex (sTEC), recognizable through its characteristic pattern of initial reduction in DNA extension followed by rapid partial increase (‘scrunching’). ( 2 ) Free components are washed out and ( 3 ) MMR components introduced. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events.
Figure Legend Snippet: Effect of RNAP roadblock on single molecule strand incision. Time-trace of incision assay carried out on the Rb-pREP4 construct using 2.5 nM MutS, 10 nM MutL, 10 nM MutH and RNA polymerase (RNAP) stalled between the bulge and the GATC site. RNAP addition ( 1 ) is followed by formation of a stalled elongation complex (sTEC), recognizable through its characteristic pattern of initial reduction in DNA extension followed by rapid partial increase (‘scrunching’). ( 2 ) Free components are washed out and ( 3 ) MMR components introduced. Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap. Red arrows indicate incision events.

Techniques Used: Construct

Shortening of MutL linker regions prevents roadblock bypass. Time courses for GATC site incision by 50 nM MutS, 50 nM MutLΔ4 and 25 nM MutH on 0.5 nM GT#2 substrate (panel A) and 0.5 nM GT#2b (panel B) in the absence and presence of a dCas9 roadblock as indicated. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs show quantification of product fractions containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments. Time-traces of incision assays carried out on the Rb-pREP4 construct using 2.5 nM MutS, 10 nM MutLΔ4, and 10 nM MutH ( C ) in the absence or ( D ) in the presence of stalled RNAP. Red arrows show incision events, green line the supercoiling pattern imposed in the magnetic trap. RNAP addition (D 1 ) is rapidly followed by formation of a stalled elongation complex (sTEC). (D 2 ) Free components are washed out and (D 3 ) MMR components introduced. Here no incision is observed. When (D 4 ) the roadblock is lifted and (D 5 ) MutS, MutLΔ4 and MutH are introduced, incision can be observed (red arrows). Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap.
Figure Legend Snippet: Shortening of MutL linker regions prevents roadblock bypass. Time courses for GATC site incision by 50 nM MutS, 50 nM MutLΔ4 and 25 nM MutH on 0.5 nM GT#2 substrate (panel A) and 0.5 nM GT#2b (panel B) in the absence and presence of a dCas9 roadblock as indicated. Reaction products are separated using gel electrophoresis under denaturing conditions and visualized using the Alexa 647 fluorophore. Graphs show quantification of product fractions containing no nick (gray), a nick at GATC site 1 (light blue), a nick at GATC site 2 (orange) and 2 nicks (dark blue). Data points with error bars represent the mean values and range of three independent experiments. Time-traces of incision assays carried out on the Rb-pREP4 construct using 2.5 nM MutS, 10 nM MutLΔ4, and 10 nM MutH ( C ) in the absence or ( D ) in the presence of stalled RNAP. Red arrows show incision events, green line the supercoiling pattern imposed in the magnetic trap. RNAP addition (D 1 ) is rapidly followed by formation of a stalled elongation complex (sTEC). (D 2 ) Free components are washed out and (D 3 ) MMR components introduced. Here no incision is observed. When (D 4 ) the roadblock is lifted and (D 5 ) MutS, MutLΔ4 and MutH are introduced, incision can be observed (red arrows). Blue points show bead position sampled at 31 Hz, red points show raw data with ∼1 s averaging. Green line represents the stepwise increase in supercoiling imposed on the DNA via the magnetic trap.

Techniques Used: Nucleic Acid Electrophoresis, Construct

38) Product Images from "Gene Expression Profiles of Mst1r-Deficient Mice during Nickel-Induced Acute Lung Injury"

Article Title: Gene Expression Profiles of Mst1r-Deficient Mice during Nickel-Induced Acute Lung Injury

Journal:

doi: 10.1165/rcmb.2005-0093OC

Northern analyses of granzymes C, D, F, and G. Total RNA was isolated from the lung, thymus, liver and spleen of untreated Mst1r TK +/+ and Mst1r TK −/− mice. Relative levels of mRNA for granzymes C, D, F, and G were assessed
Figure Legend Snippet: Northern analyses of granzymes C, D, F, and G. Total RNA was isolated from the lung, thymus, liver and spleen of untreated Mst1r TK +/+ and Mst1r TK −/− mice. Relative levels of mRNA for granzymes C, D, F, and G were assessed

Techniques Used: Northern Blot, Isolation, Mouse Assay

Diagnostic restriction enzyme analysis. ( A ) Diagram of select diagnostic restriction enzymes in granzyme D, E, F, and H. ( B and C ) Reverse transcriptase PCR was performed on RNA from lung tissue of untreated Mst1r TK −/− mice using one
Figure Legend Snippet: Diagnostic restriction enzyme analysis. ( A ) Diagram of select diagnostic restriction enzymes in granzyme D, E, F, and H. ( B and C ) Reverse transcriptase PCR was performed on RNA from lung tissue of untreated Mst1r TK −/− mice using one

Techniques Used: Diagnostic Assay, Polymerase Chain Reaction, Mouse Assay

39) Product Images from "Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿"

Article Title: Choline Induces Transcriptional Repression and Proteasomal Degradation of the Malarial Phosphoethanolamine Methyltransferase ▿

Journal:

doi: 10.1128/EC.00229-07

(A) Real-time RT-PCR analysis of levels of Pf PMT transcripts. Total RNA extracted from wild-type 3D7 parasites cultured in medium containing 0, 25, 50, 100, 200, 500, or 1,000 μM choline for one generation was reverse transcribed and analyzed
Figure Legend Snippet: (A) Real-time RT-PCR analysis of levels of Pf PMT transcripts. Total RNA extracted from wild-type 3D7 parasites cultured in medium containing 0, 25, 50, 100, 200, 500, or 1,000 μM choline for one generation was reverse transcribed and analyzed

Techniques Used: Quantitative RT-PCR, Cell Culture

40) Product Images from "Chlamydophila felis CF0218 Is a Novel TMH Family Protein with Potential as a Diagnostic Antigen for Diagnosis of C. felis Infection ▿ Infection ▿ †"

Article Title: Chlamydophila felis CF0218 Is a Novel TMH Family Protein with Potential as a Diagnostic Antigen for Diagnosis of C. felis Infection ▿ Infection ▿ †

Journal:

doi: 10.1128/CVI.00134-08

RT-PCR analysis of cf0218 expression in infected cells. Specific messages for ompA and cf0218 were detected from total RNA of HeLa cells infected with C. felis at the times indicated (including noninfected cells as negative control). DNase I-treated total
Figure Legend Snippet: RT-PCR analysis of cf0218 expression in infected cells. Specific messages for ompA and cf0218 were detected from total RNA of HeLa cells infected with C. felis at the times indicated (including noninfected cells as negative control). DNase I-treated total

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing, Infection, Negative Control

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Ligation:

Article Title: Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life
Article Snippet: .. After heat inactivation for 15 min at 70°C, end-repaired DNA (2.5 μL, 20–100 ng/μL) was ligated into pCR-Blunt (0.5 μL, 25 ng/μL) vector (Thermo Fisher Scientific, K270040, following provider’s instructions, 5 μL reaction volume) for 5 h at 20°C before heat inactivation at 70°C for 20 min. We then transformed 2 × 25 μL NEB 10-beta electrocompetent E. coli (DH10B) cells with 2 × 2 μL of the ligation product to create two cells libraries for each sample (two libraries for reproducibility). .. Cells were grown overnight in 10 mL LB medium supplemented with kanamycin (50 μg/mL) for clones amplification ( ).

Subcloning:

Article Title: Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants
Article Snippet: .. Sub-cloning of PCR products PCR products amplified from F0 DNA showing complex sequencing traces were sub-cloned using a Zero-Blunt PCR Cloning Kit (Invitrogen). ..

Construct:

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A
Article Snippet: .. The Sp1 sites deleted MAO A 2-kb-luc or the core promoter of MAO A (−303/−64 bp, 0.24 kb)-luciferase construct (MAO A 0.24-kb-luc) were generated by PCR, and the PCR product was subcloned into Zero Blunt TOPO vector (PCR cloning kit, Invitrogen) for verifying DNA sequencing. .. Subsequently, the PCR product was subcloned into pGL2-Basic luciferase reporter vector by restriction enzyme digestion (XhoI/Hind III) using self-ligation.

Polymerase Chain Reaction:

Article Title: DNA vaccine constructs against enterovirus 71 elicit immune response in mice
Article Snippet: .. 2.1 Construction and validation of DNA vaccine The VP1 gene of EV71 isolate 410/4 (genotype B4) and EV71 isolate S2/86/1 (genotype B4) which have been cloned into pCR® Blunt Vector (Zero Blunt™ PCR Cloning Kit, Invitrogen) were provided by Prof. Mary Jane Cardosa, Universiti Malaysia Sarawak (UNIMAS). .. The VP1 gene of both isolates was amplified by polymerase chain reaction (PCR).

Article Title: Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants
Article Snippet: .. Sub-cloning of PCR products PCR products amplified from F0 DNA showing complex sequencing traces were sub-cloned using a Zero-Blunt PCR Cloning Kit (Invitrogen). ..

Article Title: Live Attenuated Influenza Vaccine contains Substantial and Unexpected Amounts of Defective Viral Genomic RNA
Article Snippet: .. Bands representing PCR products amplified directly from the Fluenz™ Tetra vaccine batch CH2020 and derived from full-length RNAs and putative DI RNAs were extracted from the gel, cloned using the Zero Blunt PCR cloning kit (Invitrogen, Paisley, Scotland, UK), and sequenced. .. This confirmed that influenza A and B segment 1–3 RNAs were derived from the relevant master donor strain [ , ].

Article Title: The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster
Article Snippet: .. Finally, the PCR product of 5arm-AID-EGFP-3arm was cloned into the pCR-Blunt vector (ThermoFisher, k275020, Waltham, MA, USA) through blunt-end cloning. .. For chiRNAs construction, the two target site sequences were cloned into the pU6-BbsI-chiRNA plasmid (Addgene, #45946, Cambridge, MA, USA) as previously described [ ] using the following oligonucleotides: PAM1-sense: 5’-CTTCGACCAGACACAGCACGGGGAT-3’ PAM1-antisense: 5’-AAACATCCCCGTGCTGTGTCTGGTC-3’ PAM2-sense: 5’-CTTCGTCAGCAGCAACTGCGGGTG-3’ PAM2-antisense: 5’-AAACCACCCGCAGTTGCTGCTGAC-3’ The donor and pU6-BbsI-chiRNA plasmids were mixed and injected into embryos of the nos-Cas9 attp2 fly line (Rainbow Transgenic Flies, Inc., Camarillo, CA, USA).

Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
Article Snippet: .. Mutants identified from CAPS assay and SSCP were further confirmed by Sanger sequencing of amplicons (single copy genes) or performing PCR cloning with Zero-Blunt PCR cloning kit (Life Technologies, catalogue no. K 2700–20) prior to sequencing. .. The mutation effect was analysed by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/ ) with default parameters [ ].

Article Title: DNA vaccine constructs against enterovirus 71 elicit immune response in mice
Article Snippet: .. 6 Acknowledgements We would like to thank Prof. Mary Jane Cardosa from Universiti Malaysia Sarawak (UNIMAS) for kindly providing us the VP1 gene of EV71 isolate 410/4 (genotype B4) and EV71 isolate S2/86/1 (genotype B4) which have been cloned into pCR® Blunt Vector (Zero Blunt™ PCR Cloning Kit, Invitrogen). ..

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A
Article Snippet: .. The Sp1 sites deleted MAO A 2-kb-luc or the core promoter of MAO A (−303/−64 bp, 0.24 kb)-luciferase construct (MAO A 0.24-kb-luc) were generated by PCR, and the PCR product was subcloned into Zero Blunt TOPO vector (PCR cloning kit, Invitrogen) for verifying DNA sequencing. .. Subsequently, the PCR product was subcloned into pGL2-Basic luciferase reporter vector by restriction enzyme digestion (XhoI/Hind III) using self-ligation.

Article Title: Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life
Article Snippet: .. After heat inactivation for 15 min at 70°C, end-repaired DNA (2.5 μL, 20–100 ng/μL) was ligated into pCR-Blunt (0.5 μL, 25 ng/μL) vector (Thermo Fisher Scientific, K270040, following provider’s instructions, 5 μL reaction volume) for 5 h at 20°C before heat inactivation at 70°C for 20 min. We then transformed 2 × 25 μL NEB 10-beta electrocompetent E. coli (DH10B) cells with 2 × 2 μL of the ligation product to create two cells libraries for each sample (two libraries for reproducibility). .. Cells were grown overnight in 10 mL LB medium supplemented with kanamycin (50 μg/mL) for clones amplification ( ).

Generated:

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A
Article Snippet: .. The Sp1 sites deleted MAO A 2-kb-luc or the core promoter of MAO A (−303/−64 bp, 0.24 kb)-luciferase construct (MAO A 0.24-kb-luc) were generated by PCR, and the PCR product was subcloned into Zero Blunt TOPO vector (PCR cloning kit, Invitrogen) for verifying DNA sequencing. .. Subsequently, the PCR product was subcloned into pGL2-Basic luciferase reporter vector by restriction enzyme digestion (XhoI/Hind III) using self-ligation.

Plasmid Preparation:

Article Title: DNA vaccine constructs against enterovirus 71 elicit immune response in mice
Article Snippet: .. 2.1 Construction and validation of DNA vaccine The VP1 gene of EV71 isolate 410/4 (genotype B4) and EV71 isolate S2/86/1 (genotype B4) which have been cloned into pCR® Blunt Vector (Zero Blunt™ PCR Cloning Kit, Invitrogen) were provided by Prof. Mary Jane Cardosa, Universiti Malaysia Sarawak (UNIMAS). .. The VP1 gene of both isolates was amplified by polymerase chain reaction (PCR).

Article Title: The auxin-inducible degradation system enables conditional PERIOD protein depletion in the nervous system of Drosophila melanogaster
Article Snippet: .. Finally, the PCR product of 5arm-AID-EGFP-3arm was cloned into the pCR-Blunt vector (ThermoFisher, k275020, Waltham, MA, USA) through blunt-end cloning. .. For chiRNAs construction, the two target site sequences were cloned into the pU6-BbsI-chiRNA plasmid (Addgene, #45946, Cambridge, MA, USA) as previously described [ ] using the following oligonucleotides: PAM1-sense: 5’-CTTCGACCAGACACAGCACGGGGAT-3’ PAM1-antisense: 5’-AAACATCCCCGTGCTGTGTCTGGTC-3’ PAM2-sense: 5’-CTTCGTCAGCAGCAACTGCGGGTG-3’ PAM2-antisense: 5’-AAACCACCCGCAGTTGCTGCTGAC-3’ The donor and pU6-BbsI-chiRNA plasmids were mixed and injected into embryos of the nos-Cas9 attp2 fly line (Rainbow Transgenic Flies, Inc., Camarillo, CA, USA).

Article Title: DNA vaccine constructs against enterovirus 71 elicit immune response in mice
Article Snippet: .. 6 Acknowledgements We would like to thank Prof. Mary Jane Cardosa from Universiti Malaysia Sarawak (UNIMAS) for kindly providing us the VP1 gene of EV71 isolate 410/4 (genotype B4) and EV71 isolate S2/86/1 (genotype B4) which have been cloned into pCR® Blunt Vector (Zero Blunt™ PCR Cloning Kit, Invitrogen). ..

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A
Article Snippet: .. The Sp1 sites deleted MAO A 2-kb-luc or the core promoter of MAO A (−303/−64 bp, 0.24 kb)-luciferase construct (MAO A 0.24-kb-luc) were generated by PCR, and the PCR product was subcloned into Zero Blunt TOPO vector (PCR cloning kit, Invitrogen) for verifying DNA sequencing. .. Subsequently, the PCR product was subcloned into pGL2-Basic luciferase reporter vector by restriction enzyme digestion (XhoI/Hind III) using self-ligation.

Article Title: Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life
Article Snippet: .. After heat inactivation for 15 min at 70°C, end-repaired DNA (2.5 μL, 20–100 ng/μL) was ligated into pCR-Blunt (0.5 μL, 25 ng/μL) vector (Thermo Fisher Scientific, K270040, following provider’s instructions, 5 μL reaction volume) for 5 h at 20°C before heat inactivation at 70°C for 20 min. We then transformed 2 × 25 μL NEB 10-beta electrocompetent E. coli (DH10B) cells with 2 × 2 μL of the ligation product to create two cells libraries for each sample (two libraries for reproducibility). .. Cells were grown overnight in 10 mL LB medium supplemented with kanamycin (50 μg/mL) for clones amplification ( ).

Sequencing:

Article Title: Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants
Article Snippet: .. Sub-cloning of PCR products PCR products amplified from F0 DNA showing complex sequencing traces were sub-cloned using a Zero-Blunt PCR Cloning Kit (Invitrogen). ..

Article Title: TILLING by sequencing to identify induced mutations in stress resistance genes of peanut (Arachis hypogaea)
Article Snippet: .. Mutants identified from CAPS assay and SSCP were further confirmed by Sanger sequencing of amplicons (single copy genes) or performing PCR cloning with Zero-Blunt PCR cloning kit (Life Technologies, catalogue no. K 2700–20) prior to sequencing. .. The mutation effect was analysed by SIFT (Sorting Intolerant from Tolerant, http://sift.jcvi.org/ ) with default parameters [ ].

Transformation Assay:

Article Title: Nasal Resistome Development in Infants With Cystic Fibrosis in the First Year of Life
Article Snippet: .. After heat inactivation for 15 min at 70°C, end-repaired DNA (2.5 μL, 20–100 ng/μL) was ligated into pCR-Blunt (0.5 μL, 25 ng/μL) vector (Thermo Fisher Scientific, K270040, following provider’s instructions, 5 μL reaction volume) for 5 h at 20°C before heat inactivation at 70°C for 20 min. We then transformed 2 × 25 μL NEB 10-beta electrocompetent E. coli (DH10B) cells with 2 × 2 μL of the ligation product to create two cells libraries for each sample (two libraries for reproducibility). .. Cells were grown overnight in 10 mL LB medium supplemented with kanamycin (50 μg/mL) for clones amplification ( ).

Derivative Assay:

Article Title: Live Attenuated Influenza Vaccine contains Substantial and Unexpected Amounts of Defective Viral Genomic RNA
Article Snippet: .. Bands representing PCR products amplified directly from the Fluenz™ Tetra vaccine batch CH2020 and derived from full-length RNAs and putative DI RNAs were extracted from the gel, cloned using the Zero Blunt PCR cloning kit (Invitrogen, Paisley, Scotland, UK), and sequenced. .. This confirmed that influenza A and B segment 1–3 RNAs were derived from the relevant master donor strain [ , ].

DNA Sequencing:

Article Title: R1, a Novel Repressor of the Human Monoamine Oxidase A
Article Snippet: .. The Sp1 sites deleted MAO A 2-kb-luc or the core promoter of MAO A (−303/−64 bp, 0.24 kb)-luciferase construct (MAO A 0.24-kb-luc) were generated by PCR, and the PCR product was subcloned into Zero Blunt TOPO vector (PCR cloning kit, Invitrogen) for verifying DNA sequencing. .. Subsequently, the PCR product was subcloned into pGL2-Basic luciferase reporter vector by restriction enzyme digestion (XhoI/Hind III) using self-ligation.

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