Structured Review

Promega hpa ii msp
Methylation-sensitive amplified polymorphism (MSAP) band patterning determined by <t>Hpa</t> II/ <t>Msp</t> I isoschizomers and correspondence with methylation status of CCGG sites. Black squares indicate methylated cytosines.
Hpa Ii Msp, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 10567 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum"

Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Journal: Ecology and Evolution

doi: 10.1002/ece3.758

Methylation-sensitive amplified polymorphism (MSAP) band patterning determined by Hpa II/ Msp I isoschizomers and correspondence with methylation status of CCGG sites. Black squares indicate methylated cytosines.
Figure Legend Snippet: Methylation-sensitive amplified polymorphism (MSAP) band patterning determined by Hpa II/ Msp I isoschizomers and correspondence with methylation status of CCGG sites. Black squares indicate methylated cytosines.

Techniques Used: Methylation, Amplification

2) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

3) Product Images from "Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes"

Article Title: Rapid and efficient genome-wide characterization of Xanthomonas TAL effector genes

Journal: Scientific Reports

doi: 10.1038/srep13162

Msc I partial digestion of talC-Bam HI fragment cloned in pUC19. 2 μg plasmid DNA was digested with 5 u Msc I enzyme. The digestion times are given in minutes above the gel. M, 100 bp DNA ladder makers.
Figure Legend Snippet: Msc I partial digestion of talC-Bam HI fragment cloned in pUC19. 2 μg plasmid DNA was digested with 5 u Msc I enzyme. The digestion times are given in minutes above the gel. M, 100 bp DNA ladder makers.

Techniques Used: Clone Assay, Plasmid Preparation

4) Product Images from "Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications"

Article Title: Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications

Journal: Nucleic Acids Research

doi:

Strategy used to construct DNA molecules. A 229 bp PCR fragment that contains Eco RI and Bam HI cleavage sites in the middle was generated from pUC19 by 20 rounds of PCR. Primer sequences are given in Materials and Methods. After PCR the sample was purified on an agarose gel (Zymoclean) and digested with Eco RI and Bam HI to release a 17 bp fragment (insert 1, gray). The 5′-fragment (101 bp) containing an Eco RI sticky end (white) and 3′-fragment (103 bp) containing a Bam HI sticky end (black) were gel purified. The desired insert (insert 2, gray) with a Mfe I site on one end and a Bcl I site on the other was inserted between the 5′- and 3′-fragments using T4 DNA ligase. Inserts used in these experiments contained unmodified dsDNA, single bulged A-containing dsDNA or ribose-containing dsDNA. All inserts were 20 bp and had four 5′-dangling nucleotides on each end with a 5′-phosphate, denoted with a p. Since the desired product with a single insert has lost all four restriction sites, it can be enriched from the four side-products by digestion with the four restriction enzymes, followed by agarose gel purification (Zymoclean). Ten rounds of thermal cycling were used to recycle the side-products and increase the overall yield.
Figure Legend Snippet: Strategy used to construct DNA molecules. A 229 bp PCR fragment that contains Eco RI and Bam HI cleavage sites in the middle was generated from pUC19 by 20 rounds of PCR. Primer sequences are given in Materials and Methods. After PCR the sample was purified on an agarose gel (Zymoclean) and digested with Eco RI and Bam HI to release a 17 bp fragment (insert 1, gray). The 5′-fragment (101 bp) containing an Eco RI sticky end (white) and 3′-fragment (103 bp) containing a Bam HI sticky end (black) were gel purified. The desired insert (insert 2, gray) with a Mfe I site on one end and a Bcl I site on the other was inserted between the 5′- and 3′-fragments using T4 DNA ligase. Inserts used in these experiments contained unmodified dsDNA, single bulged A-containing dsDNA or ribose-containing dsDNA. All inserts were 20 bp and had four 5′-dangling nucleotides on each end with a 5′-phosphate, denoted with a p. Since the desired product with a single insert has lost all four restriction sites, it can be enriched from the four side-products by digestion with the four restriction enzymes, followed by agarose gel purification (Zymoclean). Ten rounds of thermal cycling were used to recycle the side-products and increase the overall yield.

Techniques Used: Construct, Polymerase Chain Reaction, Generated, Purification, Agarose Gel Electrophoresis

5) Product Images from "Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II"

Article Title: Single-molecule FRET method to investigate the dynamics of transcription elongation through the nucleosome by RNA polymerase II

Journal: Methods (San Diego, Calif.)

doi: 10.1016/j.ymeth.2019.01.009

The schematics of the single-molecule FRET system to investigate the dynamics of the nucleosome structure during transcription elongation. (A) Nucleosome assembled on the shown template is complexed with RNAPII (yeast Pol II). Rpb1 CTD antibody is immobilized on a microscope slide surface via biotinylated Protein A that is conjugated to streptavidin coated on the slide. A FRET pair (Cy3 and Atto647N) is labeled at the +34 th and +112 th nucleotide. This FRET pair location is sensitive to the nucleosomal dynamics at the proximal-dimer/DNA contact region. (B) The sequence of the upper and lower strands of the transcription template illustrated in A .
Figure Legend Snippet: The schematics of the single-molecule FRET system to investigate the dynamics of the nucleosome structure during transcription elongation. (A) Nucleosome assembled on the shown template is complexed with RNAPII (yeast Pol II). Rpb1 CTD antibody is immobilized on a microscope slide surface via biotinylated Protein A that is conjugated to streptavidin coated on the slide. A FRET pair (Cy3 and Atto647N) is labeled at the +34 th and +112 th nucleotide. This FRET pair location is sensitive to the nucleosomal dynamics at the proximal-dimer/DNA contact region. (B) The sequence of the upper and lower strands of the transcription template illustrated in A .

Techniques Used: Microscopy, Labeling, Sequencing

6) Product Images from "Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 ▿Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 ▿ †"

Article Title: Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 ▿Systematic Assembly of a Full-Length Infectious Clone of Human Coronavirus NL63 ▿ †

Journal:

doi: 10.1128/JVI.01804-08

Verification of the marker mutation in rescued virus from icNL63 and icNL63gfp. A silent BstAPI site introduced into both clones at the NL63-D and NL63-E or NL63-Egfp junctions was used to verify that the viruses rescued from the transfection flasks were
Figure Legend Snippet: Verification of the marker mutation in rescued virus from icNL63 and icNL63gfp. A silent BstAPI site introduced into both clones at the NL63-D and NL63-E or NL63-Egfp junctions was used to verify that the viruses rescued from the transfection flasks were

Techniques Used: Marker, Mutagenesis, Clone Assay, Transfection

7) Product Images from "Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome"

Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome

Journal: G3: Genes|Genomes|Genetics

doi: 10.1534/g3.119.400613

Florescence assisted cell sorting (FACS) of NeuN stained nuclei. Representative image showing FACS separation of NeuN+ and NeuN- DAPI stained nuclei. Boxes indicate gating for populations of nuclei that were collecting for gDNA isolation. Only NeuN+ gDNA was utilized for this work.
Figure Legend Snippet: Florescence assisted cell sorting (FACS) of NeuN stained nuclei. Representative image showing FACS separation of NeuN+ and NeuN- DAPI stained nuclei. Boxes indicate gating for populations of nuclei that were collecting for gDNA isolation. Only NeuN+ gDNA was utilized for this work.

Techniques Used: FACS, Staining, Isolation

Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.
Figure Legend Snippet: Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.

Techniques Used: Sequencing, Isolation, Polymerase Chain Reaction, Construct

8) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

9) Product Images from "The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus"

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040192

DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
Figure Legend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

Techniques Used: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.
Figure Legend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

Techniques Used: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography

10) Product Images from "Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay"

Article Title: Sensitive, multiplex and direct quantification of RNA sequences using a modified RASL assay

Journal: Nucleic Acids Research

doi: 10.1093/nar/gku636

Measurement of T4 DNA Ligase and Rnl2 probe joining activity on DNA and RNA templates. ( A ) 500 nM of each adapter-M13 probe was annealed on the indicated 100 nM template (60°C for 10 min, 45°C for 30 min) in 1× T4 DNA ligase buffer. Complexes were then diluted 1000-fold into a 20 μl ligation reaction containing 5 U of T4 DNA ligase and incubated at 37°C for 30 min. Ligation products were then diluted 1000-fold into SYBR green master mix before undergoing PCR. qPCR signals are expressed as percent of the ssDNA template condition. ( B ) Capillary electrophoresis of ligation products. A FAM-labeled donor probe at 1 μM and the indicated hybrid diribo- or deoxyribo-acceptor oligo (10 μM) were annealed on a synthetic GAPDH RNA template (10 μM) or no template and then diluted 10-fold into the indicated ligation reaction and incubated for 30 min at 37°C. The product of this ligation was then diluted 10-fold in stopping buffer and measured by capillary electrophoresis. ( C ) Seven GAPDH probe sets were tested with the indicated ligase in the RASL assay, using 50 ng of prostate RNA as template in a 20 μl reaction. After 10 cycles of pre-amplification, the product of each probe set was separately analyzed by qPCR using probe set-specific nested detection primers. ( D ) Sensitivity of an Rnl2-based RASL assay, determined by serial dilution of a synthetic M13 RNA template into a background of 50 ng prostate RNA (the equivalent of ∼2000 cells). Data reported are the mean of at least three independent replicates; error bars are S.E.M. ND, not detected.
Figure Legend Snippet: Measurement of T4 DNA Ligase and Rnl2 probe joining activity on DNA and RNA templates. ( A ) 500 nM of each adapter-M13 probe was annealed on the indicated 100 nM template (60°C for 10 min, 45°C for 30 min) in 1× T4 DNA ligase buffer. Complexes were then diluted 1000-fold into a 20 μl ligation reaction containing 5 U of T4 DNA ligase and incubated at 37°C for 30 min. Ligation products were then diluted 1000-fold into SYBR green master mix before undergoing PCR. qPCR signals are expressed as percent of the ssDNA template condition. ( B ) Capillary electrophoresis of ligation products. A FAM-labeled donor probe at 1 μM and the indicated hybrid diribo- or deoxyribo-acceptor oligo (10 μM) were annealed on a synthetic GAPDH RNA template (10 μM) or no template and then diluted 10-fold into the indicated ligation reaction and incubated for 30 min at 37°C. The product of this ligation was then diluted 10-fold in stopping buffer and measured by capillary electrophoresis. ( C ) Seven GAPDH probe sets were tested with the indicated ligase in the RASL assay, using 50 ng of prostate RNA as template in a 20 μl reaction. After 10 cycles of pre-amplification, the product of each probe set was separately analyzed by qPCR using probe set-specific nested detection primers. ( D ) Sensitivity of an Rnl2-based RASL assay, determined by serial dilution of a synthetic M13 RNA template into a background of 50 ng prostate RNA (the equivalent of ∼2000 cells). Data reported are the mean of at least three independent replicates; error bars are S.E.M. ND, not detected.

Techniques Used: Activity Assay, Ligation, Incubation, SYBR Green Assay, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Electrophoresis, Labeling, Amplification, Serial Dilution

Probe length analysis and design pipeline. ( A ) and ( B ) Independent probe sets targeting GAPDH and M13 were synthesized as both donor and acceptor oligos ranging in target sequence length from 12 to 22 nucleotides; the junction between acceptor and donor probes was kept constant. These oligos were pooled such that the total concentration of each pooled probe set was 5 nM in the final RASL-seq assay. The lengths of correctly (on target) ligated donor or acceptor probes were determined by deep sequencing, and each length's contribution to the total number of observed read counts was tabulated. Data from the GAPDH_1 probe sets are shown when prostate RNA, yeast RNA or no template was included in the assay. ( C ) The contribution of each probe length to off-target ligations (defined as inappropriate ligation between different probe sets) is shown for the prostate RNA template condition. The scale is different because the off-target ligations are relatively rare compared to the on-target ligations. ( D ) The probe design pipeline takes the 1 kb 3′-sequence upstream of the poly(A) and uses Primer3 to generate candidate 36 nt RASL probe sequences (reverse complement of mRNA sense strand). After extension of 4 nt in the poly(A) direction, splitting of the 40mer into acceptor and donor, and appending of appropriate adapters, the properties of the adaptor-appended probe oligos are calculated. Probes are then filtered through a quality control (QC) step (‘Materials and Methods’ section), which removes ∼75% of the candidates (based on analysis of 1000 transcripts). Successfully filtered probes are finally ranked by a combination of proximity to poly(A) and Primer3 penalty (based on the original 36 nt candidate probe), such that the best probe sets can be selected for production. Gray boxes denote processes and white boxes denote the outcomes.
Figure Legend Snippet: Probe length analysis and design pipeline. ( A ) and ( B ) Independent probe sets targeting GAPDH and M13 were synthesized as both donor and acceptor oligos ranging in target sequence length from 12 to 22 nucleotides; the junction between acceptor and donor probes was kept constant. These oligos were pooled such that the total concentration of each pooled probe set was 5 nM in the final RASL-seq assay. The lengths of correctly (on target) ligated donor or acceptor probes were determined by deep sequencing, and each length's contribution to the total number of observed read counts was tabulated. Data from the GAPDH_1 probe sets are shown when prostate RNA, yeast RNA or no template was included in the assay. ( C ) The contribution of each probe length to off-target ligations (defined as inappropriate ligation between different probe sets) is shown for the prostate RNA template condition. The scale is different because the off-target ligations are relatively rare compared to the on-target ligations. ( D ) The probe design pipeline takes the 1 kb 3′-sequence upstream of the poly(A) and uses Primer3 to generate candidate 36 nt RASL probe sequences (reverse complement of mRNA sense strand). After extension of 4 nt in the poly(A) direction, splitting of the 40mer into acceptor and donor, and appending of appropriate adapters, the properties of the adaptor-appended probe oligos are calculated. Probes are then filtered through a quality control (QC) step (‘Materials and Methods’ section), which removes ∼75% of the candidates (based on analysis of 1000 transcripts). Successfully filtered probes are finally ranked by a combination of proximity to poly(A) and Primer3 penalty (based on the original 36 nt candidate probe), such that the best probe sets can be selected for production. Gray boxes denote processes and white boxes denote the outcomes.

Techniques Used: Synthesized, Sequencing, Concentration Assay, Ligation

11) Product Images from "Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae"

Article Title: Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae

Journal: BMC Microbiology

doi: 10.1186/1471-2180-9-239

Schematic illustrating the strategy used to modify the nfsB coding region . Each numbered arrow corresponds to the procedures summarized below: 1: PCR using primers NfsBsmI-3F and NfsBsmI-2R to introduce a Bsm I recognition sequence and to alter a poly-A tract. 2: Treatment with S1 nuclease to create blunt ends, polynucleotide kinase to phosphorylate 5' ends, and T4 DNA ligase. E. coli were transformed using this construct (pEC2). Plasmid DNA was isolated by alkaline lysis. 3: Treatment with Bsm I to generate pEC1. Digestion product was ligated with T4 DNA ligase. The construct was transformed into E. coli . 4: pEC1 was amplified with primers dwnstrm-F and dwnstrm-R. The product was ligated to the omega fragment, a PCR product of pHP45Σ with the OmegaABC primer, to generate pEC3. The omega fragment is symmetric, so one primer amplifies in both directions
Figure Legend Snippet: Schematic illustrating the strategy used to modify the nfsB coding region . Each numbered arrow corresponds to the procedures summarized below: 1: PCR using primers NfsBsmI-3F and NfsBsmI-2R to introduce a Bsm I recognition sequence and to alter a poly-A tract. 2: Treatment with S1 nuclease to create blunt ends, polynucleotide kinase to phosphorylate 5' ends, and T4 DNA ligase. E. coli were transformed using this construct (pEC2). Plasmid DNA was isolated by alkaline lysis. 3: Treatment with Bsm I to generate pEC1. Digestion product was ligated with T4 DNA ligase. The construct was transformed into E. coli . 4: pEC1 was amplified with primers dwnstrm-F and dwnstrm-R. The product was ligated to the omega fragment, a PCR product of pHP45Σ with the OmegaABC primer, to generate pEC3. The omega fragment is symmetric, so one primer amplifies in both directions

Techniques Used: Polymerase Chain Reaction, Introduce, Sequencing, Transformation Assay, Construct, Plasmid Preparation, Isolation, Alkaline Lysis, Amplification

12) Product Images from "Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma"

Article Title: Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma

Journal: PLoS ONE

doi: 10.1371/journal.pone.0150479

Schematic overview of generation of human/mouse chimeric anti-Id Ab ganglidiximab. The human/mouse chimeric anti-Id Ab ganglidiximab is composed of GD 2 mimicking variable regions (VH, VL) of murine anti-Id ganglidiomab and human IgG1 constant regions. Coding sequences of VH and VL were synthesized and inserted into mammalian expression plasmids containing DNA sequences for human IgG1 heavy (p3-IgG1-HC) and light chain (p3-IgG1-LC), respectively. For ganglidiximab production, CHO cells were stably co-transfected with the two generated expression plasmids (p3-ganglidiximab-HC and p3-ganglidiximab-LC).
Figure Legend Snippet: Schematic overview of generation of human/mouse chimeric anti-Id Ab ganglidiximab. The human/mouse chimeric anti-Id Ab ganglidiximab is composed of GD 2 mimicking variable regions (VH, VL) of murine anti-Id ganglidiomab and human IgG1 constant regions. Coding sequences of VH and VL were synthesized and inserted into mammalian expression plasmids containing DNA sequences for human IgG1 heavy (p3-IgG1-HC) and light chain (p3-IgG1-LC), respectively. For ganglidiximab production, CHO cells were stably co-transfected with the two generated expression plasmids (p3-ganglidiximab-HC and p3-ganglidiximab-LC).

Techniques Used: Synthesized, Expressing, Stable Transfection, Transfection, Generated

Amplification of DNA fragments encoding for GD 2 -mimicking paratopes of ganglidiximab. ( A ) Visualization of coding sequences of GD 2 -mimicking variable heavy (VH; 440 bp) and light chain (VL; 420 bp) amplified by RT-PCR. RNA was isolated from hybridoma cells producing murine anti-Id ganglidiomab. PCR products were analyzed by agarose gel electrophoresis. Representative image is shown. NTC—no-template-control. M—Marker (100-bp). ( B ) PCR products were cloned into pCR ® 2.1-TOPO ® plasmids and analyzed by restriction enzyme digest to excise DNA sequences encoding for VH and VL (product sizes 427 bp and 407 bp, respectively). Resulting DNA fragments were analyzed by agarose gel electrophoresis. Representative image is shown. M—Marker (2-log, 0.1–10.0 kbp).
Figure Legend Snippet: Amplification of DNA fragments encoding for GD 2 -mimicking paratopes of ganglidiximab. ( A ) Visualization of coding sequences of GD 2 -mimicking variable heavy (VH; 440 bp) and light chain (VL; 420 bp) amplified by RT-PCR. RNA was isolated from hybridoma cells producing murine anti-Id ganglidiomab. PCR products were analyzed by agarose gel electrophoresis. Representative image is shown. NTC—no-template-control. M—Marker (100-bp). ( B ) PCR products were cloned into pCR ® 2.1-TOPO ® plasmids and analyzed by restriction enzyme digest to excise DNA sequences encoding for VH and VL (product sizes 427 bp and 407 bp, respectively). Resulting DNA fragments were analyzed by agarose gel electrophoresis. Representative image is shown. M—Marker (2-log, 0.1–10.0 kbp).

Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Clone Assay

13) Product Images from "Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum"

Article Title: Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

Journal: Ecology and Evolution

doi: 10.1002/ece3.758

Methylation-sensitive amplified polymorphism (MSAP) band patterning determined by Hpa II/ Msp I isoschizomers and correspondence with methylation status of CCGG sites. Black squares indicate methylated cytosines.
Figure Legend Snippet: Methylation-sensitive amplified polymorphism (MSAP) band patterning determined by Hpa II/ Msp I isoschizomers and correspondence with methylation status of CCGG sites. Black squares indicate methylated cytosines.

Techniques Used: Methylation, Amplification

14) Product Images from "Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil"

Article Title: Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02573-16

Complementation of S. cerevisiae YH1-DHFR::KanMX4 with the p414GPD.LauDHFR-TS plasmid. Transformant isolates were grown on rich YEPD medium supplemented with Geneticin in the presence or absence of dTMP. The plates were incubated at 30°C for 5 days. Only clones containing the p414GPD.LauDHFR-TS plasmid grew in the absence of TMP.
Figure Legend Snippet: Complementation of S. cerevisiae YH1-DHFR::KanMX4 with the p414GPD.LauDHFR-TS plasmid. Transformant isolates were grown on rich YEPD medium supplemented with Geneticin in the presence or absence of dTMP. The plates were incubated at 30°C for 5 days. Only clones containing the p414GPD.LauDHFR-TS plasmid grew in the absence of TMP.

Techniques Used: Plasmid Preparation, Incubation, Clone Assay

15) Product Images from "Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation"

Article Title: Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

Journal: Molecules and Cells

doi: 10.14348/molcells.2016.0159

Characterization of ura1 mutants resistant to oncogenic Agrobacterium tumefaciens A208. (A) Sterile root segments of wild-type (WT), ura1 mutant, and F1 progeny plants were infected with A. tumefaciens A208 (OD 600 = 1.0). After 2 days co-cultivation, tumors were induced on MS basal medium containing carbenicillin (100 μg/ml) for 4 weeks. (B) Phenotypes of 4-week-old WT and ura1 mutant plants. Leaf variegation showing pale green area is associated with the ura1 mutation. (C) Genomic organization of the AtVDAC1 locus composed of six-exons (grey boxes) and a T-DNA (black triangle) insertion site at the 6th exon of the AtVDAC1 gene leading to the ura1 mutant. (D) Expression of AtVDAC1 in WT and ura1 mutant plants. (E) Organ-specific expression of AtVDAC1 . rRNA was used as a loading control. F, flowers; L, leaves; R, root tissue.
Figure Legend Snippet: Characterization of ura1 mutants resistant to oncogenic Agrobacterium tumefaciens A208. (A) Sterile root segments of wild-type (WT), ura1 mutant, and F1 progeny plants were infected with A. tumefaciens A208 (OD 600 = 1.0). After 2 days co-cultivation, tumors were induced on MS basal medium containing carbenicillin (100 μg/ml) for 4 weeks. (B) Phenotypes of 4-week-old WT and ura1 mutant plants. Leaf variegation showing pale green area is associated with the ura1 mutation. (C) Genomic organization of the AtVDAC1 locus composed of six-exons (grey boxes) and a T-DNA (black triangle) insertion site at the 6th exon of the AtVDAC1 gene leading to the ura1 mutant. (D) Expression of AtVDAC1 in WT and ura1 mutant plants. (E) Organ-specific expression of AtVDAC1 . rRNA was used as a loading control. F, flowers; L, leaves; R, root tissue.

Techniques Used: Mutagenesis, Infection, Mass Spectrometry, Expressing

Overexpression of AtVDAC1 enhanced transient T-DNA gene expression. Sterile root segments of WT, ura1 mutant and transgenic lines overexpressing AtVDAC1 were infected with Agrobacterium GV3101 containing pBISN1. Standard (A; OD 600 = 1.0) and diluted Agrobacterium suspensions (B; OD 600 = 0.1). Two days after co-cultivation, the root segments were stained with X-gluc and the percentage of root segments showing GUS activity was determined. Asterisks indicate a significant difference from WT plants using a t -test ( * p
Figure Legend Snippet: Overexpression of AtVDAC1 enhanced transient T-DNA gene expression. Sterile root segments of WT, ura1 mutant and transgenic lines overexpressing AtVDAC1 were infected with Agrobacterium GV3101 containing pBISN1. Standard (A; OD 600 = 1.0) and diluted Agrobacterium suspensions (B; OD 600 = 0.1). Two days after co-cultivation, the root segments were stained with X-gluc and the percentage of root segments showing GUS activity was determined. Asterisks indicate a significant difference from WT plants using a t -test ( * p

Techniques Used: Over Expression, Expressing, Mutagenesis, Transgenic Assay, Infection, Staining, Activity Assay

Effects of phytohormone pretreatment on transient T-DNA gene expression and expression profiles of AtVDAC gene family. (A) Sterile root segments of WT, ura1 mutant, and transgenic lines overexpressing AtVDAC1 were incubated on callus inducing medium (CIM) containing phytohormones for 2 days. As described in Materials and Methods, root segments were inoculated with Agrobacterium GV3101containing pBISN1. Two days after co-cultivation, the root segments were stained with X-gluc to determine the efficiency of transient GUS expression. Bar = 0.5 cm. (B) Total RNAs were isolated from whole Arabidopsis WT plants cultivated in CIM for indicated periods. RNA blot analysis was conducted using each AtVDAC full length cDNA as a probe.
Figure Legend Snippet: Effects of phytohormone pretreatment on transient T-DNA gene expression and expression profiles of AtVDAC gene family. (A) Sterile root segments of WT, ura1 mutant, and transgenic lines overexpressing AtVDAC1 were incubated on callus inducing medium (CIM) containing phytohormones for 2 days. As described in Materials and Methods, root segments were inoculated with Agrobacterium GV3101containing pBISN1. Two days after co-cultivation, the root segments were stained with X-gluc to determine the efficiency of transient GUS expression. Bar = 0.5 cm. (B) Total RNAs were isolated from whole Arabidopsis WT plants cultivated in CIM for indicated periods. RNA blot analysis was conducted using each AtVDAC full length cDNA as a probe.

Techniques Used: Expressing, Mutagenesis, Transgenic Assay, Incubation, Staining, Isolation, Northern blot

16) Product Images from "Enterohemorrhagic Escherichia coli O157:H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia"

Article Title: Enterohemorrhagic Escherichia coli O157:H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004889

EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation. Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures ) at 37°C in 5% CO 2 . Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO 2 . Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p
Figure Legend Snippet: EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation. Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures ) at 37°C in 5% CO 2 . Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO 2 . Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p

Techniques Used: Inhibition, Cell Culture, Infection, Western Blot, Staining, Activation Assay, Software, Plasmid Preparation, Positive Control

17) Product Images from "Analysis of RP2 and RPGR Mutations in Five X-Linked Chinese Families with Retinitis Pigmentosa"

Article Title: Analysis of RP2 and RPGR Mutations in Five X-Linked Chinese Families with Retinitis Pigmentosa

Journal: Scientific Reports

doi: 10.1038/srep44465

( A ) Schematic diagram of the self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR. The blue line denoted the known sequence in intron3 of RP2 gene. The unknown region is a particular region to be investigated containing the breakpoints of the deletion. Capital E represents the exon. Small arrows with the letter P represent the sites of the primer. ( B ) After digestion with BglII and then circularization with T4 DNA ligase followed by inverse PCR, an approximately 750 bp fragment (A band) was produced from an affected male (AF) and a female carrier (FC), while an approximately 1.2 kb fragment (B band) was produced from the female carrier and a normal control (NC).
Figure Legend Snippet: ( A ) Schematic diagram of the self-ligation of restriction endonuclease-digested DNA fragments with long-distance inverse PCR. The blue line denoted the known sequence in intron3 of RP2 gene. The unknown region is a particular region to be investigated containing the breakpoints of the deletion. Capital E represents the exon. Small arrows with the letter P represent the sites of the primer. ( B ) After digestion with BglII and then circularization with T4 DNA ligase followed by inverse PCR, an approximately 750 bp fragment (A band) was produced from an affected male (AF) and a female carrier (FC), while an approximately 1.2 kb fragment (B band) was produced from the female carrier and a normal control (NC).

Techniques Used: Ligation, Inverse PCR, Sequencing, Produced

18) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Journal: Nucleic Acids Research

doi:

Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

19) Product Images from "Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *"

Article Title: Reconstitution of Uracil DNA Glycosylase-initiated Base Excision Repair in Herpes Simplex Virus-1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.010413

Reconstitution of the initial stages of BER. Storage phosphorimage showing the products of the reconstitution reaction. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Lane 1 , UL2, APE, and UL30; lane 2 , APE and UL30; lane 3 , UL2 and UL30; lane 4 , UL2 and APE; lane 5 , DNA only; lane 6 , E. coli UDG, APE and Pol β; lane 7 , APE and Pol β; lane 8 , E. coli UDG and Pol β; lane 9 , E. coli UDG and APE. The position of a linear 100-mer marker is as indicated.
Figure Legend Snippet: Reconstitution of the initial stages of BER. Storage phosphorimage showing the products of the reconstitution reaction. Reactions were performed as described under “Experimental Procedures” with the indicated proteins. Lane 1 , UL2, APE, and UL30; lane 2 , APE and UL30; lane 3 , UL2 and UL30; lane 4 , UL2 and APE; lane 5 , DNA only; lane 6 , E. coli UDG, APE and Pol β; lane 7 , APE and Pol β; lane 8 , E. coli UDG and Pol β; lane 9 , E. coli UDG and APE. The position of a linear 100-mer marker is as indicated.

Techniques Used: Marker

Ligase IIIα-XRCC1 confers specificity onto the BER reaction. Storage phosphor image of reactions performed with either ligase IIIα-XRCC1 ( lanes 1–6 ) or ligase I ( lanes 7–12 ). Standard reactions ( lanes 1 and 7 ) contained UL2, APE, and UL30. Lanes 2 and 8 , standard reactions with UL42; lanes 3 and 9 , substitution of UL2 with E. coli UDG; lanes 4 and 10 , substitution of UL30 with Pol β; lanes 5 and 11 , substitution of UL30 with exonuclease-deficient Klenow Pol; lanes 6 and 12 , substitution of UL30 with Pol δ. The positions of nicked ( N ) and ligated ( L ) products are as indicated.
Figure Legend Snippet: Ligase IIIα-XRCC1 confers specificity onto the BER reaction. Storage phosphor image of reactions performed with either ligase IIIα-XRCC1 ( lanes 1–6 ) or ligase I ( lanes 7–12 ). Standard reactions ( lanes 1 and 7 ) contained UL2, APE, and UL30. Lanes 2 and 8 , standard reactions with UL42; lanes 3 and 9 , substitution of UL2 with E. coli UDG; lanes 4 and 10 , substitution of UL30 with Pol β; lanes 5 and 11 , substitution of UL30 with exonuclease-deficient Klenow Pol; lanes 6 and 12 , substitution of UL30 with Pol δ. The positions of nicked ( N ) and ligated ( L ) products are as indicated.

Techniques Used:

20) Product Images from "Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications"

Article Title: Efficient construction of long DNA duplexes with internal non-Watson-Crick motifs and modifications

Journal: Nucleic Acids Research

doi:

Strategy used to construct DNA molecules. A 229 bp PCR fragment that contains Eco RI and Bam HI cleavage sites in the middle was generated from pUC19 by 20 rounds of PCR. Primer sequences are given in Materials and Methods. After PCR the sample was purified on an agarose gel (Zymoclean) and digested with Eco RI and Bam HI to release a 17 bp fragment (insert 1, gray). The 5′-fragment (101 bp) containing an Eco RI sticky end (white) and 3′-fragment (103 bp) containing a Bam HI sticky end (black) were gel purified. The desired insert (insert 2, gray) with a Mfe I site on one end and a Bcl I site on the other was inserted between the 5′- and 3′-fragments using T4 DNA ligase. Inserts used in these experiments contained unmodified dsDNA, single bulged A-containing dsDNA or ribose-containing dsDNA. All inserts were 20 bp and had four 5′-dangling nucleotides on each end with a 5′-phosphate, denoted with a p. Since the desired product with a single insert has lost all four restriction sites, it can be enriched from the four side-products by digestion with the four restriction enzymes, followed by agarose gel purification (Zymoclean). Ten rounds of thermal cycling were used to recycle the side-products and increase the overall yield.
Figure Legend Snippet: Strategy used to construct DNA molecules. A 229 bp PCR fragment that contains Eco RI and Bam HI cleavage sites in the middle was generated from pUC19 by 20 rounds of PCR. Primer sequences are given in Materials and Methods. After PCR the sample was purified on an agarose gel (Zymoclean) and digested with Eco RI and Bam HI to release a 17 bp fragment (insert 1, gray). The 5′-fragment (101 bp) containing an Eco RI sticky end (white) and 3′-fragment (103 bp) containing a Bam HI sticky end (black) were gel purified. The desired insert (insert 2, gray) with a Mfe I site on one end and a Bcl I site on the other was inserted between the 5′- and 3′-fragments using T4 DNA ligase. Inserts used in these experiments contained unmodified dsDNA, single bulged A-containing dsDNA or ribose-containing dsDNA. All inserts were 20 bp and had four 5′-dangling nucleotides on each end with a 5′-phosphate, denoted with a p. Since the desired product with a single insert has lost all four restriction sites, it can be enriched from the four side-products by digestion with the four restriction enzymes, followed by agarose gel purification (Zymoclean). Ten rounds of thermal cycling were used to recycle the side-products and increase the overall yield.

Techniques Used: Construct, Polymerase Chain Reaction, Generated, Purification, Agarose Gel Electrophoresis

21) Product Images from "Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity †"

Article Title: Stolbur Phytoplasma Genome Survey Achieved Using a Suppression Subtractive Hybridization Approach with High Specificity †

Journal:

doi: 10.1128/AEM.72.5.3274-3283.2006

PCR amplification of SSH orphan sequences. PCRs were performed on healthy periwinkle DNA (lane 1) and stolbur phytoplasma-infected periwinkle DNA (lane 2) for HincII-SSH libraries and RsaI-SSH libraries. Clone names are indicated according to libraries.
Figure Legend Snippet: PCR amplification of SSH orphan sequences. PCRs were performed on healthy periwinkle DNA (lane 1) and stolbur phytoplasma-infected periwinkle DNA (lane 2) for HincII-SSH libraries and RsaI-SSH libraries. Clone names are indicated according to libraries.

Techniques Used: Polymerase Chain Reaction, Amplification, Infection

22) Product Images from "Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins"

Article Title: Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00555-19

SDS-PAGE and spot test analysis of induced, affinity-purified ORF28 lysin. (A) SDS-PAGE analysis of cellular proteins from IPTG-induced culture of E. coli BL21/DE3(pGEX4T2±ORF28) harboring either empty vector (pGEX4T2) or recombinant plasmid (pGEX4T2-ORF28) containing ORF28. Lanes: M, molecular mass standards; 1, sonic extract (SE) of induced E. coli BL21/DE3(pGEX4T2) harboring empty vector pGEXT4t2 (control; note overexpressed glutathione S -transferase [GST] protein band [plus linker oligopeptide] at ∼26.5 kDa); 2, SE of induced E. coli BL21/DE3(pGEX4T2-ORF28) harboring the recombinant plasmid pGEX4T2-ORF28 containing ORF28 (note overexpressed 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST [minus linker oligopeptide]); 3, flowthrough (unbound proteins) of glutathione affinity column following application of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) culture (note relative depletion of 72.5-kDa and ∼24.0-kDa proteins compared to initial SE); 4 and 5, affinity-purified proteins from induced E. coli BL21/DE3(pGEX4T2-ORF28) SE. SE were applied to the glutathione affinity column, and after extensive washing, bound proteins were desorbed by the addition of glutathione solution (note the 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST band); 6 and 7, protein recovered from affinity column following treatment of bound 72.5-kDa fusion protein with thrombin. The linkage between the ORF28 protein and the GST in the 72.5-kDa fusion protein is thrombin sensitive. The ORF28 protein was thereby released from the GST, which remained bound to the affinity column (note the single 46.1-kDa protein band recovered). (B) Lytic activity, in E. faecalis JH2-2 lawn, of the crude SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) (lane 2 in SDS-PAGE), affinity-purified ORF28-GST fusion protein (material seen in lanes 4 and 5 of SDS-PAGE), and affinity-purified ORF28 released from the affinity column by thrombin digestion of bound ORF28-GST fusion protein (material seen in lanes 6 and 7 of SDS-PAGE). (a and b) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2) (empty vector negative control). (c and d) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28). (e and f) Spot test of affinity-purified ORF28-GST fusion protein. (g and h) Spot tests of affinity-purified ORF28 lysin released from ORF28-GST fusion protein by thrombin.
Figure Legend Snippet: SDS-PAGE and spot test analysis of induced, affinity-purified ORF28 lysin. (A) SDS-PAGE analysis of cellular proteins from IPTG-induced culture of E. coli BL21/DE3(pGEX4T2±ORF28) harboring either empty vector (pGEX4T2) or recombinant plasmid (pGEX4T2-ORF28) containing ORF28. Lanes: M, molecular mass standards; 1, sonic extract (SE) of induced E. coli BL21/DE3(pGEX4T2) harboring empty vector pGEXT4t2 (control; note overexpressed glutathione S -transferase [GST] protein band [plus linker oligopeptide] at ∼26.5 kDa); 2, SE of induced E. coli BL21/DE3(pGEX4T2-ORF28) harboring the recombinant plasmid pGEX4T2-ORF28 containing ORF28 (note overexpressed 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST [minus linker oligopeptide]); 3, flowthrough (unbound proteins) of glutathione affinity column following application of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) culture (note relative depletion of 72.5-kDa and ∼24.0-kDa proteins compared to initial SE); 4 and 5, affinity-purified proteins from induced E. coli BL21/DE3(pGEX4T2-ORF28) SE. SE were applied to the glutathione affinity column, and after extensive washing, bound proteins were desorbed by the addition of glutathione solution (note the 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST band); 6 and 7, protein recovered from affinity column following treatment of bound 72.5-kDa fusion protein with thrombin. The linkage between the ORF28 protein and the GST in the 72.5-kDa fusion protein is thrombin sensitive. The ORF28 protein was thereby released from the GST, which remained bound to the affinity column (note the single 46.1-kDa protein band recovered). (B) Lytic activity, in E. faecalis JH2-2 lawn, of the crude SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) (lane 2 in SDS-PAGE), affinity-purified ORF28-GST fusion protein (material seen in lanes 4 and 5 of SDS-PAGE), and affinity-purified ORF28 released from the affinity column by thrombin digestion of bound ORF28-GST fusion protein (material seen in lanes 6 and 7 of SDS-PAGE). (a and b) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2) (empty vector negative control). (c and d) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28). (e and f) Spot test of affinity-purified ORF28-GST fusion protein. (g and h) Spot tests of affinity-purified ORF28 lysin released from ORF28-GST fusion protein by thrombin.

Techniques Used: SDS Page, Spot Test, Affinity Purification, Plasmid Preparation, Recombinant, Affinity Column, Activity Assay, Negative Control

23) Product Images from "Producing Recombinant mTEX101; a Murine Testis Specific Protein"

Article Title: Producing Recombinant mTEX101; a Murine Testis Specific Protein

Journal: Journal of Reproduction & Infertility

doi:

Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.
Figure Legend Snippet: Double digestion of pGEM-T easy vector containing mTEX101 fragment with EcoRI and NotI restriction enzymes. 1: digested vector with mTEX101 750 bp insert cut out of the vector, 2: DNA ladder VIII.

Techniques Used: Plasmid Preparation

Coloy PCR on transformed JM109 clones by pGEM-T Easy vector carrying mTEX101 gene. 1-5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (PCR product on testis cDNA), 8: DNA ladder VIII (Roche).
Figure Legend Snippet: Coloy PCR on transformed JM109 clones by pGEM-T Easy vector carrying mTEX101 gene. 1-5: 5 selected white colonies, 6: negative control (blue colony), 7: positive control (PCR product on testis cDNA), 8: DNA ladder VIII (Roche).

Techniques Used: Polymerase Chain Reaction, Transformation Assay, Clone Assay, Plasmid Preparation, Negative Control, Positive Control

24) Product Images from "Genome-wide analysis of diamondback moth, Plutella xylostella L., from Brassica crops and wild host plants reveals no genetic structure in Australia"

Article Title: Genome-wide analysis of diamondback moth, Plutella xylostella L., from Brassica crops and wild host plants reveals no genetic structure in Australia

Journal: Scientific Reports

doi: 10.1038/s41598-020-68140-w

Heat maps showing pairwise comparisons of genetic distance measured as Weir and Cockerham’s (1984) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST (top panels) and geographic distance in kilometres (bottom panels) among P. xylostella populations collected from Australia in 2014 (left panels) and 2015 (right panels). Within each year, populations on x and y -axes are sorted geographically from north-western to north-eastern Australia in an arc following the southern coast. Visual comparison of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST and geographic distance heat maps within each year shows no congruence between genetic and geographic distance among population pairs in 2014 or 2015.
Figure Legend Snippet: Heat maps showing pairwise comparisons of genetic distance measured as Weir and Cockerham’s (1984) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST (top panels) and geographic distance in kilometres (bottom panels) among P. xylostella populations collected from Australia in 2014 (left panels) and 2015 (right panels). Within each year, populations on x and y -axes are sorted geographically from north-western to north-eastern Australia in an arc following the southern coast. Visual comparison of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST and geographic distance heat maps within each year shows no congruence between genetic and geographic distance among population pairs in 2014 or 2015.

Techniques Used: Western Blot

25) Product Images from "Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules"

Article Title: Conformational changes of the phenyl and naphthyl isocyanate-DNA adducts during DNA replication and by minor groove binding molecules

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt608

( A ) DNA sequences forming an X/dT or X/dG pair at the 3′-end of the junction used for the T4 DNA ligase reaction. The 24mer strand to be ligated was labeled with Cy3 at the 5′-end, and the 11mer strand had a phosphate group (denoted by p) at the 5′-end. ( B ) PAGE representing the fluorescent DNAs after the ligation reaction for 5 min (arrows indicate the ligated DNA strand), and the ligation rate constants for the DNA sequences forming an X/dT or X/dG pair.
Figure Legend Snippet: ( A ) DNA sequences forming an X/dT or X/dG pair at the 3′-end of the junction used for the T4 DNA ligase reaction. The 24mer strand to be ligated was labeled with Cy3 at the 5′-end, and the 11mer strand had a phosphate group (denoted by p) at the 5′-end. ( B ) PAGE representing the fluorescent DNAs after the ligation reaction for 5 min (arrows indicate the ligated DNA strand), and the ligation rate constants for the DNA sequences forming an X/dT or X/dG pair.

Techniques Used: Labeling, Polyacrylamide Gel Electrophoresis, Ligation

26) Product Images from "Cyclization of the Intrinsically Disordered ?1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function *"

Article Title: Cyclization of the Intrinsically Disordered ?1S Dihydropyridine Receptor II-III Loop Enhances Secondary Structure and in Vitro Function *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M110.205476

Expression and characterization of the cyclic II-III loop. A , the pNW1120 skDHPR II-III loop expression vector with the His 6 tag ( gray box ) and nine-residue linker ( white box ). The location of Intein N and Intein C in the vector is shown by the red arrows
Figure Legend Snippet: Expression and characterization of the cyclic II-III loop. A , the pNW1120 skDHPR II-III loop expression vector with the His 6 tag ( gray box ) and nine-residue linker ( white box ). The location of Intein N and Intein C in the vector is shown by the red arrows

Techniques Used: Expressing, Plasmid Preparation

27) Product Images from "Enterohemorrhagic Escherichia coli O157:H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia"

Article Title: Enterohemorrhagic Escherichia coli O157:H7 Gene Expression Profiling in Response to Growth in the Presence of Host Epithelia

Journal: PLoS ONE

doi: 10.1371/journal.pone.0004889

EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation. Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures ) at 37°C in 5% CO 2 . Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO 2 . Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p
Figure Legend Snippet: EHEC O157∶H7 gene Z1787 is a virulence factor responsible for inhibition of STAT-1 tyrosine phosphorylation. Cultured epithelial cells were infected with wild-type EHEC O157∶H7, strains CL56 and strain EDL 933, EHEC ΔZ1787 (in strain EDL 933) and EPEC O127∶H6 (MOI 100∶1) for 6 hr (or as described in the Experimental procedures ) at 37°C in 5% CO 2 . Washed cells were then stimulated with interferon (IFN)-γ (50 ng/mL) for 0.5 hr at 37°C in 5% CO 2 . Whole cell protein extracts were collected and immunoblots probed with either anti-latent-STAT-1 or anti-phospho-STAT-1 and anti-β-actin primary antibodies, followed by respective secondary antibodies. [Panel A] Positively staining bands were detected by using an infrared scanner. Lanes 1 2: uninfected epithelial cells in the absence and presence of interferon-γ, respectively; Lane 3: EHEC O157∶H7, strain CL56 inhibited STAT-1 tyrosine phosphorylation; Lanes 4: EPEC did not disrupt STAT-1 tyrosine phosphorylation. Lane 5: Wild-type EHEC O157∶H7, strain EDL933 inhibited IFNγ stimulated STAT-1 activation. Lane 6: Gene disruption of Z1787 in EDL 933 prevented EHEC subversion of STAT-1 signaling in response to IFNγ. [Panel B] Densitometry of positively stained bands was quantified using software imbedded in the infrared scanner. Quantification of STAT-1 tyrosine phosphorylation in epithelial cells infected with EHEC O157∶H7 complemented with gene Z1787 using the pGEM-T vector also was determined. As a positive control, levels of STAT-1 tyrosine phosphorylation also were determined in epithelial cells treated with non-pathogenic E. coli strain HB101: wild-type bacteria did not inhibit STAT-1 activation, while HB101+gene Z1787 (inserted on the pGEM-T vector) resulted in partially reduced levels of STAT-1 tyrosine phosphorylation (n = 1–4; ANOVA, *p

Techniques Used: Inhibition, Cell Culture, Infection, Western Blot, Staining, Activation Assay, Software, Plasmid Preparation, Positive Control

28) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

29) Product Images from "CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions"

Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

Journal: PLoS ONE

doi: 10.1371/journal.pone.0023572

DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
Figure Legend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

Techniques Used: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis

30) Product Images from "The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus"

Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040192

DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.
Figure Legend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

Techniques Used: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.
Figure Legend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

Techniques Used: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography

31) Product Images from "Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins"

Article Title: Bacteriophage φEf11 ORF28 Endolysin, a Multifunctional Lytic Enzyme with Properties Distinct from All Other Identified Enterococcus faecalis Phage Endolysins

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00555-19

SDS-PAGE and spot test analysis of induced, affinity-purified ORF28 lysin. (A) SDS-PAGE analysis of cellular proteins from IPTG-induced culture of E. coli BL21/DE3(pGEX4T2±ORF28) harboring either empty vector (pGEX4T2) or recombinant plasmid (pGEX4T2-ORF28) containing ORF28. Lanes: M, molecular mass standards; 1, sonic extract (SE) of induced E. coli BL21/DE3(pGEX4T2) harboring empty vector pGEXT4t2 (control; note overexpressed glutathione S -transferase [GST] protein band [plus linker oligopeptide] at ∼26.5 kDa); 2, SE of induced E. coli BL21/DE3(pGEX4T2-ORF28) harboring the recombinant plasmid pGEX4T2-ORF28 containing ORF28 (note overexpressed 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST [minus linker oligopeptide]); 3, flowthrough (unbound proteins) of glutathione affinity column following application of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) culture (note relative depletion of 72.5-kDa and ∼24.0-kDa proteins compared to initial SE); 4 and 5, affinity-purified proteins from induced E. coli BL21/DE3(pGEX4T2-ORF28) SE. SE were applied to the glutathione affinity column, and after extensive washing, bound proteins were desorbed by the addition of glutathione solution (note the 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST band); 6 and 7, protein recovered from affinity column following treatment of bound 72.5-kDa fusion protein with thrombin. The linkage between the ORF28 protein and the GST in the 72.5-kDa fusion protein is thrombin sensitive. The ORF28 protein was thereby released from the GST, which remained bound to the affinity column (note the single 46.1-kDa protein band recovered). (B) Lytic activity, in E. faecalis JH2-2 lawn, of the crude SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) (lane 2 in SDS-PAGE), affinity-purified ORF28-GST fusion protein (material seen in lanes 4 and 5 of SDS-PAGE), and affinity-purified ORF28 released from the affinity column by thrombin digestion of bound ORF28-GST fusion protein (material seen in lanes 6 and 7 of SDS-PAGE). (a and b) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2) (empty vector negative control). (c and d) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28). (e and f) Spot test of affinity-purified ORF28-GST fusion protein. (g and h) Spot tests of affinity-purified ORF28 lysin released from ORF28-GST fusion protein by thrombin.
Figure Legend Snippet: SDS-PAGE and spot test analysis of induced, affinity-purified ORF28 lysin. (A) SDS-PAGE analysis of cellular proteins from IPTG-induced culture of E. coli BL21/DE3(pGEX4T2±ORF28) harboring either empty vector (pGEX4T2) or recombinant plasmid (pGEX4T2-ORF28) containing ORF28. Lanes: M, molecular mass standards; 1, sonic extract (SE) of induced E. coli BL21/DE3(pGEX4T2) harboring empty vector pGEXT4t2 (control; note overexpressed glutathione S -transferase [GST] protein band [plus linker oligopeptide] at ∼26.5 kDa); 2, SE of induced E. coli BL21/DE3(pGEX4T2-ORF28) harboring the recombinant plasmid pGEX4T2-ORF28 containing ORF28 (note overexpressed 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST [minus linker oligopeptide]); 3, flowthrough (unbound proteins) of glutathione affinity column following application of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) culture (note relative depletion of 72.5-kDa and ∼24.0-kDa proteins compared to initial SE); 4 and 5, affinity-purified proteins from induced E. coli BL21/DE3(pGEX4T2-ORF28) SE. SE were applied to the glutathione affinity column, and after extensive washing, bound proteins were desorbed by the addition of glutathione solution (note the 72.5-kDa ORF28-GST fusion protein and ∼24.0-kDa GST band); 6 and 7, protein recovered from affinity column following treatment of bound 72.5-kDa fusion protein with thrombin. The linkage between the ORF28 protein and the GST in the 72.5-kDa fusion protein is thrombin sensitive. The ORF28 protein was thereby released from the GST, which remained bound to the affinity column (note the single 46.1-kDa protein band recovered). (B) Lytic activity, in E. faecalis JH2-2 lawn, of the crude SE from induced E. coli BL21/DE3(pGEX4T2-ORF28) (lane 2 in SDS-PAGE), affinity-purified ORF28-GST fusion protein (material seen in lanes 4 and 5 of SDS-PAGE), and affinity-purified ORF28 released from the affinity column by thrombin digestion of bound ORF28-GST fusion protein (material seen in lanes 6 and 7 of SDS-PAGE). (a and b) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2) (empty vector negative control). (c and d) Spot tests of SE from induced E. coli BL21/DE3(pGEX4T2-ORF28). (e and f) Spot test of affinity-purified ORF28-GST fusion protein. (g and h) Spot tests of affinity-purified ORF28 lysin released from ORF28-GST fusion protein by thrombin.

Techniques Used: SDS Page, Spot Test, Affinity Purification, Plasmid Preparation, Recombinant, Affinity Column, Activity Assay, Negative Control

32) Product Images from "Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase"

Article Title: Rapid single step subcloning procedure by combined action of type II and type IIs endonucleases with ligase

Journal: Journal of Biological Engineering

doi: 10.1186/1754-1611-1-7

Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.
Figure Legend Snippet: Schematic overview of the subcloning procedure . The upper box contains the two vectors the reaction starts with, i.e. the entry vector with its two key elements flanking an insert and the destination vector with its NheI recognition site. By the enzymatic action (arrows) of Esp3I and NheI these vectors are linearized to form linear intermediate products as shown in the central box. These intermediates are subject to T4 ligase activity and can be ligated to yield a range of products: the initial vectors (upper box), circular intermediate products (lower box) and the desired product vector. Note that all circular intermediate products are again substrate for Esp3I and thus again linearized. There is a sole stable product in this reaction system, which is the desired product vector (circular products only shown if carrying at least one resistance marker). Shaded boxes termed KEY: key element as shown in Fig. 1.

Techniques Used: Subcloning, Plasmid Preparation, Activity Assay, Marker

33) Product Images from "Enzyme-guided DNA Sewing Architecture"

Article Title: Enzyme-guided DNA Sewing Architecture

Journal: Scientific Reports

doi: 10.1038/srep17722

Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.
Figure Legend Snippet: Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.

Techniques Used: Ligation, Activity Assay, Sequencing, Concentration Assay

34) Product Images from "Enzyme-guided DNA Sewing Architecture"

Article Title: Enzyme-guided DNA Sewing Architecture

Journal: Scientific Reports

doi: 10.1038/srep17722

Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.
Figure Legend Snippet: Schematic drawing of major ligation factors and evaluation of T4 ligase activity. ( a ) A schematic of DNA sewing material preparation. Each overhang sequence of WY-, EY- and CY-DNA blocks is ligated by T4 ligase. ( b ) Depiction of the ligation mechanism and three major ligation factors. These major factors were characterized by impact on ligation efficiency. ( c ) Various molar concentrations of Y-DNAs were tested with fixed amounts of adenosine triphosphate (1 mM) and T4 ligase (30 Weiss units). ( d ) Molar ratios of EY-DNA were changed under the fixed amount of WY-CY, which means the mixed solution of WY-DNA and CY-DNA in determined molar ratio. The concentration of WY-CY was fixed to 6 μM in ligation solution. The ratios of WY-CY to EY-DNA were 1:0.5, 1:1, 1:2 and 1:4 in sequence. Blue, red and green bars represent T-DNA, partial T-DNA and unreacted Y-DNA, respectively. ( e ) Various salt concentrations (15, 50, 100, 200 and 400 mM) were tested. Each data point represents the mean of triplicate experiments; error bars represent the SD.

Techniques Used: Ligation, Activity Assay, Sequencing, Concentration Assay

35) Product Images from "A Novel Virulence Strategy for Pseudomonas aeruginosa Mediated by an Autotransporter with Arginine-Specific Aminopeptidase Activity"

Article Title: A Novel Virulence Strategy for Pseudomonas aeruginosa Mediated by an Autotransporter with Arginine-Specific Aminopeptidase Activity

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002854

Cartoon Model illustrating a selection of the potential roles AaaA may have within a chronic wound. In Panel A , the P. aeruginosa WT scenario is depicted, where AaaA (black dots) is present on the surface of P. aeruginosa cells colonising a host. Panel B shows infection with an AaaA deficient mutant that only has non-AaaA proteins on its surface (grey dots). It is possible that AaaA may: Panel I degrade a protein on the surface of P. aeruginosa , causing activation that aids infection (represented by removal of the black outline around the grey dots in Panel A, but not in Panel B), Panel II degrade a host protein/peptide, that may be a component of the host immune system ( Panel III ) by removing an aminoterminal arginine (R in circle). These activities may be sufficient to aid pathogenicity, however they may serve to liberate arginine that can be catabolised by the bacteria ( Panel IV ) resulting in growth promotion in Panel A that is not evident in the absence of AaaA (Panel B). This may provide a fitness advantage to the bacteria that improves virulence. In conditions where oxygen is limited, the arginine may provide a particular advantage ( Panel V ), potentially enabling formation of biofilms that could both serve to promote colonisation and provide resistance against the immune system. If only some of the released arginine is utilized by the bacteria, local arginine levels may rise in the host ( Panel VI ). This could induce arginase production in host cells (depicted by dark grey box and solid black arrows in Panel A:VI). The arginase enzymes will degrade the arginine, reducing its availability as a substrate for iNOS (indicated by pale grey box and dashed grey arrows in Panel A:VI). Consequently, there will be lower levels of nitric oxide (NO) and P. aeruginosa will be able to successfully establish an infection. Alternatively, in Panel B:VI, AaaA is absent from the invading P. aeruginosa , so there is no degradation of proteins and peptides. This maintains the limited arginine concentration and avoids induction of arginase in host cells. Consequently, arginine would be available to serve as a substrate for iNOS, and the nitric oxide generated could disable the bacterial cells and promote wound healing.
Figure Legend Snippet: Cartoon Model illustrating a selection of the potential roles AaaA may have within a chronic wound. In Panel A , the P. aeruginosa WT scenario is depicted, where AaaA (black dots) is present on the surface of P. aeruginosa cells colonising a host. Panel B shows infection with an AaaA deficient mutant that only has non-AaaA proteins on its surface (grey dots). It is possible that AaaA may: Panel I degrade a protein on the surface of P. aeruginosa , causing activation that aids infection (represented by removal of the black outline around the grey dots in Panel A, but not in Panel B), Panel II degrade a host protein/peptide, that may be a component of the host immune system ( Panel III ) by removing an aminoterminal arginine (R in circle). These activities may be sufficient to aid pathogenicity, however they may serve to liberate arginine that can be catabolised by the bacteria ( Panel IV ) resulting in growth promotion in Panel A that is not evident in the absence of AaaA (Panel B). This may provide a fitness advantage to the bacteria that improves virulence. In conditions where oxygen is limited, the arginine may provide a particular advantage ( Panel V ), potentially enabling formation of biofilms that could both serve to promote colonisation and provide resistance against the immune system. If only some of the released arginine is utilized by the bacteria, local arginine levels may rise in the host ( Panel VI ). This could induce arginase production in host cells (depicted by dark grey box and solid black arrows in Panel A:VI). The arginase enzymes will degrade the arginine, reducing its availability as a substrate for iNOS (indicated by pale grey box and dashed grey arrows in Panel A:VI). Consequently, there will be lower levels of nitric oxide (NO) and P. aeruginosa will be able to successfully establish an infection. Alternatively, in Panel B:VI, AaaA is absent from the invading P. aeruginosa , so there is no degradation of proteins and peptides. This maintains the limited arginine concentration and avoids induction of arginase in host cells. Consequently, arginine would be available to serve as a substrate for iNOS, and the nitric oxide generated could disable the bacterial cells and promote wound healing.

Techniques Used: Selection, Infection, Mutagenesis, Activation Assay, Concentration Assay, Generated

AaaA promotes the ability of P. aeruginosa to respire dipeptides with N-terminal arginine except when adjacent to Arginine or Lysine. P. aeruginosa PAO1 and its derived aaaA deficient mutant were inoculated into nitrogen minimal media (NMM) alone or NMM containing the indicated nitrogen source. Cellular respiration/metabolic activity is reported via reduction of tetrazolium dye and plotted against time. The area under the curve (AUC) for a selection of nitrogen sources following 24 h incubation in each condition is plotted here. The values have been normalised by subtraction of the AUC of the control (no nitrogen source added) on the respective Biolog plate. Relative respiration is calculated by the difference between the normalised AUC of wild type and mutant divided by their sum and multiplied by 100. The fold induction was calculated by dividing the normalised AUC of the mutant by that of the wild type, so a value of 1.0 is no change. Biolog Phenotype microarray plates PM03B and PM06-08 were used as indicated, and each condition performed in duplicate (results from one are shown).
Figure Legend Snippet: AaaA promotes the ability of P. aeruginosa to respire dipeptides with N-terminal arginine except when adjacent to Arginine or Lysine. P. aeruginosa PAO1 and its derived aaaA deficient mutant were inoculated into nitrogen minimal media (NMM) alone or NMM containing the indicated nitrogen source. Cellular respiration/metabolic activity is reported via reduction of tetrazolium dye and plotted against time. The area under the curve (AUC) for a selection of nitrogen sources following 24 h incubation in each condition is plotted here. The values have been normalised by subtraction of the AUC of the control (no nitrogen source added) on the respective Biolog plate. Relative respiration is calculated by the difference between the normalised AUC of wild type and mutant divided by their sum and multiplied by 100. The fold induction was calculated by dividing the normalised AUC of the mutant by that of the wild type, so a value of 1.0 is no change. Biolog Phenotype microarray plates PM03B and PM06-08 were used as indicated, and each condition performed in duplicate (results from one are shown).

Techniques Used: Derivative Assay, Mutagenesis, Activity Assay, Selection, Incubation, Microarray

The activity of AaaA enables P. aeruginosa to grow using the tripeptide arg-gly-asp as the sole source of carbon and nitrogen. P. aeruginosa PAO1 (closed circles) and its derived aaaA deficient mutant (Δ aaaA , open circles) alone or bearing pME6032 (vector, open triangles) or pME6032:: aaaA (complemented, closed triangles) were grown to mid-exponential phase before th e induction of AaaA production by 1 mM IPTG. Cells were resuspended in MMP to OD 600 of 1, and subsequently 20 µl of this solution diluted into 200 µl of MMP containing arginine at 10 mM ( Panel A ), or 10 mM of the tripeptide arg-gly-asp ( Panel B ). The graph shows the subsequent growth in the Tecan monitored by observing the increase in OD 492 over time. The data is representative of 3 independent repetitions of this experiment.
Figure Legend Snippet: The activity of AaaA enables P. aeruginosa to grow using the tripeptide arg-gly-asp as the sole source of carbon and nitrogen. P. aeruginosa PAO1 (closed circles) and its derived aaaA deficient mutant (Δ aaaA , open circles) alone or bearing pME6032 (vector, open triangles) or pME6032:: aaaA (complemented, closed triangles) were grown to mid-exponential phase before th e induction of AaaA production by 1 mM IPTG. Cells were resuspended in MMP to OD 600 of 1, and subsequently 20 µl of this solution diluted into 200 µl of MMP containing arginine at 10 mM ( Panel A ), or 10 mM of the tripeptide arg-gly-asp ( Panel B ). The graph shows the subsequent growth in the Tecan monitored by observing the increase in OD 492 over time. The data is representative of 3 independent repetitions of this experiment.

Techniques Used: Activity Assay, Derivative Assay, Mutagenesis, Plasmid Preparation

AaaA can remove arginine from p -nitroanilide. Panel A. The P. aeruginosa Δ aaaA mutant alone (open triangles) or bearing either the empty plasmid pME6032 (open circles) or its derivative carrying aaaA (pME6032:: aaaA : closed circles) were treated as described in Figure S2B except arginine- p -nitroanilide was used as a substrate. WT PAO1 cells were treated similarly (closed triangles), and activities (measured as changes in A 405 nm ) are compared against a growth media blank (crosses). Panel B. E. coli DH5α bearing either the empty plasmid pME6032 (open circles) or its derivative carrying aaaA (pME6032:: aaaA : closed circles) were grown in LB until exponential phase, induced with 1 mM IPTG, and then incubated with arginine- p -nitroanilide as described in Figure S2B . Activities are compared against a growth media blank (crosses). Error bars are+/−1 S.D. (n = 15). All measurements have been corrected for differential growth of bacteria by normalising to an initial OD 600 nm of 0.1.
Figure Legend Snippet: AaaA can remove arginine from p -nitroanilide. Panel A. The P. aeruginosa Δ aaaA mutant alone (open triangles) or bearing either the empty plasmid pME6032 (open circles) or its derivative carrying aaaA (pME6032:: aaaA : closed circles) were treated as described in Figure S2B except arginine- p -nitroanilide was used as a substrate. WT PAO1 cells were treated similarly (closed triangles), and activities (measured as changes in A 405 nm ) are compared against a growth media blank (crosses). Panel B. E. coli DH5α bearing either the empty plasmid pME6032 (open circles) or its derivative carrying aaaA (pME6032:: aaaA : closed circles) were grown in LB until exponential phase, induced with 1 mM IPTG, and then incubated with arginine- p -nitroanilide as described in Figure S2B . Activities are compared against a growth media blank (crosses). Error bars are+/−1 S.D. (n = 15). All measurements have been corrected for differential growth of bacteria by normalising to an initial OD 600 nm of 0.1.

Techniques Used: Mutagenesis, Plasmid Preparation, Incubation

The passenger and β-barrel domains of AaaA remain connected and are tethered to the cell surface. E. coli LEMO21 bearing the empty vector pET21a or pET21a:: aaaA was grown to mid exponential phase in LB, and induced with 1 mM IPTG for 1 h. Following harvesting, washing and resuspension in PBS-Hepes, half of the cells were lysed by sonication. The whole and lysed cells were split into three aliquots and incubated with (T) or without (−) trypsin according to the Materials and Methods . Trypsin inhibitor was added at the same time as trypsin to one of the aliquots (T+I). Proteins were separated through a 9% SDS PAGE and stained with Coomassie Blue (Panel A) or subjected to immunoblotting with either α-AaaA (Panel B, top), or α-IscS (Panel B, bottom) antisera. A parallel experiment was performed with P. aeruginosa Δ aaaA bearing either pME6032 or pME6032:: aaaA . LB overnight cultures were diluted 1∶100 in fresh LB, grown for 3 h at 37°C, and induced with 1 mM IPTG for 1 h. The immunoblot of the P. aeruginosa proteins is shown in Panel C, with the cytoplasmic control protein being detected with α-RpoS in the bottom panel. The sizes of molecular weight markers are shown in kDa on the left, and the position of AaaA is indicated. In Panels B and C, densitometry was used to estimate the quantity of the cytoplasmic protein and the full length AaaA (indicated with the asterisk) detected in the immunoblots using imageJ software. The fold change of AaaA, IscS and RpoS are shown below the images of the respective immunoblots. The images in Panels D and E were captured by confocal fluorescent microscopy. P. aeruginosa Δ aaaA (pME6032:: aaaA ) was grown and induced as described for Panel C, probed with FM1-43 and either α-AaaA (Panel E) or pre-immune serum (Panel D). Incubation with donkey α-rabbit alexa fluor 680-conjugated secondary antibody (red) was performed before images were captured at either the apex or cross section of individual cells (as indicated in the dotted lines of the cartoon). Green fluorescence from FM1-43 (top Panel, green circle in cartoon), red fluorescence from alexa fluor 680 (middle Panel, red stars in cartoon), merged 2D and merged 3D shadowed images are shown.
Figure Legend Snippet: The passenger and β-barrel domains of AaaA remain connected and are tethered to the cell surface. E. coli LEMO21 bearing the empty vector pET21a or pET21a:: aaaA was grown to mid exponential phase in LB, and induced with 1 mM IPTG for 1 h. Following harvesting, washing and resuspension in PBS-Hepes, half of the cells were lysed by sonication. The whole and lysed cells were split into three aliquots and incubated with (T) or without (−) trypsin according to the Materials and Methods . Trypsin inhibitor was added at the same time as trypsin to one of the aliquots (T+I). Proteins were separated through a 9% SDS PAGE and stained with Coomassie Blue (Panel A) or subjected to immunoblotting with either α-AaaA (Panel B, top), or α-IscS (Panel B, bottom) antisera. A parallel experiment was performed with P. aeruginosa Δ aaaA bearing either pME6032 or pME6032:: aaaA . LB overnight cultures were diluted 1∶100 in fresh LB, grown for 3 h at 37°C, and induced with 1 mM IPTG for 1 h. The immunoblot of the P. aeruginosa proteins is shown in Panel C, with the cytoplasmic control protein being detected with α-RpoS in the bottom panel. The sizes of molecular weight markers are shown in kDa on the left, and the position of AaaA is indicated. In Panels B and C, densitometry was used to estimate the quantity of the cytoplasmic protein and the full length AaaA (indicated with the asterisk) detected in the immunoblots using imageJ software. The fold change of AaaA, IscS and RpoS are shown below the images of the respective immunoblots. The images in Panels D and E were captured by confocal fluorescent microscopy. P. aeruginosa Δ aaaA (pME6032:: aaaA ) was grown and induced as described for Panel C, probed with FM1-43 and either α-AaaA (Panel E) or pre-immune serum (Panel D). Incubation with donkey α-rabbit alexa fluor 680-conjugated secondary antibody (red) was performed before images were captured at either the apex or cross section of individual cells (as indicated in the dotted lines of the cartoon). Green fluorescence from FM1-43 (top Panel, green circle in cartoon), red fluorescence from alexa fluor 680 (middle Panel, red stars in cartoon), merged 2D and merged 3D shadowed images are shown.

Techniques Used: Plasmid Preparation, Sonication, Incubation, SDS Page, Staining, Molecular Weight, Western Blot, Software, Microscopy, Fluorescence

The AaaA deficient mutant is less virulent in the chronic mouse wound model. Either the P. aeruginosa wild type PAO1 (black bars), the Δ aaaA mutant (white bars), or the complemented Δ aaaA mutant PAJL2 (grey bars) was inoculated (10 4 CFU) into a chronic wound in each of 9 mice. After 2 (3 mice per group) or 8 (7 mice per group) days, wound tissue was removed and the bacterial load was estimated by calculating the colony forming units ( Panel A ). Chronically-wounded mice were euthanized at post infection day 2 (3 mice per group) or day 8 (7 mice per group), and wound tissue was harvested for qRT-PCR to detect the mRNA of the indicated cytokines and other host enzymes in the infected wound tissue as described in the materials and methods ( Panel B and C ). Tissue from the wounds of the 2 day infected mice ( Panels D,G,J ) or 8 day infected mice ( Panels E,F,H,I,K,L ) was stained with H E and is shown at 100× magnification. Images of the P. aeruginosa wild type PAO1 ( Panels D,E,F ), Δ aaaA mutant ( Panels G,H,I ), and the complemented Δ aaaA mutant PAJL2 ( Panels J,K,L ) are shown with infiltrating neutrophils indicated by white arrows, elongated fibroblasts with a red arrow, single bacterial cells with white arrow heads and clumps of bacteria with a white asterisk. Panels D–E,G–H,J–K are representative of the wound site and Panels F,I,L are representative of the site of infection below the wound.
Figure Legend Snippet: The AaaA deficient mutant is less virulent in the chronic mouse wound model. Either the P. aeruginosa wild type PAO1 (black bars), the Δ aaaA mutant (white bars), or the complemented Δ aaaA mutant PAJL2 (grey bars) was inoculated (10 4 CFU) into a chronic wound in each of 9 mice. After 2 (3 mice per group) or 8 (7 mice per group) days, wound tissue was removed and the bacterial load was estimated by calculating the colony forming units ( Panel A ). Chronically-wounded mice were euthanized at post infection day 2 (3 mice per group) or day 8 (7 mice per group), and wound tissue was harvested for qRT-PCR to detect the mRNA of the indicated cytokines and other host enzymes in the infected wound tissue as described in the materials and methods ( Panel B and C ). Tissue from the wounds of the 2 day infected mice ( Panels D,G,J ) or 8 day infected mice ( Panels E,F,H,I,K,L ) was stained with H E and is shown at 100× magnification. Images of the P. aeruginosa wild type PAO1 ( Panels D,E,F ), Δ aaaA mutant ( Panels G,H,I ), and the complemented Δ aaaA mutant PAJL2 ( Panels J,K,L ) are shown with infiltrating neutrophils indicated by white arrows, elongated fibroblasts with a red arrow, single bacterial cells with white arrow heads and clumps of bacteria with a white asterisk. Panels D–E,G–H,J–K are representative of the wound site and Panels F,I,L are representative of the site of infection below the wound.

Techniques Used: Mutagenesis, Mouse Assay, Infection, Quantitative RT-PCR, Staining

36) Product Images from "A dual functioning small RNA/Riboswitch controls the expression of the methionine biosynthesis regulator SahR in Desulfovibrio vulgaris Hildenborough"

Article Title: A dual functioning small RNA/Riboswitch controls the expression of the methionine biosynthesis regulator SahR in Desulfovibrio vulgaris Hildenborough

Journal: bioRxiv

doi: 10.1101/803072

Structural analysis of the DseA riboswitch. ( A ) Spontaneous cleavage pattern of DseA in the absence or presence of SAM, methionine (Met), or SAH as indicated. The location of some of the guanosine residues (G) cleaved by RNase T1 is indicated. NR: no reaction; T1: RNase T1 ladder; -OH: alkaline hydrolysis ladder. ( B ) Lane profiles as determined by the program ImageQuant (GE Healthcare) of in-line probing gel. The numbers match to the same numbered areas of the gel. The lane profile of the T1 ladder is plotted in the bottom panel and represents the G residues as labeled. ( C ) Predicted secondary structure of DseA. When SAM concentrations are high an intrinsic terminator (T) is predicted to form. When SAM concentrations drop the anti-terminator (AT) forms instead. Bases colored blue are involved in forming the antiterminator.
Figure Legend Snippet: Structural analysis of the DseA riboswitch. ( A ) Spontaneous cleavage pattern of DseA in the absence or presence of SAM, methionine (Met), or SAH as indicated. The location of some of the guanosine residues (G) cleaved by RNase T1 is indicated. NR: no reaction; T1: RNase T1 ladder; -OH: alkaline hydrolysis ladder. ( B ) Lane profiles as determined by the program ImageQuant (GE Healthcare) of in-line probing gel. The numbers match to the same numbered areas of the gel. The lane profile of the T1 ladder is plotted in the bottom panel and represents the G residues as labeled. ( C ) Predicted secondary structure of DseA. When SAM concentrations are high an intrinsic terminator (T) is predicted to form. When SAM concentrations drop the anti-terminator (AT) forms instead. Bases colored blue are involved in forming the antiterminator.

Techniques Used: Labeling

DseA riboswtich expression platform response. ( A ) In vitro transcription termination assay of the riboswitch region. Percent termination was determined by the amount of termination product divided by the sum of total transcription products. ( B ) β-galactosidase activity of pRS415-DseA and the negative control vector pRS415. Values represent the mean of three experiments. Activity is represented by Miller Units. Error bars represent standard deviation.
Figure Legend Snippet: DseA riboswtich expression platform response. ( A ) In vitro transcription termination assay of the riboswitch region. Percent termination was determined by the amount of termination product divided by the sum of total transcription products. ( B ) β-galactosidase activity of pRS415-DseA and the negative control vector pRS415. Values represent the mean of three experiments. Activity is represented by Miller Units. Error bars represent standard deviation.

Techniques Used: Expressing, In Vitro, Activity Assay, Negative Control, Plasmid Preparation, Standard Deviation

37) Product Images from "Extended and dynamic linker histone-DNA interactions control chromatosome compaction"

Article Title: Extended and dynamic linker histone-DNA interactions control chromatosome compaction

Journal: bioRxiv

doi: 10.1101/2020.10.10.334474

H1-DNA contacts at the dyad are responsible for symmetric compaction. Unzipping traces obtained from 3’ and 5’ unzipping experiments with the H1K69A (magenta) and H1ΔCTD (green) mutants were analyzed and are presented as a) mean rupture force of regions of interactions, b-c) dwell time histograms and d-e) average force-position curves. Dwell time histograms of nucleosomes (black) and chromatosomes formed with WT H1 (blue) are identical to those in Fig.1g and shown for reference. n 5’-H1 =46, n 5’nuc+H1 =21, n 3’-H1 = 53, n 3’nuc+H1 =41, n 5’nuc+H1K69A =22, n 3’ nuc+H1K69A = 27, n 5’nuc+H1ΔCTD =22, n 3’nuc+ H1ΔCTD =20. * P
Figure Legend Snippet: H1-DNA contacts at the dyad are responsible for symmetric compaction. Unzipping traces obtained from 3’ and 5’ unzipping experiments with the H1K69A (magenta) and H1ΔCTD (green) mutants were analyzed and are presented as a) mean rupture force of regions of interactions, b-c) dwell time histograms and d-e) average force-position curves. Dwell time histograms of nucleosomes (black) and chromatosomes formed with WT H1 (blue) are identical to those in Fig.1g and shown for reference. n 5’-H1 =46, n 5’nuc+H1 =21, n 3’-H1 = 53, n 3’nuc+H1 =41, n 5’nuc+H1K69A =22, n 3’ nuc+H1K69A = 27, n 5’nuc+H1ΔCTD =22, n 3’nuc+ H1ΔCTD =20. * P

Techniques Used:

Dynamic mapping uncovers extended interactions of H1 CTD and their role in nucleosome compaction. a) Schematic description of the reversible unzipping assay to probe H1 interactions with linker DNA. Progression of the unzipping fork displaces H1 directionally, starting with (1) the CTD, followed by (2) the GD and (3) H3-NTD, until reaching an H2A/H2B dimer (4). Next, the tension is relaxed and dsDNA is reformed, after which the unzipping cycle is repeated (10 times, 8 s per cycle). b) Repetitive unzipping force-extension curves of the WT (blue), H1ΔCTD (green) and H1T153E(orange) chromatosomes or nucleosomes without H1 (black), all unzipped from the 3’ direction. The WT chromatosomes were probed under STO conditions. The H1ΔCTD and H1T153E binding to nucleosomes was unstable overtime (Supp. Fig. 2c) and hence investigated using MTO conditions. The clusters of detected interactions are highlighted with arrows. The interactions measured in each unzipping cycle (see Methods) were quantified and presented as c) mean rupture forces as a function of time within an 8 s window, and d) mean rupture forces. Data are shown as mean±s.e.m.; n nuc =307, n nuc+WT H1 =252, n nuc+H1ΔCTD =41, n nuc+H1T153E =54. * P
Figure Legend Snippet: Dynamic mapping uncovers extended interactions of H1 CTD and their role in nucleosome compaction. a) Schematic description of the reversible unzipping assay to probe H1 interactions with linker DNA. Progression of the unzipping fork displaces H1 directionally, starting with (1) the CTD, followed by (2) the GD and (3) H3-NTD, until reaching an H2A/H2B dimer (4). Next, the tension is relaxed and dsDNA is reformed, after which the unzipping cycle is repeated (10 times, 8 s per cycle). b) Repetitive unzipping force-extension curves of the WT (blue), H1ΔCTD (green) and H1T153E(orange) chromatosomes or nucleosomes without H1 (black), all unzipped from the 3’ direction. The WT chromatosomes were probed under STO conditions. The H1ΔCTD and H1T153E binding to nucleosomes was unstable overtime (Supp. Fig. 2c) and hence investigated using MTO conditions. The clusters of detected interactions are highlighted with arrows. The interactions measured in each unzipping cycle (see Methods) were quantified and presented as c) mean rupture forces as a function of time within an 8 s window, and d) mean rupture forces. Data are shown as mean±s.e.m.; n nuc =307, n nuc+WT H1 =252, n nuc+H1ΔCTD =41, n nuc+H1T153E =54. * P

Techniques Used: Binding Assay

H1 dynamically interacts with both linkers to stabilize the on-dyad conformation. a) Molecular constructs used for repetitive unzipping experiments. 3’ unz/WT nuc – WT nucleosome unzipped from 3’ end. 5’ unz/WT nuc – WT nucleosome unzipped from 5’ end. 3’ unz/Δ5’ nuc – nucleosome harboring a full 3’ linker to be unzipped, but with a 3 bp long 5’ linker, designed to abolish interactions with GD, CTD1 and CTD2. b) Representative traces of WT or single-linker chromatosomes unzipped partially from the 3’ or 5’ direction. Corresponding control experiments without H1 are shown in black. c-d) Average dwell time histograms constructed from partially unzipped traces (~10 cycles, 8 s each) for Δ5’ chromatosomes (c, green) and WT chromatosomes probed from the 3’ (c, blue) and 5’ (d, red) direction. All traces were low-passed filtered to 150 Hz and binned to 1 bp. The clusters of detected interactions are highlighted with arrows. e) Rupture forces and f) binding probabilities calculated for the clustered interactions, as shown in Fig. 4 c,d . Data are shown as mean±s.e.m.; n 5’nuc =136, n 3’nuc =187, n Δ5’ nuc =60, n 5’nuc+H1 =139, n 3’nuc+H1 =242, n Δ5’ nuc+H1 =60. * P
Figure Legend Snippet: H1 dynamically interacts with both linkers to stabilize the on-dyad conformation. a) Molecular constructs used for repetitive unzipping experiments. 3’ unz/WT nuc – WT nucleosome unzipped from 3’ end. 5’ unz/WT nuc – WT nucleosome unzipped from 5’ end. 3’ unz/Δ5’ nuc – nucleosome harboring a full 3’ linker to be unzipped, but with a 3 bp long 5’ linker, designed to abolish interactions with GD, CTD1 and CTD2. b) Representative traces of WT or single-linker chromatosomes unzipped partially from the 3’ or 5’ direction. Corresponding control experiments without H1 are shown in black. c-d) Average dwell time histograms constructed from partially unzipped traces (~10 cycles, 8 s each) for Δ5’ chromatosomes (c, green) and WT chromatosomes probed from the 3’ (c, blue) and 5’ (d, red) direction. All traces were low-passed filtered to 150 Hz and binned to 1 bp. The clusters of detected interactions are highlighted with arrows. e) Rupture forces and f) binding probabilities calculated for the clustered interactions, as shown in Fig. 4 c,d . Data are shown as mean±s.e.m.; n 5’nuc =136, n 3’nuc =187, n Δ5’ nuc =60, n 5’nuc+H1 =139, n 3’nuc+H1 =242, n Δ5’ nuc+H1 =60. * P

Techniques Used: Construct, Binding Assay

DNA unzipping reveals nucleosome compaction by linker histone. a) Schematic description of the experimental assay. The nucleosome is tethered between Streptavidin (S), and anti-Digoxigenin (D) coated beads captured by dual-trap optical tweezers. The trapped nucleosome is exposed to [5 nM] H1 to form a chromatosome and then subjected to DNA unzipping in the H1-free channel. b) Representation of the DNA unzipping reaction through a chromatosome, based on the crystal structure of the GD bound to a 197 bp palindromic 601L nucleosome (PDB: 5NL0) 26 . Hypothetical positions for H1 CTD and NTD domains are shown for clarity. Two strands of the DNA are connected to DNA handles bound to the trapped S and D beads. Moving one trapped bead away from the other creates tension, leading to the conversion of dsDNA to ssDNA, which allows probing the position and strength of major histone-DNA interactions (circled). c) Representative unzipping curves for nucleosomes reconstituted using the 601 DNA without (black) or in the presence of H1 (blue). ‘Naked’ (i.e. no nucleosome or H1) 601 DNA (grey) is shown for reference. The unzipping reaction starts at ~-360 bp from the dyad, proceeds through the fixed ‘alignment segment’, reaching histone-DNA interactions in a chromatosome as highlighted in Fig.1b . d,e) Mean rupture forces for H3-NTD, H2A/H2B, and H3/H4 interactions, shown for 601 (d) and Cga (e) nucleosomes. Data shown as mean±s.e.m.; n 601 =13, n 601+H1 =15, n Cga =14, n Cga +H1 =12. * P
Figure Legend Snippet: DNA unzipping reveals nucleosome compaction by linker histone. a) Schematic description of the experimental assay. The nucleosome is tethered between Streptavidin (S), and anti-Digoxigenin (D) coated beads captured by dual-trap optical tweezers. The trapped nucleosome is exposed to [5 nM] H1 to form a chromatosome and then subjected to DNA unzipping in the H1-free channel. b) Representation of the DNA unzipping reaction through a chromatosome, based on the crystal structure of the GD bound to a 197 bp palindromic 601L nucleosome (PDB: 5NL0) 26 . Hypothetical positions for H1 CTD and NTD domains are shown for clarity. Two strands of the DNA are connected to DNA handles bound to the trapped S and D beads. Moving one trapped bead away from the other creates tension, leading to the conversion of dsDNA to ssDNA, which allows probing the position and strength of major histone-DNA interactions (circled). c) Representative unzipping curves for nucleosomes reconstituted using the 601 DNA without (black) or in the presence of H1 (blue). ‘Naked’ (i.e. no nucleosome or H1) 601 DNA (grey) is shown for reference. The unzipping reaction starts at ~-360 bp from the dyad, proceeds through the fixed ‘alignment segment’, reaching histone-DNA interactions in a chromatosome as highlighted in Fig.1b . d,e) Mean rupture forces for H3-NTD, H2A/H2B, and H3/H4 interactions, shown for 601 (d) and Cga (e) nucleosomes. Data shown as mean±s.e.m.; n 601 =13, n 601+H1 =15, n Cga =14, n Cga +H1 =12. * P

Techniques Used:

A model for dynamic compaction of a chromatosome particle. H1 binds the canonical nucleosome in an ‘on-dyad’ conformation. Binding of the GD domain to the dyad induces a conformational change leading to compaction of both H2A/H2B dimers. The CTD dynamically couples both linkers at two positions, ~±110 and ~±140 bp from the dyad, to stabilize GD-linker interactions. Mechanical invasion into the linker DNA up to the GD-DNA contact triggers H1 repositioning to the less condensed and dynamic ‘off-dyad’ conformation.
Figure Legend Snippet: A model for dynamic compaction of a chromatosome particle. H1 binds the canonical nucleosome in an ‘on-dyad’ conformation. Binding of the GD domain to the dyad induces a conformational change leading to compaction of both H2A/H2B dimers. The CTD dynamically couples both linkers at two positions, ~±110 and ~±140 bp from the dyad, to stabilize GD-linker interactions. Mechanical invasion into the linker DNA up to the GD-DNA contact triggers H1 repositioning to the less condensed and dynamic ‘off-dyad’ conformation.

Techniques Used: Binding Assay

38) Product Images from "Genome-wide analysis of diamondback moth, Plutella xylostella L., from Brassica crops and wild host plants reveals no genetic structure in Australia"

Article Title: Genome-wide analysis of diamondback moth, Plutella xylostella L., from Brassica crops and wild host plants reveals no genetic structure in Australia

Journal: Scientific Reports

doi: 10.1038/s41598-020-68140-w

Heat maps showing pairwise comparisons of genetic distance measured as Weir and Cockerham’s (1984) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST (top panels) and geographic distance in kilometres (bottom panels) among P. xylostella populations collected from Australia in 2014 (left panels) and 2015 (right panels). Within each year, populations on x and y -axes are sorted geographically from north-western to north-eastern Australia in an arc following the southern coast. Visual comparison of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST and geographic distance heat maps within each year shows no congruence between genetic and geographic distance among population pairs in 2014 or 2015.
Figure Legend Snippet: Heat maps showing pairwise comparisons of genetic distance measured as Weir and Cockerham’s (1984) \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST (top panels) and geographic distance in kilometres (bottom panels) among P. xylostella populations collected from Australia in 2014 (left panels) and 2015 (right panels). Within each year, populations on x and y -axes are sorted geographically from north-western to north-eastern Australia in an arc following the southern coast. Visual comparison of the \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$F_{\text {ST}}$$\end{document} F ST and geographic distance heat maps within each year shows no congruence between genetic and geographic distance among population pairs in 2014 or 2015.

Techniques Used: Western Blot

39) Product Images from "Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers"

Article Title: Rapid SNP Discovery and Genetic Mapping Using Sequenced RAD Markers

Journal: PLoS ONE

doi: 10.1371/journal.pone.0003376

Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.
Figure Legend Snippet: Sequenced RAD marker mapping. (A) A native saltwater stickleback population, Rabbit Slough (RS), have complete lateral plate armor (brackets) while these structures are absent in the derived, freshwater Bear Paw (BP) population. The freshwater fish also have a reduction in pelvic structure (arrow) compared to the oceanic population. These two phenotypes segregate independently in an F 2 mapping cross. Using Sbf I (B) or EcoR I (C), we mapped polymorphic RAD markers from RS (red) and BP (green) parental fish along the 21 stickleback linkage groups. The apparent size differences of the linkage groups between (B) and (C) reflect the fact that the EcoR I recognition sequence occurs more frequently than Sbf I. Red and green bars above the linkage groups are measures of lateral plate linkage in the F 2 progeny, indicating the number of tightly linked markers in the local region. (D) Sequence reads per barcoded F 2 individual used to create (C). Variable numbers of reads were obtained from each of the 96 individuals used in our analysis, reflecting different concentrations of starting DNA template. 68% of individuals had between 50 K and 150 K RAD tags sequenced (∼0.4–1.0× coverage of the ∼150 K tags present in the genome). Only 2 individuals had less than 10,000 reads (red). (E) A close-up of the boxed region from (C) showing recombination breakpoints in six informative low plate F 2 fish on LGIV. Black tick marks are 1 Mb apart in physical distance. (F) F 2 individuals were repooled in silico based on the pelvic structure phenotype (A, arrow). Linkage was determined as in (B, C), mapping the locus for a reduction in pelvic structure to the end of LGVII.

Techniques Used: Marker, Derivative Assay, Fluorescence In Situ Hybridization, Sequencing, In Silico

40) Product Images from "Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products"

Article Title: Exploration of Human ORFeome: High-Throughput Preparation of ORF Clones and Efficient Characterization of Their Protein Products

Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

doi: 10.1093/dnares/dsn004

ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.
Figure Legend Snippet: ORF transfer in the Flexi ® Vector cloning system. ( A ) Flanking sequences of ORF in Flexi clones. Recognition sequences of Sgf I and Pme I are indicated as green and red characters, respectively. The nucleotide sequence corresponding to the ribosomal binding site is underlined. The amino acid sequence encoded in the frame in the flanking regions of the ORF is indicated as a three-letter code. Recognition sequences of Bst BI and Sna BI, arising in the vector of Flexi_RBS type are indicated as blue characters. ( B ) Transfer of the ORF from the pF1K clone to multiple expression vectors. The ORF sequence in the pF1K clone can be easily transferred to a variety of other expression vectors with the correct orientation after digestion by Sgf I and Pme I. For construction of a C-terminal tag-fusion clone, Sgf I– Pme I ORF sequence must be cloned into Sgf I and Eco ICRI sites of the expression vector to omit a stop codon arising in the Pme I site. The appropriate promoter is indicated as an orange arrow in the vectors.

Techniques Used: Plasmid Preparation, Clone Assay, Sequencing, Binding Assay, Expressing

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Polymerase Chain Reaction:

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Sequencing:

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Article Snippet: .. Neither decreasing or increasing the DNA input improved the sequencing output, due to too few adapter‐DNA molecules, or too many free DNA molecules potentially interfering with the sequencing reaction. .. Assuming that 2.9 µg input DNA was the equivalent of 0.2 pmol (recommended concentration as per the ONT protocol LSK108 that was used), we estimate a mean DNA fragment length of 23 kb for our sample preparation.

Purification:

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    Promega t4 dna ligase buffer
    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
    T4 Dna Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega t4 ligase buffer
    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with <t>T4</t> DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.
    T4 Ligase Buffer, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase buffer/product/Promega
    Average 92 stars, based on 3 article reviews
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    t4 ligase buffer - by Bioz Stars, 2021-01
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    DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Journal: PLoS ONE

    Article Title: CK2 Phosphorylation of Schistosoma mansoni HMGB1 Protein Regulates Its Cellular Traffic and Secretion but Not Its DNA Transactions

    doi: 10.1371/journal.pone.0023572

    Figure Lengend Snippet: DNA supercoiling and bending assays by phosphorylated SmHMGB1. (A) Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I with 1 µg of recombinant SmHMGB1-FL or SmHMGB1-S172A/S174A that were phosphorylated (lanes 3–5) or not (lanes 6–8 and 9–11), by CK2. Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gels with ethidium bromide. Form I, supercoiled DNA; form II, relaxed circular DNA. (B) Top panel: autoradiography; bottom panel: Coomassie staining. (C) A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of recombinant proteins, that were phosphorylated (lanes 7–9) or not (lanes 4–6, 10–12, 13–15 and 16–18), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Controls are as follows: FL(c1): SmHMGB1-FL without CK2; FL(c2): SmHMGB1-FL without phosphate; FL(c3): SmHMGB1-FL without CK2 buffer. Linear: linear DNA; Lm: linear multimers. (D) Top panel: autoradiography; bottom panel: Coomassie staining. These experiments were repeated four times.

    Article Snippet: Briefly, a 32 P-labeled-66-bp or a 32 P-labeled-123-bp DNA fragments (1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (50 ng), total (10 µg), nuclear (4 µg) or cytoplasmic (4 µg) adult worm extracts, in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µl.

    Techniques: Plasmid Preparation, Incubation, Recombinant, Staining, Autoradiography, Labeling, Ligation, DNA Ligation, Electrophoresis

    DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA transactions by recombinant AaHMGB1 proteins. (A) Preferential binding of AaHMGB1 protein to supercoiled DNA. An equimolar mixture of supercoiled and linearized plasmid pTZ19R (∼10 nM) was pre-incubated with increasing amounts of AaHMGB1 (0.5–1 µM) and the DNA–protein complexes were resolved on a 1% agarose gel, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; L, Linear DNA; Form II, relaxed circular DNA; (B) DNA supercoiling by AaHMGB1 and its truncated forms. Circular relaxed plasmid pTZ19R DNA was incubated in the presence of topoisomerase I (Topo I) and AaHMGB1 recombinant proteins (7–14 µM). Deproteinized DNA topoisomers were resolved on 1% agarose gels, followed by staining of the gel with ethidium bromide. Form I, supercoiled DNA; Form II, relaxed circular DNA. (C) DNA bending by AaHMGB1 and its truncated forms. A 32 P-labeled 123-bp DNA fragment (∼1 nM) was pre-incubated with recombinant proteins (25–50 nM) followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. Exo III, exonuclease III. These experiments were repeated three to five times each.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Recombinant, Binding Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Staining, Labeling, Ligation, DNA Ligation, Electrophoresis, Autoradiography

    DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Journal: PLoS ONE

    Article Title: The Dengue Vector Aedes aegypti Contains a Functional High Mobility Group Box 1 (HMGB1) Protein with a Unique Regulatory C-Terminus

    doi: 10.1371/journal.pone.0040192

    Figure Lengend Snippet: DNA bending assays by posphorylated AaHMGB1. A 32 P-labelled 123-bp DNA fragment (∼1 nM) was pre-incubated with 50 ng of AaHMGB1 that were phosphorylated by PKA (panels A and B, lanes 5 and 2, respectively) or not (panels A and B, lanes 4 and 3, respectively), or by PKC (panels C and D, lanes 5 and 2, respectively) or not (panels C and D, lanes 4 and 3, respectively), followed by ligation with T4 DNA ligase. Exonuclease III was used to verify the identity of DNA circles. The deproteinized DNA ligation products were subjected to electrophoresis on 6% non-denaturing polyacrylamide gels and visualized by autoradiography. Lm: linear multimers. These experiments were repeated five times.

    Article Snippet: Briefly, a 32 P-labeled 123-bp DNA fragment (∼1 nM) with cohesive BamHI ends were pre-incubated on ice for 20 min with appropriate amounts of recombinant proteins (25–50 nM) or total protein extracts from adult mosquitos (4 µg) in 1× T4 DNA ligase buffer (30 mM Tris–HCl, pH 7.8, 10 mM MgCl2 , 10 mM dithiothreitol, and 0.5 mM ATP; Promega) in a final volume of 20 µL.

    Techniques: Incubation, Ligation, DNA Ligation, Electrophoresis, Autoradiography