c elegans genomic dna  (New England Biolabs)


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    New England Biolabs c elegans genomic dna
    HinfI
    HinfI 25 000 units
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    Average 93 stars, based on 83 article reviews
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    Images

    1) Product Images from "The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1"

    Article Title: The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.256388

    TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.
    Figure Legend Snippet: TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.

    Techniques Used: Southern Blot, Agarose Gel Electrophoresis, Mutagenesis, Fluorescence In Situ Hybridization, Staining

    Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value
    Figure Legend Snippet: Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value

    Techniques Used: Construct, Sequencing

    TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value
    Figure Legend Snippet: TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value

    Techniques Used: Quantitation Assay, Sequencing

    2) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    3) Product Images from "Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae"

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-015-0580-8

    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product
    Figure Legend Snippet: NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Techniques Used: Methylation, Amplification, Polymerase Chain Reaction

    4) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    5) Product Images from "Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern"

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158663

    Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.
    Figure Legend Snippet: Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Techniques Used: Southern Blot, Isolation

    6) Product Images from "Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern"

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158663

    Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.
    Figure Legend Snippet: Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Techniques Used: Southern Blot, Isolation

    7) Product Images from "Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer"

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-28-125

    Imprinting analysis of IGF2 in gastric cancer . DNA (G1) and RT-PCR (G3) amplification using primers P1 and P3 and DNA amplification by PCR with primers P2 and P3 (G2) represented 1.4 kb, 1.12 kb and 292 bp respectively (see details in methods section). G1, G2 and G3 are PCR products of the same normal tissue. ApaI- and HinfI-digested normal tissue DNA PCR (Gn) from primers P2 and P3 displayed two bands of 256 and 231 bp indicating heterozygosity. The digested nested PCR product from primers P2 and P3 using the 1.12 kb RT-PCR product as a template showed monoallelic expression of IGF2 in normal (Rn1, Rn2) and biallelic expression in tumor (Rt1, Rt2) tissues.
    Figure Legend Snippet: Imprinting analysis of IGF2 in gastric cancer . DNA (G1) and RT-PCR (G3) amplification using primers P1 and P3 and DNA amplification by PCR with primers P2 and P3 (G2) represented 1.4 kb, 1.12 kb and 292 bp respectively (see details in methods section). G1, G2 and G3 are PCR products of the same normal tissue. ApaI- and HinfI-digested normal tissue DNA PCR (Gn) from primers P2 and P3 displayed two bands of 256 and 231 bp indicating heterozygosity. The digested nested PCR product from primers P2 and P3 using the 1.12 kb RT-PCR product as a template showed monoallelic expression of IGF2 in normal (Rn1, Rn2) and biallelic expression in tumor (Rt1, Rt2) tissues.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction, Nested PCR, Expressing

    8) Product Images from "Loop-Mediated Isothermal Amplification Method for Differentiation and Rapid Detection of Taenia Species ▿"

    Article Title: Loop-Mediated Isothermal Amplification Method for Differentiation and Rapid Detection of Taenia Species ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01573-08

    Nucleotide sequence alignment of the target region of clp genes (A) and LAMP results (B). (A) The locations of the primer recognition sites are indicated by arrows and the restriction enzyme HinfI recognition site in the LAMP products of T. asiatica clp
    Figure Legend Snippet: Nucleotide sequence alignment of the target region of clp genes (A) and LAMP results (B). (A) The locations of the primer recognition sites are indicated by arrows and the restriction enzyme HinfI recognition site in the LAMP products of T. asiatica clp

    Techniques Used: Sequencing

    9) Product Images from "S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas"

    Article Title: S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6602188

    RFLP-cmRT–PCR and –PCR analysis of S100A2 in NSCLC. A region of the S100A2 gene containing a cSNP was amplified from DNA and cDNA by PCR and RT–PCR, respectively. Reaction products were digested with Hinf I and run on a 3% agarose gel ( A ). Products were also run on an Agilent bioanalyser DNA 1000 chip. Peak heights of the noncutting allele (upper product on the agarose gel) were divided by those of the cutting allele (lower band on agarose gel) to give a relative value for the cutting allele compared to the noncutting allele ( B ). Patient 218 shows an imbalance in the cDNA (allelic expression imbalance) of the tumour sample compared to the normal tissue.
    Figure Legend Snippet: RFLP-cmRT–PCR and –PCR analysis of S100A2 in NSCLC. A region of the S100A2 gene containing a cSNP was amplified from DNA and cDNA by PCR and RT–PCR, respectively. Reaction products were digested with Hinf I and run on a 3% agarose gel ( A ). Products were also run on an Agilent bioanalyser DNA 1000 chip. Peak heights of the noncutting allele (upper product on the agarose gel) were divided by those of the cutting allele (lower band on agarose gel) to give a relative value for the cutting allele compared to the noncutting allele ( B ). Patient 218 shows an imbalance in the cDNA (allelic expression imbalance) of the tumour sample compared to the normal tissue.

    Techniques Used: Polymerase Chain Reaction, Amplification, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Chromatin Immunoprecipitation, Expressing

    Identification of S100A2 exon 3 polymorphism. ( A ) WAVE analysis of exon 3 in lung tumours: 5 and 6 left panel, 13 and 14 right panel. Arrows identify heteroduplex fragments. ( B ) RFLP analysis of exon 3 in lung tumours 13, 14, 5 and 6. Digestion with Hinf I reveals the presence of the polymorphism in tumours 5 and 14.
    Figure Legend Snippet: Identification of S100A2 exon 3 polymorphism. ( A ) WAVE analysis of exon 3 in lung tumours: 5 and 6 left panel, 13 and 14 right panel. Arrows identify heteroduplex fragments. ( B ) RFLP analysis of exon 3 in lung tumours 13, 14, 5 and 6. Digestion with Hinf I reveals the presence of the polymorphism in tumours 5 and 14.

    Techniques Used:

    10) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    11) Product Images from "The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1"

    Article Title: The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.256388

    TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.
    Figure Legend Snippet: TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.

    Techniques Used: Southern Blot, Agarose Gel Electrophoresis, Mutagenesis, Fluorescence In Situ Hybridization, Staining

    Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value
    Figure Legend Snippet: Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value

    Techniques Used: Construct, Sequencing

    POT-1 links the ds telomere binders to the ss telomere. (A) Y2H assay with full length TEBP-1, TEBP-2, and POT-1 fusions to the activation or DNA binding domains of Gal4. Growth on TRP LEU HIS plates demonstrates interaction. Growth on high stringency TRP LEU HIS ADE medium suggests strong interaction. (B-C) Volcano plots showing quantitative proteomic analysis of either TEBP-1::3xFLAG (B) or TEBP-2::GFP (C) IPs in embryos. IPs were performed in quadruplicates. Enriched proteins (threshold: 2-fold, p-value
    Figure Legend Snippet: POT-1 links the ds telomere binders to the ss telomere. (A) Y2H assay with full length TEBP-1, TEBP-2, and POT-1 fusions to the activation or DNA binding domains of Gal4. Growth on TRP LEU HIS plates demonstrates interaction. Growth on high stringency TRP LEU HIS ADE medium suggests strong interaction. (B-C) Volcano plots showing quantitative proteomic analysis of either TEBP-1::3xFLAG (B) or TEBP-2::GFP (C) IPs in embryos. IPs were performed in quadruplicates. Enriched proteins (threshold: 2-fold, p-value

    Techniques Used: Y2H Assay, Activation Assay, Binding Assay

    TEBP-1 and TEBP-2 are expressed throughout C. elegans development and localize to telomeres in vivo. (A) Western blot of TEBP-1::3xFLAG and TEBP-2::GFP expression in different developmental stages of C. elegans . 35 µg of extract from either N2 or a double transgenic line carrying TEBP-1::3xFLAG and TEBP-2::GFP were used. Actin was used as loading control. (B-C) Maximum intensity projections of representative confocal z stacks of an embryo (B), or oocytes (C) expressing endogenously tagged TEBP-1::GFP and transgenic POT-1::mCherry. Scale bars, 10 µm. (D E) Maximum intensity projections of representative confocal z-stacks of an adult animal (D), or embryo (E) expressing both endogenously tagged TEBP-2::GFP and transgenic POT-1::mCherry. Insets show nuclear co localization in meiotic germ cell nuclei (I), an oocyte (II), spermatozoa (III), and embryonic cells (IV). Scale bars, 20 µm (overview) and 4 µm (insets). All microscopy images were deconvoluted using Huygens remote manager.
    Figure Legend Snippet: TEBP-1 and TEBP-2 are expressed throughout C. elegans development and localize to telomeres in vivo. (A) Western blot of TEBP-1::3xFLAG and TEBP-2::GFP expression in different developmental stages of C. elegans . 35 µg of extract from either N2 or a double transgenic line carrying TEBP-1::3xFLAG and TEBP-2::GFP were used. Actin was used as loading control. (B-C) Maximum intensity projections of representative confocal z stacks of an embryo (B), or oocytes (C) expressing endogenously tagged TEBP-1::GFP and transgenic POT-1::mCherry. Scale bars, 10 µm. (D E) Maximum intensity projections of representative confocal z-stacks of an adult animal (D), or embryo (E) expressing both endogenously tagged TEBP-2::GFP and transgenic POT-1::mCherry. Insets show nuclear co localization in meiotic germ cell nuclei (I), an oocyte (II), spermatozoa (III), and embryonic cells (IV). Scale bars, 20 µm (overview) and 4 µm (insets). All microscopy images were deconvoluted using Huygens remote manager.

    Techniques Used: In Vivo, Western Blot, Expressing, Transgenic Assay, Microscopy

    TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value
    Figure Legend Snippet: TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value

    Techniques Used: Quantitation Assay, Sequencing

    12) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    13) Product Images from "Internal Transcribed Spacer rDNA and TEF-1α Gene Sequencing of Pathogenic Dermatophyte Species and Differentiation of Closely Related Species Using PCR-RFLP of The Topoisomerase II"

    Article Title: Internal Transcribed Spacer rDNA and TEF-1α Gene Sequencing of Pathogenic Dermatophyte Species and Differentiation of Closely Related Species Using PCR-RFLP of The Topoisomerase II

    Journal: Cell Journal (Yakhteh)

    doi: 10.22074/cellj.2020.6372

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) electrophoretic patterns of dermatophytes species by amplification of topoisomerase II gene and digestion of the Hinf I enzyme. Lane M; 100 bp DNA ladder, Lane 1; T. tonsurans, Lane 2; T. tonsurans (CBS 130924), Lane 3; T. interdigitale , Lane 4; T. mentagrophytes, Lane 5; T. rubrum, Lane 6; T. rubrum (PFCC 51431), Lane 7; E. floccosum , and Lane 8; E. floccosum (CBS 767.73).
    Figure Legend Snippet: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) electrophoretic patterns of dermatophytes species by amplification of topoisomerase II gene and digestion of the Hinf I enzyme. Lane M; 100 bp DNA ladder, Lane 1; T. tonsurans, Lane 2; T. tonsurans (CBS 130924), Lane 3; T. interdigitale , Lane 4; T. mentagrophytes, Lane 5; T. rubrum, Lane 6; T. rubrum (PFCC 51431), Lane 7; E. floccosum , and Lane 8; E. floccosum (CBS 767.73).

    Techniques Used: Polymerase Chain Reaction, Amplification

    14) Product Images from "Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece"

    Article Title: Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093923

    Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus grape isolates. Restriction digestion patterns (designated as A , B and C ) of ribosomal 5.8S-ITS DNA amplicons from various Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I and Hinf I . 50 bp ( l ) and 100 bp (L) DNA ladders are also shown.
    Figure Legend Snippet: Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus grape isolates. Restriction digestion patterns (designated as A , B and C ) of ribosomal 5.8S-ITS DNA amplicons from various Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I and Hinf I . 50 bp ( l ) and 100 bp (L) DNA ladders are also shown.

    Techniques Used:

    Restriction digestion patterns of sequenced ribosomal 5.8S-ITS region amplicons. Restriction digestion patterns (designated as A , B , C and D ) of five sequenced ribosomal 5.8S-ITS DNA amplicons from five different Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I , Hinf I and Rsa I . Each isolate is a representative of the five different Aspergillus species characterized in this study. L : DNA ladder.
    Figure Legend Snippet: Restriction digestion patterns of sequenced ribosomal 5.8S-ITS region amplicons. Restriction digestion patterns (designated as A , B , C and D ) of five sequenced ribosomal 5.8S-ITS DNA amplicons from five different Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I , Hinf I and Rsa I . Each isolate is a representative of the five different Aspergillus species characterized in this study. L : DNA ladder.

    Techniques Used:

    Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus reference strains. Restriction digestion patterns (designated as A and B ) of ribosomal 5.8S-ITS DNA amplicons from Aspergillus reference strains, after digestion with the restriction endonucleases Hinf I , Hha I and Rsa I . Ac : Aspergillus carbonarius , An : Aspergillus niger , At : Aspergillus tubingensis , Aw : Aspergillus westerdijkiae , Ao : Aspergillus ochraceus , L : Low molecular weight DNA ladder (molecular sizes are 766, 500, 350, 300, 250, 200, 150, 100, 75, 50 and 25 bp respectively).
    Figure Legend Snippet: Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus reference strains. Restriction digestion patterns (designated as A and B ) of ribosomal 5.8S-ITS DNA amplicons from Aspergillus reference strains, after digestion with the restriction endonucleases Hinf I , Hha I and Rsa I . Ac : Aspergillus carbonarius , An : Aspergillus niger , At : Aspergillus tubingensis , Aw : Aspergillus westerdijkiae , Ao : Aspergillus ochraceus , L : Low molecular weight DNA ladder (molecular sizes are 766, 500, 350, 300, 250, 200, 150, 100, 75, 50 and 25 bp respectively).

    Techniques Used: Molecular Weight

    15) Product Images from "The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner"

    Article Title: The Caulobacter crescentus DNA-(adenine-N6)-methyltransferase CcrM methylates DNA in a distributive manner

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr768

    Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.
    Figure Legend Snippet: Various substrates used for studying CcrM processivity. ( A ) Substrate used by Berdis et al. ( 14 ) to study CcrM processivity, referred to as N 6 60/66-mer. Two GANTC target sites are present, hemimethylated on the upper strand. HindII target sites (GTYRAC) coupled to CcrM target sites were used to screen for methylation on the lower strand. However, only one of the two HindII sites is present, making it impossible to probe the methylation state of the second site. ( B ) The distribution of GANTC sequences (shown as HinfI target sequences) throughout the pUC19 plasmid. The position of each sequence is indicated relative to the plasmid's replication origin. The vector contains a single NdeI target site, which was used in conjunction with HinfI for vector linearization, to facilitate viewing of the progression toward fully methylated state. ( C ) 129-mer substrate containing two CcrM target sites. The expected size of the fragments obtained after HinfI digestion of completely unmethylated, partially methylated and fully methylated substrates are indicated. ( D ) 129-mer_HM substrate used to probe CcrM activity over hemimethylated GANTC sites. A M.TaqI methylation site (TCGA), as well as a HincII restriction site (GTYRAC) were linked to the GANTC site. M.TaqI-established methylation occurs as shown earlier, creating two GANTC sites hemimethylated on the lower strand. CcrM-catalyzed methylation of the upper strand was probed through protection from HincII digestion, which is blocked by hemimethylation.

    Techniques Used: Methylation, Plasmid Preparation, Sequencing, Activity Assay

    CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.
    Figure Legend Snippet: CcrM processivity assayed using pUC19 ( Figure 1 B) as substrate. A double digestion with HinfI and NdeI was performed to assess the methylation state of the plasmid. pUC19 plasmid linearized by NdeI digestion was used as a control (lane marked C). A large number of incompletely methylated intermediates are formed throughout the duration of the experiment, supporting the conclusion that CcrM is a distributive, rather than a processive methyltransferase. The marker lane (lane marked M) contains the GeneRuler molecular weight marker, provided by Fermentas. The sizes of the major bands are indicated on the left.

    Techniques Used: Methylation, Plasmid Preparation, Marker, Molecular Weight

    CcrM processivity assays using the 129-mer DNA as substrate ( Figure 2 C) and conducted in the absence ( A ) and with the addition ( B ) of competitor DNA. The expected running distance of partially protected intermediates is indicated on the right by asterisk. Undigested 129-mer DNA was used as control, indicating the expected running distance of fully protected DNA. The marker lane (indicated by M) contained PCR marker provided by New England Biolabs. The sizes of the bands are indicated on the left. ( A ) Initially all of the DNA is efficiently digested by HinfI (time point 0.1 min). Increasing protection from HinfI digestion is established over time, with nearly complete protection being achieved after 120 min. The 96 bp intermediate is present in large amounts, as well as low levels of the 54 bp intermediate, indicating distributive methylation by CcrM, as well as a preference for one target sequence over the other. ( B ) Competitor DNA is added after 3 min (indicated by the arrow). The protection state of the 129-mer substrate remains the same after supplementation with competitor, suggesting dissociation of CcrM from incompletely methylated DNA. The weak band appearing in the last lanes at low molecular weights corresponds to the 23-mer competitor which has become methylated and, thereby, protected against HinfI cleavage.
    Figure Legend Snippet: CcrM processivity assays using the 129-mer DNA as substrate ( Figure 2 C) and conducted in the absence ( A ) and with the addition ( B ) of competitor DNA. The expected running distance of partially protected intermediates is indicated on the right by asterisk. Undigested 129-mer DNA was used as control, indicating the expected running distance of fully protected DNA. The marker lane (indicated by M) contained PCR marker provided by New England Biolabs. The sizes of the bands are indicated on the left. ( A ) Initially all of the DNA is efficiently digested by HinfI (time point 0.1 min). Increasing protection from HinfI digestion is established over time, with nearly complete protection being achieved after 120 min. The 96 bp intermediate is present in large amounts, as well as low levels of the 54 bp intermediate, indicating distributive methylation by CcrM, as well as a preference for one target sequence over the other. ( B ) Competitor DNA is added after 3 min (indicated by the arrow). The protection state of the 129-mer substrate remains the same after supplementation with competitor, suggesting dissociation of CcrM from incompletely methylated DNA. The weak band appearing in the last lanes at low molecular weights corresponds to the 23-mer competitor which has become methylated and, thereby, protected against HinfI cleavage.

    Techniques Used: Marker, Polymerase Chain Reaction, Methylation, Sequencing

    16) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    17) Product Images from "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1"

    Article Title: Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800121

    Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.
    Figure Legend Snippet: Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.

    Techniques Used: Transfection, Agarose Gel Electrophoresis, Amplification, Positive Control

    18) Product Images from "The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals"

    Article Title: The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35499-w

    Schematic presentation of the assembled sequence for RP13-870H17 produced from PacBio SMRT and Sanger sequencing. ( a ) The RP13-870H17 assembly from SMRT sequencing results of whole BAC clone (NG-6867) and Hinf I MUC2 PTS-TR2 fragment sequence (NG-7351) assembled with Sanger sequencing. ( b ) Schematic picture of MUC6 and MUC2 gene organization showing the gene orientation and exon and intron distribution. ( c,d ) Resulting protein domain organization for MUC6 and MUC2. VWD = von Willebrand like domain type D, VWC = von Willebrand like domain type C, CK = C -terminal cystine knot.
    Figure Legend Snippet: Schematic presentation of the assembled sequence for RP13-870H17 produced from PacBio SMRT and Sanger sequencing. ( a ) The RP13-870H17 assembly from SMRT sequencing results of whole BAC clone (NG-6867) and Hinf I MUC2 PTS-TR2 fragment sequence (NG-7351) assembled with Sanger sequencing. ( b ) Schematic picture of MUC6 and MUC2 gene organization showing the gene orientation and exon and intron distribution. ( c,d ) Resulting protein domain organization for MUC6 and MUC2. VWD = von Willebrand like domain type D, VWC = von Willebrand like domain type C, CK = C -terminal cystine knot.

    Techniques Used: Sequencing, Produced, BAC Assay

    19) Product Images from "Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿"

    Article Title: Functional Analysis of the M.HpyAIV DNA Methyltransferase of Helicobacter pylori ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00108-07

    Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.
    Figure Legend Snippet: Purified M.HpyAIV protects a GANTC-containing DNA fragment from HinfI digestion. Increasing concentrations of M.HpyAIV protein incubated with a 778-bp PCR fragment containing one GANTC site and S -adenosylmethionine. HinfI digestion of the GANTC-containing DNA fragment resulted in two fragments of 540 bp and 238 bp. The increased amount of undigested PCR products as a consequence of an increased M.HpyAIV concentration illustrates the in vitro capability of M.HpyAIV to protect GANTC sites from digestion in a concentration-dependent manner. L, ladder (samples in duplicate with increasing amounts of M.HpyAIV added [0, 200, 400, 800, and 1,200 nM]); UC, uncut control.

    Techniques Used: Purification, Incubation, Polymerase Chain Reaction, Concentration Assay, In Vitro

    20) Product Images from "Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome"

    Article Title: Ligation of high-melting-temperature 'clamp' sequence extends the scanning range of rare point-mutational analysis by constant denaturant capillary electrophoresis (CDCE) to most of the human genome

    Journal: Nucleic Acids Research

    doi:

    Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).
    Figure Legend Snippet: Restriction digestion by AhdI and HinfI generates the human HPRT target (cDNA bp 222–318) with a natural clamp (cDNA bp 141–217). Restriction digestion by ApoI, followed by ligation of a GC-base-rich clamp to the ApoI restriction end ( HPRT cDNA bp 218–221), generates the target with a ligated clamp. The filled bars indicate the positions of the PCR primers (P3, L1 and P1). The solid and dotted lines represent the melting profiles of the target wild-type with the natural and ligated clamps, respectively. These profiles were constructed using WinMelt™ 2.0 (Medprobe, Norway).

    Techniques Used: Ligation, Polymerase Chain Reaction, Construct

    Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.
    Figure Legend Snippet: Flow diagram of rare point-mutational analysis by CDCE/hifiPCR: natural versus ligated clamp. The copy numbers of the wild-type and of a mutant added at an initial fraction of 5 × 10 –5 are shown after each step. Restriction digestion by BstNI and DraI liberates the HPRT target-embedded fragment of 438 bp from genomic DNA. The lines in this fragment indicate the positions of the G to A transition and of the G to T transversion carried by the internal standards of the 438 bp PCR fragment and of the HPRT Munich cells, respectively. The restriction-recognition sites of AhdI, ApoI and HinfI are also indicated by the lines. The open and filled bars indicate the positions of the probes (Probe 1 and Probe 2) used for target isolation and of the PCR primers (P3 and P1), respectively.

    Techniques Used: Flow Cytometry, Mutagenesis, Polymerase Chain Reaction, Isolation

    21) Product Images from "DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients"

    Article Title: DNA Methylation of the ABO Promoter Underlies Loss of ABO Allelic Expression in a Significant Proportion of Leukemic Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004788

    Loss of A expression by RT-PCR and restriction enzyme digestion. (A) Schematic representation of ABO allelic expression analysis. Kpn I digestion results in a 130 bp band if the O allele is present and no digestion of the A or B allele. BstE II digestion results in a 130 bp band if the A or B allele is present and no digestion of the O allele. (B) Lane M is the pUC19/ Hpa II marker while lane 1 is the uncut ABO RT-PCR product. Lanes 2, 4, 6 and 8 are digested with Kpn I while lanes 3, 5, 7 and 9 are digested with BstE II. Lanes 2 and 3 are from cDNA of patient F7, lanes 4 and 5 from F11, lanes 6 and 7 from F15 and lanes 8 and 9 from F17. F7 and F11 are AO patients with loss of the A allele, F17 is an AO patient with no loss of ABO allelic expression. Patient F15 has an A 1 A 2 genotype, hence no cutting with Kpn I was expected. (C) Lanes 1, 3 and 5 are ABO RT-PCR product digested with Kpn I while lanes 2, 4 and 6 are digests with BstE II. Lanes 1 and 2 are from cDNA of patient F23, an A 2 B genotype, hence no cutting with Kpn I was expected. Lanes 3 and 4 are F53, an A 1 O 1 patient with loss of A at the mRNA level. Lanes 5 and 6 are S8, which is a patient with an A 1 O 1 genotype with loss of A allelic expression. (D) The ABO CpG island promoter region assessed for methylation. The methylated and bisulfite modified sequence is shown and the primer sequences are double underlined. The capital Ts identify thymines that are a result of bisulfite modification of cytosines and the CpGs are shown in bold. The start of transcription is marked with +1. The different restriction enzymes used for assessing methylation by digestion are as follows: eight BstU I sites ( cg/cg ), two Taq I ( T/cga ) sites (however one is found in the primer and hence will cut regardless of methylation status), one Hinf I ( g/aTTc ) site. Regions 161–173 and 198–210 harbor Sp1 sites [55] .
    Figure Legend Snippet: Loss of A expression by RT-PCR and restriction enzyme digestion. (A) Schematic representation of ABO allelic expression analysis. Kpn I digestion results in a 130 bp band if the O allele is present and no digestion of the A or B allele. BstE II digestion results in a 130 bp band if the A or B allele is present and no digestion of the O allele. (B) Lane M is the pUC19/ Hpa II marker while lane 1 is the uncut ABO RT-PCR product. Lanes 2, 4, 6 and 8 are digested with Kpn I while lanes 3, 5, 7 and 9 are digested with BstE II. Lanes 2 and 3 are from cDNA of patient F7, lanes 4 and 5 from F11, lanes 6 and 7 from F15 and lanes 8 and 9 from F17. F7 and F11 are AO patients with loss of the A allele, F17 is an AO patient with no loss of ABO allelic expression. Patient F15 has an A 1 A 2 genotype, hence no cutting with Kpn I was expected. (C) Lanes 1, 3 and 5 are ABO RT-PCR product digested with Kpn I while lanes 2, 4 and 6 are digests with BstE II. Lanes 1 and 2 are from cDNA of patient F23, an A 2 B genotype, hence no cutting with Kpn I was expected. Lanes 3 and 4 are F53, an A 1 O 1 patient with loss of A at the mRNA level. Lanes 5 and 6 are S8, which is a patient with an A 1 O 1 genotype with loss of A allelic expression. (D) The ABO CpG island promoter region assessed for methylation. The methylated and bisulfite modified sequence is shown and the primer sequences are double underlined. The capital Ts identify thymines that are a result of bisulfite modification of cytosines and the CpGs are shown in bold. The start of transcription is marked with +1. The different restriction enzymes used for assessing methylation by digestion are as follows: eight BstU I sites ( cg/cg ), two Taq I ( T/cga ) sites (however one is found in the primer and hence will cut regardless of methylation status), one Hinf I ( g/aTTc ) site. Regions 161–173 and 198–210 harbor Sp1 sites [55] .

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Methylation, Modification, Sequencing

    22) Product Images from "The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1"

    Article Title: The double-stranded DNA-binding proteins TEBP-1 and TEBP-2 form a telomeric complex with POT-1

    Journal: bioRxiv

    doi: 10.1101/2020.08.19.256388

    TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.
    Figure Legend Snippet: TEBP-1 and TEBP-2 regulate telomere length. (A) Southern blot analysis of C. elegans telomeres. DNA from four different strains ( tebp-1(xf133), tebp-2(xf131) , N2, and pot-2(tm1400) ) was digested and separated by agarose gel electrophoresis. DNA was transferred to a positively charged nylon membrane and hybridized with a radiolabeled (GCCTAA) 3 oligonucleotide. Brightness and contrast of the membrane read out were adjusted using Fiji. Telomere restriction fragments (TRFs) are indicated in the Fig.. (B-E) Representative maximum projection z stacks of a qFISH assay using dissected adult germlines of the following C. elegans mutant strains: tebp-1(xf133), tebp-2(xf131), pot-2(tm1400) , and wild type N2. The telomeres of dissected worms of the respective strains were visualized by hydridization with a telomeric PNA-FISH-probe. Nuclei were stained with DAPI. Scale bars, 15 µm. (F) Barplot depicting analysis of qFISH images of the strains in (B-C) and (E). Average telomere length is indicated by arbitrary units of relative integrated density, with wild type N2 set to 1. The plot on the left shows the tebp-2(xf131) and N2 values zoomed-in.

    Techniques Used: Southern Blot, Agarose Gel Electrophoresis, Mutagenesis, Fluorescence In Situ Hybridization, Staining

    Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value
    Figure Legend Snippet: Conservation of tebp genes in the Caenorhabditis genus. (A) On the left is a phylogenetic tree constructed with IQ-TREE (v1.6.12), using a MAFFT (v7.452) multiple sequence alignment of the protein sequences of TEBP orthologs (see Supplemental Table 2, sheet 2). Values on the nodes represent bootstrapping values for 10000 replicates, set to 100. On the right, the table provides an overview of whether specific tebp orthologs are in synteny with tebp-1 or tebp-2 of C. elegans . A “+” indicates regional synteny, while a “-“ is lack of synteny. (B) Volcano plot of telomere DNA pulldown, as in Fig. 1A , of gravid adult nuclear extracts from C. briggsae . Here, pulldowns were performed in quadruplicates, per condition. Enriched proteins (enrichment threshold > 2-fold, p-value

    Techniques Used: Construct, Sequencing

    POT-1 links the ds telomere binders to the ss telomere. (A) Y2H assay with full length TEBP-1, TEBP-2, and POT-1 fusions to the activation or DNA binding domains of Gal4. Growth on TRP LEU HIS plates demonstrates interaction. Growth on high stringency TRP LEU HIS ADE medium suggests strong interaction. (B-C) Volcano plots showing quantitative proteomic analysis of either TEBP-1::3xFLAG (B) or TEBP-2::GFP (C) IPs in embryos. IPs were performed in quadruplicates. Enriched proteins (threshold: 2-fold, p-value
    Figure Legend Snippet: POT-1 links the ds telomere binders to the ss telomere. (A) Y2H assay with full length TEBP-1, TEBP-2, and POT-1 fusions to the activation or DNA binding domains of Gal4. Growth on TRP LEU HIS plates demonstrates interaction. Growth on high stringency TRP LEU HIS ADE medium suggests strong interaction. (B-C) Volcano plots showing quantitative proteomic analysis of either TEBP-1::3xFLAG (B) or TEBP-2::GFP (C) IPs in embryos. IPs were performed in quadruplicates. Enriched proteins (threshold: 2-fold, p-value

    Techniques Used: Y2H Assay, Activation Assay, Binding Assay

    TEBP-1 and TEBP-2 are expressed throughout C. elegans development and localize to telomeres in vivo. (A) Western blot of TEBP-1::3xFLAG and TEBP-2::GFP expression in different developmental stages of C. elegans . 35 µg of extract from either N2 or a double transgenic line carrying TEBP-1::3xFLAG and TEBP-2::GFP were used. Actin was used as loading control. (B-C) Maximum intensity projections of representative confocal z stacks of an embryo (B), or oocytes (C) expressing endogenously tagged TEBP-1::GFP and transgenic POT-1::mCherry. Scale bars, 10 µm. (D E) Maximum intensity projections of representative confocal z-stacks of an adult animal (D), or embryo (E) expressing both endogenously tagged TEBP-2::GFP and transgenic POT-1::mCherry. Insets show nuclear co localization in meiotic germ cell nuclei (I), an oocyte (II), spermatozoa (III), and embryonic cells (IV). Scale bars, 20 µm (overview) and 4 µm (insets). All microscopy images were deconvoluted using Huygens remote manager.
    Figure Legend Snippet: TEBP-1 and TEBP-2 are expressed throughout C. elegans development and localize to telomeres in vivo. (A) Western blot of TEBP-1::3xFLAG and TEBP-2::GFP expression in different developmental stages of C. elegans . 35 µg of extract from either N2 or a double transgenic line carrying TEBP-1::3xFLAG and TEBP-2::GFP were used. Actin was used as loading control. (B-C) Maximum intensity projections of representative confocal z stacks of an embryo (B), or oocytes (C) expressing endogenously tagged TEBP-1::GFP and transgenic POT-1::mCherry. Scale bars, 10 µm. (D E) Maximum intensity projections of representative confocal z-stacks of an adult animal (D), or embryo (E) expressing both endogenously tagged TEBP-2::GFP and transgenic POT-1::mCherry. Insets show nuclear co localization in meiotic germ cell nuclei (I), an oocyte (II), spermatozoa (III), and embryonic cells (IV). Scale bars, 20 µm (overview) and 4 µm (insets). All microscopy images were deconvoluted using Huygens remote manager.

    Techniques Used: In Vivo, Western Blot, Expressing, Transgenic Assay, Microscopy

    TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value
    Figure Legend Snippet: TEBP-1 (R06A4.2) and TEBP-2 (T12E12.3) are double stranded telomere binders in C. elegans . (A) Volcano plot representing label free proteomic quantitation of pulldowns with biotinylated, concatenated oligonucleotide baits of telomeric DNA sequence (TTAGGC) n or control DNA sequence (AGGTCA) n . Pulldowns were performed with nuclear extracts from synchronized gravid adult animals, in octuplicates per condition (two biological replicates, each with four technical replicates). Log 2 fold enrichment of proteins in one condition over the other is presented on the x-axis. The y-axis shows -log 10 p-value (Welch t test) of enrichment across replicates. More than 4-fold enriched proteins with p value

    Techniques Used: Quantitation Assay, Sequencing

    23) Product Images from "The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals"

    Article Title: The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-35499-w

    Schematic presentation of the assembled sequence for RP13-870H17 produced from PacBio SMRT and Sanger sequencing. ( a ) The RP13-870H17 assembly from SMRT sequencing results of whole BAC clone (NG-6867) and Hinf I MUC2 PTS-TR2 fragment sequence (NG-7351) assembled with Sanger sequencing. ( b ) Schematic picture of MUC6 and MUC2 gene organization showing the gene orientation and exon and intron distribution. ( c,d ) Resulting protein domain organization for MUC6 and MUC2. VWD = von Willebrand like domain type D, VWC = von Willebrand like domain type C, CK = C -terminal cystine knot.
    Figure Legend Snippet: Schematic presentation of the assembled sequence for RP13-870H17 produced from PacBio SMRT and Sanger sequencing. ( a ) The RP13-870H17 assembly from SMRT sequencing results of whole BAC clone (NG-6867) and Hinf I MUC2 PTS-TR2 fragment sequence (NG-7351) assembled with Sanger sequencing. ( b ) Schematic picture of MUC6 and MUC2 gene organization showing the gene orientation and exon and intron distribution. ( c,d ) Resulting protein domain organization for MUC6 and MUC2. VWD = von Willebrand like domain type D, VWC = von Willebrand like domain type C, CK = C -terminal cystine knot.

    Techniques Used: Sequencing, Produced, BAC Assay

    24) Product Images from "The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions"

    Article Title: The DNA end-binding protein Ku associates with human telomeres primarily via protein-protein interactions

    Journal: bioRxiv

    doi: 10.1101/2019.12.11.873422

    Myc-Ku80 α5 mutant or Myc-Ku80 DEB expression compromises cell viability but does not lead to telomere shortening or t-circle formation in short term cultures. (A) Representative Southern blot analysis to measure telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku80 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku80 siRNA was used as positive and negative controls. KD indicates siRNA-mediated knockdown. Telomere Southern blots were quantified via TeloTool software and the red dot in each lane denotes mean telomere length. Additional representative experiment included in Supplemental Figure S3 (B) Same as A except telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku70 transgenes. Additional representative experiment included in Supplemental Figure S3 (C) 2D gel electrophoresis of HinfI/RsaI digested genomic DNA prepared from indicated cell lines that were subjected to either Ku70 or Ku80 knockdown. U2OS control cell line showing t-circles (D) Analysis of cell viability by Annexin V staining of indicated cell lines expressing Myc-Ku80 or Myc-Ku70 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku70 or Ku80 siRNA was used as controls.
    Figure Legend Snippet: Myc-Ku80 α5 mutant or Myc-Ku80 DEB expression compromises cell viability but does not lead to telomere shortening or t-circle formation in short term cultures. (A) Representative Southern blot analysis to measure telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku80 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku80 siRNA was used as positive and negative controls. KD indicates siRNA-mediated knockdown. Telomere Southern blots were quantified via TeloTool software and the red dot in each lane denotes mean telomere length. Additional representative experiment included in Supplemental Figure S3 (B) Same as A except telomere lengths of indicated cell lines uninduced or induced to express Myc-Ku70 transgenes. Additional representative experiment included in Supplemental Figure S3 (C) 2D gel electrophoresis of HinfI/RsaI digested genomic DNA prepared from indicated cell lines that were subjected to either Ku70 or Ku80 knockdown. U2OS control cell line showing t-circles (D) Analysis of cell viability by Annexin V staining of indicated cell lines expressing Myc-Ku80 or Myc-Ku70 transgenes. Empty vector cell line transfected with scrambled (scr) or Ku70 or Ku80 siRNA was used as controls.

    Techniques Used: Mutagenesis, Expressing, Southern Blot, Plasmid Preparation, Transfection, Software, Two-Dimensional Gel Electrophoresis, Electrophoresis, Staining

    25) Product Images from "Detection of circular telomeric DNA without 2-D gel electrophoresis"

    Article Title: Detection of circular telomeric DNA without 2-D gel electrophoresis

    Journal: DNA and cell biology

    doi: 10.1089/dna.2008.0741

    Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior
    Figure Legend Snippet: Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior

    Techniques Used: Incubation, Hybridization

    Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound
    Figure Legend Snippet: Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound

    Techniques Used: Labeling

    The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part
    Figure Legend Snippet: The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis

    26) Product Images from "Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1"

    Article Title: Transformation-induced stress at telomeres is counteracted through changes in the telomeric proteome including SAMHD1

    Journal: Life Science Alliance

    doi: 10.26508/lsa.201800121

    Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.
    Figure Legend Snippet: Co-depletion of TRF1 and SAMHD1 does not lead to rapid telomere shortening. (A) TRF analysis of genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids. Genomic DNA was digested overnight with HinfI and RsaI and fractionated on an agarose gel. (B) Phi29-dependent telomeric circles (T-circles) amplification assay. Genomic DNA prepared from HeLa cells transfected with indicated pSuper plasmids was digested overnight with HinfI and RsaI, and 0.75 μg of DNA was used for phi29-dependent amplification reaction. Genomic DNA from U2OS cell line was used as a positive control. Arrows indicate T-circle amplification products.

    Techniques Used: Transfection, Agarose Gel Electrophoresis, Amplification, Positive Control

    27) Product Images from "Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells"

    Article Title: Expression and Differentiation between OCT4A and Its Pseudogenes in Human ESCs and Differentiated Adult Somatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0089546

    Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.
    Figure Legend Snippet: Schematic representation of restriction sites in 646-PCR amplicon. Specific restriction sites were used to distinguish between embryonic OCT4A transcript and different pseudogenes. Red arrows show restriction sites. ApaI restriction site is present only in embryonic OCT4A and can be used to distinguish embryonic form from all six pseudogenes; after restriction, a 146 bp and 500 bp long fragments are produced. HinfI digestion results in several smaller fragments among which the 434 bp fragment is specific only for OCT4-pg1. BglI digests only OCT4-pg3 into two fragments of 412 bp and 232 bp. XhoI does not digest OCT4-pg4.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced

    28) Product Images from "WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †"

    Article Title: WRN Controls Formation of Extrachromosomal Telomeric Circles and Is Required for TRF2ΔB-Mediated Telomere Shortening ▿-Mediated Telomere Shortening ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01364-07

    Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Telomeric circles are present in telomerase-positive WS fibroblasts in the absence of TRF2 ΔB . DNA isolated from normal (A) and WS (C) fibroblasts transduced with lentiviruses expressing the indicated proteins was digested with HinfI and RsaI, separated by size and shape, blotted, and probed with a telomeric (CCCTAA) repeat probe. Arrows indicate arcs of telomeric DNA circles. Circularized λ × HindIII DNA fragments were used as molecular size markers (the 23- and 4.4-kb fragments have one cos end and do not circularize). Samples shown in panels A and C were run and processed in parallel under the same hybridization and washing conditions. (B) DNA isolated from ALT fibroblasts was separated by 2DGE and probed with a telomeric (CCCTAA) 4 probe. The data shown are representative of at least three independent experiments. The approximate level of telomeric circles (expressed as a percentage of the total telomeric DNA) present in each sample was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Isolation, Transduction, Expressing, Hybridization

    TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB -induced cell senescence, TIFs, and telomere shortening are reconstituted in WS fibroblasts genetically complemented with wild-type WRN but not enzymatically deficient WRN variants. (A) Expression of wild-type and mutant forms of WRN in WS cells. WS cells were infected with lentiviruses for the expression wild-type, helicase-deficient, exonuclease-deficient, and helicase- and exonuclease-deficient forms of WRN and cultured for 2 weeks. The parental and genetically complemented cells lines were then transduced with a control virus or a virus for the expression of Flag-TRF2 ΔB or Flag-TRF2. Analysis of protein expression was performed by preparation of nuclear extracts, followed by Western blotting with anti-WRN (top panel), antitubulin (middle panel), and anti-Flag (bottom panel) antibodies. (B) Detection of SA-βgal activity. Telomerase-positive WS fibroblasts transduced with the indicated lentiviruses were cultured for 8 days, fixed, and stained for SA-βgal. Five hundred cells of each line were analyzed in duplicate plates. Each bar represents the mean ± the standard deviation of three independent experiments ( n = 3) carried out in duplicate. WT, wild type. (C) Detection of 53BP1 and TRF1 in WS fibroblasts consecutively transduced with lentiviruses expressing the indicated proteins with antibodies against 53BP1 (green) and TRF1 (red) 1 day after the second transduction. For the quantitation of 53BP1 foci and 53BP1 and TRF1 colocalization, see Fig. S1 in the supplemental material. (D) The parental and genetically complemented cell lines were transduced with control lentivirus (lanes 1 and 4) and lentiviruses for the expression of Flag-TRF2 ΔB (lanes 2 and 5) or Flag-TRF2 (lanes 3 and 6). Cells were harvested 8 days after lentivirus transduction, and genomic DNA was isolated and digested with HinfI and RsaI. Equal amounts (2 μg) of digested genomic DNA were separated by electrophoresis on a 0.8% agarose gel, followed by Southern blot analysis with a radiolabeled (TTAGGG) 3 probe. Southern blot analyses were performed on three independent samples of WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Mutagenesis, Infection, Cell Culture, Transduction, Western Blot, Activity Assay, Staining, Standard Deviation, Quantitation Assay, Isolation, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation

    TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.
    Figure Legend Snippet: TRF2 ΔB induces telomere shortening in normal but not WS fibroblasts. Normal and WS fibroblasts expressing Flag-TRF2 ΔB (lanes 2, 5, 9, and 11) and Flag-TRF2 (lanes 3 and 6), along with normal and WS fibroblasts transduced with control viruses (lanes 1, 4, 8, and 10), were harvested 8 days after lentivirus transduction. Equal amounts of genomic DNA digested with HinfI and RsaI were separated by electrophoresis on a 0.8% agarose gel and analyzed by Southern blotting with a radiolabeled (TTAGGG) 3 probe. The molecular mass standards shown on right side were generated by digestion of lambda DNA with restriction endonuclease HindIII. Southern blot analyses were performed on three independent samples of normal and WS cells transduced with lentiviruses expressing the indicated proteins. The telomeric signal was normalized to the H1.1 gene probe for all lanes (see Fig. S2 in the supplemental material), and the normalized values ± standard deviations, expressed as the telomeric signal relative to the vector control for each cell line, from three independent experiments ( n = 3) are shown below the blots.

    Techniques Used: Expressing, Transduction, Electrophoresis, Agarose Gel Electrophoresis, Southern Blot, Generated, Lambda DNA Preparation, Plasmid Preparation

    Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.
    Figure Legend Snippet: Expression of wild-type WRN but not enzymatically deficient WRN variants in WS fibroblasts leads to a reduction in telomeric circles, which are reformed upon the overexpression of TRF2 ΔB . (A) DNA isolated from WS fibroblasts transduced with a vector control lentivirus or a lentivirus expressing WRN was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. (B) DNA isolated from WS fibroblasts transduced with a lentivirus expressing a WRN variant lacking either exonuclease or helicase activity was digested with HinfI and RsaI, separated by 2DGE, blotted, and probed with a telomeric (CCCTAA) 4 probe. Arrows show arcs of telomeric DNA circles. (C) DNA isolated from WRN-complemented WS fibroblasts was transduced with a control lentivirus or a lentivirus expressing TRF2 ΔB , digested with HinfI and RsaI, separated by 2DGE, and probed with a telomeric (CCCTAA) 4 probe. The samples shown in each panel were run and processed in parallel under the same hybridization and washing conditions. The approximate level of telomeric circles present in each sample (expressed as a percentage of the total telomeric DNA) was estimated (see Fig. S4 in the supplemental material) and is shown in the upper right corner of each panel. The samples shown in each panel were blotted, hybridized, washed, and analyzed simultaneously.

    Techniques Used: Expressing, Over Expression, Isolation, Transduction, Plasmid Preparation, Variant Assay, Activity Assay, Hybridization

    29) Product Images from "Impact of Oil on Bacterial Community Structure in Bioturbated Sediments"

    Article Title: Impact of Oil on Bacterial Community Structure in Bioturbated Sediments

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065347

    Bacterial community structure. nMDS analysis based on T-RFLP profiles (HaeIII and HinfI enzymes) from 16S rRNA gene (black) and 16S rRNA transcripts (gray) from microcosm sediment sampled at day 2, 7, 30, 90, 180 and 270 of incubation. CTRL: blank circle; BAL: filled circle; NEREIS: blank triangle; NEREIS+BAL: filled triangle.
    Figure Legend Snippet: Bacterial community structure. nMDS analysis based on T-RFLP profiles (HaeIII and HinfI enzymes) from 16S rRNA gene (black) and 16S rRNA transcripts (gray) from microcosm sediment sampled at day 2, 7, 30, 90, 180 and 270 of incubation. CTRL: blank circle; BAL: filled circle; NEREIS: blank triangle; NEREIS+BAL: filled triangle.

    Techniques Used: Incubation

    30) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    31) Product Images from "Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia"

    Article Title: Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia

    Journal: Genes and immunity

    doi: 10.1038/gene.2012.40

    a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).
    Figure Legend Snippet: a. Combined bisulfite restriction analysis of LINE-1 in symptomatic BPH TURP samples, asymptomatic BPH, and donors. Lane 1 is a 100 base pair (bp) molecular weight marker (mwm), lane 2 is the calibrator, lanes 3–5 are symptomatic BPH TURP samples, lanes 6–8 are asymptomatic BPH, lanes 9–11 are donor and lane 12 is undigested PCR product. DNA samples were bisulfite treated, subjected to Line1 PCR and subsequent digestion with HinfI. The undigested top band at 450bp represents unmethylated DNA whereas the digestion products at 275bp and 180bp indicate methylated DNA. b. Quantification of the percent methylation compared to the calibrator of 11 symptomatic BPH from TURP, 10 asymptomatic BPH and 9 donors. Data is represented as a box and whisker plot. A statistically significant difference exists between symptomatic BPH and donors groups (p = 0.040, Mann Whitney), whereas there is no statistically significant difference between symptomatic BPH and asymptomatic BPH. c. APOBEC3G real time PCR results for symptomatic BPH, asymptomatic BPH, and donor samples. The Mann-Whitney rank-sum test was used to determine the statistically significant p value of 0.011 between symptomatic BPH and donors and the t-test was used to determine the statistically significant difference between symptomatic BPH and donors (p = 0.043).

    Techniques Used: Molecular Weight, Marker, Polymerase Chain Reaction, Methylation, Whisker Assay, MANN-WHITNEY, Real-time Polymerase Chain Reaction

    32) Product Images from "Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei"

    Article Title: Telomere length regulation and transcriptional silencing in KU80-deficient Trypanosoma brucei

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh991

    Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.
    Figure Legend Snippet: Telomere shortening in Δ tb KU80 trypanosomes. Genomic DNA from wild-type cells, tb KU80 single allele knockout cells ( tb KU80 +/− ), tb KU80-deficient cells (Δ tb KU80) and from a tb KU80-deficient cell line, which expressed an ectopic copy of GFP– tb KU80 (Δ tb KU80 + GFP– tb KU80) was prepared every other week for a period of 8 weeks. The DNA was digested with AluI, HinfI and RsaI, and separated by agarose gel electrophoresis, Southern-blotted and probed with a radiolabeled telomeric (TTAGGG) 27 probe. Distinguishable bands of Δ tb KU80 telomeric DNA were used to estimate telomere shortening rates during 8 weeks.

    Techniques Used: Knock-Out, Agarose Gel Electrophoresis

    33) Product Images from "Discovery of DNA methylation markers in cervical cancer using relaxation ranking"

    Article Title: Discovery of DNA methylation markers in cervical cancer using relaxation ranking

    Journal: BMC Medical Genomics

    doi: 10.1186/1755-8794-1-57

    Representative COBRA on 3 gene promoters ( SST , AUTS2 and SYCP3 ) . A: schematic representation of of the restriction enzyme sites in the virtual hypermethylated BSP nucleotide sequence after bisulfite treatment.(B: BstUI , T: TaqI and H: HinfI ). Bars represent CG site and arrow is TSS (retrieved from Ensembl). B: Result of COBRA analysis of BSP products of tumour samples (T1-T10) and 5 normal cervices (N1-N5), in vitro methylated DNA as a positive control (IV) and leukocyte DNA as a negative (unmethylated) control (L); lane B is water blank.
    Figure Legend Snippet: Representative COBRA on 3 gene promoters ( SST , AUTS2 and SYCP3 ) . A: schematic representation of of the restriction enzyme sites in the virtual hypermethylated BSP nucleotide sequence after bisulfite treatment.(B: BstUI , T: TaqI and H: HinfI ). Bars represent CG site and arrow is TSS (retrieved from Ensembl). B: Result of COBRA analysis of BSP products of tumour samples (T1-T10) and 5 normal cervices (N1-N5), in vitro methylated DNA as a positive control (IV) and leukocyte DNA as a negative (unmethylated) control (L); lane B is water blank.

    Techniques Used: Combined Bisulfite Restriction Analysis Assay, Sequencing, In Vitro, Methylation, Positive Control

    34) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    35) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.
    Figure Legend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Techniques Used: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).
    Figure Legend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    36) Product Images from "GyrA ser83 and ParC trp106 Mutations in Salmonella enterica Serovar Typhi Isolated from Typhoid Fever Patients in Tertiary Care Hospital"

    Article Title: GyrA ser83 and ParC trp106 Mutations in Salmonella enterica Serovar Typhi Isolated from Typhoid Fever Patients in Tertiary Care Hospital

    Journal: Journal of Clinical and Diagnostic Research : JCDR

    doi: 10.7860/JCDR/2016/17677.8153

    Representative RFLP electrophoretic gel showing the gyrA PCR product digested with HinfI.
    Figure Legend Snippet: Representative RFLP electrophoretic gel showing the gyrA PCR product digested with HinfI.

    Techniques Used: Polymerase Chain Reaction

    37) Product Images from "A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns"

    Article Title: A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0060547

    Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions over one year. (A) Methylation-sensitive endonuclease digested PCR amplification of ERF6 upstream region. Top: schematic diagram of the identification of a methylation-sensitive BstX I digenstion site (CCANNNNNNTGG) at −275 bp of the ERF6 promoter. The bold C indicates a CHH site with annual methylation pattern change, corresponding to the cytosine labelled with red triangles in Figure 3A . Bottom: PCR amplification using genomic DNA with (+) or without (−) BstX I digestion. (B) Southern blot of genomic DNA harvested at different times of the year, first digested by a methylation non-sensitive endonuclease Mbo II (TCTTC) to obtain a full length fragment of 605 bp from −621 to −15 of ERF6 upstream regions, then digested thoroughly with BstX I, and probed with the fragment from −263 to −21 nt. The signal intensities of the band of BstX I-cleaved 244 bp changed at different time-of-year (see Table S8 ), indicating the methlytion levels of this CHH site were different, consistent with the bisulfite sequencing data in Figure 3A and methylation-sensitive endonuclease digested PCR results in Figure 4A . The same methylation-sensitive endonuclease digested PCR experiments were performed for the upstream regions of SUR4 (C) and KCS13 (E), except the methylation-sensitive endonucleases used were HinF I and Bsl I, respectively. Further, the same methylation-sensitive endonuclease digested Southern experiments were performed for the upstream regions of SUR4 (D) and KCS13 (F), except the genomic DNA were first digested by Bcl I (TGATCA) and NSi I (ATGCAT), then digested by methylation-sensitive endonucleases HinF I (GANTC) and Bsl I (CCNNNNNNNGG), respectively. The signal intensities of HinF I- and Bsl I-cleaved 330 bp and 837 bp changed similarly (see Table S8 ).
    Figure Legend Snippet: Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions over one year. (A) Methylation-sensitive endonuclease digested PCR amplification of ERF6 upstream region. Top: schematic diagram of the identification of a methylation-sensitive BstX I digenstion site (CCANNNNNNTGG) at −275 bp of the ERF6 promoter. The bold C indicates a CHH site with annual methylation pattern change, corresponding to the cytosine labelled with red triangles in Figure 3A . Bottom: PCR amplification using genomic DNA with (+) or without (−) BstX I digestion. (B) Southern blot of genomic DNA harvested at different times of the year, first digested by a methylation non-sensitive endonuclease Mbo II (TCTTC) to obtain a full length fragment of 605 bp from −621 to −15 of ERF6 upstream regions, then digested thoroughly with BstX I, and probed with the fragment from −263 to −21 nt. The signal intensities of the band of BstX I-cleaved 244 bp changed at different time-of-year (see Table S8 ), indicating the methlytion levels of this CHH site were different, consistent with the bisulfite sequencing data in Figure 3A and methylation-sensitive endonuclease digested PCR results in Figure 4A . The same methylation-sensitive endonuclease digested PCR experiments were performed for the upstream regions of SUR4 (C) and KCS13 (E), except the methylation-sensitive endonucleases used were HinF I and Bsl I, respectively. Further, the same methylation-sensitive endonuclease digested Southern experiments were performed for the upstream regions of SUR4 (D) and KCS13 (F), except the genomic DNA were first digested by Bcl I (TGATCA) and NSi I (ATGCAT), then digested by methylation-sensitive endonucleases HinF I (GANTC) and Bsl I (CCNNNNNNNGG), respectively. The signal intensities of HinF I- and Bsl I-cleaved 330 bp and 837 bp changed similarly (see Table S8 ).

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Southern Blot, Methylation Sequencing

    Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions in ROS1 RNAi lines. (A) Analysis of relative ERF6 transcription in ovules from ROS1 RNAi lines by qRT-PCR. The level of ERF6 transcripts in ovules from the empty vector line (V) was arbitrarily defined as 1. (B) Southern blot analysis of genomic DNA prepared from ROS1 RNAi lines digested thoroughly with BstX I. See detailed information in Figure 4 legend. Similar qRT-PCR experiments were performed for SUR4 (C) and KCS13 (E) transcriptions, as well as similar Southern experiments for SUR4 (D) and KCS13 (F), respectively. Note the reduced intensities of the BstX I-, HinF I- and Bsl I-cleaved bands in all three RNAi lines compared to the vector line.
    Figure Legend Snippet: Methylation-sensitive endonuclease digested PCR and Southern analysis of ERF6 , SUR4 , and KCS13 upstream regions in ROS1 RNAi lines. (A) Analysis of relative ERF6 transcription in ovules from ROS1 RNAi lines by qRT-PCR. The level of ERF6 transcripts in ovules from the empty vector line (V) was arbitrarily defined as 1. (B) Southern blot analysis of genomic DNA prepared from ROS1 RNAi lines digested thoroughly with BstX I. See detailed information in Figure 4 legend. Similar qRT-PCR experiments were performed for SUR4 (C) and KCS13 (E) transcriptions, as well as similar Southern experiments for SUR4 (D) and KCS13 (F), respectively. Note the reduced intensities of the BstX I-, HinF I- and Bsl I-cleaved bands in all three RNAi lines compared to the vector line.

    Techniques Used: Methylation, Polymerase Chain Reaction, Quantitative RT-PCR, Plasmid Preparation, Southern Blot

    38) Product Images from "Detection of circular telomeric DNA without 2-D gel electrophoresis"

    Article Title: Detection of circular telomeric DNA without 2-D gel electrophoresis

    Journal: DNA and cell biology

    doi: 10.1089/dna.2008.0741

    Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior
    Figure Legend Snippet: Klenow treatment prior to Bal31 incubation preserves the telomeric signal. Bal31 degradation of 10 μg of RsaI/HinfI-digested genomic DNA from VA-13 cells resulted in a faint signal after (TTAGGG) 4 hybridization. A Klenow fill-in reaction prior

    Techniques Used: Incubation, Hybridization

    Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound
    Figure Legend Snippet: Klenwow/Bal31 treatment does not generate a product from linear telomeric DNA. After digesting 40μg of genomic DNA with RsaI and HinfI a biotin-labeled C-rich oligo was annealed to the 3′single-stranded overhang. Pulling down oligo-bound

    Techniques Used: Labeling

    The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part
    Figure Legend Snippet: The Klenow/Bal31 treatment of ALT cell DNA generates molecules that run as a single arc in 2D gel electrophoresis. 20 μg of RsaI/HinfI-digested genomic DNA from telomerase-positive SW39 (upper part of the figure) and VA13 ALT cells (lower part

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis

    39) Product Images from "Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola"

    Article Title: Development and application of loop-mediated isothermal amplification (LAMP) for detection of Plasmopara viticola

    Journal: Scientific Reports

    doi: 10.1038/srep28935

    Restriction enzyme digestion of the positive LAMP products. ( A ) Schematic representation of the anticipated restricted DNA products. B+, B−, F+, F−,+ and-in the first row represent the same regions described by Notomi (Notomi et al . 4 ). ( B ) LAMP products were digested with Ase I and Hinf I, and the fragments were observed by 2.0% agarose gel electrophoresis.
    Figure Legend Snippet: Restriction enzyme digestion of the positive LAMP products. ( A ) Schematic representation of the anticipated restricted DNA products. B+, B−, F+, F−,+ and-in the first row represent the same regions described by Notomi (Notomi et al . 4 ). ( B ) LAMP products were digested with Ase I and Hinf I, and the fragments were observed by 2.0% agarose gel electrophoresis.

    Techniques Used: Agarose Gel Electrophoresis

    40) Product Images from "Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology"

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    Journal: Nature protocols

    doi: 10.1038/nprot.2018.012

    Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.
    Figure Legend Snippet: Optimization of CRISPR-EZ conditions for editing efficiency and embryo viability. (a) A diagram illustrates the NHEJ and HDR editing strategies for exon 1 of the Tyr gene. A successful NHEJ editing ablates a HinfI site and disrupts T yr gene function. A successful HDR editing replaces the HinfI site with an EcoRI site, introducing a frameshift mutation that abolishes Tyr gene function. (b) Representative RFLP results of Tyr edited mice indicate successful NHEJ editing (top) and HDR editing (bottom). (c) Since bi-allelic Tyr deficiency causes albinism in edited mice, the extent of albinism correlates the extent of Tyr editing that disrupts the genes function. Coat color (left) and viability (right) of C57B/6J edited mice generated from 2, 4, 6 or 8 pulse CRISPR-EZ conditions. Viability is defined as the percentage of live animals born out of total embryos transferred. The 6-pulse condition maximizes editing efficiency while minimally impacting pup viability. (d) Comparison of editing efficiency between C57B/6J and C57B/6N mouse strain using 2 or 6-pulse electroporation conditions. The 6-pulse CRISPR-EZ condition is equally effective in both strains. (e-i) Representative images are shown for the coat color of edited mice from experiments shown in (b-d). All animal procedures were approved by the Institutional Animal Care and Use Committee of UC Davis.

    Techniques Used: CRISPR, Non-Homologous End Joining, Mutagenesis, Mouse Assay, Generated, Electroporation

    Related Articles

    Nucleic Acid Electrophoresis:

    Article Title: S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas
    Article Snippet: .. RFLP-RT–PCR and RFLP-PCR A 10 μ l aliquot of PCR or RT–PCR product was digested with 5 U of Hinf I (NEB) in a 40 μ l reaction containing 1 × manufacturer's restriction enzyme buffer at 37°C for 3 h. Digests were visualised by gel electrophoresis and quantified by running on DNA 100 LabChips® in an Agilent 2100 Bioanalyser (Agilent Technologies). .. WAVE mutation analysis PCR products from S100A2 exons 2 and 3 were screened for mutation using denaturing high-performance liquid chromatography ( ) using a WAVE® system (Model 3500A Transgenomics Limited, Crewe, UK).

    Agarose Gel Electrophoresis:

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection
    Article Snippet: .. Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel. .. The samples were run in duplicates for hybridization with C-rich and G-rich telomeric probes in parallel.

    Southern Blot:

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: .. Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C. .. The digested DNA was separated on a 0.8% agarose gel and blotted onto a positively charged nylon membrane (GE Healthcare Biosciences).

    Polymerase Chain Reaction:

    Article Title: S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas
    Article Snippet: .. RFLP-RT–PCR and RFLP-PCR A 10 μ l aliquot of PCR or RT–PCR product was digested with 5 U of Hinf I (NEB) in a 40 μ l reaction containing 1 × manufacturer's restriction enzyme buffer at 37°C for 3 h. Digests were visualised by gel electrophoresis and quantified by running on DNA 100 LabChips® in an Agilent 2100 Bioanalyser (Agilent Technologies). .. WAVE mutation analysis PCR products from S100A2 exons 2 and 3 were screened for mutation using denaturing high-performance liquid chromatography ( ) using a WAVE® system (Model 3500A Transgenomics Limited, Crewe, UK).

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: .. Identification of a full length Aphenenchoides besseyi GH5 gene by inverse and RACE PCR Nematode genomic DNA (~50 ng) was digested with Hinf I (NEB, California, USA, #Ro155S) and self-ligated with T4 DNA Ligase (GeneMark, Taipei, Taiwan) according to the manufacturer’s protocol. .. The two inverse primers FmGH5i-F and FmGH5i-R ( ) were designed and synthesized on the basis of the sequence for genomic DNA fragments obtained with the degenerate primers through the LASERGENE® PrimerSelect™ program, and the PCR reaction was started with a 3 min denaturation at 94°C, followed by 35 cycles of 94°C for 1 min, 53°C for 1 min, and 72°C for 1 min 30 s, and a final extension at 72°C for 5 min.

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae
    Article Snippet: .. PCR products were digested with HpyCH4IV or HinfI restriction enzymes (New England Biolabs), respectively. .. The tRNAAsp(GTC) 72bp PCR product is digested by Hpy CH4IV resulting in two digestion products of 35bp and 37bp if C38 is methylated, and is undigested if C38 is non-methylated and thus converted to T38 by bisulfite treatment, causing the HpyCH4IV restriction enzyme site to be lost.

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer
    Article Snippet: .. The PCR products were subject to RsaI, ApaI and HinfI and RsaI (New England Biolabs, Beverly, Mass, USA) enzyme digestion at 37°C overnight, run through 12% acrylamide gel and stained with ethidium bromide respectively. .. The expected size of the PCR fragment of the LIT1 gene is 410 bp.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: S100A2 is strongly expressed in airway basal cells, preneoplastic bronchial lesions and primary non-small cell lung carcinomas
    Article Snippet: .. RFLP-RT–PCR and RFLP-PCR A 10 μ l aliquot of PCR or RT–PCR product was digested with 5 U of Hinf I (NEB) in a 40 μ l reaction containing 1 × manufacturer's restriction enzyme buffer at 37°C for 3 h. Digests were visualised by gel electrophoresis and quantified by running on DNA 100 LabChips® in an Agilent 2100 Bioanalyser (Agilent Technologies). .. WAVE mutation analysis PCR products from S100A2 exons 2 and 3 were screened for mutation using denaturing high-performance liquid chromatography ( ) using a WAVE® system (Model 3500A Transgenomics Limited, Crewe, UK).

    Acrylamide Gel Assay:

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer
    Article Snippet: .. The PCR products were subject to RsaI, ApaI and HinfI and RsaI (New England Biolabs, Beverly, Mass, USA) enzyme digestion at 37°C overnight, run through 12% acrylamide gel and stained with ethidium bromide respectively. .. The expected size of the PCR fragment of the LIT1 gene is 410 bp.

    Staining:

    Article Title: Loss of imprinting of insulin-like growth factor 2 is associated with increased risk of lymph node metastasis and gastric corpus cancer
    Article Snippet: .. The PCR products were subject to RsaI, ApaI and HinfI and RsaI (New England Biolabs, Beverly, Mass, USA) enzyme digestion at 37°C overnight, run through 12% acrylamide gel and stained with ethidium bromide respectively. .. The expected size of the PCR fragment of the LIT1 gene is 410 bp.

    In Situ Hybridization:

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern
    Article Snippet: .. Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C. .. The digested DNA was separated on a 0.8% agarose gel and blotted onto a positively charged nylon membrane (GE Healthcare Biosciences).

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    New England Biolabs hinfi restriction enzymes
    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a <t>HinfI</t> restriction site when C2268 is methylated. Below: Cleavage of <t>PCR</t> amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product
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    NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Journal: BMC Plant Biology

    Article Title: Conservation of tRNA and rRNA 5-methylcytosine in the kingdom Plantae

    doi: 10.1186/s12870-015-0580-8

    Figure Lengend Snippet: NSUN5 methylates C2268 in Arabidopsis nuclear LSU 25S rRNA. a Genomic origins of methylated and non-methylated rRNA species. Methylated rRNAs were detected from all three genomes (3 biological replicates). b Left: Heatmap showing percentage methylation of cytosines in nuclear (N), chloroplast (C) and mitochondrial (M) rRNA sequences in wild type and mutants nop2a-2 , nsun5-1 , nop2b-1 and nop2c-1 . Cytosine positions are indicated next to rRNAs (3 biological replicates). Right: Partial secondary structure of 25S nuclear LSU rRNA helix 70 (domain IV) showing the cytosine position 2268 in red, which is methylated by NSUN5. c Genomic structure of nop2b , nop2c and nsun5 mutants showing T-DNA insertions (triangles) in exons (filled boxes). d Analysis of RNA methylation by NSUN5 at position C2268 on BS treated nuclear LSU 25S rRNA template. Above: Restriction maps of dCAPS amplified products showing the expected digest patterns of methylated and non-methylated template. The 25S_rRNA_F dCAPS primer contains a G mismatch at position four to generate a HinfI restriction site when C2268 is methylated. Below: Cleavage of PCR amplified product by HinfI confirms C2268 methylation in wild type as opposed to non-methylated C2268 in nsun5-1 results in loss of HinfI restriction site. Loading control is undigested PCR product

    Article Snippet: PCR products were digested with HpyCH4IV or HinfI restriction enzymes (New England Biolabs), respectively.

    Techniques: Methylation, Amplification, Polymerase Chain Reaction

    TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    doi: 10.1093/nar/gky597

    Figure Lengend Snippet: TbUMSBP2 knockdown altered the amount of single stranded telomeric DNA. DNA samples (1 μg) of uninduced cells (−) and cells at day 3 post TbUMSBP2 RNAi induction (+), were digested with HinfI and AluI restriction endonucleases and analyzed by in-gel hybridization to C-probe (AACCCT) 3 or G-probe (AGGGTT) 3 , first under native conditions ( A ) and then re-hybridized again to the same probes after denaturation ( B ), as described under ‘Materials and Methods’. ( C and D ) the histograms represent the relative amounts of native signal (corresponding to single-stranded telomeric DNA) normalized to the denatured (total) signals. The uninduced control samples were set as 1.

    Article Snippet: Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel.

    Techniques: Hybridization

    TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    doi: 10.1093/nar/gky597

    Figure Lengend Snippet: TbUMSBP2 knockdown decreased G-overhangs and increased C-overhangs and telomeric circles. Equal amounts of DNA samples (5 μg), prepared from uninduced cells (−Tet) and cells at 3 days post TbUMSBP2 RNAi induction (+Tet), were digested with HinfI and analyzed in duplicates by neutral-neutral 2D gel electrophoresis. ( A ) The gels were dried and hybridized in-gel under native assay conditions with a radioactively labeled C-probe, (AACCCT) 3 , or G-probe, (AGGGTT) 3 , to detect single-stranded G-rich or C-rich telomeric repeats, respectively. ( B ) The DNA was subsequently denatured in situ and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Note that after denaturation the hybridization signal was stronger; much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) A shorter exposure of the gels in (B), showing comparable amounts of telomeric DNA. Schemes on the right illustrate the different arches of telomeric DNA observed by hybridization to native or denatured DNA, following ( 62 , 63 ). Indicated are G- and C- overhangs associated with linear dsDNA, ssDNA (SS-G and SS-C), telomere circles (t-circles) and a subset of t-circles containing gaps in the G-strand and single stranded regions of the C-strand (termed here as C-circles).

    Article Snippet: Genomic DNA samples (1–1.5 μg) were digested with restriction endonucleases HinfI or HinfI and AluI (NEB Inc.) and separated on a 0.7% agarose gel.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, In Situ, Hybridization

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Journal: bioRxiv

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    doi: 10.1101/2020.01.29.924951

    Figure Lengend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Article Snippet: Around 2.5 mg of DNA was digested overnight with 750 units of HinfI and MspI (NEB).

    Techniques: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Journal: PLoS ONE

    Article Title: Glycoside Hydrolase (GH) 45 and 5 Candidate Cellulases in Aphelenchoides besseyi Isolated from Bird’s-Nest Fern

    doi: 10.1371/journal.pone.0158663

    Figure Lengend Snippet: Identification of the Abe GH5-1 gene by Southern blotting. Genomic DNA isolated from the five Aphelenchoides besseyi isolates were digested with Hinf I , and it was hybridized with an AbeFm-GH5-specific DNA probe.

    Article Snippet: Southern blotting and in situ hybridization of the Abe GH5-1 gene Approximately 10 to 20 μg of genomic DNA from the 5 different A . besseyi isolates was digested with Hinf I (NEB, California, USA) overnight at 37°C.

    Techniques: Southern Blot, Isolation