t4 polynucleotide kinase  (Thermo Fisher)


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    Name:
    T4 Polynucleotide Kinase 10 U µL
    Description:
    Thermo Scientific T4 Polynucleotide Kinase T4 PNK catalyzes the transfer of the gamma phosphate from ATP to the 5 OH group of single and double stranded DNAs and RNAs oligonucleotides or nucleoside 3 monophosphates forward reaction The reaction is reversible In the presence of ADP T4 Polynucleotide Kinase exhibits 5 phosphatase activity and catalyzes the exchange of phosphate groups between 5 P oligo polynucleotides and ATP exchange reaction The enzyme is also a 3 phosphatase Highlights • Active in Thermo Scientific restriction enzyme RT and T4 DNA ligase buffers Applications • Labeling 5 termini of nucleic acids to be used as Probes for hybridization Probes for transcript mapping Markers for gel electrophoresis Primers for DNA sequencing Primers for PCR • 5 phosphorylation of oligonucleotides PCR products other DNA or RNA prior to ligation • Phosphorylation of PCR primers • Detection of DNA modification by the 32P postlabeling assay • Removal of 3 phosphate groups Notes • The 5 termini of nucleic acids can be labeled by either the forward or the exchange reaction • Polyethylene glycol PEG and spermidine improve the rate and efficiency of the phosphorylation reaction PEG is used in the exchange reaction mixture • Since T4 Polynucleotide Kinase is inhibited by ammonium ions use sodium acetate to precipitate DNA prior to phosphorylation
    Catalog Number:
    ek0031
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher t4 polynucleotide kinase
    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by <t>T4</t> polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    Thermo Scientific T4 Polynucleotide Kinase T4 PNK catalyzes the transfer of the gamma phosphate from ATP to the 5 OH group of single and double stranded DNAs and RNAs oligonucleotides or nucleoside 3 monophosphates forward reaction The reaction is reversible In the presence of ADP T4 Polynucleotide Kinase exhibits 5 phosphatase activity and catalyzes the exchange of phosphate groups between 5 P oligo polynucleotides and ATP exchange reaction The enzyme is also a 3 phosphatase Highlights • Active in Thermo Scientific restriction enzyme RT and T4 DNA ligase buffers Applications • Labeling 5 termini of nucleic acids to be used as Probes for hybridization Probes for transcript mapping Markers for gel electrophoresis Primers for DNA sequencing Primers for PCR • 5 phosphorylation of oligonucleotides PCR products other DNA or RNA prior to ligation • Phosphorylation of PCR primers • Detection of DNA modification by the 32P postlabeling assay • Removal of 3 phosphate groups Notes • The 5 termini of nucleic acids can be labeled by either the forward or the exchange reaction • Polyethylene glycol PEG and spermidine improve the rate and efficiency of the phosphorylation reaction PEG is used in the exchange reaction mixture • Since T4 Polynucleotide Kinase is inhibited by ammonium ions use sodium acetate to precipitate DNA prior to phosphorylation
    https://www.bioz.com/result/t4 polynucleotide kinase/product/Thermo Fisher
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    Images

    1) Product Images from "Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum"

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.208306

    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    Figure Legend Snippet: Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum"

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.208306

    Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate
    Figure Legend Snippet: Principle of the cRT-PCR method used to determine the ends of RNA molecules. Total RNA is dephosphorylated by shrimp alkaline phosphatase ( SAP ) and then 5′-monophosphorylated by T4 polynucleotide kinase ( T4 PNK ). The resulting 5′-phosphate

    Techniques Used: Polymerase Chain Reaction

    3) Product Images from "Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA"

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq572

    Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).
    Figure Legend Snippet: Workflow of PEP. Double-stranded template DNA is blunt-end repaired using T4 DNA polymerase and T4 polynucleotide kinase (not depicted). ( I ) Using T4 DNA ligase, biotinylated adapters are attached to both ends of the template molecules. The blunt end ligation reaction also produces adapter dimers, which are subsequently removed by size selective purification. ( II ) 5′-tailed primers carrying the 454 ‘B’ sequence (shown in blue) are hybridized to the overhanging 3′-ends of the adapters. Primer extension is carried out under reaction conditions optimal for the assayed polymerase. Unless second-strand synthesis stops prematurely, due to a blocking lesion, a nick or random polymerase stalling, the flanking adapter sequence (shown in red) is copied. ( III ) Primer extension products are captured on streptavidine beads to remove excess primers and extension products from nicked template strands. Extension products are released by heat denaturation. ( IV ) A 454 sequencing library is created by attaching single-stranded adapters with the 454 ‘A’ sequence (shown in green) to the 3′-ends. The sequencing library is converted to double-stranded form (not depicted) to allow for efficient removal of excess A-adapters. The 454 sequencing is initiated from the A-adapter. If primer extensions were complete, sequences will start with an 8-bp adapter sequence, which serves as the end-of-template recognition sequence (framed by rectangles).

    Techniques Used: Ligation, Purification, Sequencing, Blocking Assay

    4) Product Images from "Characterization of oligodeoxyribonucleotide synthesis on glass plates"

    Article Title: Characterization of oligodeoxyribonucleotide synthesis on glass plates

    Journal: Nucleic Acids Research

    doi:

    Assay of oligonucleotide synthesis using a termination nucleophosphoramidite, 5′-MeO-T, to probe the presence of available sites for coupling with a phosphoramidite at different reaction stages. Note that sequences terminated with 5′-MeO-T are not observed, since they cannot be 32 P-labeled at the 5′-OH using T4 polynucleotide kinase. ( A ) Illustration of regular T 3 synthesis. ( B ) Illustration of the use of the termination monomer. T on a glass plate is coupled with MeO-T, resulting in formation of a terminated dimer T-T(OMe), which cannot undergo further chain growth. ( C ) Illustration of the hypothesis for reaction with more hindered surface sites in several continued reaction cycles. If these sites exist oligonucleotides can be synthesized even after applying MeO-T in the coupling step. ( D ) 32 P gel electrophoresis analysis of the experiments using the terminator 5′-MeO-T at different stages of oligonucleotide synthesis. Lane 1, sequences from a synthesis which used MeO-T in the first step of coupling, followed by coupling with DMT-T. The sites that failed to couple with MeO-T would produce regular sequences, such as T 3 . This sequence is clearly present in a significant ratio along with T 2 and T 1 fragments. Lane 2, sequences from a synthesis which used MeO-T at the second step of coupling, followed by coupling with DMT-T. The monomer T sites that failed to couple with MeO-T would produce regular sequences, such as T 1 –T 3 or T 4 . Little T 4 was observed in this experiment. The surface OH sites that failed to couple with DMT-T in the first step would also be responsible for the observed T 1–3 sequences. Lane 3, sequences from a synthesis which used MeO-T at the third step, followed by coupling with DMT-T. T 5 and T 4 were not observed. There are reduced amounts of overall sequences and short T n fragments. Lane 4, sequences from a synthesis which used MeO-T at the fourth step, followed by coupling with DMT-T. Only little T 1–3 was observed. Lane 6, regular synthesis of T 6 as a control.
    Figure Legend Snippet: Assay of oligonucleotide synthesis using a termination nucleophosphoramidite, 5′-MeO-T, to probe the presence of available sites for coupling with a phosphoramidite at different reaction stages. Note that sequences terminated with 5′-MeO-T are not observed, since they cannot be 32 P-labeled at the 5′-OH using T4 polynucleotide kinase. ( A ) Illustration of regular T 3 synthesis. ( B ) Illustration of the use of the termination monomer. T on a glass plate is coupled with MeO-T, resulting in formation of a terminated dimer T-T(OMe), which cannot undergo further chain growth. ( C ) Illustration of the hypothesis for reaction with more hindered surface sites in several continued reaction cycles. If these sites exist oligonucleotides can be synthesized even after applying MeO-T in the coupling step. ( D ) 32 P gel electrophoresis analysis of the experiments using the terminator 5′-MeO-T at different stages of oligonucleotide synthesis. Lane 1, sequences from a synthesis which used MeO-T in the first step of coupling, followed by coupling with DMT-T. The sites that failed to couple with MeO-T would produce regular sequences, such as T 3 . This sequence is clearly present in a significant ratio along with T 2 and T 1 fragments. Lane 2, sequences from a synthesis which used MeO-T at the second step of coupling, followed by coupling with DMT-T. The monomer T sites that failed to couple with MeO-T would produce regular sequences, such as T 1 –T 3 or T 4 . Little T 4 was observed in this experiment. The surface OH sites that failed to couple with DMT-T in the first step would also be responsible for the observed T 1–3 sequences. Lane 3, sequences from a synthesis which used MeO-T at the third step, followed by coupling with DMT-T. T 5 and T 4 were not observed. There are reduced amounts of overall sequences and short T n fragments. Lane 4, sequences from a synthesis which used MeO-T at the fourth step, followed by coupling with DMT-T. Only little T 1–3 was observed. Lane 6, regular synthesis of T 6 as a control.

    Techniques Used: Oligonucleotide Synthesis, Labeling, Synthesized, Nucleic Acid Electrophoresis, Sequencing

    5) Product Images from "Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform"

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0101812

    Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.
    Figure Legend Snippet: Library preparation using the CapSMART method. A) The protocol used either poly A+ (0.50–10 µg) or total (10–200 µg) RNA. B) De-phosphorylation of mono-, di-, and tri- phosphate groups from non-capped 5′ end molecules using alkaline phosphatase. C) Phosphorylation to add mono-phosphate to the non-capped 5′ end molecules using T4 Polynucleotide Kinase. D) Ligation of STOP oligos. A total of three kinds of oligonucleotides ( Table 2 : STOP1: iGiCiG, STOP2: iCiGiC, STOPMix: mixture of STOP1 and STOP2) were used in the present study. E) First-strand cDNA synthesis. F) Second-strand cDNA amplification by PCR with biotinylated 5′ end primers. G) Fragmentation of cDNA using a Bioruptor and collection of biotinylated 5′ ends using beads. H) Illumina sequencing library preparation.

    Techniques Used: De-Phosphorylation Assay, Ligation, Amplification, Polymerase Chain Reaction, Sequencing

    6) Product Images from "Characterization of oligodeoxyribonucleotide synthesis on glass plates"

    Article Title: Characterization of oligodeoxyribonucleotide synthesis on glass plates

    Journal: Nucleic Acids Research

    doi:

    Assay of oligonucleotide synthesis using a termination nucleophosphoramidite, 5′-MeO-T, to probe the presence of available sites for coupling with a phosphoramidite at different reaction stages. Note that sequences terminated with 5′-MeO-T are not observed, since they cannot be 32 P-labeled at the 5′-OH using T4 polynucleotide kinase. ( A ) Illustration of regular T 3 synthesis. ( B ) Illustration of the use of the termination monomer. T on a glass plate is coupled with MeO-T, resulting in formation of a terminated dimer T-T(OMe), which cannot undergo further chain growth. ( C ) Illustration of the hypothesis for reaction with more hindered surface sites in several continued reaction cycles. If these sites exist oligonucleotides can be synthesized even after applying MeO-T in the coupling step. ( D ) 32 P gel electrophoresis analysis of the experiments using the terminator 5′-MeO-T at different stages of oligonucleotide synthesis. Lane 1, sequences from a synthesis which used MeO-T in the first step of coupling, followed by coupling with DMT-T. The sites that failed to couple with MeO-T would produce regular sequences, such as T 3 . This sequence is clearly present in a significant ratio along with T 2 and T 1 fragments. Lane 2, sequences from a synthesis which used MeO-T at the second step of coupling, followed by coupling with DMT-T. The monomer T sites that failed to couple with MeO-T would produce regular sequences, such as T 1 –T 3 or T 4 . Little T 4 was observed in this experiment. The surface OH sites that failed to couple with DMT-T in the first step would also be responsible for the observed T 1–3 sequences. Lane 3, sequences from a synthesis which used MeO-T at the third step, followed by coupling with DMT-T. T 5 and T 4 were not observed. There are reduced amounts of overall sequences and short T n fragments. Lane 4, sequences from a synthesis which used MeO-T at the fourth step, followed by coupling with DMT-T. Only little T 1–3 was observed. Lane 6, regular synthesis of T 6 as a control.
    Figure Legend Snippet: Assay of oligonucleotide synthesis using a termination nucleophosphoramidite, 5′-MeO-T, to probe the presence of available sites for coupling with a phosphoramidite at different reaction stages. Note that sequences terminated with 5′-MeO-T are not observed, since they cannot be 32 P-labeled at the 5′-OH using T4 polynucleotide kinase. ( A ) Illustration of regular T 3 synthesis. ( B ) Illustration of the use of the termination monomer. T on a glass plate is coupled with MeO-T, resulting in formation of a terminated dimer T-T(OMe), which cannot undergo further chain growth. ( C ) Illustration of the hypothesis for reaction with more hindered surface sites in several continued reaction cycles. If these sites exist oligonucleotides can be synthesized even after applying MeO-T in the coupling step. ( D ) 32 P gel electrophoresis analysis of the experiments using the terminator 5′-MeO-T at different stages of oligonucleotide synthesis. Lane 1, sequences from a synthesis which used MeO-T in the first step of coupling, followed by coupling with DMT-T. The sites that failed to couple with MeO-T would produce regular sequences, such as T 3 . This sequence is clearly present in a significant ratio along with T 2 and T 1 fragments. Lane 2, sequences from a synthesis which used MeO-T at the second step of coupling, followed by coupling with DMT-T. The monomer T sites that failed to couple with MeO-T would produce regular sequences, such as T 1 –T 3 or T 4 . Little T 4 was observed in this experiment. The surface OH sites that failed to couple with DMT-T in the first step would also be responsible for the observed T 1–3 sequences. Lane 3, sequences from a synthesis which used MeO-T at the third step, followed by coupling with DMT-T. T 5 and T 4 were not observed. There are reduced amounts of overall sequences and short T n fragments. Lane 4, sequences from a synthesis which used MeO-T at the fourth step, followed by coupling with DMT-T. Only little T 1–3 was observed. Lane 6, regular synthesis of T 6 as a control.

    Techniques Used: Oligonucleotide Synthesis, Labeling, Synthesized, Nucleic Acid Electrophoresis, Sequencing

    7) Product Images from "Cleavage of poly(A) tails on the 3?-end of RNA by ribonuclease E of Escherichia coli"

    Article Title: Cleavage of poly(A) tails on the 3?-end of RNA by ribonuclease E of Escherichia coli

    Journal: Nucleic Acids Research

    doi:

    Cleavage of an A 40 oligonucleotide substrate by RNase E is dependent on the presence of a 5′- but not a 3′-monophosphate group. The A 40 substrate in ( A ) was labelled at the 5′-end using [γ- 32 P]ATP and T4 polynucleotide kinase (PNK), whereas the same oligonucleotide in ( B ) and ( C ) was labelled at the 3′-end using [5′- 32 P]pCp and T4 RNA ligase. The oligonucleotide in (B) was in addition monophosphorylated at the 5′-end using PNK and an excess of non-radioactive ATP. The oligonucleotide in ( D ) (A 29 :G 21 ) had a 5′-segment of 29 A residues linked to a 3′-segment of 21 G residues and was labelled at the 5′-end. In each case the concentrations of full-length RNase E and substrate were ∼50 and 750 nM, respectively. Samples were removed immediately prior to the addition of enzyme (lane 1) and after incubation at 37°C for 2, 5, 10 and 20 min (lanes 2–5, respectively) and were analysed on standard (45 cm length) 10% (w/v) polyacrylamide sequencing-type gels. The numbers on the left of (A) to (C) indicate the sizes of 5′-labelled species produced by RNase E cleavage of A 40 . Similarly, those on the right of (D) indicate the sizes of 5′-labelled species corresponding to A 29 :G 21 . The latter were determined from a series of gels in which the 5′-labelled products were run against RNAs of known size (data not shown). The results shown in (D) and those in (A) to (C) were derived from different gels; therefore, the mobility of the bands cannot be compared directly. Each of the panels is derived from a phosphorimage.
    Figure Legend Snippet: Cleavage of an A 40 oligonucleotide substrate by RNase E is dependent on the presence of a 5′- but not a 3′-monophosphate group. The A 40 substrate in ( A ) was labelled at the 5′-end using [γ- 32 P]ATP and T4 polynucleotide kinase (PNK), whereas the same oligonucleotide in ( B ) and ( C ) was labelled at the 3′-end using [5′- 32 P]pCp and T4 RNA ligase. The oligonucleotide in (B) was in addition monophosphorylated at the 5′-end using PNK and an excess of non-radioactive ATP. The oligonucleotide in ( D ) (A 29 :G 21 ) had a 5′-segment of 29 A residues linked to a 3′-segment of 21 G residues and was labelled at the 5′-end. In each case the concentrations of full-length RNase E and substrate were ∼50 and 750 nM, respectively. Samples were removed immediately prior to the addition of enzyme (lane 1) and after incubation at 37°C for 2, 5, 10 and 20 min (lanes 2–5, respectively) and were analysed on standard (45 cm length) 10% (w/v) polyacrylamide sequencing-type gels. The numbers on the left of (A) to (C) indicate the sizes of 5′-labelled species produced by RNase E cleavage of A 40 . Similarly, those on the right of (D) indicate the sizes of 5′-labelled species corresponding to A 29 :G 21 . The latter were determined from a series of gels in which the 5′-labelled products were run against RNAs of known size (data not shown). The results shown in (D) and those in (A) to (C) were derived from different gels; therefore, the mobility of the bands cannot be compared directly. Each of the panels is derived from a phosphorimage.

    Techniques Used: Incubation, Sequencing, Produced, Derivative Assay

    8) Product Images from "The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus"

    Article Title: The three major types of CRISPR-Cas systems function independently in CRISPR RNA biogenesis in Streptococcus thermophilus

    Journal: Molecular microbiology

    doi: 10.1111/mmi.12644

    Sth crRNA 5′ and 3′ chemical end groups. (A) 5′ end analysis was performed by incubating total RNA from Sth in the absence or presence of Terminator 5′-Phosphate-Dependent Exonuclease (TEX) followed by Northern blotting using probes for the RNAs indicated below each panel. 1.01 is the leader proximal crRNA from CRISPR 1, 2.01 is the leader proximal crRNA from CRISPR 2, etc. (B) For 3′ chemical end analysis of crRNAs from CRISPRs 1–3, gel extracted sRNAs from Sth were incubated in the absence or presence of E. coli poly(A) polymerase (PAP) followed by Northern blotting. The chemical end groups present on crRNAs from CRISPR4 were determined by Northern analysis of Sth total RNA following combinations of treatments with Thermosensitive Alkaline Phosphatase (TSAP), T4 polynucleotide kinase (PNK), and E. coli poly(A) polymerase (PAP). Mature crRNAs are indicated by an asterisk. A very minor ~37 nt form detected for 2.01 crRNA discussed in the text is indicated by an arrow.
    Figure Legend Snippet: Sth crRNA 5′ and 3′ chemical end groups. (A) 5′ end analysis was performed by incubating total RNA from Sth in the absence or presence of Terminator 5′-Phosphate-Dependent Exonuclease (TEX) followed by Northern blotting using probes for the RNAs indicated below each panel. 1.01 is the leader proximal crRNA from CRISPR 1, 2.01 is the leader proximal crRNA from CRISPR 2, etc. (B) For 3′ chemical end analysis of crRNAs from CRISPRs 1–3, gel extracted sRNAs from Sth were incubated in the absence or presence of E. coli poly(A) polymerase (PAP) followed by Northern blotting. The chemical end groups present on crRNAs from CRISPR4 were determined by Northern analysis of Sth total RNA following combinations of treatments with Thermosensitive Alkaline Phosphatase (TSAP), T4 polynucleotide kinase (PNK), and E. coli poly(A) polymerase (PAP). Mature crRNAs are indicated by an asterisk. A very minor ~37 nt form detected for 2.01 crRNA discussed in the text is indicated by an arrow.

    Techniques Used: Northern Blot, CRISPR, Incubation

    9) Product Images from "An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides"

    Article Title: An In Vitro DNA Double-Strand Break Repair Assay Based on End-Joining of Defined Duplex Oligonucleotides

    Journal: Methods in molecular biology (Clifton, N.J.)

    doi: 10.1007/978-1-61779-998-3_33

    The sequences and duplex formation scheme for a typical undamaged control duplex oligonucleotide end-joining substrate. T4-PNK T4-polynucleotide kinase.
    Figure Legend Snippet: The sequences and duplex formation scheme for a typical undamaged control duplex oligonucleotide end-joining substrate. T4-PNK T4-polynucleotide kinase.

    Techniques Used:

    10) Product Images from "A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing"

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    Journal: Molecular Microbiology

    doi: 10.1111/mmi.12810

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).
    Figure Legend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Techniques Used: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis

    11) Product Images from "Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff"

    Article Title: Conversion of Topoisomerase I Cleavage Complexes on the Leading Strand of Ribosomal DNA into 5?-Phosphorylated DNA Double-Strand Breaks by Replication Runoff

    Journal: Molecular and Cellular Biology

    doi:

    ) by annealing primer 1 (P1) to denatured genomic DNA. After primer extension and in vitro phosphorylation of the 5′-OH termini with T4 polynucleotide kinase, ligation to the double-stranded linker was performed. Thereafter, rRNA gene-specific DNA fragments were amplified with Taq DNA polymerase using the linker-primer and a nested, gene-specific PCR primer. After 26 cycles of PCR, a third primer (5′ end labeled with 32 P; star) was used for two primer extension cycles before the samples were separated in 7% denaturing polyacrylamide gels. In the assay for replication-mediated DNA double-strand breaks, collision between a replication fork and a top1 cleavage complex is proposed to lead to replication runoff, with generation of a DNA double-strand break (upper right). Because of in vivo 5′-end phosphorylation of replication-mediated DNA double-strand breaks, ligation to the linker could be performed without prior T4 polynucleotide kinase reaction. The following reaction steps were the same as for the detection of top1-induced DNA single-strand breaks. Note that the single-strand break assay detects both single- and double-strand breaks.
    Figure Legend Snippet: ) by annealing primer 1 (P1) to denatured genomic DNA. After primer extension and in vitro phosphorylation of the 5′-OH termini with T4 polynucleotide kinase, ligation to the double-stranded linker was performed. Thereafter, rRNA gene-specific DNA fragments were amplified with Taq DNA polymerase using the linker-primer and a nested, gene-specific PCR primer. After 26 cycles of PCR, a third primer (5′ end labeled with 32 P; star) was used for two primer extension cycles before the samples were separated in 7% denaturing polyacrylamide gels. In the assay for replication-mediated DNA double-strand breaks, collision between a replication fork and a top1 cleavage complex is proposed to lead to replication runoff, with generation of a DNA double-strand break (upper right). Because of in vivo 5′-end phosphorylation of replication-mediated DNA double-strand breaks, ligation to the linker could be performed without prior T4 polynucleotide kinase reaction. The following reaction steps were the same as for the detection of top1-induced DNA single-strand breaks. Note that the single-strand break assay detects both single- and double-strand breaks.

    Techniques Used: In Vitro, Ligation, Amplification, Polymerase Chain Reaction, Labeling, In Vivo

    12) Product Images from "The 5?-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex"

    Article Title: The 5?-end heterogeneity of adenovirus virus-associated RNAI contributes to the asymmetric guide strand incorporation into the RNA-induced silencing complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp764

    The phosphorylation status of mivaRNAIs derived from VA RNAI(A) and VA RNAI(G). The RISC associated RNAs from pVA RNAI(A) or pVA RNAI(G) transfected cells were treated with combinations of calf intestinal alkaline phosphatase (CIAP), T4 polynucleotide kinase (PNK) and Terminator 5′-exonuclease (TE). After enzyme treatment, the 5′- ( A ) or 3′-strands ( B ) of mivaRNAI were analyzed by northern blotting.
    Figure Legend Snippet: The phosphorylation status of mivaRNAIs derived from VA RNAI(A) and VA RNAI(G). The RISC associated RNAs from pVA RNAI(A) or pVA RNAI(G) transfected cells were treated with combinations of calf intestinal alkaline phosphatase (CIAP), T4 polynucleotide kinase (PNK) and Terminator 5′-exonuclease (TE). After enzyme treatment, the 5′- ( A ) or 3′-strands ( B ) of mivaRNAI were analyzed by northern blotting.

    Techniques Used: Derivative Assay, Transfection, Northern Blot

    13) Product Images from "Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro"

    Article Title: Reconstitution of a Staphylococcal Plasmid-Protein Relaxation Complex In Vitro

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.11.3374-3383.2004

    The pC221 cop903 nic 5′ end is resistant to phosphorylation. (A) Overview of expected BstXI/BstUI fragment of pC221 cop903 after cleavage with BsrFI or nicking by MobA subsequently 5′ end labeled with T4 polynucleotide kinase; (B) autoradiograph
    Figure Legend Snippet: The pC221 cop903 nic 5′ end is resistant to phosphorylation. (A) Overview of expected BstXI/BstUI fragment of pC221 cop903 after cleavage with BsrFI or nicking by MobA subsequently 5′ end labeled with T4 polynucleotide kinase; (B) autoradiograph

    Techniques Used: Labeling, Autoradiography

    14) Product Images from "A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA"

    Article Title: A novel synthesis and detection method for cap-associated adenosine modifications in mouse mRNA

    Journal: Scientific Reports

    doi: 10.1038/srep00126

    T4 polynucleotide kinase does not preferentially label m 6 Am or Am containing ends. The oligonucleotides SK-526 (m 6 Am) and SK-524 (Am) were mixed in ratios of 5:1, 2:1, 1:1. 1:2 and 1:5, end labelled using T4 polynucleotide kinase, digested to 5' phosphate mononucleotides and separated by TLC. The relative intensities of the resulting spots were quantified using phosphorimaging. The calculated percentage of m 6 Am closely matched the actual m 6 Am percentage.
    Figure Legend Snippet: T4 polynucleotide kinase does not preferentially label m 6 Am or Am containing ends. The oligonucleotides SK-526 (m 6 Am) and SK-524 (Am) were mixed in ratios of 5:1, 2:1, 1:1. 1:2 and 1:5, end labelled using T4 polynucleotide kinase, digested to 5' phosphate mononucleotides and separated by TLC. The relative intensities of the resulting spots were quantified using phosphorimaging. The calculated percentage of m 6 Am closely matched the actual m 6 Am percentage.

    Techniques Used: Thin Layer Chromatography

    15) Product Images from "Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate"

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    Journal: Nature

    doi: 10.1038/s41586-018-0557-5

    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).
    Figure Legend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Techniques Used: Thin Layer Chromatography, Generated, Incubation

    Related Articles

    Ancient DNA Assay:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Ligation:

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    Article Snippet: .. The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Labeling:

    Article Title: Characterization of oligodeoxyribonucleotide synthesis on glass plates
    Article Snippet: .. A portion of the sample (3 µl) was labeled with [γ-32 P]ATP (5 µCi, 3000 Ci/mmol) using T4 polynucleotide kinase (1 U) and the conditions recommended by the manufacturer (Gibco). ..

    Purification:

    Article Title: DNA-guided DNA interference by a prokaryotic Argonaute
    Article Snippet: .. Purified nucleic acids were [γ-32 P]ATP labelled with T4 PNK (Fermentas) in exchange- or forward-labelling reactions and thereafter separated from free [γ-32 P] ATP using a Sephadex G-25 column (GE). .. Labelled nucleic acids were incubated with nucleases (DNase-free RNase A (Fermentas),RQ1 RNase-free DNaseI (Promega) or P1 nuclease (Sigma)) for 1 h at 37 °C.

    Sequencing:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Incubation:

    Article Title: Sequence and Generation of Mature Ribosomal RNA Transcripts in Dictyostelium discoideum
    Article Snippet: .. After precipitation, the RNA was incubated with 20 units of T4 polynucleotide kinase (Fermentas) and 20 units of RiboLock RNase Inhibitor (Fermentas) in 30 μl of 50 m m Tris-HCl (pH 7.6), 10 m m MgCl2 , 5 m m DTT, 100 μ m spermidine, 1 m m ATP for 30 min at 37 °C. .. After protein extraction with phenol and chloroform, RNA was precipitated with ethanol.

    Article Title: Four Methods of Preparing mRNA 5? End Libraries Using the Illumina Sequencing Platform
    Article Snippet: .. The products were then treated with T4 Polynucleotide Kinase to add mono-phosphate to non-capped mRNA to ready it for ligation; a reaction mixture consisting of 1 µl of T4 Polynucleotide Kinase (Fermentas, # EK0032), 2 µl of RNA Ligase Reaction Buffer (New England Biolabs), 0.5 µl of RNaseOUT (Invitrogen, #10777-019), 1 µl of 100 mM ATP solution (Fermentas, #R0441), and 15.5 µl of alkaline phosphatase-treated RNA was incubated for 30 minutes at 37°C. .. Next, 20 µl of T4 Polynucleotide Kinase-treated RNA were incubated with 2.5 µl of nuclease-free water, 1 µl of RNA Ligase Reaction Buffer (New England Biolabs), 4.5 µl of PEG8000 (New England Biolabs), 1 µl of STOP oligo { , STOP1 (50 µM): iGiCiG, STOP2 (50 µM): iCiGiC, STOP Mix (50 µM): mixture of STOP1 and STOP2, synthesized by Metabion, Germany}, and 1 µl of T4 RNA Ligase (New England Biolabs, M0204S) for 16 hours at 16°C to ligate STOP oligos to the non-capped mRNA.

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

    Mass Spectrometry:

    Article Title: Detection of Ligation Products of DNA Linkers with 5?-OH Ends by Denaturing PAGE Silver Stain
    Article Snippet: .. A quality inspection report of T4 DNA ligase from Fermentas showed that T4 PNK could not be detected in their T4 DNA ligase ( ); (iii) PNK could not be detected in T4 DNA ligase (Fermentas) by using mass spectrometry (MS) analysis ( and ); (iv) PNK is abundant in mammalian cells but absent in E. coli cells . .. Therefore, the endogenous PNK should be absent in the host E. coli cells that carry plasmids enabling T4 or E. coli DNA ligase high expression; (v) The ligation of linkers A–B and E–F could not be significantly inhibited by (NH4 )2 SO4 , a strong inhibitor of T4 PNK ( , and ); and (vi) T4 PNK requires ATP for activity.

    Polymerase Chain Reaction:

    Article Title: Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA
    Article Snippet: .. PEP assays and sequencing For blunt end repair, ∼15 ng of PCR product pool, 2 ng of fragmented horse DNA, 4 ng of UV-irradiated horse DNA, 10 µl ancient DNA extract or a water sample were incubated for 15 min at 12°C and 15 min at 25°C in a 40 µl reaction containing in final concentrations 1× Tango buffer, 0.1 U/µl T4 DNA polymerase, 0.5 U/µl T4 polynucleotide kinase (all Fermentas), 1 mM ATP and 0.1 mM dNTP. .. Reactions were purified using the MinElute PCR Purification kit.

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    Thermo Fisher t4 pnk
    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by <t>T4</t> PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.
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    RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: The RNA-Protein Interactome of Differentiated Kidney Tubular Epithelial Cells

    doi: 10.1681/ASN.2018090914

    Figure Lengend Snippet: RNA-interactome capture identifies novel RNA binders in mIMCD-3 cells. (A) Table of novel, mIMCD-3–specific RBPs, previously not identified as mouse or human mRNA-interacting proteins. Depicted are the gene names, protein names according to Uniprot and MGI, and the selection criteria. The top 19 proteins (#) were significant in the performed t test (Perseus software). The bottom six proteins (*) were measured at least four times in the crosslinked samples (+CL) and not more than once in the noncrosslinked samples (−CL). (B) List of proteins selected for biochemical confirmation of RNA-binding capacity. The table contains information on gene name, protein name, presence in previous RIC studies as summarized for mouse (Mm) and human (Hs) datasets in the Hentze compendium, classification in the mIMCD-3 RBPome (class), and t test significance. (C) Cellular localization pattern of MFAP1, GADD45GIP1, and HIC2. MFAP1: HEK293T cells expressing an integrated, single copy of the human MFAP1 CDS fused to eGFP, using the TALEN approach, were subjected to fluorescent imaging. GADD45GIP1 and HIC2: HEK293T cells transiently expressing the human CDS of GADD45GIP1 or HIC2 fused to triple FLAG were subjected to immunofluorescent imaging. DAPI was used as a nuclear counterstain. Scale bar, 20 µ m. (D) Biochemical validation of Mfap1a/b, Hic2, and Gadd45Gip1 as RBPs. Briefly, the human CDS of MFAP1, HIC2, and GADD45GIP1 were cloned into the 3xFLAG-pcDNA6 and transiently expressed in HEK293T cell. FLAG-tagged proteins were immunoprecipitated from crosslinked (+) and noncrosslinked (−) samples and the associated RNA was labeled by T4 PNK with 32P. The protein-RNA complexes were separated on PAA-gels and blotted onto nitrocellulose membranes. PNK-assay: autoradiograph of the membrane containing the indicated protein with the associated RNA labeled with 32P. Western blot: visualization of FLAG-tagged protein by western blotting with the anti-FLAG antibody. Hs, homo sapiens; Mm, mus musculus; n.d., not detected.

    Article Snippet: Beads were resuspended in PNK buffer containing 5 mM DTT, 0.2 μ Ci/ μ l ( γ 32 P)ATP (Hartmann-Analytic), and 1 U/ μ l T4 PNK (ThermoFisher).

    Techniques: Selection, Software, RNA Binding Assay, Expressing, Imaging, Clone Assay, Immunoprecipitation, Labeling, Autoradiography, Western Blot

    Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Journal: Molecular Microbiology

    Article Title: A comparison of key aspects of gene regulation in Streptomyces coelicolor and Escherichia coli using nucleotide-resolution transcription maps produced in parallel by global and differential RNA sequencing

    doi: 10.1111/mmi.12810

    Figure Lengend Snippet: Processing at the −43 site of 16S rRNA.A. Differential RNA-seq data for the 3′ end of 16S rRNA. The screenshot is for the rrnE operon, which is representative of all seven rRNA operons in E. coli . The tracks from top to bottom show the genome position, location of the 3′ end of 16S rRNA and positions of processing sites as identified by differential RNA-seq in the absence of TAP treatment. The positions of the −43 site, sites of known cleavage by RNase III and P and a site of cleavage by an unknown RNase (labelled ‘?’) are indicated. The numbers at the left of the RNA-seq track indicates the scale of the sequencing reads. The schematic at the bottom of panel indicates the position of a BstEII site that was used to confirm the identity of an 88 bp amplicon produced by RLM-RT-PCR analysis of the −43 site (see B and C). The numbers indicate the sizes (bp) of the predicted products of cleavage at the BstEII site. It should be noted that the products have the equivalent of half a base-pair as BstEII generates 5 nt overhangs. Arrows indicate the position of primers used for PCR. Segments of the amplicon corresponding to the 5′ adaptor are drawn at an angle, while those corresponding to the 3′ end of 16S rRNA are drawn horizontally.B. RLM-RT-PCR analysis of RNA isolated from strain BW25113 (labelled wt) growing exponentially (labelled Exp.) and a congenic Δ mazF strain growing exponentially or in stationary phase (labelled Stat.). Prior to RLM-RT-PCR analysis, an aliquot of each sample was treated with T4 polynucleotide kinase (labelled P). Aliquots of untreated samples (labelled U) were also analysed. Labelling on the left indicates the sizes of molecular markers from Invitrogen (labelled M). The amplicon corresponding to the −43 cleavage site is indicated (labelled 88 bp) on the right. Products were analysed using a 10% polyacrylamide gel and stained with ethidium bromide. No amplicons were produced in the absence of reverse transcription (data not shown).C. Restriction enzyme analysis of amplicons produced from BW25113 RNA not treated with PNK. The substrate (labelled U) was incubated with BstEII and along with the resulting products (labelled B) analysed using gel electrophoresis as described in (B). Labelling on the right indicates the positions of resolvable substrate (labelled S) and products (labelled P).

    Article Snippet: Specific E. coli transcripts were probed using complementary oligonucleotides (see ) labelled at their 5′ ends with 32 P using T4 polynucleotide kinase (Thermo Scientific) and γ-32 P-ATP (3000 Ci mmol−1 , 10 mCi ml−1 , 250 μCi, Perkin Elmer).

    Techniques: RNA Sequencing Assay, Sequencing, Amplification, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Isolation, Staining, Incubation, Nucleic Acid Electrophoresis

    Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Journal: Nature

    Article Title: Ring nucleases deactivate Type III CRISPR ribonucleases by degrading cyclic oligoadenylate

    doi: 10.1038/s41586-018-0557-5

    Figure Lengend Snippet: Sso2081 and Sso1393 cA 4 degradation mechanism investigated by TLC. Csm generated cOA (lane 1) was incubated with 2 μM Sso2081 dimer at 60 °C to determine the intermediate (Y) and final (X) reaction product over time (lanes 2-10). Lanes 11-13 show reaction product seen in lanes 2, 4 and 6, 5’-end phosphorylated using T4 polynucleotide kinase (PNK) for identification of reaction intermediates and products by comparison to 5’-end phosphorylated MazF nuclease generated HO-A 2 > P and HO-A 4 > P standards. Lanes 14 15 show the reaction products of 2 μM Sso1393 dimer incubated with cOA at 70 °C for 20 and 120 min, respectively. Reaction product from lanes 14 15 are 5’-end phosphorylated by PNK for comparison to P-A 2 > P and P-A 4 > P standards. Comparison of PNK treated reaction product to standards showed the presence of a low amount of intermediate (P-Y) during the Sso2081 cA 4 cleavage reaction, which migrated similarly to the P-A 4 > P standard and did not change in abundance over time, whereas the abundance of the final product (P-X) increased over time. In contrast, comparison of Sso1393 PNK treated 20 min and 120 min reaction products showed a decrease of the intermediate (P-Y) over time and increase of product (P-X).

    Article Snippet: For use as standards, A2 > P and A4 > P linear oligoadenylates were 5’-end labelled using 32 P-γ-ATP and T4 Polynucleotide Kinase (PNK; Thermo Fisher Scientific) via its forward reaction.

    Techniques: Thin Layer Chromatography, Generated, Incubation