Structured Review

ICN Biomedicals α 32 p atp
Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. <t>UTP/CTP/ATP</t> and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.
α 32 P Atp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/α 32 p atp/product/ICN Biomedicals
Average 86 stars, based on 6 article reviews
Price from $9.99 to $1999.99
α 32 p atp - by Bioz Stars, 2020-04
86/100 stars

Images

1) Product Images from "Formation of the conserved pseudouridine at position 55 in archaeal tRNA"

Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl530

Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.
Figure Legend Snippet: Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

Techniques Used: Modification, Sequencing, Labeling, Thin Layer Chromatography, Mutagenesis, Incubation, Autoradiography, Migration

2) Product Images from "Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures"

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures

Journal: Nucleic Acids Research

doi:

Affinity-purified T.thermophilus TrmI protein methylates A58 of in vitro transcribed T.thermophilus tRNA Asp . ( A ) Nucleotide sequence of T.thermophilus tRNA Asp ). m 1 A58 is shown in a gray box. The adenosines 5′-adjacent to a guanosine are indicated by an asterisk. ( B ) Autoradiograms of 2-dimensional chromatograms of P1 or T2 hydrolysates of [α- 32 P]ATP- or [α- 32 P]GTP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of the purified TrmI protein (see Materials and Methods for details). In the case of the chromatogram designated GTP/T2*, the T2 hydrolysis was carried out in the absence of carrier yeast tRNA. Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers. ( C ) Kinetics of in vitro formation of m 1 A58 in T.thermophilus tRNA Asp in the presence (closed circles) or absence (open circles) of the purified TrmI protein.
Figure Legend Snippet: Affinity-purified T.thermophilus TrmI protein methylates A58 of in vitro transcribed T.thermophilus tRNA Asp . ( A ) Nucleotide sequence of T.thermophilus tRNA Asp ). m 1 A58 is shown in a gray box. The adenosines 5′-adjacent to a guanosine are indicated by an asterisk. ( B ) Autoradiograms of 2-dimensional chromatograms of P1 or T2 hydrolysates of [α- 32 P]ATP- or [α- 32 P]GTP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of the purified TrmI protein (see Materials and Methods for details). In the case of the chromatogram designated GTP/T2*, the T2 hydrolysis was carried out in the absence of carrier yeast tRNA. Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers. ( C ) Kinetics of in vitro formation of m 1 A58 in T.thermophilus tRNA Asp in the presence (closed circles) or absence (open circles) of the purified TrmI protein.

Techniques Used: Affinity Purification, In Vitro, Sequencing, Labeling, Incubation, Purification, Migration

Generation of a T.thermophilus mutant lacking TrmI activity, by homologous recombination. ( A ) Plasmid construction in E.coli for T.thermophilus trmI gene inactivation (see Materials and Methods for details). The trmI gene is represented by a solid bar, the thermostable knt gene (Km r ) by an open bar and the 16S promoter by a horizontal arrow. Restriction sites are indicated as follows: B, Bsu 36I; E, Eco RI; M, Mlu I. ( B ) Autoradiography of 2-dimensional chromatograms of P1 hydrolysates of [α- 32 P]ATP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of a crude extract of the T.thermophilus wild-type strain (HB27) or trmI mutant (RD1). Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers.
Figure Legend Snippet: Generation of a T.thermophilus mutant lacking TrmI activity, by homologous recombination. ( A ) Plasmid construction in E.coli for T.thermophilus trmI gene inactivation (see Materials and Methods for details). The trmI gene is represented by a solid bar, the thermostable knt gene (Km r ) by an open bar and the 16S promoter by a horizontal arrow. Restriction sites are indicated as follows: B, Bsu 36I; E, Eco RI; M, Mlu I. ( B ) Autoradiography of 2-dimensional chromatograms of P1 hydrolysates of [α- 32 P]ATP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of a crude extract of the T.thermophilus wild-type strain (HB27) or trmI mutant (RD1). Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers.

Techniques Used: Mutagenesis, Activity Assay, Homologous Recombination, Plasmid Preparation, Autoradiography, Labeling, Incubation, Migration

3) Product Images from "A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase"

Article Title: A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkh191

A deaminase present in a crude P.furiosus extract transforms m 1 A57 preformed in yeast tRNA Asp by the P.abyssi TrmI enzyme into m 1 I57 and does not deaminate the m 1 A58 preformed in T.thermophilus tRNA Asp . [α- 32 P]ATP-labelled yeast tRNA Asp (A and C) and [α- 32 P]ATP- labelled T.thermophilus tRNA Asp (B and D) were incubated for 1 h at 60°C in the presence of the purified P.abyssi TrmI enzyme and of AdoMet. The transcripts were then recovered and incubated in a crude P.abyssi S30 extract for different periods of time as shown. At the end of the incubation time the transcripts were recovered, digested by nuclease P1 and analysed by 1D-TLC using solvent B (see Materials and Methods) on cellulose plates followed by autoradiography ( A and B ). ( C ) and ( D ) correspond to the autoradiograms of 2D-TLC of the samples incubated for 60 min in the presence of the P.furiosus extract.
Figure Legend Snippet: A deaminase present in a crude P.furiosus extract transforms m 1 A57 preformed in yeast tRNA Asp by the P.abyssi TrmI enzyme into m 1 I57 and does not deaminate the m 1 A58 preformed in T.thermophilus tRNA Asp . [α- 32 P]ATP-labelled yeast tRNA Asp (A and C) and [α- 32 P]ATP- labelled T.thermophilus tRNA Asp (B and D) were incubated for 1 h at 60°C in the presence of the purified P.abyssi TrmI enzyme and of AdoMet. The transcripts were then recovered and incubated in a crude P.abyssi S30 extract for different periods of time as shown. At the end of the incubation time the transcripts were recovered, digested by nuclease P1 and analysed by 1D-TLC using solvent B (see Materials and Methods) on cellulose plates followed by autoradiography ( A and B ). ( C ) and ( D ) correspond to the autoradiograms of 2D-TLC of the samples incubated for 60 min in the presence of the P.furiosus extract.

Techniques Used: Incubation, Purification, Thin Layer Chromatography, Autoradiography

Related Articles

Clone Assay:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 . .. For kinase assays using recombinant and purified INCA1, human GST- INCA and GST- INCA -del75–99 were cloned into the baculovirus-shuttle vector pDEST20, shuttled to the baculovirus via the Bac-to-Bac baculovirus expression system (Invitrogen), and transfected into Sf9 insect cells.

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: Paragraph title: Cloning and T7 in vitro transcription of the T.thermophilus tRNAAsp gene ... [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: Paragraph title: Cloning and T7 in vitro transcription of the B. subtilis tRNASer (GGA) gene ... [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

Transfection:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: For kinase assays performed with lysates of Sf9 insect cells, cells transfected baculovirally with human INCA1 or cyclin A1/CDK2 were lysed and subjected to kinase reactions using the conditions as described previously ( , ). .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 .

Amplification:

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: The sequence coding for T.thermophilus tRNAAsp was amplified by PCR using the ML-1 and ML-2 oligonucleotides and Pwo DNA polymerase (Roche Diagnostics). .. [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: Cloning and T7 in vitro transcription of the B. subtilis tRNASer (GGA) gene The sequence encoding the B. subtilis tRNASer (GGA) was amplified by PCR using oligonucleotides tser-1 and tser-2 ( ) and Pwo DNA polymerase. .. [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

In Vitro:

Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA
Article Snippet: Radioactive (32 P) in vitro transcripts were prepared as described elsewhere ( ) using MvaI (for generating tRNAAsp substrate) or NarI (for generating tRNAAsp -3′CA substrate) digested plasmids as templates. .. [α-32 P]UTP, [α-32 P]CTP, [α-32 P]ATP and [α-32 P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics.

Article Title: A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase
Article Snippet: Paragraph title: T7 in vitro transcription of tRNA genes ... [α-32 P]ATP, [α-32 P]GTP and [α-32 P]UTP were purchased from ICN Biomedicals and T7-RNA polymerase was purchased from Roche Diagnostics.

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: Paragraph title: In Vitro Kinase Reactions ... Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 .

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: Paragraph title: Cloning and T7 in vitro transcription of the T.thermophilus tRNAAsp gene ... [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: Paragraph title: Cloning and T7 in vitro transcription of the B. subtilis tRNASer (GGA) gene ... [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: A precursor RNA containing the aI5β intron RNA was generated by in vitro transcription of the paI5FL vector, which yields a 1604 nt aI5β pre-RNA that includes a 12 nt 5’ exon, the catalytic core, an open reading frame encoding a degenerate homing endonuclease, and the 3’ SS followed by an 18 nt 3’ exon. .. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP.

Expressing:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 . .. This was then incubated for 30 min in 1× kinase buffer (10 μ m ATP, 50 m m Hepes, pH 7.5, 1 m m DTT, 10 m m MgCl2 , 0.1 m m Na3 VO4 , 1 m m NaF).

Synthesized:

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP. .. High specific activity transcripts used in filter binding experiments were synthesized by adding 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole) to a transcription mix containing 0.04 mM ATP and 0.4 mM of each remaining NTP.

Footprinting:

Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded
Article Snippet: Paragraph title: Potassium Permanganate and ExoIII Footprinting. ... EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position.

Incubation:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: Briefly, lysates were incubated overnight with glutathione beads at 4 °C. .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 .

Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded
Article Snippet: EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position. .. After the complex was eluted from Ni-NTA-agarose with 50 mM EDTA, the DNA in the supernatant was precipitated in the presence of 10 μg of calf thymus DNA as a carrier, incubated in 100 μl of 10% piperidine for 15 min at 90°C, reprecipitated, and lyophilized.

Purification:

Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA
Article Snippet: [α-32 P]UTP, [α-32 P]CTP, [α-32 P]ATP and [α-32 P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics. .. Radioactive transcripts were purified by 10% polyacrylamide gel electrophoresis.

Article Title: A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase
Article Snippet: [α-32 P]ATP, [α-32 P]GTP and [α-32 P]UTP were purchased from ICN Biomedicals and T7-RNA polymerase was purchased from Roche Diagnostics. .. Radioactive transcripts were purified by 10% polyacrylamide gel electrophoresis.

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: The fusion proteins GST-RB and GST-B-Myb as substrates for kinase assays were expressed with pGEX-5X-2 vectors in E. coli BL21-DE3 overnight at 30 °C and purified according to the manufacturer's recommendations (GST Gene Fusion System, Amersham Biosciences) using glutathione-agarose beads (Sigma). .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 .

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: End-labeled RNAs were transcribed and 32 P-labeled at the 5’ end and purified as described. .. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP.

Concentration Assay:

Article Title: Transcriptional arrest: Escherichia coli RNA polymerase translocates backward, leaving the 3? end of the RNA intact and extruded
Article Snippet: EC11 (the numerical index denotes the length of the transcript) was treated with 10 units of T4 polynucleotide kinase (New England Biolabs) and 50 μCi of [α-32 P]ATP (4500 Ci/mmol; ICN Biomedicals, Costa Mesa, CA) for 10 min to label the DNA, washed with TB, and walked to the desired position. .. KMnO4 (1 mM final concentration) was added to 10 μl of the EC.

Generated:

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: A precursor RNA containing the aI5β intron RNA was generated by in vitro transcription of the paI5FL vector, which yields a 1604 nt aI5β pre-RNA that includes a 12 nt 5’ exon, the catalytic core, an open reading frame encoding a degenerate homing endonuclease, and the 3’ SS followed by an 18 nt 3’ exon. .. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP.

Polymerase Chain Reaction:

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: The PCR product was cloned into the Sma I site of the pUC18 vector, giving plasmid pML1 in which the tRNA sequence is flanked by a 5′ T7 promoter and a 3′ Mva I restriction site. .. [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: The PCR product was cloned into the SmaI site of the pUC18 vector, giving plasmid pUC-tser in which the tRNA sequence is flanked by a 5′ T7 promoter and a 3′ MvaI restriction site. .. [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

Activity Assay:

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: .. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP. .. High specific activity transcripts used in filter binding experiments were synthesized by adding 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole) to a transcription mix containing 0.04 mM ATP and 0.4 mM of each remaining NTP.

Polyacrylamide Gel Electrophoresis:

Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA
Article Snippet: [α-32 P]UTP, [α-32 P]CTP, [α-32 P]ATP and [α-32 P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics. .. Radioactive transcripts were purified by 10% polyacrylamide gel electrophoresis.

Article Title: A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase
Article Snippet: [α-32 P]ATP, [α-32 P]GTP and [α-32 P]UTP were purchased from ICN Biomedicals and T7-RNA polymerase was purchased from Roche Diagnostics. .. Radioactive transcripts were purified by 10% polyacrylamide gel electrophoresis.

Sequencing:

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: The PCR product was cloned into the Sma I site of the pUC18 vector, giving plasmid pML1 in which the tRNA sequence is flanked by a 5′ T7 promoter and a 3′ Mva I restriction site. .. [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: The plasmid insert was verified by sequencing. .. [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

Staining:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: After washing, 20 μl of the slurry were run on an SDS-polyacrylamide gel, to control the preparations for equal concentrations and protein degradation, and stained with Coomassie Brilliant Blue. .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 .

Binding Assay:

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP. .. High specific activity transcripts used in filter binding experiments were synthesized by adding 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole) to a transcription mix containing 0.04 mM ATP and 0.4 mM of each remaining NTP.

Recombinant:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 . .. For kinase assays using recombinant and purified INCA1, human GST- INCA and GST- INCA -del75–99 were cloned into the baculovirus-shuttle vector pDEST20, shuttled to the baculovirus via the Bac-to-Bac baculovirus expression system (Invitrogen), and transfected into Sf9 insect cells.

BAC Assay:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 . .. For kinase assays using recombinant and purified INCA1, human GST- INCA and GST- INCA -del75–99 were cloned into the baculovirus-shuttle vector pDEST20, shuttled to the baculovirus via the Bac-to-Bac baculovirus expression system (Invitrogen), and transfected into Sf9 insect cells.

Plasmid Preparation:

Article Title: Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling *
Article Snippet: .. Briefly, 5 μCi of [α-32 P]ATP (ICN Biomedicals) were added to 15 μl of GST fusion beads and 6 μg of insect cell lysate expressing cyclin and CDK2 as well as insect cell lysates expressing control vector or INCA1 . .. This was then incubated for 30 min in 1× kinase buffer (10 μ m ATP, 50 m m Hepes, pH 7.5, 1 m m DTT, 10 m m MgCl2 , 0.1 m m Na3 VO4 , 1 m m NaF).

Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures
Article Snippet: Radioactive (32 P) in vitro transcripts were obtained using Mva I-digested pML1 plasmid as a template. .. [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

Article Title: The YqfN protein of Bacillus subtilis is the tRNA: m1A22 methyltransferase (TrmK)
Article Snippet: Radioactive (32 P) in vitro transcripts were obtained using MvaI digested pUC-tser plasmid as template. .. [α-32 P]-ATP and [α-32 P]-GTP were from ICN Biomedicals, Irvine, USA and T7 RNA polymerase from Roche Diagnostics.

Article Title: A DExH/D-box protein coordinates the two steps of splicing in a group I intron
Article Snippet: The pCOX3UTL plasmid was used to transcribe the COX3 5’UTL RNA. .. High specific activity transcripts used in the cross-linking reactions were transcribed in the presence of 4 µCi/µL [α-32 P]ATP (3,000 Ci/mmole; ICN Biomedicals, Irvine, CA), 0.8 mM 4-thioUTP (Ambion, Austin, TX), 0.04 mM ATP, and 0.4 mM of each remaining NTP.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    ICN Biomedicals α 32 p atp
    Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. <t>UTP/CTP/ATP</t> and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.
    α 32 P Atp, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 86/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/α 32 p atp/product/ICN Biomedicals
    Average 86 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    α 32 p atp - by Bioz Stars, 2020-04
    86/100 stars
      Buy from Supplier

    Image Search Results


    Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

    Journal: Nucleic Acids Research

    Article Title: Formation of the conserved pseudouridine at position 55 in archaeal tRNA

    doi: 10.1093/nar/gkl530

    Figure Lengend Snippet: Analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. ( A ) Cloverleaf structure of tRNA Asp of P.furiosus used as the wild-type tRNA substrate in this work. The universal numbering system for nucleotides in tRNA corresponds to that of ( 4 ). U55 is indicated. C75 and A76 (missing in −3′CA substrates) are indicated by a dashed box. The portion of the tRNA sequence included in the miniS substrate is indicated by the plain box. ( B ) The expected patterns of nuclease P1 and RNase T2 cleavage in the U55 region of an [α- 32 P]UTP- and [α- 32 P]CTP-labeled tRNA, respectively. Nuclease P1 generates 5′-phosphate-nucleosides while RNase T2 generates 3′phosphate-nucleosides. ( C ) 2D-TLC analysis of pfu Cbf5 and pfu Pus10 modification of various tRNA substrates. WT indicates wild-type pfu tRNA Asp (panels 1–4, 9–12); U55C indicates U55 replacement mutant (panels 5,6,13,14); miniS indicates mini-substrate (panels 7,15) and −3′CA indicates 3′ terminal CA deletion (panels 8,16). See text for more details. UTP/CTP/ATP and GTP refer to the ( 32 P)-labeled nucleotide incorporated at transcription. Incubation was for 1 h at 70°C in the presence of 0.12 nmol (5 µg) of pfu Cbf5 (panels 1–8) and 0.01 nmol (0.5 µg) of pfu Pus10 (panels 9–16). After incubation, the RNA was digested by nuclease P1 or RNase T2 (as indicated in each panel) and the resulting nucleotides were analyzed by 2D-TLC on cellulose plates and autoradiography. Circles in dotted lines show the migration of the canonical nucleotides used as UV markers.

    Article Snippet: [α-32 P]UTP, [α-32 P]CTP, [α-32 P]ATP and [α-32 P]GTP (400 Ci/mmol) were from ICN Biomedicals and T7 RNA polymerase was from Roche Diagnostics.

    Techniques: Modification, Sequencing, Labeling, Thin Layer Chromatography, Mutagenesis, Incubation, Autoradiography, Migration

    Affinity-purified T.thermophilus TrmI protein methylates A58 of in vitro transcribed T.thermophilus tRNA Asp . ( A ) Nucleotide sequence of T.thermophilus tRNA Asp ). m 1 A58 is shown in a gray box. The adenosines 5′-adjacent to a guanosine are indicated by an asterisk. ( B ) Autoradiograms of 2-dimensional chromatograms of P1 or T2 hydrolysates of [α- 32 P]ATP- or [α- 32 P]GTP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of the purified TrmI protein (see Materials and Methods for details). In the case of the chromatogram designated GTP/T2*, the T2 hydrolysis was carried out in the absence of carrier yeast tRNA. Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers. ( C ) Kinetics of in vitro formation of m 1 A58 in T.thermophilus tRNA Asp in the presence (closed circles) or absence (open circles) of the purified TrmI protein.

    Journal: Nucleic Acids Research

    Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures

    doi:

    Figure Lengend Snippet: Affinity-purified T.thermophilus TrmI protein methylates A58 of in vitro transcribed T.thermophilus tRNA Asp . ( A ) Nucleotide sequence of T.thermophilus tRNA Asp ). m 1 A58 is shown in a gray box. The adenosines 5′-adjacent to a guanosine are indicated by an asterisk. ( B ) Autoradiograms of 2-dimensional chromatograms of P1 or T2 hydrolysates of [α- 32 P]ATP- or [α- 32 P]GTP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of the purified TrmI protein (see Materials and Methods for details). In the case of the chromatogram designated GTP/T2*, the T2 hydrolysis was carried out in the absence of carrier yeast tRNA. Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers. ( C ) Kinetics of in vitro formation of m 1 A58 in T.thermophilus tRNA Asp in the presence (closed circles) or absence (open circles) of the purified TrmI protein.

    Article Snippet: [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

    Techniques: Affinity Purification, In Vitro, Sequencing, Labeling, Incubation, Purification, Migration

    Generation of a T.thermophilus mutant lacking TrmI activity, by homologous recombination. ( A ) Plasmid construction in E.coli for T.thermophilus trmI gene inactivation (see Materials and Methods for details). The trmI gene is represented by a solid bar, the thermostable knt gene (Km r ) by an open bar and the 16S promoter by a horizontal arrow. Restriction sites are indicated as follows: B, Bsu 36I; E, Eco RI; M, Mlu I. ( B ) Autoradiography of 2-dimensional chromatograms of P1 hydrolysates of [α- 32 P]ATP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of a crude extract of the T.thermophilus wild-type strain (HB27) or trmI mutant (RD1). Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers.

    Journal: Nucleic Acids Research

    Article Title: Cloning and characterization of tRNA (m1A58) methyltransferase (TrmI) from Thermus thermophilus HB27, a protein required for cell growth at extreme temperatures

    doi:

    Figure Lengend Snippet: Generation of a T.thermophilus mutant lacking TrmI activity, by homologous recombination. ( A ) Plasmid construction in E.coli for T.thermophilus trmI gene inactivation (see Materials and Methods for details). The trmI gene is represented by a solid bar, the thermostable knt gene (Km r ) by an open bar and the 16S promoter by a horizontal arrow. Restriction sites are indicated as follows: B, Bsu 36I; E, Eco RI; M, Mlu I. ( B ) Autoradiography of 2-dimensional chromatograms of P1 hydrolysates of [α- 32 P]ATP-labeled T.thermophilus tRNA Asp transcripts incubated for 1 h at 60°C in the presence of a crude extract of the T.thermophilus wild-type strain (HB27) or trmI mutant (RD1). Circles of dotted lines show the migration of the pG, pC and pU nucleotides used as UV markers.

    Article Snippet: [α-32 P]ATP and [α-32 P]GTP were purchased from ICN Biomedicals (Costa Mesa, CA) and T7 RNA polymerase was purchased from Roche Diagnostics.

    Techniques: Mutagenesis, Activity Assay, Homologous Recombination, Plasmid Preparation, Autoradiography, Labeling, Incubation, Migration

    A deaminase present in a crude P.furiosus extract transforms m 1 A57 preformed in yeast tRNA Asp by the P.abyssi TrmI enzyme into m 1 I57 and does not deaminate the m 1 A58 preformed in T.thermophilus tRNA Asp . [α- 32 P]ATP-labelled yeast tRNA Asp (A and C) and [α- 32 P]ATP- labelled T.thermophilus tRNA Asp (B and D) were incubated for 1 h at 60°C in the presence of the purified P.abyssi TrmI enzyme and of AdoMet. The transcripts were then recovered and incubated in a crude P.abyssi S30 extract for different periods of time as shown. At the end of the incubation time the transcripts were recovered, digested by nuclease P1 and analysed by 1D-TLC using solvent B (see Materials and Methods) on cellulose plates followed by autoradiography ( A and B ). ( C ) and ( D ) correspond to the autoradiograms of 2D-TLC of the samples incubated for 60 min in the presence of the P.furiosus extract.

    Journal: Nucleic Acids Research

    Article Title: A primordial RNA modification enzyme: the case of tRNA (m1A) methyltransferase

    doi: 10.1093/nar/gkh191

    Figure Lengend Snippet: A deaminase present in a crude P.furiosus extract transforms m 1 A57 preformed in yeast tRNA Asp by the P.abyssi TrmI enzyme into m 1 I57 and does not deaminate the m 1 A58 preformed in T.thermophilus tRNA Asp . [α- 32 P]ATP-labelled yeast tRNA Asp (A and C) and [α- 32 P]ATP- labelled T.thermophilus tRNA Asp (B and D) were incubated for 1 h at 60°C in the presence of the purified P.abyssi TrmI enzyme and of AdoMet. The transcripts were then recovered and incubated in a crude P.abyssi S30 extract for different periods of time as shown. At the end of the incubation time the transcripts were recovered, digested by nuclease P1 and analysed by 1D-TLC using solvent B (see Materials and Methods) on cellulose plates followed by autoradiography ( A and B ). ( C ) and ( D ) correspond to the autoradiograms of 2D-TLC of the samples incubated for 60 min in the presence of the P.furiosus extract.

    Article Snippet: [α-32 P]ATP, [α-32 P]GTP and [α-32 P]UTP were purchased from ICN Biomedicals and T7-RNA polymerase was purchased from Roche Diagnostics.

    Techniques: Incubation, Purification, Thin Layer Chromatography, Autoradiography