α-32 p Search Results


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  • 95
    GE Healthcare α 32 p dctp
    cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 <t>P]dCTP</t> (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.
    α 32 P Dctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 9686 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p dctp
    Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of <t>dCTP</t> α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.
    α 32 P Dctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 2852 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer datp
    Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c <t>(hcytc-</t> His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM <t>dATP</t> and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.
    Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 1692 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare α 32 p utp
    Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 <t>P]UTP,</t> the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.
    α 32 P Utp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 1828 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer dgtp
    A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing <t>telomerase</t> reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 <t>P-dGTP</t> for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.
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    99
    GE Healthcare α 32 p datp
    RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 <t>P]dATP.</t> K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.
    α 32 P Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2440 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare α 32 p ctp
    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 <t>P]CTP</t> during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.
    α 32 P Ctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 939 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    GE Healthcare α 32 p atp
    (A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α- 32 <t>P]ATP</t> was hydrolyzed to [α- 32 P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α- 32 P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32 P-labeled template RNA was loaded as a size control (lane 2).
    α 32 P Atp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 97/100, based on 703 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours α 32 p dctp
    DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 <t>P]dCTP-labeled</t> cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.
    α 32 P Dctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene α 32 p dctp
    Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 <t>P]dCTP</t> labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.
    α 32 P Dctp, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare α 32 p gtp
    Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 <t>P]GTP</t> and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.
    α 32 P Gtp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 498 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim α 32 p dctp
    T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 <t>P]dCTP</t> ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.
    α 32 P Dctp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    PerkinElmer α 32 p dttp
    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 <t>P]dTTP</t> end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.
    α 32 P Dttp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 96/100, based on 356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours α 32 p utp
    In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] <t>UTP</t> as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.
    α 32 P Utp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 258 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science α 32 p dctp
    Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 <t>-P]dCTP-labeled</t> jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.
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    92
    DuPont de Nemours α 32 p datp
    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 <t>P]dATP.</t> The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.
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    92
    Valiant α 32 p dctp
    (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 <t>P]dCTP.</t> The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.
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    96
    GE Healthcare α 32 p dttp
    Identification of 4A′-bound nucleotides and their dissociation kinetics. ( A ) 4A′ (2 μM hexamer) and [α- 32 <t>P]dTTP</t> (200 μM) were mixed at 18°C for 30 s and nonradiolabeled dTTP (10 mM) was added at time zero. After varying chase times (30 s to 20 min), aliquots were filtered through NC membranes. A total of 3.7 ± 0.87 nt (error calculated from seven independent measurements) were bound to 4A′ at zero chase time (•). After addition of dTTP chase, the 2–3 4A′-bound nucleotides exchanged at 0.008 ± 0.002 s −1 (○). ( B ) To identity the tightly bound nucleotides, NC membranes were extracted and the eluted nucleotides were analyzed by polyethyleneimine-cellulose TLC. Here the PhosphorImager scan of the TLC plate is shown. Lane 1, dTDP (20%) and dTTP (80%) bound to 4A′ before chase was added. Lanes 2–9, nucleotides bound after 0.5, 3, 6, 9, 12, 15, 20, 25 min of chase. In all lanes, dTDP is 6–17% and dTTP is 94–83%. Lane 10, dTDP bound to 4A′ after 90 min of incubation. Lane 11, almost no dTDP remained bound after 30-s chase with unlabeled dTTP. ( C ) The exchange of 4A′-bound dTMP-PCP was measured by preincubating 4A′ (0.83 μM hexamer) with [α- 32 P]dTMP-PCP (98 μM) and adding 5 mM dTTP as chase. Three to four dTMP-PCP are bound before chase was added (•). Two dTMP-PCPs exchanged with unlabeled dTTP in the medium (○) with a rate constant of 0.002 ± 0.0004 s −1 ; one dTMP-PCP did not exchange even after 50-min chase time. ( Inset ) dTTP hydrolysis in the presence of dTMP-PCP. Reaction conditions were same as in C , except 4A′ was preincubated with dTMP-PCP and hydrolysis of 5 mM [α- 32 P]dTTP was measured. dTTP was hydrolyzed without lag at 0.1 ± 0.001 s −1 in the presence of dTMP-PCP (▴) and at 0.14 ± 0.003 s −1 in the absence of dTMP-PCP (♦).
    α 32 P Dttp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 138 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.

    Journal: Plant Physiology

    Article Title: Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1 [w]

    doi: 10.1104/pp.104.056010

    Figure Lengend Snippet: cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.

    Article Snippet: A 0.75-kb Sal I/ Eco RI fragment of CSZ-7 was labeled with [ α -32 P]dCTP (10 μ Ci μ L−1 ; New Megaprime Random Labeling kit, Amersham Biosciences, Uppsala).

    Techniques: Hybridization, Labeling, Agarose Gel Electrophoresis, Molecular Weight

    Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

    doi: 10.1093/nar/gkx900

    Figure Lengend Snippet: Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.

    Article Snippet: For splicing analysis, template cDNA was amplified by Taq polymerase (NEB) using Tnp1 -specific intron-flanking primers, along with control primers against the reference gene TUBB , in a multiplexed PCR reaction containing 33 nM α-32 P-dCTP (3000 Ci/mmol, Perkin Elmer).

    Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Amplification, Multiplex Assay, Plasmid Preparation

    Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Journal: Nucleic Acids Research

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

    doi: 10.1093/nar/gkr563

    Figure Lengend Snippet: Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Article Snippet: Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Techniques: Inhibition, Modification, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Concentration Assay, MTT Assay

    Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc- His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.

    Journal: The Journal of Cell Biology

    Article Title: The two cytochrome c species, DC3 and DC4, are not required for caspase activation and apoptosis in Drosophila cells

    doi: 10.1083/jcb.200408054

    Figure Lengend Snippet: Effect of recombinant DC3 or DC4 on caspase activity in BG2 cell lysates. (A) Absorption spectrum of human cytochrome c (hcytc- His6 ), DC3 His6 , and DC4 His6 . Absorption was measured between 400 and 600 nm at a scanning speed of 1 nm/s. (B) Immunoblot analysis of cytochrome c proteins with anti–cytochrome c antibody detects recombinant DC3 His6 , DC4 His6 , and hcytc- His6 . White line indicates that intervening lanes have been spliced out. (C) BG2 and 293T cytosolic (S100) extracts immunoblotted with anti–cytochrome c antibody confirm absence of cytochrome c in these fractions. Expression of DRICE and caspase-3 is shown in bottom panel. (D) Purified recombinant DC3 His6 , DC4 His6 , or hcytc His6 (10 μM) were incubated with BG2 S100 (top) or 293T S100 (bottom) lysates together with 1 mM dATP and caspase activity measured on DEVD-amc. Values represent the mean ± SEM from three independent experiments.

    Article Snippet: Where indicated, S100 lysates were incubated with 10 μM of purified DC3His6 , DC4His6 , or human cytochrome c (hcytc ), and 1 mM dATP, in caspase assay buffer ( ) at RT and activity was measured over time using a fluorometric plate reader (PerkinElmer) (excitation 385 nm, emission 460 nm).

    Techniques: Recombinant, Activity Assay, Expressing, Purification, Incubation

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker

    Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 P]UTP, the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Effect of cre (2C)mut1 on VPg uridylylation in reconstituted reactions. VPg uridylylation was measured in reconstituted reaction mixtures containing 3D pol , VPg, [α- 32 P]UTP, the indicated RNAs, and 3CD as described in Materials and Methods (lanes 2 to 5). 32 P-labeled VPgpUpU synthesized in these reactions was resolved by SDS-PAGE (9 to 18% polyacrylamide). [ 35 S]methionine-labeled poliovirus proteins were used as markers and are shown in lane 1.

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Labeling, Synthesized, SDS Page

    Synthesis of uridylylated VPg in PIRCs. HeLa S10 translation-RNA replication reaction mixtures containing 2 mM guanidine HCl were prepared with PV1 RNA and PV1VPgY3F RNA. VPg uridylylation was measured in PIRCs resuspended in reaction mixtures containing [α- 32 P]UTP. The labeled VPgpUpU synthesized in these reactions was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods and resolved by SDS-PAGE. [ 35 S]methionine-labeled poliovirus proteins were used as markers (lane 7).

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Synthesis of uridylylated VPg in PIRCs. HeLa S10 translation-RNA replication reaction mixtures containing 2 mM guanidine HCl were prepared with PV1 RNA and PV1VPgY3F RNA. VPg uridylylation was measured in PIRCs resuspended in reaction mixtures containing [α- 32 P]UTP. The labeled VPgpUpU synthesized in these reactions was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods and resolved by SDS-PAGE. [ 35 S]methionine-labeled poliovirus proteins were used as markers (lane 7).

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Labeling, Synthesized, Immunoprecipitation, SDS Page

    Effect of cre (2C)mut1 on VPg uridylylation in PIRCs. Viral protein synthesis was measured in HeLa S10 translation-replication reaction mixtures containing the indicated RNAs and [ 35 S]methionine (1.2 mCi/ml) (lanes 1 to 4). VPg uridylylation was measured in PIRCs isolated from identical HeLa S10 translation-replication reaction mixtures containing each of the indicated RNAs. Reaction mixtures containing the PIRCs and [α- 32 P]UTP were incubated at 37°C for 1 h. Labeled VPgpUpU was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods (lanes 5 to 12). The labeled viral proteins and immunoprecipitated products were resolved by SDS-PAGE (9 to 18% polyacrylamide).

    Journal: Journal of Virology

    Article Title: Poliovirus cre(2C)-Dependent Synthesis of VPgpUpU Is Required for Positive- but Not Negative-Strand RNA Synthesis

    doi: 10.1128/JVI.77.9.5136-5144.2003

    Figure Lengend Snippet: Effect of cre (2C)mut1 on VPg uridylylation in PIRCs. Viral protein synthesis was measured in HeLa S10 translation-replication reaction mixtures containing the indicated RNAs and [ 35 S]methionine (1.2 mCi/ml) (lanes 1 to 4). VPg uridylylation was measured in PIRCs isolated from identical HeLa S10 translation-replication reaction mixtures containing each of the indicated RNAs. Reaction mixtures containing the PIRCs and [α- 32 P]UTP were incubated at 37°C for 1 h. Labeled VPgpUpU was immunoprecipitated with anti-VPg antibody (Ab) as described in Materials and Methods (lanes 5 to 12). The labeled viral proteins and immunoprecipitated products were resolved by SDS-PAGE (9 to 18% polyacrylamide).

    Article Snippet: The uridylylation reactions were identical to those described above for the RNA replication assays, except the reaction mixtures contained 5 μM UTP, which was provided by the addition of 100 μCi of [α-32 P]UTP (400 Ci/mmol; Amersham) and 250 μM (each) ATP, CTP, and GTP.

    Techniques: Isolation, Incubation, Labeling, Immunoprecipitation, SDS Page

    A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Journal: The EMBO Journal

    Article Title: RNA/DNA hybrid binding affinity determines telomerase template-translocation efficiency

    doi: 10.1038/emboj.2011.363

    Figure Lengend Snippet: A 5-bp RNA/DNA hybrid competitor inhibits repeat-addition processivity. Native template-containing telomerase reconstituted in 293FT cells was used in the pulse-chase/challenge assay. Two RNA/DNA duplexes (5 and 7 bp) were used as competitors to challenge the processive telomerase during the 90-min chase reaction at 4°C. The pulse reaction was carried out in the presence of α- 32 P-dGTP for 15 min to radioactively label the telomeric DNA primer (TTAGGG) 3 ). DNA size markers were generated by 3′-end labelling of the 5 and 7-base DNA oligonucleotide (M 5 : TAGGG and M 7 : GTTAGGG) using α- 32 P-dGTP and terminal deoxynucleotidyl transferase.

    Article Snippet: In brief, 2–3 μl of in vitro reconstituted telomerase was assayed in a 10-μl reaction containing 1 × telomerase reaction buffer, 1 mM dTTP, 1 mM dATP, 2 μM dGTP, 0.165 μM α-32 P-dGTP (3000 Ci/mmol, 10 mCi/ml, Perkin-Elmer) and 1 μM (TTAGGG)3 DNA primer.

    Techniques: Pulse Chase, Generated

    Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Journal: Scientific Reports

    Article Title: A cancer-associated point mutation disables the steric gate of human PrimPol

    doi: 10.1038/s41598-018-37439-0

    Figure Lengend Snippet: Ribonucleotides are valid substrates for the Y100H variant during primer synthesis. ( a ) Scheme on the top shows PrimPol in complex with the GTCA template oligonucleotide and the two nucleotides forming the initial dimer. The autoradiograph shows dimer formation (primase activity) either by wild-type (WT) PrimPol or Y100H (400 nM) using [α- 32 P]dATP (upper panel) or [γ- 32 P] ATP (lower panel) as the 5′-site nucleotide (16 nM), and increasing concentrations of either dGTP or GTP as the incoming 3′-site nucleotide (0, 10, 50, 100 µM). ( b ) Binary complex formation, measured by EMSA, between WT PrimPol or Y100H and labeled 60-mer DNA template GTCC (1 nM), using the indicated PrimPol concentration (2.5, 5, 10, 20, 40 and 80 nM) ( c ) Pre-ternary complex formation measured by EMSA between WT PrimPol or Y100H (1 µM), 60-mer DNA template GTCC and either [α- 32 P]dGTP or [α- 32 P] GTP (16 nM). ( d ) DNA or RNA primers synthesized using as template 5′-T 20 ACGACAGACTGT 29 -3′ to allow elongation beyond the dimer. Products were labeled with [γ- 32 P] ATP . The autoradiographs shown in this figure are representative of at least 3 independent experiments.

    Article Snippet: Radiolabeled nucleotides [γ-32 P] ATP , [α-32 P]dATP and [α-32 P]dGTP (3000 Ci/mmol) were obtained from Perkin Elmer (Waltham, MA, USA).

    Techniques: Variant Assay, Autoradiography, Activity Assay, Labeling, Concentration Assay, Synthesized

    RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 P]dATP. K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Replication and Transcription Activator Regulates Viral and Cellular Genes via Interferon-Stimulated Response Elements

    doi: 10.1128/JVI.79.9.5640-5652.2005

    Figure Lengend Snippet: RTA binds to ISRE. A. The purified RTA protein binds to K14 ISRE. The probe was labeled with [α- 32 P]dATP. K14 ISRE, mK14 ISRE, ISG15 ISRE, Tap-2 ISRE, and mTap2-ISRE were used as cold competitors. Cold competitors were added at a 50-fold molar excess over hot probe. Various amounts of Ni 2+ -NTA agarose beads (1.5, 5, and 15 μl) were used to remove the histidine-tagged RTA proteins in an EMSA. Fifteen microliters of GST beads was used as a control. Rabbit polyclonal anti-RTA or K15 serum was also used. ns, nonspecific. B. RTA binds to known ISREs. The probe was ISRE-1 from the vIL-6 promoter. The cold competitors, Ni 2+ -NTA agarose beads, GST beads, and antisera used are shown. Specific protein-DNA complexes are shown.

    Article Snippet: The probes were obtained by first annealing complementary oligonucleotides and then labeling them with [α-32 P]dATP (Amersham) using DNA polymerase Klenow fragment (Fermentas).

    Techniques: Purification, Labeling

    Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 P]CTP during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: Comparison of His 6 -tagged and untagged PrfA proteins and the effect of a His 6 tag on formation of CI, CII, and CIII complexes and in vitro transcription. Binding affinity of 230 nM PrfA Lm (P), 230 nM nontagged PrfA Lm (P nt ), 12 nM PrfA* Lm (P*), and 12 nM nontagged PrfA* Lm (P* nt ) to 5 nM of the hly and the actA DNA promoter fragments of L. monocytogenes P hly Lm (A) and P actA Lm (C). The RNAP concentration is 1.5 nM. The graphs to the right show the band intensities relative to that of the CII band in lane 2. Transcriptional activity of 1.75 nM RNAP with increasing concentrations (1.6, 4, 8, and 16 nM) of the different PrfA proteins was detected with 19 nM of the two promoter fragments P hly Lm (B) and P actA Lm (D). The mRNA was labeled with [α- 32 P]CTP during transcription. The graphs to the right show the increasing transcriptional initiations compared to that from the lane without PrfA. The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro, Binding Assay, Concentration Assay, Activity Assay, Labeling

    In vitro transcription assays with the hly and actA promoters of L. monocytogenes (P hly Lm and P actA Lm ), L. ivanovii (i-P hly and i-P actA ), and L. seeligeri (s-P hly and s-P actA ). Results are from an in vitro transcription assay with increasing concentrations of the PrfA proteins PrfA Lm , PrfA* Lm , PrfA Ls , and PrfA Li using 16 nM promoter template DNA and 1.3 nM (when using P hly ) or 1.9 nM (when using P actA ) RNA polymerase. The runoff transcripts were radioactively marked by adding [α- 32 P]CTP to the in vitro assay. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. Values represent the relative ratios of the measured radioactivities and the molar concentrations of PrfA protein in the range of linear dependency. The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: In vitro transcription assays with the hly and actA promoters of L. monocytogenes (P hly Lm and P actA Lm ), L. ivanovii (i-P hly and i-P actA ), and L. seeligeri (s-P hly and s-P actA ). Results are from an in vitro transcription assay with increasing concentrations of the PrfA proteins PrfA Lm , PrfA* Lm , PrfA Ls , and PrfA Li using 16 nM promoter template DNA and 1.3 nM (when using P hly ) or 1.9 nM (when using P actA ) RNA polymerase. The runoff transcripts were radioactively marked by adding [α- 32 P]CTP to the in vitro assay. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. Values represent the relative ratios of the measured radioactivities and the molar concentrations of PrfA protein in the range of linear dependency. The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro

    Replacement of the C-terminal 38 amino acids of PrfA Ls with those of PrfA Lm . (A) ClustalW alignment of the PrfA proteins of L. monocytogenes (PrfA Lm ), L. ivanovii (PrfA Li ), and L. seeligeri (PrfA Ls ). Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Amino acid substitutions leading to a constitutively active PrfA are marked. (B) In vitro transcription assay with increasing concentrations of PrfA Lm , the hybrid PrfA Lsm , and PrfA Ls using 16 nM promoter template DNA and 1.9 nM RNA polymerase. The mRNA was marked with [α- 32 P]CTP during transcription. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. The graphs in the lower part of the panel below show the amounts of CI and CII measured in EMSAs. The components were 5 nM 32 P-marked promoter DNA (P hly Lm ) and 1.5 nM RNA polymerase, and the PrfA concentration is given in the figure. Quantification of CI and CII complexes was performed using ImageMaster (Amersham). The data shown here represent the results of one of three independently performed experiments.

    Journal: Journal of Bacteriology

    Article Title: Species-Specific Differences in the Activity of PrfA, the Key Regulator of Listerial Virulence Genes ▿

    doi: 10.1128/JB.00473-06

    Figure Lengend Snippet: Replacement of the C-terminal 38 amino acids of PrfA Ls with those of PrfA Lm . (A) ClustalW alignment of the PrfA proteins of L. monocytogenes (PrfA Lm ), L. ivanovii (PrfA Li ), and L. seeligeri (PrfA Ls ). Identical amino acids are shaded in black, and similar amino acids are shaded in gray. Amino acid substitutions leading to a constitutively active PrfA are marked. (B) In vitro transcription assay with increasing concentrations of PrfA Lm , the hybrid PrfA Lsm , and PrfA Ls using 16 nM promoter template DNA and 1.9 nM RNA polymerase. The mRNA was marked with [α- 32 P]CTP during transcription. The transcription-activating potentials of the different PrfA proteins compared to that of PrfA Lm are given in the graphs to the right. The graphs in the lower part of the panel below show the amounts of CI and CII measured in EMSAs. The components were 5 nM 32 P-marked promoter DNA (P hly Lm ) and 1.5 nM RNA polymerase, and the PrfA concentration is given in the figure. Quantification of CI and CII complexes was performed using ImageMaster (Amersham). The data shown here represent the results of one of three independently performed experiments.

    Article Snippet: After 5 min of incubation at room temperature, the reaction was started by the addition of 2 μl [α-32 P]CTP (5 μCi, 3,000 Ci mmol−1 ; Amersham) and incubated at 37°C for 5 min before 2 μl of a heparin solution (10 g/liter) was added.

    Techniques: In Vitro, Concentration Assay

    (A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α- 32 P]ATP was hydrolyzed to [α- 32 P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α- 32 P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32 P-labeled template RNA was loaded as a size control (lane 2).

    Journal: Journal of Clinical Microbiology

    Article Title: Immunoassay Targeting Nonstructural Protein 5 To Differentiate West Nile Virus Infection from Dengue and St. Louis Encephalitis Virus Infections and from Flavivirus Vaccination

    doi: 10.1128/JCM.41.9.4217-4223.2003

    Figure Lengend Snippet: (A) WNV genome structure. The recombinant proteins used in this study are shaded. (B and C) Purified NTPase/helicase domain of NS3 and full-length NS5 were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; gels were stained with Coomassie blue. (D) ATPase activity of the recombinant NTPase/helicase domain of WNV NS3. In the presence of recombinant NS3, [α- 32 P]ATP was hydrolyzed to [α- 32 P]ADP and phosphate (lane 2). No ATP is hydrolyzed in the absence of NS3 (lane 1). (E) RDRP activity of the recombinant NS5. The RDRP activity of NS5 was assayed with a WNV subgenomic RNA transcript (890 nt) containing a large deletion from nucleotide 269 to 10408. The reaction products (RXT) were labeled with [α- 32 P]UTP, and the products of 1× and 2X forms of RNA were analyzed on a denaturing polyacrylamide gel followed by autoradiography (lane 1). A 32 P-labeled template RNA was loaded as a size control (lane 2).

    Article Snippet: The NTPase assay was performed in a 10-μl reaction volume containing 20 mM Tris (pH 7.5), 2.5 mM MgCl2 , 2 mM dithiothreitol, 1 mM cold ATP spiked with 1 μCi of corresponding [α-32 P]ATP (2,000 Ci/mmol) (Amersham, Piscataway, N.J.), and 0.8 μM recombinant NS3.

    Techniques: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, Staining, Activity Assay, Labeling, Autoradiography

    DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

    doi:

    Figure Lengend Snippet: DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.

    Article Snippet: Specific probes were generated by labeling reamplified or cloned cDNA fragments with [α-32 P]dCTP (Dupont/NEN) with a random primer DNA labeling kit (Life Technologies).

    Techniques: Derivative Assay, Northern Blot, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Labeling, Hybridization

    Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

    doi:

    Figure Lengend Snippet: Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.

    Article Snippet: Specific probes were generated by labeling reamplified or cloned cDNA fragments with [α-32 P]dCTP (Dupont/NEN) with a random primer DNA labeling kit (Life Technologies).

    Techniques: Agarose Gel Electrophoresis, Labeling

    Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 P]dCTP labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    doi: 10.1128/MCB.24.6.2423-2443.2004

    Figure Lengend Snippet: Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 P]dCTP labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.

    Article Snippet: The PCR-amplified fragments were gel purified and labeled with [α-32 P]dCTP by using a Prime-It RmT random primer labeling kit (Stratagene), and the labeled probes were purified with a NucTrap probe purification column (Stratagene) for detection of mRNA transcripts of blr1 , RARs, and RXRs by hybridization at 50°C overnight after a 2-h prehybridization at 42°C for blr1 and 45°C for RARs and RXRs.

    Techniques: Expressing, Isolation, Northern Blot, Derivative Assay, Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Labeling, Staining

    Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 P]GTP and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.

    Journal: Nucleic Acids Research

    Article Title: Transcription efficiency of human polymerase III genes in vitro does not depend on the RNP-forming autoantigen La

    doi:

    Figure Lengend Snippet: Analysis of functionally active pol III transcription complexes separated by a glycerol gradient. Several identical transcription reactions were performed in a volume of 25 µl each as described in Materials and Methods. After incubation they were pooled. Aliquots of 200 µl from this pool (corresponding to eight transcription reactions) were applied to a 4.2 ml glycerol gradient and centrifuged for 3 h at 50 000 r.p.m. in an SW 60 rotor. After centrifugation, fractions of 350 µl were collected. The corresponding fractions from six parallel gradients were pooled. ( A ) In vitro transcription. The fractions from the gradients and the load fraction were incubated with nucleotides, [α- 32 P]GTP and RNase Block as described above. No additional DNA was added. After incubation, the RNA in the samples was extracted as described above and loaded onto a 6% denaturing polyacrylamide gel. Lane 1, 25 µl load fraction; lanes 2–13, 50 µl glycerol gradient fractions. Fraction 1 is the top fraction ( B ) Western blot. The remaining pooled fractions were treated with Strataclean Resin (Stratagene) and loaded onto a 12.5% SDS gel. After blotting, the membrane was incubated with monoclonal anti-TBP antibodies and subsequently incubated with [ 125 I]anti-mouse antibodies as secondary antibodies. The membrane was analysed with a phosphorimager. The membrane was not stripped, but incubated with a mixture of SW5 and 3B9 antibodies and subsequently incubated with 125 I-labelled anti-mouse antibodies. The final analysis was performed with a phosphorimager and by autoradiography. Note that the band in fraction 8 at the height of the La signal was already visible after TBP incubation (data not shown). It is presumably an artefact due to the second antibody. Lane 1, 160 µl of load fraction; lanes 2–13, 1.85 ml glycerol gradient fractions; lane 14, 5 µl IIIBβ; lane 15, 5 µl of a La fraction, purified from PCA (20 ng La/µl). ( C ) In vitro transcription. The in vitro transcription was performed as described in (A) using a mixture of fractions 8 and 9 of a typical gradient. To ensure single round conditions in lanes 6–10, heparin was added at a final concentration of 600 µg/ml. Lanes 1–5, incubation without heparin for 2, 5, 10, 20 and 40 min; lanes 6–10, incubation with heparin for 2, 5, 10, 20 and 40 min. ( D ) Multiple round transcription of fractions 8 and 9 for 60 min. The gel was autoradiographed for a shorter time period as in (C). BSA (10 µg) was added to the reaction in order to avoid any protein effects. Lane 1, no additional La; lanes 2–4: addition of 15, 30 or 60 ng recombinant La.

    Article Snippet: Aliquots of 50 µl of the gradient fractions were mixed with nucleotides, 3 µCi [α-32 P]GTP (Amersham) and RNase Block and transcribed as described above in a total volume of 60 µl.

    Techniques: Incubation, Centrifugation, In Vitro, Blocking Assay, Western Blot, SDS-Gel, Autoradiography, Purification, Concentration Assay, Recombinant

    T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 P]dCTP ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 P]dCTP ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.

    Article Snippet: Ten microliters of the telomerase reaction was added to a 50-μl final volume PCR mix containing 1× modified TRAP buffer, 50 μM each dNTP, 20 pmol of TS primer, 20 pmol of CX-ext primer, 0.1 μCi/μl [α-32 P]dGTP or [α-32 P]dCTP and 1 unit of Taq polymerase (Boehringer Mannheim).

    Techniques: Labeling

    T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Article Snippet: Ten microliters of the telomerase reaction was added to a 50-μl final volume PCR mix containing 1× modified TRAP buffer, 50 μM each dNTP, 20 pmol of TS primer, 20 pmol of CX-ext primer, 0.1 μCi/μl [α-32 P]dGTP or [α-32 P]dCTP and 1 unit of Taq polymerase (Boehringer Mannheim).

    Techniques: Activity Assay, Primer Extension Assay, Incubation, Molecular Weight, Marker

    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    doi: 10.1128/MCB.24.6.2423-2443.2004

    Figure Lengend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences.

    Techniques: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

    Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Journal: The Journal of Biological Chemistry

    Article Title: Comparative Analysis of Histophilus somni Immunoglobulin-binding Protein A (IbpA) with Other Fic Domain-containing Enzymes Reveals Differences in Substrate and Nucleotide Specificities *

    doi: 10.1074/jbc.M111.227603

    Figure Lengend Snippet: Nucleotide specificity of IbpA-Fic2. A , GST-tagged and purified IbpA-Fic1, IbpA-Fic2, PfhB2-Fic1, PfhB2-Fic2, and VopS and His 6 -SUMO-tagged HYPE-Fic were incubated with Cdc42 1–179 Q61L in an in vitro reaction using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples separated by SDS-PAGE were visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). The ability of the indicated Fic enzymes to utilize different nucleotides for post-translationally modifying Cdc42 is shown. All the panels were given equal exposure times for autoradiography. The dotted line represents a break in the gels. B , reactions with His 6 -SUMO-tagged HYPE-Fic displayed in panel A were rerun on SDS-PAGE and visualized by longer exposures for autoradiography ( upper panel ) and Coomassie Blue staining ( bottom panel ). HYPE-Fic efficiently uses ATP, and CTP to a lesser degree, to modify Cdc42. C , point mutations in the IbpA-Fic2 Fic motif did not alter its affinity for nucleotides. GST-tagged and purified Pro-3718 to Gly (IbpA_Fic2-P/G) and Glu-3271 to Asp (IbpA_Fic2-E/D) mutants of IbpA-Fic2, as well as wild type IbpA-Fic2 and VopS, were incubated with Cdc42-Q61L using [α- 32 P]ATP and -GTP in an in vitro reaction. Samples were separated on SDS-PAGE and visualized by autoradiography ( top panel ) and Coomassie Blue staining ( bottom panel ). Conversion of the IbpA-Fic2 Fic motif sequence to match the corresponding residues in the Fic motif of VopS did not confer specificity for nucleotides. D , comparison of IbpA-Fic2 and VopS to target switch 1 Tyr-32 and Thr-35 mutants of Cdc42 using different nucleotides. GST-tagged IbpA-Fic2 and VopS were incubated with wild type ( W ), Y32F ( Y ), or T35A ( T ) versions of Cdc42 expressed as GST fusion proteins in bacteria in an in vitro assay using [α- 32 P]ATP, -GTP, -CTP, -UTP, or -dTTP. Samples were assessed by autoradiography ( top panel ) with exposure times adjusted for optimal visualization and by Coomassie Blue staining ( lower panel ). Mutation of T35A in Cdc42 did not alter the ability of IbpA-Fic2 to target the switch 1 Tyr-32 for modification. In contrast, the Y32F mutation in Cdc42 severely impaired VopS in modifying Thr-35 using the different nucleotide sources.

    Article Snippet: For nucleotide specificity assays, in vitro reactions were conducted as above with α-32 P-labeled ATP, GTP, CTP, UTP, or dTTP (PerkinElmer Life Sciences) containing 1 mm of each respective cold dNTP.

    Techniques: Purification, Incubation, In Vitro, SDS Page, Autoradiography, Staining, Sequencing, Mutagenesis, Modification

    In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] UTP as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.

    Journal: The New Phytologist

    Article Title: The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus

    doi: 10.1111/nph.12304

    Figure Lengend Snippet: In vitro RdRp assay with exogenous RNA templates. Approximately 50 ng of RNA template r138/40A (the 3′ UTR of Bamboo mosaic virus (BaMV)) in (a) and Ba-77 (the 3′-end 77 nts of BaMV minus-strand) in (b) were incubated with BaMV RdRp complex for the in vitro RNA synthesis in the presence of different concentrations (0–100 mM) of glutathione (GSH) as indicated. The RdRp products labeled with [α- 32 P] UTP as indicated bands were separated on a 5% acrylamide gel and quantified by a phosphorimager. (c) The relative RdRp template activities were plotted according to the data derived from (a) and (b). The banding density of the in vitro RdRp assay with either r138/40A or Ba-77 was set as 100% in the absence of GSH (0 mM). Each spot on the plot was the average ± SE of at least three independent experiments.

    Article Snippet: The RdRp assay was carried out in a 50-μl reaction containing 5 μl of 1.5% NP40-solublized RdRp fraction, 2 mM each of ATP, CTP, and GTP, 2 μM UTP, 0.066 μM [α-32 P]UTP (3000 Ci mmol−1 , Dupont-NEN), 4.8 mg ml−1 of bentonite, 10 mM dithiothreitol, 3 mM MgCl2 , and 50 ng of RNA template and incubated at 30°C water bath for 1 h. The RNA products were extracted with phenol-chloroform and precipitated with ethanol.

    Techniques: In Vitro, Incubation, Labeling, Acrylamide Gel Assay, Derivative Assay

    Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 -P]dCTP-labeled jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.

    Journal: The EMBO Journal

    Article Title: jkk-1 and mek-1 regulate body movement coordination and response to heavy metals through jnk-1 in Caenorhabditis elegans

    doi: 10.1093/emboj/20.18.5114

    Figure Lengend Snippet: Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 -P]dCTP-labeled jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.

    Article Snippet: The blots were pre-hybridized with ExpressHyb hybridization solution (Clontech) for 1 h, and then probed with random-primed (Prime-it II, Stratagene), [α-32 P]dCTP (NEN Life Science)-labeled full-length jnk-1β cDNA at 68°C for 1 h. The blots were washed under stringent conditions (65°C and 0.1% SSC/0.1% SDS) and exposed for 48 h to Kodak XAR-5 film at –70°C.

    Techniques: Clone Assay, Sequencing, Southern Blot, Labeling

    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: DNA Synthesis, Purification, Infection, Isolation, Autoradiography, Inhibition, Generated

    Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Labeling, Titration, Generated

    (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 P]dCTP. The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.

    Journal: Microbiology

    Article Title: Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

    doi: 10.1099/mic.0.033167-0

    Figure Lengend Snippet: (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 P]dCTP. The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.

    Article Snippet: [ α -32 P]dCTP was obtained from MP Biomedicals.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Produced

    (a) Diagrammatic illustration of single-crossover disruption in the vlmI gene created by plasmid pKC1139Δ vlmI , showing expected restriction fragments. (b) Southern blot analysis of Kpn I-digested genomic DNA from wild-type S. viridifaciens MG456-hF10 (WT) and S. viridifaciens MG456-hF10 ( vlmI : : pKC1139Δ vlmI ). A 478 bp internal vlmI fragment obtained by Eco RI digestion of plasmid pGEMTΔ vlmI was labelled with [ α - 32 P]dCTP and used as a probe. Kpn I sites are indicated by K .

    Journal: Microbiology

    Article Title: Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

    doi: 10.1099/mic.0.033167-0

    Figure Lengend Snippet: (a) Diagrammatic illustration of single-crossover disruption in the vlmI gene created by plasmid pKC1139Δ vlmI , showing expected restriction fragments. (b) Southern blot analysis of Kpn I-digested genomic DNA from wild-type S. viridifaciens MG456-hF10 (WT) and S. viridifaciens MG456-hF10 ( vlmI : : pKC1139Δ vlmI ). A 478 bp internal vlmI fragment obtained by Eco RI digestion of plasmid pGEMTΔ vlmI was labelled with [ α - 32 P]dCTP and used as a probe. Kpn I sites are indicated by K .

    Article Snippet: [ α -32 P]dCTP was obtained from MP Biomedicals.

    Techniques: Plasmid Preparation, Southern Blot

    Identification of 4A′-bound nucleotides and their dissociation kinetics. ( A ) 4A′ (2 μM hexamer) and [α- 32 P]dTTP (200 μM) were mixed at 18°C for 30 s and nonradiolabeled dTTP (10 mM) was added at time zero. After varying chase times (30 s to 20 min), aliquots were filtered through NC membranes. A total of 3.7 ± 0.87 nt (error calculated from seven independent measurements) were bound to 4A′ at zero chase time (•). After addition of dTTP chase, the 2–3 4A′-bound nucleotides exchanged at 0.008 ± 0.002 s −1 (○). ( B ) To identity the tightly bound nucleotides, NC membranes were extracted and the eluted nucleotides were analyzed by polyethyleneimine-cellulose TLC. Here the PhosphorImager scan of the TLC plate is shown. Lane 1, dTDP (20%) and dTTP (80%) bound to 4A′ before chase was added. Lanes 2–9, nucleotides bound after 0.5, 3, 6, 9, 12, 15, 20, 25 min of chase. In all lanes, dTDP is 6–17% and dTTP is 94–83%. Lane 10, dTDP bound to 4A′ after 90 min of incubation. Lane 11, almost no dTDP remained bound after 30-s chase with unlabeled dTTP. ( C ) The exchange of 4A′-bound dTMP-PCP was measured by preincubating 4A′ (0.83 μM hexamer) with [α- 32 P]dTMP-PCP (98 μM) and adding 5 mM dTTP as chase. Three to four dTMP-PCP are bound before chase was added (•). Two dTMP-PCPs exchanged with unlabeled dTTP in the medium (○) with a rate constant of 0.002 ± 0.0004 s −1 ; one dTMP-PCP did not exchange even after 50-min chase time. ( Inset ) dTTP hydrolysis in the presence of dTMP-PCP. Reaction conditions were same as in C , except 4A′ was preincubated with dTMP-PCP and hydrolysis of 5 mM [α- 32 P]dTTP was measured. dTTP was hydrolyzed without lag at 0.1 ± 0.001 s −1 in the presence of dTMP-PCP (▴) and at 0.14 ± 0.003 s −1 in the absence of dTMP-PCP (♦).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase

    doi:

    Figure Lengend Snippet: Identification of 4A′-bound nucleotides and their dissociation kinetics. ( A ) 4A′ (2 μM hexamer) and [α- 32 P]dTTP (200 μM) were mixed at 18°C for 30 s and nonradiolabeled dTTP (10 mM) was added at time zero. After varying chase times (30 s to 20 min), aliquots were filtered through NC membranes. A total of 3.7 ± 0.87 nt (error calculated from seven independent measurements) were bound to 4A′ at zero chase time (•). After addition of dTTP chase, the 2–3 4A′-bound nucleotides exchanged at 0.008 ± 0.002 s −1 (○). ( B ) To identity the tightly bound nucleotides, NC membranes were extracted and the eluted nucleotides were analyzed by polyethyleneimine-cellulose TLC. Here the PhosphorImager scan of the TLC plate is shown. Lane 1, dTDP (20%) and dTTP (80%) bound to 4A′ before chase was added. Lanes 2–9, nucleotides bound after 0.5, 3, 6, 9, 12, 15, 20, 25 min of chase. In all lanes, dTDP is 6–17% and dTTP is 94–83%. Lane 10, dTDP bound to 4A′ after 90 min of incubation. Lane 11, almost no dTDP remained bound after 30-s chase with unlabeled dTTP. ( C ) The exchange of 4A′-bound dTMP-PCP was measured by preincubating 4A′ (0.83 μM hexamer) with [α- 32 P]dTMP-PCP (98 μM) and adding 5 mM dTTP as chase. Three to four dTMP-PCP are bound before chase was added (•). Two dTMP-PCPs exchanged with unlabeled dTTP in the medium (○) with a rate constant of 0.002 ± 0.0004 s −1 ; one dTMP-PCP did not exchange even after 50-min chase time. ( Inset ) dTTP hydrolysis in the presence of dTMP-PCP. Reaction conditions were same as in C , except 4A′ was preincubated with dTMP-PCP and hydrolysis of 5 mM [α- 32 P]dTTP was measured. dTTP was hydrolyzed without lag at 0.1 ± 0.001 s −1 in the presence of dTMP-PCP (▴) and at 0.14 ± 0.003 s −1 in the absence of dTMP-PCP (♦).

    Article Snippet: [α-32 P]dTTP was purchased from Amersham, and its purity was checked and corrected in all the experiments.

    Techniques: Thin Layer Chromatography, Incubation

    Pre-steady-state pulse–chase kinetics of dTTP hydrolysis. 4A′ (3.3 μM hexamer) was mixed with [α- 32 P]dTTP (100–600 μM) in a rapid quench-flow instrument at 18°C, and the reactions were chased with dTTP or EDTA (see Materials and Methods ). ( A ) Representative time course of the pulse–chase kinetics at 100 μM dTTP is shown. ( Inset ) The entire pulse–chase time course to 60 s. The pulse–chase kinetics fit best to two exponentials (solid line) followed by a linear phase. The first phase amplitude is 1.09 ± 0.05 dTTP per hexamer, fast exponential rate constant is 10.0 ± 1.1 s −1 , second phase amplitude is 0.61 ± 0.07 dTTP per hexamer, second exponential rate constant is 0.23 ± 0.07 s −1 , and steady-state dTTPase turnover is 0.06 ± 0.002 s −1 . The dotted line shows the poor fit to one exponential followed by a linear equation. ( B ) The [dTTP] dependence of the first phase amplitude (▴) and rate constant [EDTA chase data (•) and dTTP chase data (♦)] are shown. The linear [dTTP] dependence of the fast exponential rate constant provided a k on of 0.07 ± 0.01 mM −1 ⋅s −1 (slope) and a k off of 0.4 ± 1.1 s −1 ( y intercept).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase

    doi:

    Figure Lengend Snippet: Pre-steady-state pulse–chase kinetics of dTTP hydrolysis. 4A′ (3.3 μM hexamer) was mixed with [α- 32 P]dTTP (100–600 μM) in a rapid quench-flow instrument at 18°C, and the reactions were chased with dTTP or EDTA (see Materials and Methods ). ( A ) Representative time course of the pulse–chase kinetics at 100 μM dTTP is shown. ( Inset ) The entire pulse–chase time course to 60 s. The pulse–chase kinetics fit best to two exponentials (solid line) followed by a linear phase. The first phase amplitude is 1.09 ± 0.05 dTTP per hexamer, fast exponential rate constant is 10.0 ± 1.1 s −1 , second phase amplitude is 0.61 ± 0.07 dTTP per hexamer, second exponential rate constant is 0.23 ± 0.07 s −1 , and steady-state dTTPase turnover is 0.06 ± 0.002 s −1 . The dotted line shows the poor fit to one exponential followed by a linear equation. ( B ) The [dTTP] dependence of the first phase amplitude (▴) and rate constant [EDTA chase data (•) and dTTP chase data (♦)] are shown. The linear [dTTP] dependence of the fast exponential rate constant provided a k on of 0.07 ± 0.01 mM −1 ⋅s −1 (slope) and a k off of 0.4 ± 1.1 s −1 ( y intercept).

    Article Snippet: [α-32 P]dTTP was purchased from Amersham, and its purity was checked and corrected in all the experiments.

    Techniques: Pulse Chase, Flow Cytometry

    Number of catalytically competent dTTPs bound to 4A′ during steady-state dTTPase turnover. In a three-syringe experimental set-up ( Materials and Methods ), 4A′ (2 μM hexamer) was mixed with [α- 32 P]dTTP (150 μM) for 60 s. Nonradiolabeled dTTP (7 mM) was added at 60 s, and the reactions were chased for 10–60 s prior to acid-quenching. As shown (▴), only one additional dTTP per hexamer was hydrolyzed to dTDP during the chase period. When reactions were not chased, the steady state hydrolysis of [α- 32 P]dTTP (150 μM) continues at 0.14 ± 0.006 s −1 (•).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase

    doi:

    Figure Lengend Snippet: Number of catalytically competent dTTPs bound to 4A′ during steady-state dTTPase turnover. In a three-syringe experimental set-up ( Materials and Methods ), 4A′ (2 μM hexamer) was mixed with [α- 32 P]dTTP (150 μM) for 60 s. Nonradiolabeled dTTP (7 mM) was added at 60 s, and the reactions were chased for 10–60 s prior to acid-quenching. As shown (▴), only one additional dTTP per hexamer was hydrolyzed to dTDP during the chase period. When reactions were not chased, the steady state hydrolysis of [α- 32 P]dTTP (150 μM) continues at 0.14 ± 0.006 s −1 (•).

    Article Snippet: [α-32 P]dTTP was purchased from Amersham, and its purity was checked and corrected in all the experiments.

    Techniques:

    Pre-steady-state acid-quenched kinetics of dTTP hydrolysis. 4A′ (3.3 μM hexamer) was mixed with [α- 32 P]dTTP (50–600 μM) in a rapid quench-flow instrument at 18°C, and reactions were quenched with 1 M HCl. ( A ) Representative dTTPase time course at 100 μM dTTP (•) is shown. The data were fit to a single exponential followed by a linear phase with a burst amplitude of 0.80 ± 0.04 dTTP per hexamer, a burst rate constant of 0.34 ± 0.04 s −1 , and a dTTPase turnover rate of 0.043 ± 0.0007 s −1 . ( B ) The [dTTP] dependence of the burst amplitude (▴) and the burst rate constant (•) is shown. The [dTTP] dependence of the burst rate constant fit to a hyberbola with a maximum dTTP hydrolysis rate constant of 0.66 ± 0.083 s −1 and K ½ of 167 ± 57 μM.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: The dTTPase mechanism of T7 DNA helicase resembles the binding change mechanism of the F1-ATPase

    doi:

    Figure Lengend Snippet: Pre-steady-state acid-quenched kinetics of dTTP hydrolysis. 4A′ (3.3 μM hexamer) was mixed with [α- 32 P]dTTP (50–600 μM) in a rapid quench-flow instrument at 18°C, and reactions were quenched with 1 M HCl. ( A ) Representative dTTPase time course at 100 μM dTTP (•) is shown. The data were fit to a single exponential followed by a linear phase with a burst amplitude of 0.80 ± 0.04 dTTP per hexamer, a burst rate constant of 0.34 ± 0.04 s −1 , and a dTTPase turnover rate of 0.043 ± 0.0007 s −1 . ( B ) The [dTTP] dependence of the burst amplitude (▴) and the burst rate constant (•) is shown. The [dTTP] dependence of the burst rate constant fit to a hyberbola with a maximum dTTP hydrolysis rate constant of 0.66 ± 0.083 s −1 and K ½ of 167 ± 57 μM.

    Article Snippet: [α-32 P]dTTP was purchased from Amersham, and its purity was checked and corrected in all the experiments.

    Techniques: Flow Cytometry