Southern blots and J-immunoblots of genomic DNA of and (bloodstream form) digested with a variety of frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> The DNA was treated as in . The blot was incubated with the J-antisera (α-J) followed by hybridization with a telomeric probe (telo). () Southern blot of genomic DNA of . () Southern blot of genomic DNA of (bloodstream form). The bands migrating at the top of the lanes 1, 2 and 4 that strongly react with the J-antisera are probably due to the 50 bp repeats as these are digested by RsaI (which was used in the lanes 3, 5, 6 and 7). Lanes 1. AluI, HpaII, BsrGI, HaeII; 2. AluI, HpaII, Sau3AI, TaqI; 3. AluI, HpaII, CfoI, RsaI; 4. BsrGI, HaeII, Sau3AI, Taq I; 5. BsrGI, HaeII, CfoI, RsaI; 6. Sau3AI, RsaI, CfoI, TaqI; 7. AluI, HpaII, BsrGI, HaeII, Sau3AI, RsaI, CfoI, TaqI. ND stands for not digested

Southern blot and J-immunoblot of genomic DNA of Friedlin, and digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> () Genomic DNA of Friedlin was digested with the enzymes AluI, BsrGI, BstUI, CfoI, HaeII, HpaII, Sau3AI and TaqI and size-fractionated by electrophoresis in an agarose gel and blotted on a nylon membrane. The blot was incubated with the J-antiserum (α-J) and after analysis, hybridized with telomeric (telo) and LST-RA radioactively labeled DNA oligonucleotide probes. () Southern blot and J-immunoblot of genomic DNA of , , and . DNA of was digested with AluI, AvaII, CfoI, HinfI, RsaI, SspI. DNA of and was digested with the enzymes listed in A. The combination of restriction enzymes was optimized to digest the greatest variety of DNA repetitive sequences. The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)

Southern blot and J-immunoblot of genomic DNA of various kinetoplastid parasites digested with frequently cutting restriction enzymes

<p><b>Copyright information:</b></p><p>Taken from "Telomeric localization of the modified DNA base J in the genome of the protozoan parasite "</p><p></p><p>Nucleic Acids Research 2007;35(7):2116-2124.</p><p>Published online 28 Feb 2007</p><p>PMCID:PMC1874636.</p><p>© 2007 The Author(s)</p> DNA was digested with the restriction enzymes AluI, AvaII, CfoI, HinfI, RsaI, SspI, size-fractionated and blotted as described in . The left panel shows the result after incubation with the J-antiserum (α-J). The right panel shows the result of the hybridization with the telomeric probe (telo)

Binding of the J‑Binding Protein to DNA Containing Glucosylated hmU (Base J) or 5‑hmC: Evidence for a Rapid Conformational Change upon DNA Binding

Base J (β-d-glucosyl-hydroxymethyluracil) was discovered in the nuclear DNA of some pathogenic protozoa, such as trypanosomes and <i>Leishmania</i>, where it replaces a fraction of base T. We have found a J-Binding Protein 1 (JBP1) in these organisms, which contains a unique J-DNA binding domain (DB-JBP1) and a thymidine hydroxylase domain involved in the first step of J biosynthesis. This hydroxylase is related to the mammalian TET enzymes that hydroxylate 5-methylcytosine in DNA. We have now studied the binding of JBP1 and DB-JBP1 to oligonucleotides containing J or glucosylated 5-hydroxymethylcytosine (glu-5-hmC) using an equilibrium fluorescence polarization assay. We find that JBP1 binds glu-5-hmC-DNA with an affinity about 40-fold lower than J-DNA (∼400 nM), which is still 200 times higher than the JBP1 affinity for T-DNA. The discrimination between glu-5-hmC-DNA and T-DNA by DB-JBP1 is about 2-fold less, but enough for DB-JBP1 to be useful as a tool to isolate 5-hmC-DNA. Pre-steady state kinetic data obtained in a stopped-flow device show that the initial binding of JBP1 to glucosylated DNA is very fast with a second order rate constant of 70 μM^–1 s^–1 and that JBP1 binds to J-DNA or glu-5-hmC-DNA in a two-step reaction, in contrast to DB-JBP1, which binds in a one-step reaction. As the second (slower) step in binding is concentration independent, we infer that JBP1 undergoes a conformational change upon binding to DNA. Global analysis of pre-steady state and equilibrium binding data supports such a two-step mechanism and allowed us to determine the kinetic parameters that describe it. This notion of a conformational change is supported by small-angle neutron scattering experiments, which show that the shape of JBP1 is more elongated in complex with DNA. The conformational change upon DNA binding may allow the hydroxylase domain of JBP1 to make contact with the DNA and hydroxylate T’s in spatial proximity, resulting in regional introduction of base J into the DNA

Plasma and tumor topotecan pharmacokinetics (PK).

<p>A, Plasma topotecan PK. <i>Abcg2^−/−</i> animals, carrying ABCG2-positive tumor T1 (Fig. 2C), were treated with either vehicle +0.5 mg topotecan (green line) or 10 mg Ko143+0.5 mg topotecan (red line) per kg body weight and plasma was collected at 15, 30, 60, 90 and 120 minutes following i.p. injection. Error bars indicate standard deviations of the mean of at least 3 animals per time point. B, Tumor topotecan PK. The tumors of the animals in A were harvested and homogenized to determine topotecan concentrations at the same time points. Error bars indicate standard deviations of the mean of at least 3 animals per time point. <i>P</i> values were calculated using the unpaired t-test.</p

Efficacy of topotecan, irinotecan and EZN-2208 in five ABCG2-positive <i>Brca1^−/−</i>;<i>p53^−/−</i> mammary tumors.

<p>A, K–M curves showing survival (%) until a tumor volume of about 1500 mm³ was reached after one regimen of five consecutive i.v. injections on days 0, 2, 4, 6 and 8 of saline- (dark blue line, N = 23), topotecan- (light blue line, 4 mg i.v., N = 32), irinotecan- (pink line, 40 mg i.v., N = 28) and EZN-2208-treatments (green line, 10 mg SN38 equivalents i.v., N = 38) per kg body weight. <i>P</i> values were calculated using the Log-rank test. B, Box and whiskers plots indicating per drug the tumor volume change (%) after two weeks of treatment. Lines represent the median response, while the whiskers show the maximum and minimum values; the small circle is an outlier. <i>P</i> values were calculated using the Mann Whitney test. C, Waterfall plots showing tumor volume change (%) after two weeks of treatment per individual mouse, with relative volumes normalized to the treatment start volume.</p

Topotecan response and ABCG2 immunoreactivity of eight individual <i>Brca1^−/−</i>;<i>p53^−/−</i> mammary tumors.

<p>A, Relative tumor volume (%) of matched control (blue lines) and topotecan-treated (green lines) tumors over time. Each arrow indicates one dosing regimen of i.p. injections of 0.5 mg topotecan per kg body weight on days 0–4 and 14–18. B, Semi-quantification of ABCG2 immunoreactivity. Representative micrographs of four classes of stained tumor cells (0%, 1–10%, 11–50% and more than 50% of counted cells in 10 independent 400x magnification fields are ABCG2-positive) are shown. C, Table indicating ABCG2 immunoreactivity of the spontaneous (spon), untreated control (control) and topotecan-treated samples per individual tumor. <i>P</i> values of the spon - control (0.865) and spon - topotecan (0.020) comparisons were calculated using the Wilcoxon rank-sum test.</p

Determination of J levels in the DNA of JBP1 null trypanosomes expressing the Lt JBP1 mutants

<p><b>Copyright information:</b></p><p>Taken from "The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase"</p><p></p><p>Nucleic Acids Research 2007;35(7):2107-2115.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874643.</p><p>© 2007 The Author(s)</p> () Genomic DNA of wild-type bloodstream form 427 line (WT), JBP1 null trypanosomes (JBP1 dKO) and JBP1 null trypanosomes expressing the wild-type Tb GFP-JBP1 fusion (Tb JBP1 WT), the wild-type Lt GFP-JBP1 fusion (Lt JBP1 WT) or the mutant Lt GFP-JBP1 fusions (H189A, D191A, H239A, R255A, V259A) were serially diluted in steps of two, denatured, spotted on a nitrocellulose membrane and incubated with a polyclonal J antiserum (left panel). The membrane was hybridized with a tubulin DNA probe as a loading control (right panel). () Western blot on lysates of the JBP1 null trypanosomes (JBP1 dKO) and the JBP1 null trypanosomes expressing the wild-type (WT) and the mutant (H189A, D191A, H239A, R255A, V259A) Lt GFP-JBP1 fusions using an Lt JBP1 antiserum

Determination of the J-binding activity of purified Lt JBP1 mutants by electromobility shift assay

<p><b>Copyright information:</b></p><p>Taken from "The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase"</p><p></p><p>Nucleic Acids Research 2007;35(7):2107-2115.</p><p>Published online 27 Mar 2007</p><p>PMCID:PMC1874643.</p><p>© 2007 The Author(s)</p> () Upper panel: Electromobility shift assay with the purified His-tagged wild-type (WT) and mutant (H189A, D191A, H239A, R255A, V259A) Lt JBP1 proteins. Proteins (10 fmol) were incubated with a radioactively labeled double-stranded oligonucleotide containing J (J-DNA) (10 fmol) and run on 4.5% native acrylamide gel. The gel was dried and the signal detected by autoradiography. The bands corresponding to the J-DNA/Lt JBP1 and the free J-DNA are indicated. Lower panel: Segment of a Coomassie-brilliant-blue-stained acrylamide gel with the His-tagged wild-type (WT) and mutant (H189A, D191A, H239A, R255A, V259A) Lt JBP1 proteins purified from . Only the segment of the gel with the His-tagged Lt JBP1 band is shown. () Example of a determination of the J-binding activity of mutant Lt JBP1 by electromobility shift assay. His-tagged wild-type (WT) and H189A + D191A mutant Lt JBP1 proteins were purified from and incubated with a radioactively labeled double-stranded oligonucleotide containing J (J) or lacking J (T) in the presence or absence of a Lt JBP1 antibody (α-Lt JBP1). Samples were run on a 4.5% native acrylamide gel. The gel was dried and the signal detected by autoradiography. Lanes without recombinant protein are indicated by ‘–’. The position of the free DNA, the J-DNA/Lt JBP1 complex and the J-DNA/Lt JBP1 + α-Lt JBP1 complex is indicated