ZtaC189S binding to meZREs in Rp is compromised <i>in vivo</i>.

<p>A. Schematic representation of the <i>BRLF1</i> gene. The black arrow indicates the primary transcript. The location of primer sets used to detect sub-regions of Rp and upstream and downstream regions are indicated relative to the transcription start site. B. HisZta and HisZtaC189S were introduced into 293-BZLF1-KO cells and chromatin prepared. Chromatin affinity purification was undertaken and binding to Rp detected with the indicated primer sets by real-time PCR. The signal was set relative to the “empty vector”, pcDNA3 (striped box), and the signal for Zta (filled box) and ZtaC189S (open box) are shown together with the standard error from duplicate experiments. C. Expression of HisZta, HisZtaC189S and a loading control, PARP, were assessed by western blot analysis.</p

Structural model of Zta bound to meZRE3.

<p>A. Alignment of Zta recognition sequences. The numbering convention is shown for base pair positions (bold italics) and individual nucleotides (plain font). Cytosines modified by methylation are indicated by a dot. B. Model of the Zta-meZRE3 complex viewed along the pseudodyad. The methylation sensitive C189 residue (red asterisk) and bidentate hydrogen bond interactions between R190^Left and Guanine^0′ (dotted black lines) are indicated. Cytosine methyl groups are semi-transparent. C. Orthogonal view. The hydrophobic contact between Cytosine^1′ and S186^Left (broken blue line) and hydrogen bond network involving S186^Left, N182^Left and Guanine2′ (dotted black lines) are shown. D. Schematic summary of contacts. van der Waals contacts involving the CpG methyl groups and Zta residues are shown as broken lines.</p

Single amino acid in basic region of Zta blocks the ability to transactivate Rp in BL cells.

<p>A. Schematic diagram showing the relationship between Zta expression and activation of the <i>BRLF1</i> promoter. B. Expression vectors for Zta, ZtaC189S and the relevant “empty” vector (pBABE) were introduced into Raji cells and their ability to activate the endogenous <i>BRLF1</i> gene determined. 24 hours after transfection, RNA was prepared, cDNA was synthesized then amplified using quantitative PCR with specific primers for the <i>BRLF1</i> transcript and a housekeeping gene, L32. Expression of <i>BRLF1</i> mRNA, following normalization for expression of L32 is shown, relative to that seen following Zta transfection (100%). C. Expression of Zta and ZtaC89S were determined by quantitative PCR and expressed relative to expression of Zta (100%).</p

All four methylation sites on ZRE3 contribute to the binding by Zta.

<p>A. Schematic representation of the four methyl-cytosine residues in ZRE3. Methylation is indicated by an asterisk and the numbering system is shown. B, C. Competition EMSA reactions were undertaken with a labeled ZRE (ZIIIB) and non-labeled Zta protein. As indicated, increasing amounts (6×, 10×, 20×, 50×, 75× and 100× excess) of unlabelled competitor ZRE3 DNA (methylated or not) was included in the EMSA reaction. D–G. EMSA competition from replicate experiments showing the ability of the indicated excess of each singly methylated ZRE3 site to compete for the binding of Zta. Experiments were undertaken in duplicate and were used to calculate the standard deviation shown in the error bars.</p

Zta and ZtaC189S interact with non-methylated Rp ZREs equivalently.

<p>A. Schematic diagram showing the location of ZREs 1–3 in Rp. Transcription of this gene occurs in a leftwards direction with respect to the viral genome. The numbering relates to the type I EBV genome accession number NC_007605. Asterixes mark the methylated Cytosine residues. B. Zta and Zta C189s were generated in an <i>in vitro</i> translation system and fractionated on SDS-PAGE, together with a non-programmed translation reaction (IVT). C.–E. Equivalent amounts of the indicated proteins were subject to EMSA analysis with the probes indicated above. A reaction with no added protein was also included, indicated probe. F. The three ZREs associated with Rp are aligned and their areas of conservation indicated by boxes. The interactions of Zta and ZtaC189S with each site are summarized.</p

ZtaC189S binding to meZREs in Rp is compromised <i>in vitro</i>.

<p>A. EMSA analysis was undertaken for meZRE2 with the indicated proteins or an unprogrammed lysate (IVT). B. Zta, ZtaC189A and ZtaC189S were generated <i>in vitro</i> and analyzed by SDS-PAGE. C. EMSA analysis of equivalent amounts of the indicated proteins was undertaken with meZRE3 as described above.</p

Evidence for C189- and S186- me cytosine⁻² interactions.

<p>A. Contacts between CpG motif 1 and Zta residues. B. EMSA analysis of unprogramed <i>in vitro</i> translation reaction (none), or Zta protein with the indicated probes was undertaken. The protein-DNA complex is shown. C. The ability of Zta and ZtaS186A to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA. D. The ability of Zta and ZtaC189S to interact with a probe that omits the methylation of cytosine⁻² was determined by EMSA.</p

260/ 280 and 260/ 230 ratios after a time course of warm (A, C) or cold (B, D) ischaemia.

<p>Values represent means ± SEM. All values were <i>N</i> = 6 except for 3 values that were <i>N</i> = 5; 260/ 280 ratio for warm ischaemia at 30 min (snap frozen), 260/ 230 ratios for warm ischaemia at 0 min (RNAlater) and 30 min (snap frozen). ^aSignificantly different from corresponding 0 min condition, <i>p</i> < 0.05. ^bSignificantly different from corresponding 90 min condition, <i>p</i> < 0.05.</p

A representative electropherogram is shown for the highest (A) and lowest (B) average RNA Integrity Number (RIN) obtained in this study. RIN of ileum mucosa specimens collected by snap-freezing in liquid nitrogen or banked using RNA stabilization solution after a time course of warm (C) or cold ischaemia (D).

<p>Values represent means ± SEM with <i>N</i> = 6. *Significantly different from corresponding snap-frozen condition, <i>p</i> < 0.05.</p

Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.

<p>Experimental plan for the collection of human ileum mucosa specimens from hemicolectomy right surgeries to examine the effects of warm or cold ischaemia and different processing methods used before banking of the tissues at -80°C.</p