Journal: Nucleic acids research

Article Title: Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice.

doi: 10.1093/nar/gkad516

Figure Lengend Snippet: Figure 4. Search for binding motifs for CD ( = A) factor in the fragment CD. (A) Sequences of fragment CD and its mutant deri vati v es. The (C)RCTG motifs in each fragment are inverted in black-and-white, in which the mutated bases are re v erted. The binding affinity of each sequence to the CD ( = A) factor, which was estimated by visual-examination of the EMSA results by three individuals, is indicated by ++ (strong), + (moderate) and – (weak) to the right of each sequence. (B, C) EMSA with nuclear extract from P19 cells and probe CD. Forty- and 200-fold molar excess of oligos were used as competitors. (D) Sequences of fragment CD and its scrambled mutants. Sequence composition ( i.e. the number of each nucleotides) is maintained e v en after shuffling the sequence. (E) EMSA with P19 cell nuclear extract and probe CD. Twenty- and eighty-fold molar excess of oligos were used as competitors. (F) Putati v e binding site for nuclear receptor-type transcription factors ( i.e. direct repeat sequence) is indicated by a pair of arrows. (G–I) EMSA with nuclear extracts from P19 or testis cells and probe F. One hundred-fold molar excess of oligos were used as competitors. Nucleotide sequences of ‘Epsi’ and ‘RARE’ oligos each carrying binding motifs for TR2 / TR4 are as follows: Epsi, 5’-CTG AGGACA C AGGTCA GCCT TGACCA A TGACTT TTA-3’ and RARE, 5’-TT GCT GT GACCT CT GCCCT T CTAGCCT CT-3’ (only the sequence of one strand is shown and binding motifs ar e underlined). In Figur e 4 H, the super-shift bands observed in the presence of antibodies against either TR2 or TR4 are indicated by an open triangle. In Figure 4 I, nuclear extracts wer e pr epar ed from P19 cells transfected with siRNA duplex es against TR2, TR4, or both. The decrease in protein le v els in these cells was v erified by western blotting. NC; non-targeting control siRNA.

Article Snippet: For super-shift assays, 1 g of above mentioned antibodies specific for either TR2 or TR4 (Santa Cruz Biotechnology) was included in the reaction mixture.

Techniques: Binding Assay, Mutagenesis, Sequencing, Transfection, Western Blot, Control

Journal: Nucleic acids research

Article Title: Five nucleotides found in RCTG motifs are essential for post-fertilization methylation imprinting of the H19 ICR in YAC transgenic mice.

doi: 10.1093/nar/gkad516

Figure Lengend Snippet: Figure 5. Generation of YAC TgM. (A) Structure of the mouse Igf2 / H19 locus. The H19 ICR is contained within a 2.9-kb Sac I (Sa)- Bam HI (B) fragment, in which CTCF binding sites and the ‘b’ region ( 33 ) are indicated by dots (1-4) and a filled box, respecti v ely. (B) The 118 bp sequence in the H19 ICR. (C)RCTG motifs are highlighted with their mutated sequences in the 5 mutant underneath. The sequences included in the LCb80 are underlined and TR2 / 4 binding site is shown by tandem arrows. (C) Structure of the 150-kb human -globin locus YAC. The LCR and -like globin genes are denoted as black and open box es, r especti v ely. Each of the H19 ICR 5 (open rectangle; 2.9 kb) and LCb80 (gray; 2.4 kb) fragment was floxed by a pair of lo xP sites [lo xP5171 (solid triangles) and lo xP2272 (open)], tandemly arranged and introduced 3’ to the LCR for employing co-placement strategy. The expected SfiI restriction enzyme fragments (thick lines) generated from the YAC transgene and probes (filled rectangles) are shown beneath the map. (D) Long range structural analysis of the 5-LCb80 YAC transgene. DNA from thymus cells was digested with SfiI in agarose plugs and separated by pulsed-field gel electrophoresis, and Southern blots were hybridized separately to probes. (E,F) In vivo Cr e-loxP r ecombination to deri v e 5 or LCb80 TgM. Recombination between two loxP5171 sites (solid) in the parental 5-LCb80 transgene, for example, would generate LCb80 allele, during which one of the loxP2272 sites (open) is concomitantly removed to pre v ent further recombination. Tail DNA from parental and daughter YAC-TgM sublines was digested with Kpn I and analyzed by Southern blotting using the probe shown in ( E ).

Article Snippet: For super-shift assays, 1 g of above mentioned antibodies specific for either TR2 or TR4 (Santa Cruz Biotechnology) was included in the reaction mixture.

Techniques: Binding Assay, Sequencing, Mutagenesis, Generated, Pulsed-Field Gel, Electrophoresis, In Vivo, Southern Blot