Journal: Molecular Cytogenetics

Article Title: Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

doi: 10.1186/1755-8166-1-28

Figure Lengend Snippet: P1 DNA molecules of different size . A) PFGE-analysis of the P1 clone RMC11P010. After incubation with Not1, pulse field gel electrophoresis showed circular DNA ( c ) and a band containing relaxed molecules ( r ) corresponding to a size of approximately 55 kb. The size marker lane ('szm', left) contains 500 ng of low range PFGE marker. B) Gel electrophoretic analysis of P1 clones containing part or all of the human NF-κB2 gene. The DNA from individual clones were digested with Not1 and separated by PFGE. All clones produced two or more restriction fragments indicating the presence of at least one Not1 site within the P1 insert. The size marker lane (szm) contains 500 ng of low range PFGE marker. C) Staining of gel-purified 'circular' P1 DNA molecules with YOYO-1 (Molecular Probes) reveals circular and randomly broken DNA molecules on the slide. Most of the DNA molecules shown here in the liquid phase prior to molecular combing are bound to the substrate with one end or via nicks in circular molecule.

Article Snippet: For these purposes, large insert DNA clones are obtained from the Dupont P1 library by screening [ , ].

Techniques: Incubation, Nucleic Acid Electrophoresis, Marker, Clone Assay, Produced, Staining, Purification

Journal: Molecular Cytogenetics

Article Title: Validation of DNA probes for molecular cytogenetics by mapping onto immobilized circular DNA

doi: 10.1186/1755-8166-1-28

Figure Lengend Snippet: High resolution physical mapping by QDFM to linear and circular DNA molecules . A)-D) Isolated molecules of RMC11P010 were hybridized with plasmid probe pADJ762 (red) and a probe DNA specific for the P1 vector pSac BII (green). The entire P1 DNA molecules were counterstained by hybridization of random-primed RMC11P010 DNA (blue). A PCR generated ~1400 bp probe that maps in the pAd10SacBII vector near the T7 promoter site was included in the hybridization mix to show the vector orientation. A linear DNA molecule is presented in A). E) Schematic representation of the hybridization result shown in B-D). The approximate location of the Not1 site in a 'normal' P1 is indicated. F) Analysis of the overlap between two linked P1 clones ('3012', '3015') that contain one or more Not1 sites in the insert. The labeling and detection scheme is described in Table 3. G)-J) The red, green and blue images of hybridization signals along a single, closed circular DNA molecule ('3012') as well as the RGB image (J) generated by superposition of images G)-I). K) Schematic diagram showing the overlap between clones '3012' and '3015' and the location of P1-vector-specific probes.

Article Snippet: For these purposes, large insert DNA clones are obtained from the Dupont P1 library by screening [ , ].

Techniques: Isolation, Plasmid Preparation, Hybridization, Random Primed, Generated, Clone Assay, Labeling

Journal: PLoS ONE

Article Title: The “speed gene” effect of myostatin arises in Thoroughbred horses due to a promoter proximal SINE insertion

doi: 10.1371/journal.pone.0205664

Figure Lengend Snippet: RNA was isolated from equine gluteus medius skeletal muscle tissue and 5'RACE was used to recover sequence data for the 5'-end of the myostatin mRNA transcript. DNA was sequenced using M13 primers by MWG eurofins. (A) Displays snapshots of the raw sequencing data obtained with transcription start sites (TSS) indicated. The SMARTer 5'RACE primer sequence is shown prior to the TSS along with 8 additional bases (*) which were added to first-strand cDNA; during reverse transcription, when the SMARTScribe reverse transcriptase reaches the 5’ end of the RNA, its terminal transferase activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA. The same set of 8 bases ( ACATGGGG ) is observed in each clone. (B) Depiction of a non-SINE insertion MSTN gene (top) and a SINE insertion MSTN gene (bottom), with experimentally determined TSS indicated. Red lines indicate the position of the SINE insertion sequence. Numbering of nucleotide bases established from the human TSS (and the in silico predicted equine transcription start site) as +1. Position of the ATG (predicted translation start site) and predicted TATA box are also marked on diagram. (C) 5'RACE PCR products were electrophoresed on a 1.5% agarose gel, 1: TT/NN non-SINE insertion sample; 2: CC/II SINE insertion sample; M1: wide range MW markers (Sigma).

Article Snippet: A sample (4 μg) of plasmid DNA (GLuc + MSTN plasmid, with SINE insertion 227 bp polymorphism and C-allele at SNP g.66493737C>T) was sent to GenScript (USA) for the SINE insertion 227 bp polymorphism sequence to be removed.

Techniques: Isolation, Sequencing, Reverse Transcription, Activity Assay, In Silico, Agarose Gel Electrophoresis

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: (A) T-DNA insertion and RNAi locations within the JMJ703 gene. (B) Comparison of the main panicle internodes between wild type and mutant plants. Lengths of the uppermost internodes were measured. (C) Comparison of stem epidermal cell lengths between wild type and mutant plants. No significant difference of cell lengths between wild type and mutant plants is detected by t-tests ( p = 0.02). Bar = 50 µm. (D) Relative expression, H3K4me2 and H3K4me3 levels of cytokinin oxidase ( CKX ) genes in wild type and mutant plants. Expression levels in wild type are set as 1.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Comparison, Mutagenesis, Expressing

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: (A) Schematic presentation of JMJ703 protein structure. Fragment spanning amino acids 139–498 was obtained for crystal growth and structural determination. The region between amino acids 113–700 was used for enzymatic assays. (B) In vivo histone H3K4 demethylase activity of JMJ703. Constructs containing the JMJ703 fragment tagged by FLAG-HA (FA-J3NCZ) were transfected into tobacco leaf cells. Nuclei expressing FA-J3NCZ (stained with anti-HA) were examined for H3K4 methylation by anti-H3K4me1/2/3 and anti-H3K27me3 (indicated by arrows). At least 30 nuclei expressing JMJ703 per transfection were observed and imaged. Bar = 25 µm. (C) In vitro demethylase activity of JMJ703. Bulk histones were incubated with three quantities of FA-J3NCZ and analyzed by Western blots using antibodies of H3K4me1/2/3. The same blots were analyzed by anti-H3 to control loadings. FA-J3NCZ levels were revealed by anti-HA. (D) Characterization of c-JMJ703 binding to H3K4me0/1/2/3, H3K9me0 and H3K36me0 peptides using surface plasmon resonance. Curves for different concentration of peptide are differentially colored and labeled.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: In Vivo, Activity Assay, Construct, Transfection, Expressing, Staining, Methylation, In Vitro, Incubation, Western Blot, Control, Binding Assay, SPR Assay, Concentration Assay, Labeling

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: (A) Cartoon representation of the overall structure of c-JMJ703. Separate domains are colored differently and labeled. (B–E) Structural comparison of JmjN domain (B), long-beta sheet domain (C), mixed domain (D), and JmjC domain (E) among c-JMJ703 (deep salmon), c-JMJD2A (tv green), and c-Rph1 (slate).

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Labeling, Comparison

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: (A) Sequence alignment of the JmjC domain of JMJ703, plant JMJ proteins (in black) and representative human/yeast JMJ proteins (in blue). Residues conserved in and specific to H3K4 demethylases are boxed in red and labeled above the alignment. (B) Structure of JmjC domain of JMJ703 showing the locations of H3K4 demethylase-specific residues. H3K4me3 peptide and H3K4 demethylase-specific residues are shown in yellow sticks and purple sticks respectively. A proposed extension path of H3K4me3 peptide toward its C-terminal is shown in dashed red line.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Sequencing, Labeling

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: Summary of H3K4 demethylation assays in tobacco cells of substitution mutants of key residues of JMJ703 catalytic domain.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Methylation

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: The interaction network of NOG (colored green), Fe(II) (colored brown), and c-JMJ703 residues (colored purple) involved in the interactions are shown. The H3K4me3 peptide is colored yellow. The secondary structure surrounding the active site is shown as a purple cartoon.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques:

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: (A) Binding of H3K4me3 peptide by c-JMJ703. (B) Binding of H3K9me3 by c-JMJD2A. (C) Binding of H3K36me3 by c-JMJD2A. c-JMJ703 and c-JMJD2A are shown as electrostatic surface and the substrate peptides are shown as yellow sticks and the residues are labeled in red. NOG and α-KG are shown as green sticks and labeled.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Binding Assay, Labeling

Journal: PLoS Genetics

Article Title: Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice

doi: 10.1371/journal.pgen.1003239

Figure Lengend Snippet: Residues interacting with (A) α-KG and (B) NOG in c-JMJ703 structures are represented as blue and green sticks, respectively. Dashed lines denote hydrogen bonds, including the solvent-mediated interactions formed by residues of c-JMJ703 and solvent molecules and bound α-KG/NOG. Fe(II) and solvent molecules are shown as deep red and red spheres, respectively. (C) Comparison of residues involved in binding to Fe(II) and α-KG/NOG in c-JMJ703, c-JMJD2A, and c-Rph1. Residues of c-JMJ703-α-KG, c-JMJ703-NOG-H3K4me3, c-JMJD2A, and c-Rph1 are colored in blue, green, light pink, and purple, respectively. Numbers for each residue in c-JMJ703, c-JMJD2A, and c-Rph1 are shown with black, light pink, and purple, respectively. Solvent molecules are not included in this panel. N404 and Y321, which adopt significant conformation shifts, are framed out by red squares and a circle.

Article Snippet: A T-DNA insertion line of JMJ703 (3A-00550) was obtained from the Postech rice mutant database ( http://www.postech.ac.kr/life/pfg/risd/ ).

Techniques: Solvent, Comparison, Binding Assay, Residue