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: Systemic Analysis of Gene Expression Profiles Identifies ErbB3 as a Potential Drug Target in Pediatric Alveolar Rhabdomyosarcoma

doi: 10.1371/journal.pone.0050819

Figure Lengend Snippet: A ) Body-wide expression profile of the ERBB3 gene across the database. Each dot represents the expression of ERBB3 in one sample. Anatomical origins of each sample are marked in color bars below the gene plot. The ERBB3 gene is highly expressed in malignant connective and muscular tissue samples (green dots). B ) Box plot analysis of the ERBB3 gene expression levels across a variety of pediatric cancer samples. ERBB3 is particularly highly expressed in alveolar rhabdomyosarcomas. NOS, not otherwise specified.

Article Snippet: RD rhabdomyosarcoma cells (ATCC CLL-136) were transfected with pcDNA3.1 ErbB3 (insert includes nucleotides 277–4305 from GenBank accession NM_001982) or an empty pcDNA3.1neo(–) vector (Invitrogen) using FuGENE 6 (Roche).

Techniques: Expressing, Gene Expression

Journal: PLoS ONE

Article Title: Systemic Analysis of Gene Expression Profiles Identifies ErbB3 as a Potential Drug Target in Pediatric Alveolar Rhabdomyosarcoma

doi: 10.1371/journal.pone.0050819

Figure Lengend Snippet: ( A ) Western analysis of ErbB3 expression in RD cells transfected or not with a plasmid encoding ErbB3 or an empty vector control (lanes 1–3), or with ERBB3 -targeting or control siRNA (lanes 4–5). Membranes were reblotted with anti-actin to control loading. ( B–D ) Immunohistochemical analysis of ErbB3 expression in normal adult human stomach ( B ) and in two samples representing pediatric alveolar rhabdomyosarcoma ( C , D ). ( E , F ) MTT proliferation analysis of the effect of ERBB3 overexpression ( E ) or siRNA-mediated ERBB3 down-regulation on the growth of RD cell transfectants. Expression of ErbB3 in the transfectants is shown in ( A ). *, P <0.001 when compared to control. Scale bar in ( B–D ), 100 µm.

Article Snippet: RD rhabdomyosarcoma cells (ATCC CLL-136) were transfected with pcDNA3.1 ErbB3 (insert includes nucleotides 277–4305 from GenBank accession NM_001982) or an empty pcDNA3.1neo(–) vector (Invitrogen) using FuGENE 6 (Roche).

Techniques: Western Blot, Expressing, Transfection, Plasmid Preparation, Control, Immunohistochemical staining, Over Expression

Journal: Diabetes

Article Title: Amylase α-2A Autoantibodies

doi: 10.2337/db08-0493

Figure Lengend Snippet: Cloning of human amylase α-2A cDNAs from λTriplEx2 human pancreas cDNA library. Seven clones of human amylase α-2A cDNAs. Their lengths and 5′-ends are shown (A in ATG is designated as 1). The top bar indicates human amylase α-2A cDNA as reported by Wise et al. , and the common regions shared by all seven clones, from codons 299 to 512, are shown in the bottom bar.

Article Snippet: The λTriplEx2 human pancreas large insert cDNA library (HL5517u) and Escherichia coli XL-1 competent cells were obtained from BD Biosciences Clontech (Palo Alto, CA).

Techniques: Clone Assay, cDNA Library Assay

Journal: Diabetes

Article Title: Amylase α-2A Autoantibodies

doi: 10.2337/db08-0493

Figure Lengend Snippet: Prevalence of autoantibody against human AMY-2A in patients with various pancreatic diseases. A : Prevalence of autoantibody against human AMY-2A in patients with AIP ( n = 15), chronic alcoholic pancreatitis (CP, n = 25), pancreatic tumor (PT, n = 25), control subjects from healthy volunteers (C, n = 100), and Hashimoto's thyroiditis (H, n = 47) was examined by ELISA, as described in research design and methods . The data are the mean of triplicate values. The dotted line shows a cutoff value. Fisher's exact test was carried out between AIP and control groups. * P < 0.001. B : Time course of anti-AMY antibody and IgG4 of AIP patients. AIP patient (A.O.), whose IgG was used to screen λTriplEx2 human pancreas cDNA library, was treated with prednisolone ( arrow ). Before and after the treatment, anti-AMY antibody (•-•) and IgG4 (○-○) were measured. In the other AIP patient (T.M.), titer of the anti-AMY antibody (▴-▴) was also measured before and after prednisolone treatment ( arrow ).

Article Snippet: The λTriplEx2 human pancreas large insert cDNA library (HL5517u) and Escherichia coli XL-1 competent cells were obtained from BD Biosciences Clontech (Palo Alto, CA).

Techniques: Enzyme-linked Immunosorbent Assay, cDNA Library Assay

Journal: The Journal of Experimental Medicine

Article Title: Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

doi: 10.1084/jem.20092238

Figure Lengend Snippet: Mutation spectrum of modified AIDs assayed in vitro on a gapped duplex lacZ target. (A) Depiction of the M13mp19 lacZ gapped duplex substrate DNA. (B) 5′-flanking nucleotide preferences of the C mutations produced by the variant AID deaminases. The spectra shown for the AID/3G chimera is from an AID 1 */3G derivative in which the asterisk denotes that the protein has been truncated at amino acid position 190 of AID (removing the nuclear export sequence) and is shown to allow comparison with the same AID variant analyzed in transfected DT40 B cells (see ). The C-terminal truncations do not detectably alter the patterns of in vitro mutational targeting. (C) Distribution of mutations over a 310-nt stretch of the single-stranded lacZ target. The numbers of independent mutations at each nucleotide position are expressed as a percentage of the total mutation database (as analyzed over the entire 475-nt single-stranded target). Nucleotide position 1 is defined as the start of the lac promoter. Mutations at C residues flanked by a 5′-purine (Pu-C) are shown in red, those flanked by a 5′-pyrimidine (Py-C) in blue. (D) Identity of the three most frequently targeted residues by each deaminase, with targeting expressed as the percentage of clones analyzed in which the relevant cytosine (underlined) was mutated.

Article Snippet: For the T7 transcription-coupled assays, semipurified GST-AID (200 ng) was incubated with 1 μg DNA substrate (either M13mp19, in which a 27-nt fragment containing the T7 promoter had been inserted upstream of lacZα [ Fig. S5 B ], or plasmid pCR-Blunt II-Topo (Invitrogen), in which a GFP gene and either 49- or 35-nt fragments [positions 115–164 or 228–263] of chicken IgVλ were placed between the lac and T7 promoters [Fig. S5C]) at 37°C in 10 μl reaction buffer (40 mM-Tris, 6 mM-MgCl 2 , 10 mM-dithiothreitol, and 2 mM-spermidine, pH 7.9) containing RNase A (0.5 μg μl −1 ), 0.6 mM rNTP mix and 2 U T7 RNA polymerase (NEB).

Techniques: Mutagenesis, Modification, In Vitro, Produced, Variant Assay, Sequencing, Comparison, Transfection, Clone Assay

Journal: The Journal of Experimental Medicine

Article Title: Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

doi: 10.1084/jem.20092238

Figure Lengend Snippet: Different IgVλ hotspots dominate the mutation spectra in B cells and in the gapped duplex assay. (A) The M13mp19-IgVλ- lacZ gapped duplex substrate DNA is depicted with more detailed information provided in Fig. S5 A . Nucleotide position 1 is equivalent to the first nucleotide of the in vivo IgVλ analyzed in . (B) The graphs compare the distribution of IgVλ mutations obtained with AID 1 or AID 1 */3G in the gapped duplex assay (shown below the line) with the distribution of IgVλ top strand C mutations obtained with the same deaminases in DT40 B cells (shown above the line). The distributions of IgVλ top strand C mutations in DT40 cells for AID 1 */3G and AID 1 derive from the same mutation databases used in and Fig. S2 , respectively, although those figures portray all C mutations (i.e., whichever DNA strand they have occurred upon). The positions of some individual hotspots are indicated in italics. Red, 5′-purine flank (Pu-C); blue, 5′-pyrimidine flank (Py-C). (C) The location and local context of the most frequently mutated C residues along the IgVλ top strand in the gapped duplex and DT40 B cell mutation assays are compared. Mutation load at each nucleotide position is represented as a percentage of the total mutations in each dataset. (D) The percentages of total C mutations in the gapped duplex assays and DT40 B cells (top strand) at C residues with each of the four possible 5′-flanking bases are compared for AID 1 and AID 1 */3G.

Article Snippet: For the T7 transcription-coupled assays, semipurified GST-AID (200 ng) was incubated with 1 μg DNA substrate (either M13mp19, in which a 27-nt fragment containing the T7 promoter had been inserted upstream of lacZα [ Fig. S5 B ], or plasmid pCR-Blunt II-Topo (Invitrogen), in which a GFP gene and either 49- or 35-nt fragments [positions 115–164 or 228–263] of chicken IgVλ were placed between the lac and T7 promoters [Fig. S5C]) at 37°C in 10 μl reaction buffer (40 mM-Tris, 6 mM-MgCl 2 , 10 mM-dithiothreitol, and 2 mM-spermidine, pH 7.9) containing RNase A (0.5 μg μl −1 ), 0.6 mM rNTP mix and 2 U T7 RNA polymerase (NEB).

Techniques: Mutagenesis, In Vivo

Journal: The Journal of Experimental Medicine

Article Title: Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

doi: 10.1084/jem.20092238

Figure Lengend Snippet: Mutation spectra from in vitro transcription-coupled mutation assays. (A) The T7- lacZ transcription-coupled substrate in which a T7 promoter is inserted upstream of the lac promoter in M13mp19 ( Fig. S5 B ). Nucleotide position 1 is defined as the start of the lac promoter. The adjacent table shows the 5′-flanking nucleotide preferences of the C mutations produced by GST-AID 1 or GST-AID 1 */3G in lac target DNA during in vitro transcription by phage T7 RNA polymerase. (B) Mutation distribution along the T7 transcription-coupled lacZ target DNA with mutation at each nucleotide position expressed as the percentage of total mutations. Two heavily mutated positions are off-scale: their percentage mutations are indicated. Because mutation analysis was restricted to Lac − plaques, this selection results in a skewing in favor of lac -inactivating mutations, although most mutated templates carried 1–3 mutations in the target region. Positions at which C deamination yields a stop codon are indicated by an asterisk. (C) The location and local context of the five most frequently mutated C residues along the transcribed lac target. All the mutated residues shown are located on the top (nontranscribed) strand. (D) Transcription-linked mutation of a T7-linked GFP-Vλ target. The substrate DNA is a derivative of plasmid pCR-Blunt II-TOPO in which a region of IgVλ (residues 115–164 or 228–263) has been inserted between the T7 promoter and a GFP reporter with T7-catalyzed transcription of the IgVλ fragment being in the same sense as IgVλ transcription in B cells ( Fig. S5 C ). For each construct (pCR-GFP-Vλ 115−164 -T7 and pCR-GFP-Vλ 228−263 -T7), the tables compare the percentage of total mutations within the target Vλ region that occur at selected individual positions with the equivalent percentage mutation values for the same positions over the same target regions in the M13 or B cell mutation assays. (E) Comparison of the mutation distributions obtained in the T7-coupled mutation assay over IgVλ residues 115–164 (for AID 1 ) or 228–263 (for AID 1 */3G) to the distributions obtained in DT40 B cells (left) or in the gapped duplex assay (right). In these comparisons, analysis is restricted to nontranscribed strand mutations. Positions of individual hotspots are indicated in italics. Red, 5′-purine flank; blue, 5′-pyrimidine flank.

Article Snippet: For the T7 transcription-coupled assays, semipurified GST-AID (200 ng) was incubated with 1 μg DNA substrate (either M13mp19, in which a 27-nt fragment containing the T7 promoter had been inserted upstream of lacZα [ Fig. S5 B ], or plasmid pCR-Blunt II-Topo (Invitrogen), in which a GFP gene and either 49- or 35-nt fragments [positions 115–164 or 228–263] of chicken IgVλ were placed between the lac and T7 promoters [Fig. S5C]) at 37°C in 10 μl reaction buffer (40 mM-Tris, 6 mM-MgCl 2 , 10 mM-dithiothreitol, and 2 mM-spermidine, pH 7.9) containing RNase A (0.5 μg μl −1 ), 0.6 mM rNTP mix and 2 U T7 RNA polymerase (NEB).

Techniques: Mutagenesis, In Vitro, Produced, Selection, Plasmid Preparation, Construct, Comparison

Journal: The Journal of Experimental Medicine

Article Title: Altering the spectrum of immunoglobulin V gene somatic hypermutation by modifying the active site of AID

doi: 10.1084/jem.20092238

Figure Lengend Snippet: AID N51A mutants retain DNA deaminase activity. (A) Bacterial mutator activity of AID variants monitored by frequency of Rif r colonies in overnight cultures with mean frequencies (x10 −8 ) from 12 independent cultures indicated. The N51A mutant of AID 1 is abbreviated N51A 1 . (B) Switching to IgG 1 in LPS+IL-4 cultures of AID-deficient B cells that have been transduced with retroviruses encoding the various AIDs as indicated together with a linked IRES-GFP. Representative flow cytometry plots are presented along with histograms showing the results of four experiments (mean and SD, with 2–3 mice per construct per experiment). AID abundance in the B cell extracts 3 d after retroviral transduction was monitored by Western blotting; the blot was reprobed with anti-GFP antibodies as a control. (C) The frequency of surface IgM-loss variants in transfectants of AID −/− ψV −/− sIgM + DT40 that coexpress the indicated AID mutant and GFP is presented both with a histogram showing the average percentage sIgM-loss variants in 12 independent clonal transfectants as well as by representative flow cytometry plots. Representative Western blots show AID abundance in the DT40 cell extracts with tubulin as loading control.

Article Snippet: For the T7 transcription-coupled assays, semipurified GST-AID (200 ng) was incubated with 1 μg DNA substrate (either M13mp19, in which a 27-nt fragment containing the T7 promoter had been inserted upstream of lacZα [ Fig. S5 B ], or plasmid pCR-Blunt II-Topo (Invitrogen), in which a GFP gene and either 49- or 35-nt fragments [positions 115–164 or 228–263] of chicken IgVλ were placed between the lac and T7 promoters [Fig. S5C]) at 37°C in 10 μl reaction buffer (40 mM-Tris, 6 mM-MgCl 2 , 10 mM-dithiothreitol, and 2 mM-spermidine, pH 7.9) containing RNase A (0.5 μg μl −1 ), 0.6 mM rNTP mix and 2 U T7 RNA polymerase (NEB).

Techniques: Activity Assay, Mutagenesis, Transduction, Flow Cytometry, Construct, Retroviral, Western Blot, Control