Journal: Nucleic Acids Research

Article Title: Transformation of isolated mammalian mitochondria by bacterial conjugation

doi: 10.1093/nar/gni140

Figure Lengend Snippet: T7RNAP-driven transcription assay after conjugation between E.coli and T7RNAP-mitochondria. E.coli S17-1 harboring pT7hpGFP (lanes 1 and 2) and pT7hpGFP+oriT (lanes 3 and 4), respectively, were used as the donors. All mating samples were treated with 200 µg/ml of ampicillin, 200 U of DNase I and 50 µg/ml of RNase A for 2 h at 37°C after 5 h mating in an agarose block. After isolating total RNA from the mating mixture, RT–PCR was carried out using GFP -specific primers (see Materials and Methods). Expected band size of the RT–PCR GFP products was 423 bp. Lane M, 1 kb plus DNA ladder (Life Technologies). Control lanes (lanes 1 and 3) in which reverse transcriptase (RT) was omitted are indicated by minus signs.

Article Snippet: RT–PCR analysis Total RNAs from the T7RNAP-mitochondria mating mixture or from electroporated mitochondria were collected by isopropyl alcohol precipitation, and residual DNA contaminants were removed using RNase-free DNase I (Promega, Madison, WI) as directed by the manufacturer.

Techniques: Conjugation Assay, Blocking Assay, Reverse Transcription Polymerase Chain Reaction

Journal: The Journal of Experimental Medicine

Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

doi:

Figure Lengend Snippet: Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.

Article Snippet: For each experiment, 3–6 × 108 cells were harvested, lysed by addition of NP-40, centrifuged through a 1.7 M sucrose cushion, and resuspended in 5 ml; 450-μl aliquots of suspended nuclei were treated with serially diluted DNase I ( Boehringer-Mannheim , Indianapolis, IN) to give final DNase I concentrations of 0–8 μg/ml.

Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, BAC Assay, Activity Assay, Sequencing, Southern Blot, Hybridization

Journal: The Journal of Experimental Medicine

Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

doi:

Figure Lengend Snippet: Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference 13 (EMBL/GenBank/DDBJ accession numbers X96607 and X96608 ), while numbering for mouse HS4 is according to reference 12 (EMBL/DDBJ/GenBank accession number S74166 ). ( A ) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. ( B ) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. ( C ) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. ( D ) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE ( 41 , 42 ). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

Article Snippet: For each experiment, 3–6 × 108 cells were harvested, lysed by addition of NP-40, centrifuged through a 1.7 M sucrose cushion, and resuspended in 5 ml; 450-μl aliquots of suspended nuclei were treated with serially diluted DNase I ( Boehringer-Mannheim , Indianapolis, IN) to give final DNase I concentrations of 0–8 μg/ml.

Techniques: Sequencing, Binding Assay, Functional Assay, Mouse Assay

Journal: The Journal of Experimental Medicine

Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

doi:

Figure Lengend Snippet: Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E ( 11 ) and 3′αE ( 9 ) are shown as vertical ovals, along with the DNase I hypersensitivity site designations ( 12 ). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome ( arrows ) that flanks HS12, according to the sequence analysis of Chauveau and Cogné ( 13 ). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units ( brackets ) and the possibility of two regions homologous to the murine LCR.

Article Snippet: For each experiment, 3–6 × 108 cells were harvested, lysed by addition of NP-40, centrifuged through a 1.7 M sucrose cushion, and resuspended in 5 ml; 450-μl aliquots of suspended nuclei were treated with serially diluted DNase I ( Boehringer-Mannheim , Indianapolis, IN) to give final DNase I concentrations of 0–8 μg/ml.

Techniques: Sequencing

Journal: The Journal of Experimental Medicine

Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

doi:

Figure Lengend Snippet: Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

Article Snippet: For each experiment, 3–6 × 108 cells were harvested, lysed by addition of NP-40, centrifuged through a 1.7 M sucrose cushion, and resuspended in 5 ml; 450-μl aliquots of suspended nuclei were treated with serially diluted DNase I ( Boehringer-Mannheim , Indianapolis, IN) to give final DNase I concentrations of 0–8 μg/ml.

Techniques: Isolation, Generated, Labeling, Sequencing, Purification

Journal: The Journal of Experimental Medicine

Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

doi:

Figure Lengend Snippet: Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. ( A ) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. ( B ) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

Article Snippet: For each experiment, 3–6 × 108 cells were harvested, lysed by addition of NP-40, centrifuged through a 1.7 M sucrose cushion, and resuspended in 5 ml; 450-μl aliquots of suspended nuclei were treated with serially diluted DNase I ( Boehringer-Mannheim , Indianapolis, IN) to give final DNase I concentrations of 0–8 μg/ml.

Techniques: Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Amplification