Journal: Gene Expression

Article Title: Homology between α2(V) and αl(lll) collagen promoters and evidence for negatively acting elements in the α2(V) first intron and 5′ flanking sequences

doi:

Figure Lengend Snippet: Promoter activities of sequences from the 5′ end of the human α2(V) gene. Arrows, beneath a partial restriction map of the 5′ portion of the α2(V) gene, denote the extent and orientation of α2(V) DNA fragments fused to the CAT gene for expression analysis. Bold arrows represent sequences, from −151 to +82, found to have strongest promoter activity. CAT activity of each plasmid is expressed relative to the conversion level achieved by the vector pCAT-control (100.0). pCAT-control (Promega, technical bulletin #81) is similar in configuration to pCAT Enhancer but contains a 200 bp Sphl-HindII fragment (128–5171) containing the SV40 early promoter, upstream of the CAT gene. (+) and (−) designate the presence or absence, respectively, of the SV40 enhancer downstream of the CAT gene in various plasmids. Data represent means ± standard deviation (in parenthesis) of from four to six independent transfection experiments. NA: not ascertained.

Article Snippet: CAT activity of each plasmid is expressed relative to the conversion level achieved by the vector pCAT-control (100.0). pCAT-control (Promega, technical bulletin #81) is similar in configuration to pCAT Enhancer but contains a 200 bp Sphl-HindII fragment (128–5171) containing the SV40 early promoter, upstream of the CAT gene. (+) and (−) designate the presence or absence, respectively, of the SV40 enhancer downstream of the CAT gene in various plasmids.

Techniques: Expressing, Activity Assay, Plasmid Preparation, Standard Deviation, Transfection

Journal: Gene therapy

Article Title: Cell-specific nuclear import of plasmid DNA

doi: 10.1038/sj.gt.3300924

Figure Lengend Snippet: Organization of cis-acting elements within the SMGA promoter. Cartoon depicting the smooth muscle specific transcription factor binding sites identified by sequence homology and experimentally within the SMGA promoter14–16,18 and constructs used in these experiments.

Article Snippet: A similar series of transcription reporter plasmids (pGL-2294 and pGL-404) were created in pGL3-basic (Promega) that express luciferase. pCAT-control contains the SV40 early promoter (nts 5171–130) and enhancer (nts 130–270) and was purchased from Promega.

Techniques: Binding Assay, Sequencing, Construct

Journal: Gene therapy

Article Title: Cell-specific nuclear import of plasmid DNA

doi: 10.1038/sj.gt.3300924

Figure Lengend Snippet: Model for plasmid DNA nuclear import. (a) Sequence-specific nuclear import of SV40 promoter/enhancer containing plasmid DNA occurs in all cell types tested. The SV40 enhancer contains binding sites for ubiquitously expressed NLS-containing transcription factors. Since these proteins are synthesized in the cytoplasm, once plasmid DNA has entered the cytoplasm by transfection, injection, or infection, the newly synthesized proteins can bind to the plasmid DNA to form a DNA–protein complex. Thus, the DNA is coated with NLSs, allowing the NLS-mediated import machinery to recognize DNA as a substrate and target it to the nucleus. (b) Smooth muscle cell-specific nuclear import is achieved by incorporating DNA sequence elements that contain binding sites for transcription factors that are expressed uniquely in SMCs, such as SRF or certain bHLH proteins that bind to E-boxes. These factors act analogously to the ubiquitous transcription factors to couple the DNA to the NLS-mediated nuclear import machinery.

Article Snippet: A similar series of transcription reporter plasmids (pGL-2294 and pGL-404) were created in pGL3-basic (Promega) that express luciferase. pCAT-control contains the SV40 early promoter (nts 5171–130) and enhancer (nts 130–270) and was purchased from Promega.

Techniques: Plasmid Preparation, Sequencing, Binding Assay, Synthesized, Transfection, Injection, Infection