Journal: Human mutation

Article Title: A Human Phospholamban Promoter Polymorphism in Dilated Cardiomyopathy Alters Transcriptional Regulation by Glucocorticoids

doi: 10.1002/humu.20692

Figure Lengend Snippet: Effect of the −36A>C genetic variant on human PLN promoter activity. A: Rat neonatal cardiomyocytes were transiently transfected with a luciferase expression vector driven by PLN-WT or PLN-MT (−36A>C) promoters, and were cultured in the absence (left) or presence (right) of 3 μM dexamethasone (Dex) for 45 hr. Transcriptional activity of the promoters was defined as a ratio of firefly luciferase activity to Renilla luciferase activity in the same cells, and normalized to the mean basal transcriptional activity of the promoter-less pGL3-basic vector. B: Sequence alignment of the normal and mutant human PLN upstream promoter regions. The relative positions of the promoter starting site (-1) and of the potential regulatory sequences (underlined) are indicated. The values are expressed as means±SEM (n=7). *P<0.05 vs. PLN-WT without Dex (two-way ANOVA and Student-Neuman-Keuls test). Polymorphism numbering is based on using the GenBank accession number AF177763.1for human PLN-sequence corresponding to proximal promoter and exon1as a reference.

Article Snippet: For promoter-reporter studies, after 24 hr incubation with serum-free medium, the myocytes were transiently cotransfected with 300 ng of each PLN luciferase test plasmid and 75 ng of phRL-TK control plasmid (Promega).

Techniques: Variant Assay, Activity Assay, Transfection, Luciferase, Expressing, Plasmid Preparation, Cell Culture, Sequencing, Mutagenesis

Journal: BMC Genomics

Article Title: Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

doi: 10.1186/1471-2164-8-99

Figure Lengend Snippet: Effect of serial 5'-deletions of the rat HL upstream region on transcriptional activity in HepG2 cells . HepG2 cells were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, cells were lysed and expression of CAT and β-galactosidase protein was determined. Data are expressed as the ratio of CAT over β-galactosidase expression. Data are means ± SD from 4–7 independent experiments, each performed in triplicate. *: P < 0.05; **: P < 0.01; n.s.: not significant.

Article Snippet: Cells were co-transfected with 2.5 μg/well of the CAT reporter test plasmid and 0.2 μg/well of control RSV β-galactosidase expression plasmid (Promega) [ ].

Techniques: Activity Assay, Transfection, Construct, Expressing

Journal: BMC Genomics

Article Title: Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

doi: 10.1186/1471-2164-8-99

Figure Lengend Snippet: Effect of serial 5'-deletions of the rat HL promoter region on transcriptional activity in HepG2 cells . Experiments were performed as described in the legends to figure 4. Data were expressed as percentage of the ratio measured in the rHL-446 CAT – transfected cells, and are means ± SD from 3–5 independent experiments, each performed in triplicate. *: P < 0.05; n.s.: not significant.

Article Snippet: Cells were co-transfected with 2.5 μg/well of the CAT reporter test plasmid and 0.2 μg/well of control RSV β-galactosidase expression plasmid (Promega) [ ].

Techniques: Activity Assay, Transfection

Journal: BMC Genomics

Article Title: Comparative genomics and experimental promoter analysis reveal functional liver-specific elements in mammalian hepatic lipase genes

doi: 10.1186/1471-2164-8-99

Figure Lengend Snippet: Transcriptional activity of the rat proximal HL promoter region in HepG2 and HeLa cells . HepG2 (hatched bars) and HeLa cells (open bars) were transiently transfected with the indicated promoter-reporter constructs. At 48 h post-transfection, transcriptional activity was determined as the ratio of CAT over β-galactosidase expression level. Data are expressed as percentage of the ratio measured in the rHL-39 CAT – transfected cells. Data are means ± SD from 3 independent experiments, each performed in triplicate. *, + : P < 0.05 compared to 100% in HepG2 and HeLa cells, respectively.

Article Snippet: Cells were co-transfected with 2.5 μg/well of the CAT reporter test plasmid and 0.2 μg/well of control RSV β-galactosidase expression plasmid (Promega) [ ].

Techniques: Activity Assay, Transfection, Construct, Expressing

Journal: Nucleic Acids Research

Article Title: Human miR-1271 is a miR-96 paralog with distinct non-conserved brain expression pattern

doi: 10.1093/nar/gkq798

Figure Lengend Snippet: Gene regulation by miR-1271 is disrupted by the HTR1B A/G 3′UTR polymorphism. ( A ) Nucleotides that are common to microRNAs, miR-1271 and miR-96 are indicated by grey shading. A dashed line indicates the ‘seed’ sequence, nucleotides 2–8. Shown below is the potential interaction of both microRNAs with an element from the serotonin receptor 1B mRNA 3′UTR. The location of the functional A>G rs13212041 SNP in the 3′UTR opposite nucleotide 3 of the microRNA is indicated by bold italic. ( B ) The dual luciferase reporter plasmid used to test microRNA response. The expression values in panels (B)–(D) are normalized for each construct to the expression value in the absence of co-transfected microRNA. Similar to miR-96, miR-1271 regulates the expression of the HTR1B rs13212041 A-element, but not the G-element. A control microRNA, miR-600[G], does not regulate the element with the same specificity. ** P < 0.005 comparing A-element to G-element in the indicated conditions, and error bars indicate ± SEM for 4-7 experiments . ( C ) MiR-96 and miR-1271 target HTR1B mRNA with similar specificity. The HTR1B 3′UTR based sequences used to test the nucleotide specificity of miR-96 and miR-1271 are described in detail in Supplementary File 1 . The position of each nucleotide change is numbered relative to the microRNA 5′ end, i.e. mutant 1 is opposite nucleotide 1 of each microRNA. + 1 to + 3 indicates HTR1B 3′ UTR nucleotides downstream from potential microRNA annealing site. The responses of each point mutant element to miR-96 and miR-1271 are significantly correlated, r = 0.94, P < 0.0001 (Pearson's correlation). Error bars indicate ± SEM for two experiments. ( D ) MiR-96 and miR-1271 gene repression is argonaute 2 dependent. Asterisk indicates P < 0.05 and error bars indicate ± SEM for six to eight experiments.

Article Snippet: The luciferase test plasmids are a modified version of the psiCHECK-2 (Promega, Madison, WI, USA) plasmid described by Felice et al . .

Techniques: Sequencing, Functional Assay, Luciferase, Plasmid Preparation, Expressing, Construct, Transfection, Control, Mutagenesis