Figure 4

Catfish Oct1 fails to drive transcription from physiologically relevant reporter constructs. A. Schematic diagram of the reporter construct pGL3/Δ56/R#2 [50]. This contains the core of the catfish enhancer in which there are 2 octamer motifs (ovals) and a μE5 site (square) upstream of a minimal TATA box (circle) followed by the luciferase gene (open rectangle). This reporter construct was used in the experiments shown in panels A&B. A. Transcriptional activation driven from the core enhancer by catfish Oct2β (pRc/CMV/Oct2β) or catfish Oct1 (pRc/CMV/Oct1) in the mouse B cell line (J558L). B. Transcriptional activation in the catfish B cell line 1G8 driven from the core enhancer construct by catfish Oct2β (pRc/CMV/Oct2β), catfish Oct1 (pRc/CMV/Oct1), human BOB.1 (pRc/CMV/hBOB.1), or by a co-transfection of catfish Oct2β with human BOB.1, or catfish Oct1 with human BOB.1. C. (Top) Schematic diagram of the reporter construct (pFVH-CAT) that contains a full fish V _H promoter (arrow) followed by the CAT gene [27]. C. (Bottom) Transcriptional activation driven from the V _H promoter (pFVH-CAT) by catfish Oct2β (pRc/CMV/Oct2β) or catfish Oct1 (pRc/CMV/Oct1) co-transfected with the reporter construct into catfish 1G8 B cells. Values are presented as mean ± SD for 12 replicates (A), 9 replicates (B), and 4 replicates (C). Statistical significance was calculated by the Student T test assuming unequal variances. One asterisk indicates a p value of less than .05 and two asterisks indicate a p value of less than .01.