(A) RT-PCR analysis of endogenous MSA transcriptional termination: Diagram shows the final MSA exon (black box), poly(A) site and 3′ flank (line). Arrows are shown, corresponding to primers A–E, and forward primer used for subsequent PCR (F) is also labeled. A scale bar is provided indicating the distance from the poly(A) site. Exact primer coordinates are provided in the Materials and Methods section. (Top panel) Agarose gel electrophoretic analysis of PCR products made from cDNAs A–F using mouse liver RNA as a template. Presence or absence of reverse transcriptase for cDNA synthesis is indicated by + or −, respectively. (Lower panel) Identical RT-PCR analysis of an in vitro transcribed, full-length MSA 3′ flanking region RNA. (B) ChIP analysis of Pol II occupancy in 5′ and 3′ regions of the MSA ORF and 3′ flank. The accompanying diagram shows the MSA gene (black bar), poly(A) site and 3′ flank (black line). The positions of the 5′, 3′, and flank primer pairs are indicated above the diagram. Distances upstream (–) and downstream (+) of the poly(A) site are also shown. Exact coordinates are given in Materials and Methods. Quantitation is represented as the fold extra signal obtained by using a Pol II-specific antibody to pull down chromatin as opposed to no antibody.
