(A) RNase protection assays of mRNA from transfected cells show that Space-75 has a normal early-to-late switch, but that as additional spacer sequences are inserted, the switch is strongly affected. Mutant names represent the clones indicated in Figure 5 and indicate the spacing between opposing poly(A) signals. For the spacing constructs shown, insertion of fragments in either orientation had the same effects (data not shown), indicating that distance but not sequence is important for the effects. Equal loading of samples was confirmed using a probe for GAPDH as an internal control, and quantitation was as described in Figure 1B. The L/E signal ratio for Space-1502 was arbitrarily set to 1, and all other ratios were compared to it. (B) Constructs with defective early-to-late switches also show lower levels of primary transcripts downstream from the late poly(A) site. Equal loading was confirmed using GAPDH as in A, and relative L/E ratios were also determined as in A.
