RNase protection with the 1M-containing probe. (A) Design of the experiment. The antisense probe contained the 1M exon (filled rectangle), the second, and part of the third exon common to all b5R transcripts (open rectangle), and portions of the vector sequence (single lines marked V). After hybridization to b5R mRNA, which contains 1M, the entire probe (except for the vector sequence) is expected to be protected from RNase digestion. In contrast, transcripts with an alternative first exon will only partially protect the probe, with generation of a smaller protected species. (B) Electrophoretic analysis of protected fragments. A total of 2 μg of poly A(+) RNA from HeLa cells (lane H) or from K562 cells induced (lane Ki) or not induced (lanes Kc) to differentiate, 17 μg of total RNA from erythroblasts (lane E), or 10 μg of total RNA from reticulocytes (lane R) or from torulla yeast (lane Y) were hybridized overnight with 40,000 dpm of 1M antisense probe at 43°C. One half of the same RNA samples (or one fifth in the case of erythroblast RNA) was hybridized to 40,000 dpm of GAPDH antisense probe (bottom row). Digestion was with 2.50 U/mL of RNase A and 100 U/mL of RNase T1 at 37°C for 1 hours. Numbers on the left of the panels indicate the size (in nucleotides) and position of Msp 1-digested pBR322 DNA marker fragments. The asterisk at the left of the panels indicates the position of the undigested probe. Some probe remained undigested in the samples shown in the middle panel. RNA from reticulocytes shows a clearly detectable partially protected probe fragment (arrowhead), predicted to be generated after hybridization to a b5R transcript with an alternative first exon.