S1 nuclease protection analyses of the mousebcl-x transcription start sites.

(A) Labeled, single-stranded (ss) DNA probe was the noncoding strand of the region shown in Figure 1B as Probe 1 (−754 through −412). The probe was hybridized to total RNA from mouse erythroblasts (FVA cells) that had been cultured for 36 hours with EPO. Lane 1: the DNA fragments that were protected from S1 nuclease. Lane 2: purified probe, not subjected to digestion (about 7000 cpm). Products of a set of sequencing reactions for a portion of the human coagulation Factor XI gene of known sequence are shown as precise size markers in the first 4 lanes (not numbered). The inset photograph to the right of lane 2 shows the triplet bands of lane 1 at higher resolution. (B) Labeled, ssDNA probe was from the clone shown in Figure 1B as Probe 3 (−433 through +26 lacking the intron). The full length of the bcl-xsequence in Probe 3 is 176 bp. Lane 1: probe hybridized with erythroblast cell RNA (36 hours of culture) and digested with S1 nuclease. Lane M: 5′ end–labeled ΦX174 HinfI fragments. Lane 2: undigested probe (1000 cpm). (C) Labeled, ssDNA probe represented the sequence shown in Figure 1B as Probe 4 (−498 through +49 lacking the intron). Sequence in Probe 4 complementary tobcl-x mRNA is 264 bp. Analyses as for (A) and (B) using S1 nuclease. Lane 1: undigested probe (1000 cpm). Lane 2: probe hybridized with yeast RNA. Lanes 3-6: probe hybridized with total RNAs from thymus, brain, mouse erythroblasts (FVA cells), mouse fetal liver (day 15 gestation). Lane M: as described previously. (D) Labeled, ssDNA was from the clone shown in Figure 1B as Probe 5 (−431 through −34 including the intron). Probe 5 contains 397 bp of sequence of the gene. Lane M: as described in the legend to panel B. Lane 1: probe hybridized to erythroblast RNA (36 hours of culture) and digested with S1 nuclease. Numbers with asterisks are fragments protected from nucleases. Numbers without asterisks are sizes of ΦX174 marker fragments.