Analysis of Dβ1 germline transcriptional start sites. (A) Location of upstream (US) or downstream (DS) primers used in the primer extension assay, as well as probe used for ribonuclease protection assay. (B) Primer extension analysis of RNA from either normal or Rag-2–deficient thymocytes or the p5424 pro-T cell line using US (left-hand gel) or DS (right-hand gel) primers. The positions of several of the major bands are indicated by arrows, with numbering relative to the first base in the Dβ1 element. (C) Ribonuclease protection assay for Dβ1 germline transcripts. A major transcriptional start site is indicated, which maps to approximately +32. The full-length protected fragment (FL) is 478 bases long and indicates the presence of transcripts initiating upstream of −202. The undigested probe is 578 bases long. Marker sizes are as indicated.