FIG. 2.

Detection of germ line transcription across the Igh D region. (A) Design of the D_H region germ line transcription reverse transcription-PCR assay. Reverse transcription antisense (to detect sense transcription) and sense (to detect antisense transcription) primers were designed 5′ and 3′ of the D_H gene RSS sequences. Nested primers were used for PCR. Similar schemes were used for intergenic regions. (B) Schematic of the DFL16.1-J1 sequence showing regions analyzed by reverse transcription-PCR. Not to scale. (C) Reverse transcription-PCR of Rag1^−/− CD19⁺ total RNA. Above lanes, with (+) or without (−) reverse transcriptase (RT). Right margin, regions amplified (shown in panel B); left margin, molecular sizes. DNA, genomic DNA positive control. (D) Sequencing of DSP genic and intergenic reverse transcription-PCR products. The number of reverse transcription-PCR products cloned and sequenced is shown for each D_H gene and intergenic region. (E) Real-time quantitative reverse transcription-PCR of Rag1^−/− CD19⁺ random-primed cDNA showing relative transcription levels for Hprt, DFL16.1, V_HJ558 intergenic, and V_HJ606 intergenic regions. The level of V_HJ558 intergenic transcription was arbitrarily set to 1.