Figure 4.

The RNA detected by CJ11–CJ12 is found in the cytoplasm and is exclusively derived from autosomal Rps12 expression. (A) Nuclear and cytoplasmic distribution of the “P₀ RNA” isolated from male (M) and female (F) fibroblasts (Fib) and ES cells, RNA was reverse transcribed and amplified with CJ11 and CJ12. Xist was amplified with primers Mix20–Mx23b (12), which spans exons 3 to 6 of Xist. (B) TaqI and HinfI restriction maps for pS12X and Rps12 fragments bounded by CJ11 and CJ12. Sizes are shown for polymorphic fragments. Asterisks indicate RFLP positions. (C) RFLP analysis of CJ11–CJ12 RT-PCR products. PCR products were diluted and extended one cycle to minimize heteroduplex formation and then digested with TaqI or HinfI. Polymorphic restriction fragments were detected by hybridization to radiolabeled nested oligonucleotide CJ10. + and − indicate the presence or absence, respectively, of restriction enzyme during incubation. (D) Sensitivity of the RFLP assay of CJ11–CJ12 amplification. A constant amount of Rps12 RT-PCR product was mixed with 10-fold dilutions of pS12X PCR product, digested with TaqI, and visualized by hybridization to CJ10 oligonucleotide. pS12X fragments were visible at 10⁻³ dilution (shown) and at 10⁻⁴ dilution on the original autoradiogram (data not shown).