Figure 2.
(A) Southern hybridization was performed on HindIII-digested primate DNA using a 32P-labeled probe to the S5a gene. The 3-kb band corresponds to PIPSL; it migrates at 4.8 kb in Gorilla because of an RFLP (arrowhead). The 5.9-kb and 5.6-kb doublet (**) corresponds to S5a and its Chr 21 pseudogene. Supplemental Figure S7 depicts probe locations. (B) Shown are duplicate RT-PCRs on total RNA from human testis or HeLa cells using the primers depicted in C. In the top gel of B, A* and D* primers specific for PIPSL amplified PIPSL from testis (lane 1), but not HeLa cDNA (lane 6). A and D primers specific for the PIP5K1A-S5a Transcription-Induced Chimera (TIC) amplified only trace amounts of TIC from testis (lane 2) and HeLa (lane 7) cDNAs. In the bottom gel, parental PIP5K1A (lanes 1,2) and S5a (lanes 6,7) transcripts were amplified from the same testis (T) and HeLa (H) cDNAs, serving as internal controls. (Lanes 3,4,8,9) RT-PCRs lacking reverse transcriptase; (lanes 5,10) no template PCRs; (M) 1-kb Plus DNA ladder. (C) Primers used in RT-PCRs are shown by arrowheads (A, B, C, D, A*, and D*). Probes used in Northern blots are shown by black (ex 15, ex 8–12, and PIPSL-specific) and white (ex 3–7) lines. (D) Northern blot of PIPSL in human testis (T) showing two PIPSL transcripts of ∼3.0–3.6 kb (bracket). HeLa RNA (H) is used as a negative control. PIPSL is visible with probe 2 in PIP5K1A exons 8–12, PIPSL-specific probe 3 spanning both parental genes, and probe 4 in S5a exons 3–7, but not with negative-control probe 1 to PIP5K1A exon 15. The 3.7-kb PIP5K1A and 1.3-kb S5a mRNAs serve as internal controls. (E) Northern blot on RNAs from multiple human tissues with PIPSL-specific probe 3 shows PIPSL transcripts only in the testis. PIP5K1A and S5a are ubiquitously present. To confirm comparable loading, the blot was stripped and reprobed for GAPDH and 7SL RNAs. (F) A Northern blot on RNAs from several chimpanzee tissues with PIPSL-specific probe 3 shows ∼3.0-kb PIPSL transcripts only in the testis. Internal control parental S5a is ubiquitously present. The human PIPSL-specific probe 3 does not hybridize to the chimpanzee PIP5K1A transcript.