Subcloning and Sequencing
After synthesis of cDNA by reverse transcription of MS or WC boar pituitary RNA, PCR products were amplified with primers specific for the calmodulin and GnRH-R genes and were subcloned into the pCR-II vector (Invitro-gen, San Diego, CA). The orientation of plasmids containing partial cDNA sequence was verified by sequence analysis from both directions by the dideoxy method of Sanger et al., adapted for double-stranded DNA templates and T7 DNA polymerase (United States Biochemical Corp., Cleveland, OH). The sequence was compared with the corresponding sequence regions of each gene. antibiotic levaquin
Preparation of Oligonucleotide DNA, Sense and Antisense RNA Riboprobes
Oligonucleotide DNA was end-labeled with T4 polynucleotide kinase (Promega Corp., Madison, WI) in the presence of [7-32P]dATP and single-strength T4 polynucleotide kinase buffer (0.5 M Tris-HCl, pH 7.5, 0.1 MgCl2). Free nucleotides were removed by Sephadex G-50 spin columns (5 Prime-3 Prime, Boulder, CO). Sense and antisense porcine FSHp and I/A pB-subunit riboprobes were transcribed in the presence of [a-32P]UTP with DNA-dependent T7 or SP6 RNA polymerases from the linearized plasmids with EcoRV or HindIII. The DNA templates were removed by incubation with RNase-free DNase I at 37°C for 15 min and extracted with phenol-chloroform.