Pituitary Adenylate Cyclase-Activating Polypeptide Gene Expression Regulated by a Testis-Specific Promoter in Germ Cells during Spermatogenesis

P. B. Daniel
2000 Endocrinology  
Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family of hormones that is widely expressed in various tissues. The PACAP messenger RNA (mRNA) and protein is expressed at high levels in the germ cells of the testis, where it locally activates cAMP-coupled receptors located in the somatic Sertoli cells. The PACAP mRNA expressed specifically in the testis is shorter than the mRNA expressed in hypothalamus and includes 127 nucleotides of novel
more » ... at the 5Ј-end, suggesting a different start site of transcription in the testes and the utilization of a tissue-specific promoter. Here we present evidence that a single PACAP gene uses a testis-specific promoter to express a mRNA containing a unique exon located 13.5 kb upstream from the first coding exon. As determined by RT-PCR analysis of testis mRNA, the expression of the first testis-specific exon is relatively specific for the Polyadenylated [poly(A) ϩ ] mRNA was prepared from adult testis RNA by oligo(deoxythymidine)-cellulose chromatography (16). Double stranded rat cDNA was prepared from poly(A) ϩ mRNA and ligated to adapters using reagents supplied with the Marathon rapid amplification of cDNA ends (RACE) kit (CLONTECH Laboratories, Inc., Palo Alto, CA). The cDNAs were diluted 1:500 for amplification by PCR. The first round of PCR amplification was performed with adapter primer 1 and PCPR1120. A second round of amplification was performed with adapter primer 2 and PCPR743 using 2 l first round product as template. Conditions for the first round of amplification were 94 C for 1 min,
doi:10.1210/en.141.3.1218 pmid:10698199 fatcat:wta7j7lfjzbyhecmzt7njecdn4