Isolation and characterization of the rat prolactin-releasing peptide gene: multiple TATA boxes in the promoter region.
The prolactin-releasing peptide (PrRP) gene is a novel bioactive peptide expressed in very restricted regions in the brain. To explore the molecular mechanism of PrRP gene expression, we cloned and characterized the gene and its promoter region. The gene spans approximately 2.4 kb and contains three exons and two introns. 3'RACE analysis showed that a polyadenylation signal 103 bp downstream from the stop codon was functional. Primer extension analysis indicated three transcriptional start sites (TSSs) 92, 199, and 325 bp upstream from the translational start site. Interestingly, in addition to the putative binding sites for SP1-1, AP-2, and Oct-2A, three characteristic TATA boxes were identified close to these TSSs. Transient transfection study using a series of deletion mutants revealed that the middle TATA box is important for the promoter activity. Furthermore, the cloned 1.6 kb promoter region was active only in neuron- and pituitary-derived cell lines, and the promoter region -1600 approximately -800 bp worked as a negative regulatory element. We demonstrated for the first time, the genomic organization and promoter function of the PrRP gene, and this knowledge will facilitate elucidation of transcriptional control of the PrRP gene.
Yamada
M
M
First Department of Internal Medicine, Gunma University School of Medicine, Maebashi, Gunma, 371-8511, Japan. myamada@med.gumma-u.ac.jp
Ozawa
A
A
Ishii
S
S
Shibusawa
N
N
Hashida
T
T
Ishizuka
T
T
Hosoya
T
T
Monden
T
T
Satoh
T
T
Mori
M
M
D020413:3' Untranslated Regions; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D001921:Brain / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D015723:Gene Library; D007028:Hypothalamic Hormones / Q000235:genetics*; D007438:Introns; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009479:Neuropeptides / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D056690:Prolactin-Releasing Hormone; D011401:Promoter Regions, Genetic; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013154:Spleen / Q000378:metabolism; D016385:TATA Box; D014158:Transcription, Genetic; D014162:Transfection
D020413:3' Untranslated Regions; D018076:DNA, Complementary; D007028:Hypothalamic Hormones; D009479:Neuropeptides; C534379:Prlh protein, rat; D056690:Prolactin-Releasing Hormone