Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene.

Abstract

A human genomic DNA segment containing the gene for the corticotropin‐releasing factor precursor has been isolated by screening a gene library with an ovine cDNA probe. The cloned DNA segment has been subjected to restriction endonuclease mapping and nucleotide sequence analysis. Comparison of the nucleotide sequence of the gene with that of the ovine cDNA indicates that an intron of 800 bp is inserted in the segment encoding the 5′‐untranslated region of the mRNA. The segment corresponding to the protein‐coding and the 3′‐untranslated region of the mRNA is uninterrupted. The mRNA and amino acid sequences of the human corticotropin‐releasing factor precursor have been deduced from the corresponding gene sequence. The deduced amino acid sequence of human corticotropin‐releasing factor exhibits seven amino acid substitutions in comparison with the ovine counterpart.

Proopiomelanocortin, corticotropin releasing hormone and corticotropin releasing hormone receptor genes are expressed in human skin

“…The presence of POMC mRNA correlates with previous detection of ACTH, fl-MSH and fl-endorphin immunoreactivity in human scalp, basal cell (1), pollution control (reaction mixture without DNA template) (2), DNA size markers (3); untreated melanocytes (4), melanocytes treated with UVB 25 mJ/cm z (5), melanocytes treated with 100 nM of TPA (6); untreated keratinocytes (7), keratinocytes treated with UVB 25 mJ/cm 2 (8), keratinocytes treated with 100 nM of TPA (9). RT-PCR detection was done using sense primer, 5'-CAC CCT CAG CCC TTG GAT TTC-Y (nt 1436 to 1456) and antisense primer, 5'-GCC CTG GCC ATT TCC AAG AC-3' (nt 1828 to 1848) for the CRH cDNA [11]. The PCR program was 35 cycles of 30 s at 96°C, 30 s at 55°C and 2 min at 72°C with final extension of 7 min at 72°C.…”

“…Only samples that were proven to be free from DNA contamination by running PCR amplification without prior RT were used for the experiments. Fragments derived from the coding regions of human POMC [10], CRH [11] and CRH-R [12,13] cDNAs were amplified using primers designed and synthetized commercially (National Bioscience, Plymouth, MN). The reaction mixtures contained 25 mM (NH4)2SO4 buffer, pH 9.0 (POMC cDNA) or pH 9.5 (CRH-R and CRH cDNAs), 2 mM MgCl 2, 0.4 mM dNTP and 4 ¢tM of primers.…”

“…POMC expression and processing are under stimulatory control, mediated mainly by CRH through the activation of CRH receptors [1][2][3]. With the use of coding sequences of the CRH gene cloned from the human brain [11] and CRHR cloned from the human pituitary and brain [12,13], primers for RT-PCR assays were designed (cf. Figures legend).…”

The role of corticotrophin-releasing hormone in the diagnosis of Cushing’s syndrome

“…The human CRH (hCRH) gene was cloned and sequenced 2 years later (8). It is a 41-amino acid peptide derived by enzymatic cleavage from a 191-amino acid preprohormone and primarily released by the paraventricular nucleus of the hypothalamus.…”

Site-specific overexpression of corticotropin-releasing hormone (CRH) in the mouse brain – modelling central CRH system hyperactivity

“…The human CRH gene contains two exons separated by one intron in its 5' untranslated region (Shibahara et al, 1983). The rat, ovine and mouse CRH gene have the same organization (Roche et al, 1988), the coding region of the CRH precursor is in exon 2, the 24 amino acids in the N-terminal contain a typical signal peptide sequences which is commonly associated with neuropeptides that undergo secretion (Thompson et al, 1987).…”