Cloning and sequence analysis of cDNA encoding urotensin I precursor.

Abstract

The primary structure of the precursor of urotensin I, a neuropeptide hormone from the caudal neurosecretory system of the carp Cyprinus carpio, has been determined by analyzing the nucleotide sequence of cloned DNA complementary to the mRNA encoding it. The precursor consists of 145 amino acid residues and the carboxyl terminus represents the 41-amino acid sequence of urotensin I, preceded by Lys-Arg and followed by Gly-Lys. Sequence homology as well as similar organization of the precursors of urotensin I and mammalian corticotropin-releasing factors suggest that they are evolutionarily related. RNA transfer blot analysis indicates that mRNA encoding the precursor of urotensin I is present only in the spinal cord and not in the brain, intestine, liver, or kidney of the carp.Urotensin I (UI) is one ofthe neuropeptides released from the urophysis, the neurohemal organ of the caudal neurosecretory system of teleosts. UI has potent hypotensive activity in mammals (1), and a relationship between this peptide and osmoregulation has been suggested for fishes (2). Recently, the primary structure of UI peptides has been determined (3, 4); a close similarity exists among UI, mammalian corticotropin-releasing factors (CRFs) (5-7), and sauvagine, a peptide isolated from the skin of a frog (8). The Identification of Carp UI Precursor cDNA. A total of 6000 transformants was screened by colony hybridization using a mixture of 32P-labeled synthetic oligonucleotides, 5' GC-CAT(T,C)TC(A,G,C,T)ATCAT 3', representing all possible cDNA sequences that correspond to the pentapeptide sequence Met-Ile-Glu-Met-Ala, amino acid residues 18-22 of carp UI (Fig. 2a). The probe was 5' end-labeled to a specific activity of 1.2 x 108 cpm/,Rg. The filters were hybridized with the probe for 46 hr at 370C in 0.6 M NaCIV60 mM sodium citrate, pH 7.4)/10x concentrated Denhardt's solution (lx Denhardt's = 0.02% bovine serum albumin/0.02% Ficoll/ 0.02% polyvinylpyrrolidone)/E. coli DNA (100 Ag/ml) and washed twice at 0C for 5 min and twice at 37TC for 20 min in 0.6 M NaCl/60 mM sodium citrate.Nucleotide Sequence Analysis. Plasmids from the positive clones were analyzed by several restriction enzymes. The inserts from the positive clones were subcloned into pUC 18 vector and various deleted plasmids were prepared by the kilosequence method (20) 308The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Cloning and sequence analysis of cDNAs for neurohypophysial hormones vasotocin and mesotocin for the hypothalamus of toad, Bufo japonicus.

“…Procedures for constructing a cDNA library from the toad diencephalons, screening of the library, analyzing the nucleotide sequences, and RNA transfer-blot analysis followed those of Ishida et al (10). In brief, the diencephalons (15 g) were collected from 300 toads captured in the breeding season in March 1985, and a total RNA (5.1 mg) was extracted from them in 4 M guanidinium thiocyanate buffer (11).…”

Cloning and sequence analysis of cDNAs encoding precursors of urotensin II-alpha and -gamma

“…Although a partial homology in amino acid sequence has been indicated previously (Pearson et al, 1980), there is homology in neither amino acid nor nucleotide sequence between the precursors of UII and somatostatin from the rat (Montominy et al, 1984). There is also no homology in amino acid or nucleotide sequences between the precursors of UII and urotensin I (Ishida et al, 1986), indicating that urotensins I and II have evolved from separate genes.…”

“…Poly(A)+RNA was purified from the total RNA by oligo(dT)-cellulose column chromatography (Aviv and Leder, 1972). A cDNA library was constructed by the metbed of Okayama and Berg (1982) with 5 pg of poly(A)'RNA and 1.6 ccg of vector/primer DNA as described by Ishida et al (1986). After transformation of E. co/i HB 101 (Morrison, 1979), the cells were plated on ampicillincontaining agar plates and replicated on nitrocellulose filters.…”

“…Urotensin I is a 41 amino acid residue peptide (Ichikawa et al, 1982; homologous to mammalian corticotropin-releasing factor (CRF; see Ichikawa, 1985). The elucidation ofthe primary structure of the precursor of carp UI (Ishida et al, 1986) has shown a sequence homology, as well as an organizational sim-ilarity, between the precursors of UI and CRF (Furutani et al, 1983;Shibahara et al, 1983), suggesting that they are evolutionarily related. The primary structure of a single UII peptide from the goby, Gillichthys mirabilis, was determined by Pearson et al (1980), followed by the identification of 2 forms of UII (UII, and UII,) from the sucker, Catostomus commersoni (McMaster and Lederis, 1983), and 3 forms of UII (UI-CU, -0, and -7) from the carp, Cyprinus carpio (Ichikawa et al, 1984).…”

“…Urotensin I is a 41 amino acid residue peptide (Ichikawa et al, 1982; homologous to mammalian corticotropin-releasing factor (CRF; see Ichikawa, 1985). The elucidation ofthe primary structure of the precursor of carp UI (Ishida et al, 1986) has shown a sequence homology, as well as an organizational sim- …”