Cloning and Sequence Analysis of cDNAs Encoding Precursors of Urotensin II-a! and -y

The primary structures of the precursors of urotensin II (UII)-a and -7, neuropeptide hormones of the caudal neurosecretory system of the carp, Cyprinus carpio, have been determined by analyzing the nucleotide sequences of cloned DNAs complementary to the mRNAs encoding them. A cDNA library, constructed with poly(A)+RNA in the preterminal spinal cord, was screened using 3ZP-labeled synthetic oligonucleotldes representing all possible cDNA sequences corresponding to the pentapeptlde common to all forms of carp UII (UII-a, -0, and 9). Twenty out of 39 positive clones were analyzed with the restriction endonucleases, Hi&III and PvuII, and classified into 4 groups. Nucleotide sequence analysis of 4 clones representing each group revealed that 2 clones encode the precursor of UIk and the other 2 that of UII-y. Both precursors consist of 125 amino acid residues, and UI1-a and -“/ exist at the carboxyl-termini preceded by Arg-Lys-Arg. The homology in both nucleotide and amino acid sequences between the precursors of UII-a and -7 is more than 90%, suggesting that the genes were generated from a common ancestral gene by duplication. There is no sequence homology between the precursors of UII and urotensin I, another peptlde hormone of the caudal neurosecretory system, nor between the precursors of UII and somatostatin-14. RNA transfer blot analysis indicated that mRNAs encoding the precursors of UI1-u and 9 are present in the spinal cord but not in the brain, intestine, liver, or kidney of the carp. In sifu hybridization using 32P-labeled synthetic oligonucleotide complementary to common sequence of mRNAs for both UII-ol and --y has detected the UII-producing neurons in the caudal spinal cord of the carp.