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Comparative Study
. 1998 Dec 22;95(26):15803-8.
doi: 10.1073/pnas.95.26.15803.

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Y Coulouarn  1 , I Lihrmann, S Jegou, Y Anouar, H Tostivint, J C Beauvillain, J M Conlon, H A Bern, H Vaudry
Affiliations
Comparative Study

Cloning of the cDNA encoding the urotensin II precursor in frog and human reveals intense expression of the urotensin II gene in motoneurons of the spinal cord

Y Coulouarn et al. Proc Natl Acad Sci U S A. .

Abstract

Urotensin II (UII) is a cyclic peptide initially isolated from the caudal neurosecretory system of teleost fish. Subsequently, UII has been characterized from a frog brain extract, indicating that a gene encoding a UII precursor is also present in the genome of a tetrapod. Here, we report the characterization of the cDNAs encoding frog and human UII precursors and the localization of the corresponding mRNAs. In both frog and human, the UII sequence is located at the C-terminal position of the precursor. Human UII is composed of only 11 amino acid residues, while fish and frog UII possess 12 and 13 amino acid residues, respectively. The cyclic region of UII, which is responsible for the biological activity of the peptide, has been fully conserved from fish to human. Northern blot and dot blot analysis revealed that UII precursor mRNAs are found predominantly in the frog and human spinal cord. In situ hybridization studies showed that the UII precursor gene is actively expressed in motoneurons. The present study demonstrates that UII, which has long been regarded as a peptide exclusively produced by the urophysis of teleost fish, is actually present in the brain of amphibians and mammals. The fact that evolutionary pressure has acted to conserve fully the biologically active sequence of UII suggests that the peptide may exert important physiological functions in humans.

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Figures

Figure 1
Figure 1
Alignment of the deduced amino acid sequences of human, frog, and carp prepro-UII. The putative signal peptide sequence is indicated in italics. Conserved amino acid residues are indicated in black. Prohormone convertase cleavage sites are marked by stars. Conserved acidic residues are denoted by a closed circle. The intrachain disulfide bridge in UII is indicated under the urotensin sequence. The amino acid residues are numbered on the right. Gaps, marked by hyphens, have been inserted to achieve optimal alignment.
Figure 2
Figure 2
Tissue distribution of frog prepro-UII mRNA. (A) Northern blot analysis of prepro-UII mRNA expression in the frog central nervous system. Total RNA (30 μg) was electrophoresed on a formaldehyde–agarose gel, transferred onto a nylon membrane and probed with the frog prepro-UII cDNA probe. (B) Analysis of prepro-UII mRNA distribution in various frog tissues by RT-PCR. Total RNA (5 μg) was reverse transcribed, amplified with prepro-UII specific primers, electrophoresed on an agarose gel and stained with ethidium bromide (Upper). The gel was then blotted and hybridized with an internal prepro-UII primer (Lower). The control lane contained no DNA. The sizes of the PCR products were evaluated by using a DNA mass ladder (M).
Figure 3
Figure 3
X-ray autoradiographs showing the distribution of prepro-UII mRNA in the frog brainstem and spinal cord. Coronal sections were hybridized with the antisense (A–E) or the sense (F) prepro-UII riboprobe and were exposed for 2 weeks onto x-ray film. IV, trochlear nucleus; VI, abducens nucleus; VII, facial nucleus; IX, glossopharyngeal nucleus; XII, hypoglossal nucleus; VH, ventral horn of the spinal cord.
Figure 4
Figure 4
Photomicrographs of sections through the frog facial motor nucleus (A), spinal cord (B), and nucleus of the reticular formation (C) hybridized with a digoxygenin-labeled prepro-UII riboprobe. Dense prepro-UII mRNA accumulation in discrete neurons is revealed by the red precipitate. (X520).
Figure 5
Figure 5
Tissue distribution of human prepro-UII mRNA. (A) Dot blot analysis of prepro-UII mRNA expression in various human tissues. The master blot (CLONTECH) consisted of 50 human-tissue poly(A) RNAs (80–448 ng per dot) normalized by using the RNA expression levels of eight housekeeping genes. Positive controls consisted of human genomic DNA. Negative controls included yeast and Escherichia coli RNA or DNA, as well as human repetitive genomic sequences. The blot was probed with the human prepro-UII cDNA probe and exposed for 2 days onto an X-Omat film (B) Northern blot analysis of prepro-UII mRNA expression in the human spinal cord. Spinal cord poly(A) mRNA (2 μg) was hybridized with the human prepro-UII cDNA probe. The molecular weight was determined by using RNA markers. (C) X-ray autoradiographs showing the distribution of prepro-UII mRNA in the human spinal cord. Coronal sections were hybridized with the antisense (Top) or sense (Bottom) prepro-UII riboprobe and exposed for 10 days onto x-ray film.
Figure 6
Figure 6
Comparison of the primary structures of urotensin II from different species. Gaps (-) were inserted into the sequences to achieve optimal alignment. Dots denote residue identity to the human sequence.
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