RESUMO
Galanin is a hormone 29 or 30 amino acids (aa) long that is widely distributed within the body and exerts numerous biological effects in vertebrates. To fully understand its physiological roles in reptiles, we analyzed preprogalanin cDNA structure and expression in the turtle pituitary. Using the Chinese soft-shell turtle (Pelodiscus sinensis order Testudines), we obtained a 672-base pair (bp) cDNA containing a 99-bp 5'-untranslated region, a 324-bp preprogalanin coding region, and a 249-bp 3'-untranslated region. The open-reading frame encoded a 108-aa preprogalanin protein with a putative 23-aa signal sequence at the NH(2) terminus. Based on the location of putative Lys-Arg dibasic cleavage sites and an amidation signal of Gly-Lys-Arg, we propose that turtle preprogalanin is processed to yield a 29-aa galanin peptide with Gly(1) and Thr(29) substitutions and a COOH-terminal amidation. Sequence comparison revealed that turtle preprogalanin and galanin-29 had 48-81% and 76-96% aa identities with those of other vertebrates, respectively, suggesting their conservative nature. Expression of the turtle galanin gene was detected in the pituitary, brain, hypothalamus, stomach, liver, pancreas, testes, ovaries, and intestines, but not in the adipose or muscle tissues, suggesting tissue-dependent differences. An in vitro study that used pituitary tissue culture indicated that treatment with 17beta-estradiol, testosterone, or gonadotropin-releasing hormone resulted in increased galanin mRNA expression with dose- or time-dependent differences, whereas leptin and neuropeptide Y reduced galanin mRNA levels. These results suggest a hormone-dependent effect on hypophyseal galanin mRNA expression.
Assuntos
Galanina/genética , Regulação da Expressão Gênica , Hipófise/química , Precursores de Proteínas/genética , Tartarugas/genética , Regiões 3' não Traduzidas , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sequência de Bases , Códon de Terminação , DNA Complementar/análise , Glicina/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Precursores de Proteínas/química , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Treonina/metabolismo , Tartarugas/fisiologiaRESUMO
The complete complementary DNA (cDNA) of proopiomelanocortin (POMC), a common precursor of opioid hormone beta-endorphin, melanotropin (MSH), and corticortropin (ACTH), was cloned and sequenced from pituitary and hypothalamus of mud turtle (Pelodiscus sinensis) by RT-PCR and rapid amplification of cDNA end (RACE) methods. Two transcripts of POMC mRNAs with different polyadenylation sites were observed. Both transcripts had an open reading frame encoding a 261-amino acid peptide containing nine dibasic amino acids (pair of Arg and Lys), putative proteolytic cleavage sites for processing to functional peptides. All the functional peptide fragments of mud turtle POMC, gamma-MSH, alpha-MSH, ACTH, beta-MSH, and beta-endorphin, are flanked by dibasic residues as found in other tetrapods, implying that it could be processed to give rise to all members of POMC-derived peptides. The deduced amino acid sequences of mud turtle POMC displays 63-67% identity with amphibian, 59% with chicken, 48-53% with mammals, and 37-59% identity with fish. However, functional peptide fragments are much more conserved than overall sequence and intervening fragments. In addition to pituitary and brain, mud turtle POMC mRNAs are also expressed in many peripheral tissues, such as skin, thyroid, and testis. This is the first report on the complete sequence of cDNA nucleotides and deduced amino acids of POMC in reptile.