RESUMO
Three distinct calmodulin (CaM)-encoding cDNAs were isolated from a reptile, the Japanese tortoise (Clemmys japonica), based on degenerative primer PCR. Because of synonymous codon usages, the deduced amino acid (aa) sequences were exactly the same in all three genes and identical to the aa sequence of vertebrate CaM. The three cDNAs, referred to as CaM-A, -B, and -C, seemed to belong to the same type as CaMI, CaMII, and CaMIII, respectively, based on their sequence identity with those of the mammalian cDNAs and the glutamate codon biases. Northern blot analysis detected CaM-A and -B as bands corresponding to 1.8 kb, with the most abundant levels in the brain and testis, while CaM-C was detected most abundantly in the brain as bands of 1.4 and 2.0 kb. Our results indicate that, in the tortoise, CaM protein is encoded by at least three non-allelic genes, and that the æmultigene-one protein' principle of CaM synthesis is applicable to all classes of vertebrates, from fishes to mammals
Assuntos
Humanos , Animais , Sequência de Aminoácidos , Calmodulina , DNA Complementar , Répteis/genética , Código Genético , Reação em Cadeia da PolimeraseRESUMO
In many reptile species, sexual differentiation of gonads is sensitive to temperature during a critical period of embryonic development (thermosensitive period, TSP). Experiments carried out with different models among which turtles, crocodilians and lizards have demonstrated the implication of estrogens and the key role played by aromatase (the enzyme complex that converts androgens to estrogens) in ovary differentiation during TSP and in maintenance of the ovarian structure after TSP. In some of these experiments, the occurrence of various degrees of gonadal intersexuality is related to weak differences in aromatase activity, suggesting subtle regulations of the aromatase gene at the transcription level. Temperature could intervene in these regulations. Present studies deal with cloning (complementary DNAs) and expression (messenger RNAs) of genes that have been shown, or are expected, to be involved in gonadal formation and/or differentiation in mammals. Preliminary results indicate that homologues of AMH, DAX1, SF1, SOX9 and WT1 genes with the same function(s) as in mammals exist in reptiles. How these genes could interact with aromatase is being examined.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Répteis/fisiologia , Diferenciação Sexual/genética , Temperatura , Androgênios/farmacologia , Androgênios/fisiologia , Animais , Aromatase/fisiologia , Temperatura Corporal , Clonagem Molecular , DNA Complementar/genética , Transtornos do Desenvolvimento Sexual/embriologia , Estrogênios/farmacologia , Estrogênios/fisiologia , Feminino , Genes Reguladores , Masculino , Mamíferos/genética , Ovário/embriologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/genética , Répteis/embriologia , Répteis/genética , Homologia de Sequência do Ácido Nucleico , Processos de Determinação Sexual , Diferenciação Sexual/efeitos dos fármacos , Especificidade da Espécie , Testículo/embriologia , Fatores de TempoRESUMO
The presence of transthyretin in mammals and birds, but not amphibia, suggested that transthyretin expression first appeared in stem reptiles. Therefore, transthyretin synthesis was studied in a lizard. Transthyretin synthesis in choroid plexus pieces from Tiliqua rugosa was demonstrated by incorporation of radiactive amino acids. Oligonucleotides corresponding to conserved regions of transthyretin were used as primers in polymerase chain reaction with lizard choroid plexus cDNA. Amplified DNA was used to screen a lizard choroid plexus cDNA library. A full-length transthyretin cDNA clone was isolated and sequenced. A three-dimensional model of lizard transthyretin was obtained by homology modeling. The central channel of transthyretin, containing the thyroxine-binding site, was found to be completely conserved between reptiles and mammals. Transthyretin expression was not detected in lizard liver. These data suggest that transthyretin first evolved in the choroid plexus of the brain. Due to a change in tissue distribution of gene expression, occurring much later during evolution, transthyretin also became a plasma protein, synthesized in the liver.