RESUMO
The zebrafish has recently been developed as a good genetic model system. We report here the use of zebrafish to study the regulation of estrogen biosynthesis. The CYP19 gene encodes cytochrome P450 aromatase, which catalyzes the synthesis of estrogens. Two cyp19 genes, termed cyp19a and cyp19b, have been isolated from zebrafish. Sequence comparison shows that Cyp19a and Cyp19b belong to two separate Cyp19 subfamilies. The cyp19a gene is expressed in the ovary, whereas cyp19b is expressed in the brain. The cyp19a and cyp19b genes are located on zebrafish chromosomes LG 18 and 25, respectively. Our data indicate that these gene loci arose through an ancient chromosomal duplication event. The expression of duplicated genes in distinct tissues may have evolutionary significance.
Assuntos
Aromatase/biossíntese , Aromatase/genética , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Encéfalo/enzimologia , DNA Complementar/genética , Feminino , Dados de Sequência Molecular , Ovário/enzimologia , Processos de Determinação Sexual , Diferenciação Sexual/genéticaRESUMO
Cytochrome P450 aromatase (Cyp19) is an enzyme catalyzing the synthesis of estrogens, thereby controlling various physiological functions of estrogens. We isolated two cyp19 cDNAs, termed cyp19a and cyp19b, respectively, from zebrafish. These genes are located in linkage groups 18 and 25, respectively. Detailed gene mapping indicated that zebrafish linkage groups 18 and 25 may have arisen from the same ancestral chromosome by a chromosome duplication event. Cyp19a is expressed mainly in the follicular cells lining the vitellogenic oocytes in the ovary during vitellogenesis. Cyp19b is expressed abundantly in the brain, at the hypothalamus and ventral telencephalon, extending to the olfactory bulbs. The expression of duplicated cyp19 genes at two different tissues highlights the evolutionary significance of maintaining two active genes on duplicated zebrafish chromosomes for specific functions in the ovary and the brain.
Assuntos
Aromatase/genética , Encéfalo/enzimologia , Ligação Genética/genética , Ovário/enzimologia , Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Aromatase/metabolismo , Encéfalo/anatomia & histologia , Encéfalo/fisiologia , Cromossomos/genética , Feminino , Duplicação Gênica , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Ovário/citologia , Ovário/fisiologia , Alinhamento de SequênciaRESUMO
Sox9 is a transcription factor required for cartilage formation and testis determination in mammals. We have cloned from zebrafish two sox9 genes, termed sox9a and sox9b. Gene phylogenies showed that both genes are orthologous to tetrapod SOX9 genes. Genetic mapping showed that these two loci reside on chromosome segments that were apparently duplicated in a large-scale genomic duplication event in ray fin fish phylogeny. Both Sox9a and Sox9b proteins bind to the HMG consensus DNA sequences in vitro. We tested different domains for transactivation potential and identified a potential activation domain located in the middle of both Sox9a and Sox9b. During embryogenesis, sox9a and sox9b expression patterns are distinct but overlap in some regions of the brain, head skeleton, and fins. Expression of sox9a/b correlates well with that of col2a1 in chondrogenic elements. In the adults, sox9a is expressed in many tissues including brain, muscle, fin, and testis, whereas sox9b expression is restricted to previtellogenic oocytes of the ovary. This expression pattern predicts that sox9a and sox9b may have unique functions in some specific tissues during development. The role of gene duplication for the evolution of developmental gene function is discussed.
Assuntos
Duplicação Gênica , Proteínas de Grupo de Alta Mobilidade/genética , Transativadores/genética , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Mapeamento Cromossômico , Clonagem Molecular , DNA/metabolismo , Ossos Faciais/metabolismo , Feminino , Ligação Genética , Gônadas/metabolismo , Masculino , Dados de Sequência Molecular , Filogenia , Fatores de Transcrição SOX9 , Crânio/metabolismo , Peixe-ZebraRESUMO
Steroid hormones are important physiological regulators in the body. Steroid hormones are mainly synthesized in the adrenal and gonads. Their synthesis is stimulated by pituitary hormones through cAMP as an intracellular mediator. The first and rate-limiting step for steroid biosynthesis is catalyzed by CYP11A1. Important regulatory elements for the control of the CYP11A1 gene expression have been characterized both in vitro and in vivo. The SF-1-binding sites are cis-acting elements controlling the basal and cAMP-stimulated gene expression. Our transgenic mouse studies showed that the 2.3kb promoter contains information controlling developmentally regulated gene expression. Finally, we present our results on the cloning of steroidogenic genes in zebrafish, a new model organism for genetic studies.
Assuntos
Regulação da Expressão Gênica , Camundongos Transgênicos/genética , Esteroides/biossíntese , Peixe-Zebra/genética , Glândulas Suprarrenais/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , AMP Cíclico/farmacologia , Gônadas/metabolismo , Camundongos , Modelos Animais , Hormônios Hipofisários/farmacologia , Regiões Promotoras GenéticasRESUMO
The cyp19 encodes P450 aromatase, the enzyme catalyzing the conversion of estrogens from androgens. Estrogens affect the dimorphic, anatomical, functional and behavioral aspects of development of both males and females. In zebrafish, two cyp19 genes, cyp19a and cyp19b were found. They are expressed in ovary and brain, respectively. Expression of cyp19b can be detected by 11 days post-fertilization (dpf) by in situ hybridization in the olfactory bulbs, ventral telencephalic region and the hypothalamus of the brain in both male and female, where it is generally known to be affecting the reproductive function and sexual behavior. COS-1 clones permanently expressing the enzymes have been isolated. Both aromatase enzymes encoded by these two genes are functional in COS-1 cells and they can use androstenedione and testosterone equally efficiently. The presence of two functional cyp19 in zebrafish has its evolutionary and physiological importance.
Assuntos
Aromatase/genética , Aromatase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Animais , Encéfalo/enzimologia , Células COS , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Feminino , Expressão Gênica , Hibridização In Situ , Masculino , Ovário/enzimologia , Filogenia , Reprodução/genética , Comportamento Sexual Animal , Distribuição Tecidual , TransfecçãoRESUMO
Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation.
