RESUMO
Vitellogenins (Vtg) are egg-yolk precursor proteins crucial for reproductive success in oviparous animals. We have cloned the first complete cichlid Vtg cDNA from the tilapia fish, Oreochromis aureus. This cDNA has the largest phosvitin (PV) domain amongst piscine Vtgs, being comparable to those of lamprey, Xenopus, and chicken. Thus, the size of PV is independent of the evolutionary advancement of a species. The closer interspecific relationship between O. aureus Vtg1 and Fundulus VtgII than the intraspecific relationship between Fundulus VtgI and II isoforms suggests that teleost ancestors had at least two Vtg isoforms. Contrary to the results of previous phylogenetic inference using Vtgs which indicate that insect lineage is most diverged and nematodes are closer to vertebrate lineage, our results show that nematodes and hexapods form two monophyletic sister groups. Another arthropod taxon, represented by a malacostracan crustacean, Penaeus japonicus, appears to be more closely related to the vertebrates than the hexapods.
Assuntos
DNA Complementar/genética , Fosvitina/genética , Tilápia/genética , Vitelogeninas/genética , Sequência de Aminoácidos , Animais , Sequência Conservada/genética , DNA Complementar/química , Proteínas do Ovo/genética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The Oreochromis aureus vitellogenin (OaVtg) gene contains three imperfect oestrogen response elements (EREs) and GATA and VBP (vitellogenin binding protein) binding sites. An analysis of the promoter indicates that the 5'-flanking region up to position -625 is sufficient to mediate E(2) control. Furthermore, transfection of deletion and mutagenised promoters indicates that both GATA and VBP synergise with ER, and thus contribute to the regulation of the endogenous OaVtg gene. These findings support the notion that the interplay of promoter elements mediates proper hormone-dependent and tissue-specific expression of the OaVtg gene, regardless of non-consensus sequence context of EREs and VBP.
Assuntos
Proteínas Aviárias , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Receptores de Estrogênio/fisiologia , Fatores de Transcrição/fisiologia , Vitelogeninas/genética , Animais , Fatores de Transcrição de Zíper de Leucina Básica , Células COS , Fator de Transcrição GATA6 , Perciformes , Regiões Promotoras Genéticas , Transcrição GênicaRESUMO
The Oreochromis aureus vitellogenin, OaVtg, gene spans 9 kb and contains 34 exons. Its transcription start site is located 15 bp upstream of the translational start codon. Although the OaVtg promoter has a nonconsensus TATA, transient transfection assay showed that this promoter is capable of driving basal transcription. Two imperfect estrogen response elements: EREp (proximal) and EREd (distal) are located in the promoter at - 532 and - 1352, respectively. In competition gel mobility-shift assays, only EREp exhibited specific binding of the recombinant estrogen receptor protein, GST-C/D OaER. Another imperfect ERE (EREexon2) was detected within exon 2 of the OaVtg gene. This is a novel finding for a vitellogenin (Vtg) gene. EREexon2 similarly showed specific recognition of GST-C/D OaER. Both EREp and EREexon2 showed comparable binding affinities as consensus ERE. In transient transfections, the OaVtg promoter, EREp and EREd elicited significant increase in estrogen-dependent synthesis of CAT protein. Hence, we propose that the non-consensus OaVtg EREs contribute to the estrogen-dependent regulation of the OaVtg gene in vivo.