RESUMO
In the Xenopus laevis oocyte there is a million fold more transcription factor IIIA (TFIIIA) and its corresponding mRNA than in a somatic cell. These high levels of TFIIIA gene expression are achieved primarily by transcriptional regulation. The TATA box along with three positive cis-elements in the control region of the TFIIIA gene located at positions -269 to -264 (E1), -235 to -220 (E2), and -669 to -636 (E3) are required for this high level of expression in oocytes. The proteins that bind E1 and E3 of the TFIIIA gene have been identified as Xenopus USF (Xl-USF) and B3 (homolog of Vg1 RBP/VERA). In this study the B2 protein was found to bind E2 in a zinc-dependent fashion and anti-human Sp1 (but not Sp2, Sp3, nor Sp4) supershifted the B2:element 2 complex. The E2 binding protein was purified by DNA affinity chromatography. Based on supershift analysis, molecular weight estimation experiments, and purified human Sp1 DNA binding affinity tests the data strongly support the idea that the B2 protein is the Xenopus ortholog of Sp1, but not Sp2, Sp3, nor Sp4. Xl-USF binds to element 1 of the TFIIIA gene which is immediately adjacent to element 2. Coimmunoprecipitation experiments using crude whole oocyte extracts revealed that Xenopus Sp1 and USF or closely related factors are present together in a high-affinity complex. This structure contributes positively to the initiation of TFIIIA gene transcription in Xenopus oocytes.
Assuntos
Proteínas de Ligação a DNA , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição TFIIA/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Ligação Competitiva , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Humanos , Immunoblotting , Peso Molecular , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Elementos de Resposta/genética , Fator de Transcrição Sp1/imunologia , Fator de Transcrição TFIIA/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores Estimuladores Upstream , Xenopus laevis/genética , Dedos de Zinco/genéticaRESUMO
Much of our current understanding of the function of genes modulating the normal process of embryonic development has come from mutant analysis. The availability of thousands of mutant lines in zebrafish that allows for identification of novel genes regulating various aspects of embryogenesis has been instrumental in establishing zebrafish as a robust and reliable genetic system. With the advances in genomic sequencing, the construction of several genetic maps, and cloning of hundreds of ESTs, positional cloning experiments in zebrafish have become more approachable. An increasing number of mutant genes have been cloned. Several zebrafish mutants are representative of known forms of human genetic diseases. The success of morpholino antisense technology in zebrafish potentially opens the door for modeling nearly any inherited developmental defect. This review highlights the strengths and limitations of using the zebrafish as an organism for elucidation of the genetic etiology of human disease. Additionally a survey of current and future zebrafish models of human disease is presented.