RESUMO
We have investigated the utility of the green fluorescent protein (GFP) as a marker for gene expression in living adult Drosophila melanogaster (Dm) and cultured plant and mammalian cells. Using Dm, we generated transgenic flies bearing a glass-responsive gfp fusion gene to test the utility of GFP as a spatial reporter. In the adult living fly, GFP is clearly visible in the ocelli and the eye. We have optimized the use of filters for distinguishing the GFP signal from abundant autofluorescence in living Dm. In addition, we have used GFP to identify photoreceptor cells in pupal eye cultures that have been fixed and stained according to standard histological procedures. GFP was also detected in individual living plant cells following transient transfection of soybean suspension cultures, demonstrating that GFP is an effective transformation marker in plant cells. Similarly, transient transfection of mammalian cells with a modified form of GFP, S65T, allowed detection of single living cells expressing the reporter. This modified form of GFP gave a robust signal that was resistant to photobleaching. We then used a CellScan system exhaustive photon reassignment (EPR) deconvolution algorithm to generate high-resolution three-dimensional images of GFP fluorescence in the living cell.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Expressão Gênica , Genes Reporter , Proteínas Luminescentes/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Citomegalovirus/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/metabolismo , Ratos , Cifozoários , Glycine max/citologia , Células Tumorais CultivadasRESUMO
We have investigated the transcriptional regulation of the Antennapedia P2 (Antp P2) promoter using nuclear extracts prepared from Drosophila embryos. Transcriptional analysis of deletion mutants reveals the presence of multiple cis-regulatory elements located both upstream and downstream of the start site. One of the elements appears to mediate negative regulation, since deletion of this element results in higher levels of transcription. Several factors that interact with these control elements have been detected and isolated. One DNA-binding protein, Drosophila Transcription Factor-1 (DTF-1), specifically recognizes the consensus sequence GCAACAT/CG/C that is reiterated four times within an upstream activating element. DTF-1 was purified by sequence-specific DNA affinity chromatography and identified as a doublet of approximately 50 kD. Purified DTF-1 enhances transcription from the Antp P2 promoter 7- to 15-fold in a binding site-dependent manner.
Assuntos
Drosophila/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Animais , Sequência de Bases , Cromatografia de Afinidade , DNA , Proteínas de Ligação a DNA , Drosophila/embriologia , Proteínas de Drosophila , Técnicas In Vitro , Dados de Sequência Molecular , Mutação , Plasmídeos , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/isolamento & purificaçãoRESUMO
A homolog of mammalian enhancer binding factor AP-1 was detected in Drosophila and was purified from embryo nuclear extracts by sequence-specific DNA affinity chromatography. The purified fraction, dAP-1, displays the sequence specificity as well as transcriptional activation properties of mammalian AP-1 and consists of two major proteins of mol. wts 40 and 70 kd. Antibody cross-reactivity experiments suggest that these proteins are Drosophila homologs of proto-oncogene products, Jun and Fos. The Drosophila Jun- and Fos-related antigens, when separated, are individually capable of sequence-specific DNA binding, and the Jun-related antigen activates transcription in vitro.
