RESUMEN
We have generated transgenic flies expressing R7 cell-specific opsins in the major class of photoreceptor cells of the Drosophila retina and characterized their spectral properties using high-resolution microspectrophotometry and sensitivity recordings. We show that the Rh3 and Rh4 opsin genes encode UV-sensitive opsins with similar spectral properties (lambda max = 345 nm and 375 nm), and that Rh3 corresponds to the R7p and R7marg class of visual pigments. We have also generated Rh3 and Rh4 isoform-specific antibodies and present an R7 cell map of the Drosophila retina. In a related set of experiments, we show that it is possible to coexpress two different visual pigments functionally in the same cell and produce photoreceptors that display the summed spectral response of the individual pigments. These findings open up the possibility of tuning an animal's visual behavior by targeted expression of combinations of opsin genes to selective types of photoreceptors.
Asunto(s)
Percepción de Color , Células Fotorreceptoras/metabolismo , Rodopsina/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila melanogaster , Células Fotorreceptoras/química , Células Fotorreceptoras/fisiología , Regiones Promotoras Genéticas , Rodopsina/análisis , Opsinas de Bastones/análisis , Opsinas de Bastones/metabolismoRESUMEN
The gene for ciliary neurotrophic factor (CNTF) was cloned from a human genomic DNA library by screening with a DNA fragment amplified from human genomic DNA using the polymerase chain reaction. A DNA sequence coding for human CNTF was placed under control of an regulatable promoter in the expression vector pJU1003 and transformed into Escherichia coli strain BL21(DE3). Induction of expression in cultures of this transformant led to the accumulation of approx. 25 mg/l per A600 unit of human CNTF. CNTF was purified to homogeneity from cell lysates via anion-exchange, cation-exchange and Zn(2+)-affinity chromatography. Purified CNTF contained less than 0.1% contaminating E. coli proteins, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis and reversed-phase high-pressure liquid chromatography (HPLC). The protein exhibited an ultraviolet absorption maximum at 279 nm with a calculated extinction coefficient of A1%(279) = 9.0. Peptide map and amino acid sequence analyses confirmed that the expressed protein has the amino acid sequence expected for human CNTF, except for the absence of the amino-terminal methionine. High-purified recombinant human CNTF supported the survival of chick embryo parasympathetic, sympathetic and sensory neurons in culture at low picomolar concentrations. These results indicate that the biological activities previously ascribed to impure CNTF preparations indeed reside in one molecule.
Asunto(s)
Factores de Crecimiento Nervioso/biosíntesis , Proteínas del Tejido Nervioso/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Embrión de Pollo , Cromatografía Líquida de Alta Presión , Factor Neurotrófico Ciliar , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Expresión Génica/fisiología , Humanos , Datos de Secuencia Molecular , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/aislamiento & purificación , Factores de Crecimiento Nervioso/farmacología , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/farmacología , Neuronas/efectos de los fármacos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
Ciliary neurotrophic factor (CNTF) is one of a small number of proteins with neurotrophic activities distinct from nerve growth factor (NGF). CNTF has now been purified and cloned and the primary structure of CNTF from rabbit sciatic nerve has been determined. Biologically active CNTF has been transiently expressed from a rabbit complementary DNA clone. CNTF is a neural effector without significant sequence homologies to any previously reported protein.
Asunto(s)
Factores de Crecimiento Nervioso/genética , Proteínas del Tejido Nervioso/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Factor Neurotrófico Ciliar , Clonación Molecular , ADN/genética , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/aislamiento & purificación , Conejos , Proteínas Recombinantes/biosíntesis , Nervio Ciático/metabolismo , TransfecciónRESUMEN
We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter.
Asunto(s)
Drosophila melanogaster/genética , Proteínas del Ojo/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Cloranfenicol O-Acetiltransferasa/genética , Proteínas de Unión al ADN/genética , Desoxirribonucleasa I , Elementos de Facilitación Genéticos , Mutación , Oligonucleótidos/genética , Regiones Promotoras Genéticas , Opsinas de Bastones , Transformación GenéticaRESUMEN
We have analyzed the cis-acting regulatory sequences of the Drosophila melanogaster Rh2 gene that encodes the protein component of a rhodopsin which is expressed in ocellar photoreceptor cells. DNA fragments containing the start point of transcription of the Rh2 gene were fused to either the Escherichia coli chloramphenicol acetyltransferase (CAT) or lacZ (beta-galactosidase) genes and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the Rh2 gene to confer upon the indicator genes the Rh2 pattern of expression. Fragments containing between 4.3 kb and 183 bp upstream of the start of transcription plus the first 32 bp of the 5'-untranslated leader were found to result in nearly identical levels of head-specific CAT expression. Deletion of Rh2 sequences distal to position -112 bp resulted in loss of detectable CAT expression from these Rh2/CAT fusion constructs. We have, therefore, defined a region essential for head-specific expression of the Rh2 gene to a region extending from -183 to -112. We have determined the DNA sequence of the Rh2 promoter from -448 to +32 and have found an 11-bp sequence which is also present in the upstream flanking sequences of two other photoreceptor-specific genes (ninaE and ninaC). By histochemical staining of beta-galactosidase expressed under the control of the Rh2 promoter and by analyzing the effect of the ocelliless mutation on the expression of an Rh2/CAT fusion gene, we have been able to demonstrate that this promoter is active in ocelli.
Asunto(s)
Drosophila melanogaster/genética , Proteínas del Ojo/genética , Genes , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Hibridación de Ácido Nucleico , Secuencias Reguladoras de Ácidos Nucleicos , Opsinas de BastonesRESUMEN
We have used P-element-mediated transformation to introduce the cloned Rh1 rhodopsin gene into the germ line of Drosophila and fully rescue the visual phenotype of mutant ninaE flies. A transcriptional fusion between the ninaE promoter and the structural gene for a minor opsin (Rh2) that is not normally expressed in the R1-R6 photoreceptor cells was used to demonstrate that Rh2 rhodopsin can photoactivate the R1-R6 transduction cascade, but with different spectral sensitivity. In addition, we show that two mutants that specifically affect the R1-R6 cells, ninaA and rdgB, do not directly affect expression of the ninaE gene.
Asunto(s)
Drosophila/genética , Proteínas del Ojo/genética , Células Fotorreceptoras/metabolismo , Pigmentos Retinianos/genética , Pigmentos Retinianos/fisiología , Rodopsina/genética , Animales , Clonación Molecular , ADN/genética , Proteínas del Ojo/fisiología , Regulación de la Expresión Génica , Mutación , Fenotipo , Regiones Promotoras Genéticas , Opsinas de Bastones , Transcripción Genética , Transfección , Transformación GenéticaRESUMEN
We have analyzed the cis-acting regulatory sequences of the ninaE gene. This gene encodes the major Drosophila melanogaster opsin, the protein component of the primary chromophore of photo-receptor cells R1-R6 of the adult eye. DNA fragments containing the start point of transcription of the ninaE gene were fused to either the Escherichia coli chloramphenicol acetyltransferase or lacZ (beta-galactosidase) gene and introduced into the Drosophila germline by P-element-mediated transformation. Expression of the E. coli genes was then used to assay the ability of various sequences from the ninaE gene to confer the ninaE pattern of expression. Fragments containing between 2.8 kb and 215 bp of the sequences upstream of the start of transcription plus the first 67 bp of the untranslated leader were able to direct nearly wild-type expression. We have identified three separable control regions in the ninaE promoter. The first, which has the properties of an enhancer element, is located between nucleotides -501 and -219. The removal of this sequence had little effect on promoter function; this sequence appears to be redundant. However, it appears to be able to substitute for the second control region which is located between nucleotides -215 and -162, and which also affects the level of output from this promoter. Removal of these two control regions resulted in a 30-fold decrease in expression; however tissue specificity was not affected. The third control region, located downstream from nucleotide -120, appears to be absolutely necessary for promoter function in the absence of the first two regulatory sequences. Examination of larvae containing fusion genes expressing beta-galactosidase suggests that the ninaE gene is also expressed in a subset of cells in the larval photoreceptor organ.