Ectopic expression of ultraviolet-rhodopsins in the blue photoreceptor cells of Drosophila: visual physiology and photochemistry of transgenic animals.

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.

Expression and characterization of recombinant human ciliary neurotrophic factor from Escherichia coli.

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.

Purification, cloning, and expression of ciliary neurotrophic factor (CNTF).

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.

Definition of cis-acting elements regulating expression of the Drosophila melanogaster ninaE opsin gene by oligonucleotide-directed mutagenesis.

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.

Analysis of the promoter of the Rh2 opsin gene in Drosophila melanogaster.

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.

Ectopic expression of a minor Drosophila opsin in the major photoreceptor cell class: distinguishing the role of primary receptor and cellular context.

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.

Analysis of the promoter of the ninaE opsin gene in Drosophila melanogaster.

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.