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Several photoreceptor-specific genes are actively transcribed in Y79 retinoblastoma (Rb) cells, making this cell line potentially useful for the study of photoreceptor metabolism. The utility of these cells is limited because commonly used methods of gene transfer into Y79 cells are inefficient and lack reproducibility. In contrast, we found that adenovirus transduction yields high efficiency gene transfer, however, generation of recombinant adenovirus is lengthy and time consuming. Here, we show that adenofection, a method of coupling adenovirus to plasmid DNA for improved gene transfer, is efficient for gene delivery into Y79 cells. Recombinant adenovirus expressing bacterial lacZ was noncovalently complexed to GFP or luciferase reporter plasmids with polyethylenimine. Efficiency of plasmid gene delivery was determined by monitoring GFP fluorescence. For comparison, calcium phosphate-mediated or cationic lipid transfection was performed in Y79 and HEK293 cells using standard protocols. The adenofection protocol yielded significantly higher efficiencies in Y79 cells than that obtained in these cells with calcium phosphate or cationic lipids. This method will facilitate any experiment requiring reproducible high-level gene transfer. Here, we show that adenofection can be used to analyze activity of the rod photoreceptor PDE6A gene promoter.

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the alpha-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an approximately 500 bp sequence upstream of the mouse Pde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putative cis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed footprints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.

The cDNA for casein kinase 1 (CK1) ofPlasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of ∼37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1–7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1–7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring ≫ schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a key enzyme in the phototransduction cascade. Lines of transgenic mice were established to determine the spatial expression pattern directed by an upstream fragment of the PDE6A gene. RT-PCR analysis showed that three of four lines analyzed transcribed the transgene predominantly in the retina and weakly in brain. The line showing no transgene transcription did not contain an intact transgene. Transcription of the transgene in the three lines was found in retina and weakly in brain, but not in heart, kidney, liver, or lung. Transcripts were most predominant in the photoreceptors of the retina. These results demonstrate that a short segment of the upstream region of the PDE6A gene comprises a functional promoter that is most active in photoreceptors.