Isolation of genes encoding photoreceptor-specific proteins by immunoscreening with antibodies directed against purified blowfly rhabdoms.
The proteins which perform and regulate key steps in phototransduction are assumed to be localized in the rhabdomeric membrane of invertebrate photoreceptor cells. We have employed antibodies raised against rhabdoms purified from blowfly eyes in order to isolate copy deoxyribonucleic acid (cDNA) clones encoding proteins that are required in the phototransduction machinery. By immunoscreening a Calliphora retinal cDNA library, we obtained clones of genes coding for five different proteins. As revealed by partial cDNA sequence analysis, three of these genes represent the Calliphora homologs of Drosophila trp, inaC and InaD, while the other two displayed no homology to known genes. Northern blot analysis confirmed that trp, inaC and InaD transcripts were present in RNA isolated from the retina, but not in RNA isolated from brain or thorax. Specific antibodies directed against trp, inaC and InaD protein were raised using recombinantly expressed proteins or synthetic peptides. Western blot analyses revealed that trp, inaC and InaD protein are specifically associated with the rhabdomeral photoreceptor membrane. Extraction of membranes with buffers of different ionic strengths suggested that the trp gene product is an integral membrane protein, whilst the inaC and InaD gene products are peripherally bound membrane proteins. This demonstrates that the immunoscreening approach used here can be successfully applied to isolate genes that code for either integral or peripheral photoreceptor membrane proteins.
Huber
A
A
Institute of Zoology I, University of Karlsruhe, Germany.
Sander
P
P
Wolfrum
U
U
Groell
C
C
Gerdon
G
G
Paulsen
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D015220:Calcium Channels / Q000235:genetics; D018076:DNA, Complementary; D004175:Diptera / Q000378:metabolism*; D029721:Drosophila Proteins; D005136:Eye Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D007301:Insect Hormones / Q000235:genetics; D019476:Insect Proteins; D008297:Male; D008969:Molecular Sequence Data; D017956:Photoreceptor Cells, Invertebrate / Q000378:metabolism*; D012333:RNA, Messenger; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D050051:Transient Receptor Potential Channels
D015220:Calcium Channels; D018076:DNA, Complementary; D029721:Drosophila Proteins; D005136:Eye Proteins; D007301:Insect Hormones; D019476:Insect Proteins; D012333:RNA, Messenger; D050051:Transient Receptor Potential Channels; C091568:inaD protein, Drosophila; C063169:trp protein, Drosophila