Molecular and biochemical analyses of iodopsin in rd chick retina.
OBJECTIVE
The results of previous immunocytochemical and electrophysiological studies of retinas of rd (retinal degeneration) chicks suggest that the iodopsin cone visual pigment may be defective in this mutant. The goal of this study was to determine if the primary structure and synthesis of this protein is normal in this animal model of inherited retinal degeneration.


METHODS
Northern cDNA sequence and western analyses were used to study rd/rd iodopsin. cDNAs encoding rd/rd iodopsin were obtained by screening an rd/rd cDNA retinal expression library and by reverse transcription PCR. Western blots were probed with either R4 or COS-1, two different monoclonal antibodies that have been shown to specifically recognize chicken iodopsin.


RESULTS
Hybridization of the +/+, +/rd, and rd/rd poly(A)+ RNA with an iodopsin cDNA probe revealed the presence of a single 1.5 kb band in each of the samples, all of which were labeled with equal intensity. No significant differences were found between the published nucleic acid sequences for normal chicken iodopsin cDNA and that determined for rd/rd iodopsin cDNA. Antibody-dependent differences in the staining intensity of the 34 kDa band containing iodopsin were observed on western blots of +/+, +/rd, and rd/rd retinal protein. R4 stained the 34 kDa band in each sample with equal intensity. COS-1 labeling of the 34 kDa band in the rd/rd sample was less intense than that observed in the +/+ and +/rd samples.


CONCLUSIONS
Based on the results of the cDNA sequence and northern blot experiments, the authors conclude that the gene encoding iodopsin and transcription of this gene are normal in the rd mutant. The results of the western blot analyses of rd/rd iodopsin suggest that post-translational processing of iodopsin may be abnormal in this mutant.
Semple-Rowland
S L
SL
Department of Neuroscience, University of Florida College of Medicine, Gainesville 32610-0244.
Green
D A
DA
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002642:Chick Embryo; D004247:DNA / Q000032:analysis; D015342:DNA Probes; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D014176:Protein Biosynthesis; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism*; D012162:Retinal Degeneration / Q000235:genetics* / Q000378:metabolism; D012168:Retinal Pigments / Q000235:genetics* / Q000378:metabolism; D017299:Rod Opsins; D014158:Transcription, Genetic
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D012333:RNA, Messenger; D012168:Retinal Pigments; D017299:Rod Opsins; C034941:iodopsin; D012313:RNA; D004247:DNA