Guanylate cyclase (GC) in the retina catalyzes the formation of cGMP, which plays an important role in visual transduction. Although the retinal enzyme has not yet been sequenced, three membrane bound rat GCs (GC-A and GC-B from brain and GC-C from intestine) have been recently characterized by cDNA cloning. Since we have shown earlier that ANF can activate retinal GC, we have investigated the expression of mRNA in the rat retina for GC-A, the isozyme shown to possess ANF receptor activity. A technique based on polymerase chain reaction (PCR) was developed for this purpose. Messenger RNA fractions were isolated from rat retina and converted to cDNA using reverse transcriptase. The cDNA preparation was used as template for PCR using oligonucleotide primers designed from the reported cDNA sequence of brain GC-A. A approximately 680 bp product was amplified when 5'-GGG AAC CTC AAG TCA TCC AAC-3' and 5'-AAA GCC CAC AAT ATC ACT GAA-3' were used as upstream and downstream primers, respectively. A product of identical size was also obtained when the experiment was performed with brain cDNA preparation as a positive control. The identity of the PCR product was verified by DNA sequence analysis. In order to do this, the PCR reaction was performed using the above primers except that one of them was biotinylated at the 5'end. The PCR product was purified by gel electrophoresis and treated with magnetic beads containing streptavidin. The bound DNA fragment was treated with NaOH to separate the strands. The strand bound to the magnetic beads and the free strand were separated and subjected to dideoxy sequencing. Sequence analysis of PCR products from both brain and retina showed 100% sequence homology to that reported for GC-A DNA. Thus, it appears that mRNA for GC-A (or a closely related form) is expressed in the rat retina.
