Vol. 182, No. 2, 1992 January 31, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS Pages 851-857

EXPRESSION OF GUANYlATE CYClASE-A mRNA IN THE RAT RETINA: DETECTION USING POLYMERASE CHAIN REACTION R. Krishnan Kutty*, R. Theodore

Fletcher+,

Gerald J. Chader+ , and Gopal Krishna*

*Section on Drug-Tissue Interaction, Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute and +Laboratory of Retinal Cell Biology and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD 20892 Received

December

18,

1991

SUMMARY: A technique based on RNA-PCR was successfully employed for the detection of guanylate cyclase-A (GC-A) mRNA in the rat retina. Three sets of primers designed from the published cDNA sequence of rat brain guanylate cyclaseA (GC-A) produced amplification products of expected sizes from the retina as well as brain. Analysis of retinal PCR products yielded a 970 bp sequence, which showed 100% homology to the cDNA sequence of GC-A (2343-3312 bp region). Northern blot analysis was not very sensitive for the detection of GC-A mRNA in the retina. The results indicate that the mRNA for GC-A (or a closely related form) is probably expressed in the retina, but at a lower level than that found in the brain. 0 1992

Press,

Academic

Inc.

Cyclic GMP plays a very important role as a mediator in visual transduction (1). Both synthesis and degradation photoreceptor

appear to control its steady state level in rod

cells in the retina (1,2). Large amounts of cyclic GMP are present in

the rod outer segments of bovine retina in combination guanylate

cyclase, the enzyme

phosphodiesterase,

responsible

with high activities of both

for synthesis

the enzyme involved in the degradation

of cyclic GMP, and of cyclic GMP (2,3).

Retinal guanylate cyclase has been recently solubilized and purified, but its amino acid sequence

has not been established

(4-6). However, a membrane

guanylate

cyclase (GC-A), which also can function as an atrial natriuretic factor (ANF) receptor, has been well characterized indications

from rat brain by cDNA cloning (7). There are several

that retina also may contain an ANF receptor

cyclase. Both ANF and high affinity ANF receptors

containing

guanylate

have been found in rat retina

(8,9). An antibody preparation against ANF receptor/guanylate

cyclase isolated from

adrenal tumor cells has been reported to react with retinal preparations

from rat,

mouse and chicken (10). Moreover, ANF has been shown to activate guanylate

851

0006-291X/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol.

182, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cyclase in bovine rod outer segments (11). It seemed possible that the ANF receptor activity detected in the retina could be that of GC-A. In support of this view, we now report that we are able to detect the presence of GC-A mRNA in rat retina by a technique

based on the polymerase

MATERIALS

chain reaction.

AND METHODS

RNA-PCR: Poly(A)+ RNA fractions were isolated from retina and brain obtained from Sprague-Dawley rats (male, 125-150 g) and reverse transcribed in the presence of oligo(dT) (cDNA cycle kit, Invitrogen, San Diego, CA). The cDNAs were used as templates for PCR (12). Several sense and antisense primer sets were designed from the cDNA sequence reported for GC-A (7). Three sets of primers, (a) 5’-biotinyl GGGAACCTCAAGTCATCCAAC and 5’AAAGCCCACAATATCACTGAA (b) 5’-ggggaattcCATCCTGGACAACCTGC and 5’ggggaattcTAGGTCCGAACCTTGCC (contain added EcoRl sites, shown in lower case letters) (c) S-CGACCCTCCATGGATCatatGAGCCACCTGGAGGAA (sequence shown in lower case letters was modified from original sequence to contain an Ndel site) and 5’AAACTTAGGTGTCCCCTGCAGTCCCCACCATCTCCA were found to be useful. The oligonucleotides were synthesized on a PCR-MATE DNA synthesizer and were purified using oligonucleotide purification cartridges (Applied Biosystem, Foster City, CA). A reaction mixture (100 PI), consisting of cDNA preparation (equivalent to 0.2 clg of poly(A)‘, 50 mM Tris-HCI, pH 9.0, 20 mM ammonium sulfate, 1.5 mM MgCI,, sense primer (1 PM), antisense primer (1 PM), and 5 units of Amplitaq (Perkin Elmer Cetus, Norwalk, CT), was layered with 70 HI of mineral oil and subjected to 35 cycles at 95’ C for 1 min, 55’ C for 1 min, and 72’ C for 2 min. The reaction mixture was then kept at 72’ C for another 10 min. An aliquot (20 ~1) was subjected to Agarose gel electrophoresis and the amplification products were visualized by ethidium bromide staining. DNA SEQUENCING: Single strand DNA template for sequencing was prepared as described by Merril and Mitchell (13). PCR was performed with primer set a, as described above. The PCR product was gel purified and eluted with Gene Clean (Bio 101, La Jolla, CA). The biotinylated and the non-biotinylated strands were separated using streptavidin attached to magnetic beads (Dynal, Inc., Great Neck, NY). Sequencing was performed by the dideoxy chain termination method using Sequenase 2.0 (USB, Cleveland, OH). A technique based on direct sequencing of PCR product also was employed (14). The purified PCR products were sequenced using dsDNA Cycle Sequencing System (GIBCO BRL, Gaithersburg, MD). NORTHERN BLOT ANALYSIS: Poly(A)+ RNA preparations (5 pg) from retina or brain were subjected to Agarose gel (1.2%) electrophoresis in the presence of formaldehyde (1.2%) and transferred onto an lmmobilon N membrane (Millipore, Bedford, MA). An RNA-PCR product generated from the rat retina was used as the hybridization probe. It was gel purified and labelled with =P using the random priming technique (15). The hybridization was carried out using this probe at 42-C for 16 h in solution containing dimethylformamide (50%) and the membrane was washed under stringent conditions (16). The membrane was then exposed to an XOmat AR film (Kodak, Rochester, NY). 852

Vol. 182, No. 2, 1992

BIOCHEMICALAND BIOPHYSICALRESEARCHCOMMUNICATIONS

RESULTS AND DISCUSSION

Three sets of primers designed from rat brain GC-A cDNA (7) were found to produce amplification

products

of expected sizes from both retina and brain by

RNA-PCR (Fig. 1). The primer pairs a, b, and c were designed to amplify 2190-2870, 2648-3361,

and 2505-3588 bp regions, respectively, of rat brain GC-A cDNA. The

PCR products obtained from retina were identical in sizes to those from brain. Thus, retina like brain may express GC-A mRNA. The possibility that the amplification was from genomic DNA, which may contaminate

the mRNA preparation,

was ruled out

since the sense and antisense primers in a particular set were parts of different exons (17).

R

B

M

R

B

M

R

- 1353 b - 1078 - 1353 b - 1078 - 872

- 872 -

B

M

- 1353 b - 1078 - 872 -

- 603 - 310

- 310

- 310

Figure 1 RNA-PCR analysis of the expression of GC-A mRNA in rat retina and brain. Poly(A)+ RNA preparations from retina or brain were reverse transcribed and used as templates for PCR. The PCR products were separated by electrophoresis on 2% Agarose gel, stained with ethidium bromide, and photographed under UV light. The amplification was performed with the following sets of primers: (a) 5’-Biotinyl GGGAACCTCAAGTCATCCAAC & 5’-AAAGCCCACAATATCACTGAA (b) 5’-GGGGAATTCCATCCTGGACAACCTGC & 5’GGGGAAT-TCTAGGTCCGAACCTTGCC (c) 5’-CGACCCTCCATGGATCATATGAGCCACCTGGAGGAA & 5’-AAACT-TAGGTGTCCCCTGCAGTCCCCACCATCTCCA R, Retina; 6, Brain; M, Molecular size markers (Hae Ill fragments of #X RF174). 853

Vol.

182, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

10 30 GCTTCGCCACCTGCCCGTGGCTCCCAAGCTGGGGATGGGGATGTGTACAGCTTTGGTATCATCCTG 70 90 CAGGAGATTGCCCTAAGAAGTGGGGTCTTCTATGTGGAAG 130 150 GAGATCATTGAGCGTGTGACTCGGGGTGAGCAGCCCCCATTCCGACCCTCCATGGATCTG 190 210 CAGAGCCACCTGGAGGAACTGGGGCAGCTGATGCAGCGGTGCTGGGCAGAGGACCCACAG 250 270 GAGCGGCCACCCTTTCAGCAGATCCGCCTGGCGCTGCGCACAGC 310 330 AGCAACATCCTGGACAACCTGCTGTCACGCATGGAGCAGTATGCT~C~CCTGGAGG~ 370 390 CTGGTAGAGGAGAGAACACAGCTTATCTGGAGGAGAAGCGCTGAGGCCTTGCTT 430 450 TACCAGATTCTGCCTCACTCCGTGGCTGAGCAGCTGAAGAGCT 490 510 GAGGCCTTTGATAGTGTTACCATCTACTTCAGTGATATTGTGGGCTTTACAGCTCTTTCA 550 570 GCAGAAAGCACACCCATGCAGGTGGTGACTCTGCTCAATGATCTGTACACCTGTTTTGAT 610 630 GCTGTCATAGACAACTTTGATGTGTACAAGGTGGTGGAGACCATTGGTGATGCTTACATGGTG 670 690 GTGTCAGGGCTCCCAGTGCGGAATGGACAACTCCACGCCACGCCCGAGAGGTGGCCCG~TGGCA 730 750 CTTGCACTACTGGATGCTGTGCGCTCCTTCCGCATCCGCCATAGGCCCCAGG~CAGCTG 790 810 CGCTTGCGCATTGGCATCCACACAGGTCCTGTGTGTGCTGGTGTGGTAGGGCT~GATG 850 870 CCCCGATACTGCCTCTTTGGAGACACAGTCAACACAGCTTTGGA 910 930 GAAGCCCTCAAGATCCACTTGTCTTCAGAGACCAAGGCTGTGGT 970 TTCGAGCTGG

Fiaure 2 Partial sequence of rat retinal GC-A described in Materials and Methods.

cDNA. PCR products

50 110 170 230 290 350 410 470 530 590 650 710 770 830 890 950

were

sequenced

To establish the identity of the retinal GC-A mRNA, we sequenced

as

selected

PCR products. For this purpose, the PCR products obtained from retina with primer sets a and b were gel purified and subjected described

to DNA sequence

in Materials and Methods. A fourth PCR product spanning

analysis as the entire

region (2190-3354 bp) of GC-A amplified by primer sets a and b was also generated from

the

retina

using

GAACClTGCCllTGCCClTCA

5’-biotinyl

GGGAACCTCAAGTCATCCAAC

and

5’-

for this purpose (data not shown). We sequenced

970 bases in this manner (Fig. 2). Comparison

of this sequence

information

by

FastA program based on the method of Pearson and Lipman (18) showed 100% homology to the 23433312

bp region of rat brain GC-A (7). The homology to cDNA

sequences reported for other forms of guanylate cyclases (GC) from rat were only: 74% to 2185-3124 bp of brain GC-B (19) 59% to 21842941

bp of GC-C (20), 57%

to 1135-1800 bp of kidney GC (21), 57% to 1185-1744 bp of 70 kD subunits ofdung soluble GC and 58% to 1744-2354 bp of lung soluble GC 80 kD subunit (22). Thus, GC-A mRNA or a closely related species seems to be present in the retina. 854

Vol.

182, No. 2, 1992

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

4kb

Fiaure 3 Northern blot analysis of the expression of GC-A mRNA in rat retina and brain. Poly(A)+ fractions (5 pg) from retina and brain were subjected to Northern Blot analysis using PCR product corresponding to 2648-3361 bp region of GC-A as hybridization probe. R, retina; B, Brain.

The cDNA sequence for GC-A has been reported to contain codons for an ANF binding domain at the 5’-end, a kinase homologous

region in the middle, and

a guanylate cyclase catalytic domain at the 3’-end (7). The region amplified from the retina by RNA-PCR is from the areas encompassing

the guanylate

cyclase and

kinase domains. Several primer sets were prepared to amplify the 5’- end region of GC-A cDNA coding the ANF domain from retina by this technique. primers did not produce any detectable amplification

However, these

products from retina or brain

by RNA-PM. The mRNA for GC-A in rat retina was barely detectable technique.

A northern blot of poly(A)’

by the northern blot

RNA fractions from retina and brain with a

retinal PCR product (2648-3361 bp region) as the hybridization

probe showed only

a weak signal for retina (Fig. 3). In contrast, a very strong band around 4 kb was obtained for brain. The molecular size of the band was similar to that reported for rat GC-A by Schulz @ al. (19).

In conclusion,

we have shown the presence of

mRNA for guanylate cyclase-A in the rat retina. Previous studies (19) have reported that the northern blotting technique using total RNA produces only a weak signal for GC-A from brain. Our study shows that use of a poly (A)+ RNA fraction reveals a 855

Vol.

182,

No.

2,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

strong signal in the brain but a comparatively

RESEARCH

COMMUNICATIONS

weak one in retina. Thus, our

approach based on RNA-PCR backed up by northern blot data appears to provide a sensitive method by which levels of expression of GC-A mRNA can be determined in any tissue and compared

to the level of expression

in brain. The need for a

similar approach is also supported by the fact that an in situ hybridization technique, although sensitive enough to detect the equivalent of GC-A mRNA in primate brain, failed to detect it in several tissues including testis, lung, and liver (23). Our study has found that, even though the retina is part of the central nervous system, its potential for GC-A expression is much lower than that of brain. It will be interesting, however, to determine whether the expression of GC-A mRNA is confined to any specific retinal cell type and thus possibly be linked to a particular step in the visual process. ACKNOWLEDGMENT We acknowledge NCI Advanced Scientific Computing Laboratory of the Frederick Cancer Research Development Center for the support received for DNA sequence analysis. REFERENCES 1. 2. 3. 4. 5. 6. 7. a. 9. 10. 11. 12. 13. 14. 15. 16.

Stryer, L. (1991) J. Biol. Chem 266, 10711-10714 Krishna, G., Krishnan, N., Fletcher, R.T., and Chader, G. (1976) J. Neurochem. 27, 717-722 Krishnan, N., Fletcher, R.T., Chader, G.J., and Krishna, G (1976) Biochim. Biophys. Acta 523, 506515 Hakki,S. and Sitaramayya, A. (1990) Biochemistry 29, 1066-1094 Hayashi,F. and Yamazaki, A. (1991) Proc. Natl. Acad. Sci. USA 88, 47464750 Koch, K.-W. (1991) J. Biol. Chem. 266, 8634-6637 Chinkers, M., Garbers, D.L., Chang, M.-S., Lowe, D.G., Chin, H., Goeddel, D.V., and Schulz, S. (1969) Nature 338, 76-83 Stone, R.A., Glembotski, C.C. (1966) Biochem. Biophys. Res. Commun. 134, 1022-1028 Fernandez-Durango, R., Sanchez, D., Gutkowska, J., Carrier, F., and Fernandez-Cruz, A. (1969) Life Sci. 44, 1637-1646 Cooper, N.G.F., Fedinec, A.A., and Sharma, R.K. (1969) Invest. Ophthalmol. Visual Sci. 30 (3 suppl.) 295 Krishna, G., Marala, R.B., and Sharma, R.K. 14th International Congress of Biochemistry, Prague, Czechoslovakia, July 1O-l 5, 1966 Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., and Ehrlich, H.A. (1968) Science 239, 467-491 Mitchell, L.G. and Merril, C.R. (1989) Anal. Biochem. 178, 239-242 Murray, V. (1969) Nucleic Acids Res. 17, 6689 Feinberg, A.P. and Vogelstein, B. (1963) Anal. Biochem. 132, 6-13 Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular cloning. A laboratory manual, pp 7.37-7.52, Cold Spring Harbor Laboratory, New York. 856

Vol.

182,

No.

2,

1992

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

17. Yamaguchi, M., Rutledge, L.J., and Garbers, D.L. (1990) J. Biol. Chem. 265, 20414-20420 18. Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85, 2444-2448 19. Schulz, S., Singh, S., Bellet, R.A., Singh, G., Tubb, D.J., Chin, H., and Garbers, D.L. (1989) Cell 58, 1155-1162 20. Schulz, S., Green, C.K., Yuen, P.S.T., and Garbers, D.L. (1990) Cell 63,941-948 21. Yuen, P.S.T., Potter, L.R., and Garbers, D.L. (1990) Biochemistry 29, 1087210878 22. Nakane, M., Arai, K., Saheki, S., Kuno, T., Buechler, W., and Murad, F. (1990) J. Biol. Chem. 265, 16841-16845 23. Wilcox, J.N., Augustine, A., Goeddel, D.V., and Lowe, D.G. (1991) Mol. Cell. Biol. 11, 3454-3462

857