Gene. 88 (1990) 227-232 Elsevier

227

GENE03~6

Isolation and characterization of eDNA encoding the gamma-subunit of eGMP phosphodiesterase in human retina (Recombinant DNA; photoreceptor degeneration; molecular cloning; sequencing mRNA; deduced amino acid sequence; chromosome assignment; somatic cell hybrids; in situ hybridization)

Narendra TuteJa ae, Michael Danciger"b, Ivan IOisak d, Renu Tuteja *, George hmna c, T. Mohandas ~, Robert S. Simrkes a and Debora B. Farber" ° Jules Stein Eye Institute and Department of Ophthalmology, UCLA School of Medicine, Los Angeles, CA 90024 (U.S.A) Tel (213)206-6800, b Department of Biology, Loyola Marymount University. Los Angeles, CA 90045 (U.S.A.) Tel. (213)642-3548. ¢ Bascom Pabner Eye Imtitu~e. University of Miami School of Medicine, Miami, FL 33 ! 36 (U.S.A.) Tel. (305)326-603 !, d Department of Medicine. UCLA School of Medicine. Los Angeles, CA 90024 (U.S.A.) Tel. (213)825-5720.and e Division of Medical Oenetics, Harbor/UCLA Medical Center. Torrance. CA 90509 (U.S.A) Tel. (213)533.3764 Received by J. Piatigursky: I August 1989 Revised: 2 December 1989 Accepted: 7 December 1989

SUMMARY

Cyclic GMP-phosphodiesterase (cGMP-PDE) plays a key role in the normal functioning of retinal rod photoreceptor cells. The enzyme is composed of 0c- and/~-catalytic subunits which are inhibited by two identical ?-subunits. A eDNA encoding the y-subunits (PDE~,) from human retina has been cloned and sequenced. The 1012-bp eDNA has a coding region of 261 bp which is highly homologous to those ofthe PDE7cDNAs from bovine and mouse retinas. Comparison of the deduced amino acid sequences of the proteins from the three species indicates that PDE7 has been very well conserved through evolution. The mRNA encoded by the cloned eDNA is 1.0 kb long, is similar in size to the corresponding mRNAs from mouse, dog and bovine retinas and is not detected in ground squirrel retina. The PDEG gene has been assigned to human chromosome 17, probably in the region q21.1.

INTRODUCTION

The 7-subunit of cGMP-phosphodiesterase (PDET) has been a target of our interest for two main reasons: (1)it is Correspondence to: Dr. D.B. Farber, Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, CA 90024-1771(U.S.A.) Tel. (213) 2066800; Fax (213)206-3652. * Current address: International Centre for Genetic Engineering and Bioteehnology, Trieste (Italy) Tel. 39-40-226-0326. Abbreviations: aa, amino acid(a); bp, base pair(s); eDNA, DNA complementary to RNA; cGMP-PDE, cyclic GMP-phosphodiesterase; DEPC, diethylpyrocarbonate; kb, kilobase(s) or 1000 bp; at, nucleotide(s); oligo, oligodeoxyribonueleotide; PDE?, ?-subunit ofcGMP-PDE; PDEG,gene encoding PDEy; rd, retinal degeneration; RP, retinitis pigmentosa; SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI/15 mM Naj" citrate, pH 7.6; UWGCG, University of Winsconsin Genetics Computer Group. 0378-1119/90/S03.50© 1990Elsevier SciencePublishers B.V.(BiomedicalDivision)

a very important component of the phototransduction process of retinal rod cells, since with its inhibitory properties it regulates the activity of cGMP-PDE (Hurley and Stryer, 1982); and (2) the concentration of PDE~, may be responsible for the decreased activity of the enzyme in some inherited retinal degenerations in animals, such as those of the Irish setter (Agnirre et al., 1978) and the collie (Woodford et al., 1982) dogs, and even in some types of human RP (Farber et al.; 1987). We have already studied and characterized the PDE7 eDNA in the disease of rd mouse (Tuteja et al., 1989) and have demonstrated that it does not constitute the primary cause of the rd disorder (Danciger et al., 1989). However, in human RP the lesion that causes a decrease in cGMP-PDE activity may have a different origin than that of the rd mouse. Therefore, we have cloned and characterized the normal human PDE~,

228 eDNA in order to compare it to that of retinas affected with RP.

MATERIALS AND METHODS

(a) eDNA library preparation and screening A human retinal eDNA library was prepared i n / g t l 1 from retinal poly(A) + RNA as described by Huynh et al. (1985). The library was screened with a mouse PDE7 eDNA that we had isolated previously (Tuteja and Farber, 1988). Eighteen of the 40000 clones plated hybridized with the PDEy eDNA (0.48 kb) labelled with [~-32p]dCTP (3000 Ci/mmol) by the random priming method (Feinberg and Vogelstein, 1983). Specific activity of the probe was approximately 5 x l0 s cpm/pg for all experiments. The conditions for prehybridization, hybridization and washing were the same as described in the legend of Fig. 3.

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CCCCTGCTGAAGTCCAGACCCTCCCCCTCCTGCCCACTMGCTkAACCCTGCTCAGGATTCC

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TGTTGAGGAGATGACCTCCCTAGCCCCAGATGGGACCTGGACACCAGGATGGGACTGCAACC

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TCAGGTCTCCCCCTACATATTAATACCAGTCACCAGGAQOCCACCACCTCCCTCTAGGATGC

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CCCCTCAGGGTGGCCAGGCCCTOCTCAACATCTGGAGACACAGGCCCACCCCTCAG'i~CTGC

519

CCACAGAGAGGCTTGGTCGGTCTCCACTCCCAGGGAGAACGGGAAGTGGACCCCAGCCCG(]G

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AGCCTGCTGGACCCCAGATCGTCCCCTCCTCCCAGCTGGAAAGCTAGGGCAGGTCTCCCCAG

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AGTGCTTCTGCACCCCAGCCCCCTGTCCTGCCTGTAAGGGGATACAGAOAAGCTCCCCGTCT

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CTC-CATCCCTTCCCAOGGGGGTGCCCTrAGTTTGGACATGCTGGGTAGCAGGACTCCAGGGG

767

GTGCACGGTGAGCAGMGAGGCCCCAAGCTCATCACACCAGGGGGCCATCCTTCTCAATACA

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GCCCGCCCTTGOAGTCCCTATTTCAAAATAAAATTAGTGTGTCCTTGCAAAAAAAAAAAAAA

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(b) Cloning and sequencing The positive clones were isolated and plaque purified. The 1.0-kb insert of one ofthese clones was subcloned into the EcoRl site of the plasmid pBluescript SK + (Stratagene, San Diego, CA) and sequenced by the dideoxy chain termination method (Sanger et al., 1977)using [~-3sS]thiodATP and Sequenase (USB) or a T7 sequencing kit (Pharmacia). T3, T7 and an internal synthetic oligo [an 18-met made from positions 247-264 of the sequence of normal mouse PDE 7 eDNA: CAGTATGGCATCATTTAG (Tuteja and Father, 1988)] were used to determine the entire sequence of one strand of DNA and most of the sequence of the complementary strand. The 18-mer primer was synthesized by the phosphoramidite method on an Applied Biosystems 380A DNA synthesizer (Beaucage and Carnthers, 1981).

RESULTS AND DISCUSSION (a) Sequence analysis of human PDEy eDNA Figure I shows the nt sequence of the isolated full-length eDNA which has 1012 bp with a poly(A) tail of 34 bp at the 3' end following 650 bp of untranslated region, a coding region of 261 bp and a 5' end of 101 bp. The termination codon TAG is in the 262-264 position. The deduced aa sequence of human PDEy is also shown in Fig. 1. The 87 aa correspond to about 9.7 kDa. (b) Nudeotide and amino acid similarities with moese and bovine A comparison of the nt in the coding sequences of human, bovine (Ovchinnikov et al., 1986) and mouse (Tuteja and Farber, 1988) PDEy cDNAs (Fig. 2) showed

Fig. !. The nt sequenceof PDE~,cDNAfromhumanretina and deduced aa sequence.The positionof the nt and of the deducedaa are indicated on the rightmargin.The $'-untranslatedregionis numbered- 10i to - I. The codingregionstarts at nt number !. The stop codonin frame with the reading frame is indicatedby TER..']'be Y-untransintedregion is numberedfollowingthe codingregion. 91.5~o identity between human and bovine and 9 0 ~ identity between human and mouse. Further, the same number of aa can be predicted from the human, bovine and mouse PDE), eDNA coding sequences. According to the deduced aa sequences the aa m7is found to be different in the three proteins: Ala, in human; Met, in bovine; and Ile, in mouse. The Ala s in the human is changed to Gly only in the mouse, and the Phe ~° in the human is changed to lie in both the bovine and mouse PDE7 proteins. Several nt differing from the corresponding nt in the human eDNA, pointed out in Fig. 2, were observed either in the bovine or mouse cDNAs, but they did not cause an aa change. Thus, PDE7 is a remarkably well conserved protein which shows 97.7~ similarity between human and bovine and 96.6~ similarity between human and mouse. As expected, the 5'- and 3'-untranslated regions of human, bovine and mouse PDE7 showed much less similarity than the coding sequences. In the 5' region, human vs bovine had 73.6~ and human vs mouse 63.4~o identity whereas in the Y-untranslated region, the species differences were even more pronounced: human vs. bovine, 61.2~o and human vs, mouse, 50.0~o identity, suggesting that there is no pressure to keep unchanged the areas ofthe gene that are not expressed.

(e) mRNA analysis The lesser similarity in the T-untranslated area also became evident when we prepared a Northern blot of

229 huron ATG AAC GTGGAA CCG CCC AAG OCT GAG TTC COG TCA GCC ACC AGO 0113 GCC GGG bovlem ATO AAC CTG ( ~ CCp~CCC AAO I ~ G A G ~ ] ] CGG TCA GCC A ~ GTG ~ muse .~TGAAOCTG G/U COAl CCC AAG G(:~IGAGI,qI"CCOG "lraa GCC ACC AGG GTG IAI"GIGGG

GGACCTGTCACCCCCAGO MA ~ G CC~~ T ~*A TTr ~ G CAeCO^ CAGACC * ~ GGAC~GTC ^CC CCCA ~ AM r.,~ c d ~ ~ T r r AAGC ~ CGplC~ *CC Ae~ G ~ C~GTC Ad~ccc AGG AAAGGGCCCCm A,*An ' r ~ CAGcGr,r..~ Ace *OG CAG~ C ^AG AGe~ G CCCCCA~ G ~ A GGCOn" C ~ G ~ n-r c~G GACGACAT(: CAG TIC AAG AGC AAG CCC Cq-CI AAG AM GGC G'I~CAA GGG TTT ~ G/(i~GACATC CAGI"rCAAG AGCAAGCCCCO...qAAG AAAC.~ G'IL~CAAGGGITrG(~ G~GACATC CCT GGA ATG GAA GGC CTG GGA ACA GAC ATC ACA GTC ATC TGC CCT TGG GAG GCC CCT GGA ATG GAA GGC CTG G ~ ACA C-=~ATCAC~ GTC Arc TGC CC_TTGG GAG GCC COTGGA ATG GAP,GGC CTG GGA ACA GAC ATC AO~GTC ATC TGC CC~ TGG GAG GCC l"rC AAC CAC CTG GAG CTG CAC GAG CTG GGCCAA TAT GGC ATC Arc TAG "FrOA/~ CAC CI~]GAG CTG CAC GAG CTG GCC C ~ T A T GGG ATC AI~ TAG "l-rC AAC CAC CTG GAG CTG CAC GAG CTG GCC CAG T.qCIGGC ATC AT(; TAG

Fig. 2. Comparison of the nt sequence of the coding region in PDE? cDNAs from human (Fig. i), bovine (Ovchinnikov et al., 1986) and mouse (Tutcja and Farber, 1988) retinas. The nt which are different from those in the human sequence are boxed.

R N A s from ~everal mouse tissues and from retinas o f different species and hybridized them f'u'st to the human and

then to the mouse PDE7 eDNA (Figs. 3a,b). The human cDNA, which has 650bp of 3°-untranslated sequence, hybridized only to a mRNA from human retina (Fig. 3a), whereas the mouse eDNA probe, which has only 100 bp of Y.untranslated region, hybridized to the corresponding message from retinas of most of the different species studied. No hybridization was observed to messages &any other tissues. Depicted in Fig. 3b are the mRNA-cDNA braids from young adult normal mouse (four-week-old) retina, nine-day-old and three-week-old diseased rd mouse l°etina, and from human, dog and bovine retina, all of which 1

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are similar in size. The reduced intensity observed with the three-week-old rdmouse retinal RNA reflects the loss of the photoreceptor cells that occurs in this retina as a result of the inherited disease that alTccts it. The rat message hybridized by the PDETcDNA is smaller than all the other mRNAs and we have not studied this in detail. Interestingly, mRNA from ground squirrel retina gave no signal with our probe. The ground squirrel retina is dominated by cones and our results, repeated in several Northern blots with different RNA preparations of ground squirrel retina, suggest that the ?-subunit of rod cGMP-PDE is considerably different from that of the cone enzyme since no cross hybridization is detected. As a control for the above data, 3

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Fig. 3. PDEy mRNA in different tissues from the mouse, and in retina From several species. Total RNA from different frozen mouse tissues and retinas of several species was isolated by the method of ChirBwin et al. (1979). The resulting RNA pellet was dissolved in 10 mM Tris-EDTA buffer, pH 7.5 (made in DEPC-treated water) and precipitated with ethanol. (a) RNA from (lanes): mouse brain (!), heart (Z), kidney (3), small intestine (4), liver ($), and muscle (6), and from retinas of 4 week-old normal mouse (7), nine-day-old diseased rd mouse (8), three-week-old diseased rd mouse (9), human (10), dos (11), ground squirrel (12), rat (13) and bovine (14) was denatured in formamide, eleetrophoresed on a i.5% agarose-2.2 M Formaldehyde 8el (Lehrach et al., 1977) and transblotted to a nylon membrane. Each lane has 15 P8 of RNA. The Nonhero blot was prehybridized for 6 to 8 h at 65°C in a solution containing 3 x SSC/2 × Denhardt's solution/denatured salmon sperm DNA (300 F~ml)/0.2~ SDS. Hybridization was carried out with human [~¢-32p]PDE?eDNA in a solution contalninB 6 × SSC/4 × Denhardt's solution/denatured salmon sperm DNA (300 pg/ml)/40 mM Tris. H ~ (pH 7.5)/0.2~ SDS at 60°C For 16-20 h. After hybridization the blot was washed twice For 20 rain each in 2 x SSC/0.1% SDS at 57°C, twice for 20 rain each in 0.3 × SSC/0.1 ~ SDS at 55 ° C and once for S rain with 0.1 x SSC/0. 1% SDS at room temperature. The blot was then exposed to Kodak XAR-5 film with an intensifyingscreen (DuPont Cronex Lightning Plus) at -80 ° C. Determination of the sizes of the hybridized RNAs was made by comparison to the migration ofeukaryotic ribosomal RNAs (28S, 18S) in the same gel and also to an RNA laddcr (BRL) consisting of 1.77, 1.52, !.28, 0.78, 0.53, 0.46 and 0.16 kb RNAs. (b)The same blot shown in (a) was stripped and reprobed with mouse [~t-3=P]PDEyeDNA (Tuteja and Father, 1988).

230 the same Northern blot was hybridized with an actin e D N A probe. All lanes showed mRNA-cDNA bands of comparable intensity indicating that the lack of hybridization in the particular lanes of Fig. 3a,b is due to lack of homology with the PDETcDNAs used and not to the quality of the RNA.

Control human and mouse DNAs digested with EcoR! and hybridized with [ 32p]PDE7 ci)NA gave clearly discernible bands on Southern blots (Fig. 5, lanes I and 2). The 6.6-kb band was used to score for the presence of PDEplike sequences in human chromosomes. Lanes 3-5 are representative of the original 15 somatic cell hybrid DNAs analyzed. All of these hybrids showed the human band. The only human chromosome present in each of the 15 hybrids was chromosome 17 and Table I shows that there are at least three discordances for the presence of PDEG in every human chromosome except for 17, for which there is perfect concordance. These data strongly suggest that the PDEG gene ~s located on chromosome 17. In order to confn'rn this assignment, we tested another hybrid clone which lacked chromosome 17. As expected, the 6.6-kb human fragment was absent (Fig. 5, lane 6).

(d) Secondary structure of human PDE~, In order to predict features of the secondary structure of human PDE~,, we used the PEPPLOT program from the UWGCG. The results are shown in Fig. 4. Part A indicates the characteristics of the aa that constitute the protein. PDE 7 is a basic protein and ten of the basic aa are present in the polypeptide region corresponding to aa 24-45, which has been shown to be important for PDE7 inhibitory activity (Morfison et al., 1987). The curves below the aa sequence (Fig. 4, part B) represent the propensity measures for formation of ~-helices and fi-sheets, analyzed according to Chou and Fasman's methods (1974). Areas of ~-helical configuration occur in regions close to the amino and C termini whereas fi-sheet configuration occurs between aa 63 and 69. Part C of Fig. 4 shows the hydropathy plot of the predicted aa sequence of PDET, analyzed according to the methods of Kyte and Doolittle (1982). There appears to be a mild hydrophobic region close to the p-sheet area and an expanse of hydrophilic aa towards the center of the molecule which includes the basic region.

(f) In situ hybridization We further confirmed the human c h r o m o s o m e 17 assignmerit of PDEG by hybridizing [ 3H]PDE7 eDNA directly to human metaphases (Fig. 6). This allowed us to determine that the PDEG gene is located on the long arm of human chromosome 17 probably in the q21.1 region, since this is where the highest peak of hybridization was observed. Interestingly, this assignment of the human gene is in very good agreement with the assignment of the mouse gene to mouse chromosome 11 (Danciger et al., 1989) since there are at least three other genes that map to mouse 11 and human 17q21.q22 (Searle et al,, 1987).

(e) Chromosomal assignment using somatic cell hybrids A panel of 15 human-mouse somatic cell hybrids was used for chromosome mapping of the human PDEG gene.

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Fig.4. Secondary structureprediction.The secondary structureand hydrophobicity analyses of PDE~ were performed on a VAX I1/780 minicomputer using the PEPPLOT program from the Sequence Analysis So/~ware package of the UWGCG. (A): Hydrophi|ic or hydrophobic nature of the aa. Hydropbilic: ~der marks above the baselinerepresentcharged residues;thinnermarks across the baselineindicateuncharged residues,the longerones containing hydroxyl groups and the shorter,amide groups. Hydrophobic: ~der marks below the basefne, the long ones being aromatic and the shorter

afphatic. Prolinesare the shortest thin lines and giycines,alaninesand cysteinesare unmarked.(B):Propensityto form a hefices(----) or ~ sheets ( ~ ) plotted according to Chon and Fasman's methods (1974). Above and below the curves the residues that are m-helix- or ~-sheet-forming (upper ones) or breaking (lower ones) are indicated. The