Gene. 88 (1990) 227-232 Elsevier

GENE03~6

227

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 a e, 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 ) 206- 6800; 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 comple- mentary 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 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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.

(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]thio- dATP 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: CAGTATGGCATCATT- TAG (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

-101 • CCQCACTCACAGCACAGCCCCCTGAGACCCGCCCTGCACTrO -80

ACCGCAGCAGGAGGGAGTCCAGGAGCCAAQGTTGCCGCGGTGTCTCCGTCAGCCTCACC -1

MG AAC CTG GAk CCG CCC AAG 001 GAG 'I'rC CGG TCA GCC ACC AGG G'I'G GCC GaG 54 Met Asn Leo ale pm Pro Lys Ala G~u Phe Arg 6er Ala ~ /ug Val Ala G~y 18

G0A CCT GTC ACC CCC AGG ~ GaG CCC CCT A/Lk TIT/tAG C^G CGA GAG ACC AGG 108 (31",/ Pro Val l'hr Pro A~O Lp Oly Pro Pro L~ Phe Lys Gin keg Gfn Thr Arg 30

CAG TTC AAG AGO .tAG CCC CCA tAG AAA GGC G'l-r CAA GaG 1TI" GaG GAC GAC ATC 162 Gin Phe Lys Sef Lys Pro Pro Lys Lys GIy Va] Gin Gly Phe Gilt ~ Asp lie 54

CCT GGA ATG GAA GGC CTG GGA ACA GAG ATC ACA GTC ATC TGC CCT TG(i GAG GCG 216 Pro GIv Mel GIu Gly Leu GI y Thr Asp lie Thr Viii fie Cys Pro Tfp Glu Nil 72

TTC AAC CAC CTG GAG CTG CAO GAG CTG GCC CkA TAT GGC ATC ATC TAG CACGAGG 271 Phe Arm His L~J GIu Leu His Geu Leu AL~ Gin Tyr Gin lie lie ff.R 87

C CCCTGCTGAAGTCCAGACCCTCCCCCTCCTGCCCACTMGCTkAACCCTGCTCAGGATTCC 333

TGTTGAGGAGATGACCTCCCTAGCCCCAGATGGGACCTGGACACCAGGATGGGACTGCAACC 395

TCAGGTCTCCCCCTACATATTAATACCAGTCACCAGGAQOCCACCACCTCCCTCTAGGATGC 457

CCCCTCAGGGTGGCCAGGCCCTOCTCAACATCTGGAGACACAGGCCCACCCCTCAG'i~CTGC 519

CC ACAGAGAGGCTTGGTCGGTCTCCACTCCCAGGGAGAACGGGAAGTGGACCCCAGCCCG(]G 581

AGC CTGCTGGACCCCAGATCGTCCC CTCCTCCCAGCTGGAAAGCTAGGGCAGGTCTCCCCAG 643

AGTGCTTCTGCACCCCAGCCCCCTGTCCTGCCTGTAAGGGGATACAGAO AAGCTCCCCGTCT 705

CTC-CATCCCTTCCCAOGGGGGTGCCCTrAGTTTGGACATGCTGGGTAGCAGGACTCCAGGGG 767

GTGC ACGGTGAGCAGMGAGGCCCCAAGCTC ATCACACCAGGGGGC CATCCTTCTCAATACA 829

GCCCGC CCTTGOAGTCCCTATTTCAAAATAAAATTAGTGTGTCCTTGCAAAAAAAAAAAAAA 891

911

Fig. !. The nt sequence of PDE~,cDNA from human retina and deduced aa sequence. The position of the nt and of the deduced aa are indicated on the right m argin. The $'-untranslated region is numbered - 10 i to - I. The coding region starts at nt number !. The stop codon in frame with the reading frame is indicated by TER..']'be Y-untransinted region is numbered following the coding region.

91.5~o identity between human and bovine and 90~ 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 m7 is 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 GTG GAA 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 .~TG AAO CTG G/U COAl CCC AAG G(:~I GAGI,qI"C COG "lraa GCC ACC AGG GTG IAI"GIGGG

GGA CCT GTC ACC CCC AGO MA ~ G CC~ ~ T ~*A TTr ~G CAe CO^ CAG ACC * ~ GGA C~GTC ̂ CC CCC A ~ AM r.,~ c d ~ ~ T r r AAG C ~ CGplC~ *CC Ae~ G ~ C~GTC Ad~ccc AGG AAA GGG CCC Cm A,*A n ' r ~ CAG cGr, r . .~ Ace *OG

CAG ~C ^AG AGe ~G CCC CCA ~ G ~ A GGC On" C ~ G ~ n-r c~G GAC GAC AT(: CAG TIC AAG AGC AAG CCC Cq-C I AAG AM GGC G'I~CAA GGG TTT ~ G/(i~GAC ATC 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-=~ATC AC~ GTC Arc TGC CC_T TGG GAG GCC COT GGA 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 GGC CAA TAT GGC ATC Arc TAG "FrO A/~ 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.qCI GGC 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.

RNAs 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

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. Inter- estingly, 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 consider- ably different from that of the cone enzyme since no cross hybridization is detected. As a control for the above data,

1 | :1 4 8 e ? a 9 10 1t 1 | t~1 14 1 II 3 4 6 •

(o) . 6 ,0kb (b)

-3,5kb

t -0 .9kb

• a M t0 11 tB 1:1 t4

I -3.Skb

| t i .O9k

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 intensifying screen (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]PDEy eDNA (Tuteja and Father, 1988).

230

the same Northern blot was hybridized with an act in e D N A

probe. All lanes showed mRNA-cDNA bands of compar- able 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.

(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.

(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.

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 repre- sentative 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).

(f) In situ hybridization We further confirmed the human c h r o m o s o m e 17 assign-

merit 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).

o , . , 2o . . . . 4 o , . . ep . . 8.o ,

" ~ " , . K . ' . , , . . ~ . v . o o . , , T r ~ , ~ o , , . K ~ . a . . o . . . . . . ~ o ~ o o . Qo~, ~o.IoLo ' ,v,~ ,,, ,, ,.~,..tL,,o l l l l * l i ' ' ' ' , ' t - ' ' • ' i , i , . . i , T P ! r ~ r ~ H I l Y O l l | l ~ O

I I I I i l l i i l I ' I I I I I I I I l l l l l S l l l l l i l l l l ~ l l l l l l i . l i i l . l l , l l l l ~ " B ; o •

"-.. . . . . ~, ,"~ f.,~.--..-_,..-'-"~ . o 2o 40 ~ 8"o

Fig. 4. Secondary structure prediction. The secondary structure and hydrophobicity analyses of PDE~ were performed on a VAX I 1/780 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 baseline represent charged residues; thinner marks across the baseline indicate uncharged residues, the longer ones containing hydroxyl groups and the shorter, amide groups. Hydrophobic: ~der marks below the basefne, the long ones being aromatic and the shorter afphatic. Prolines are the shortest thin lines and giycines, alanines and cysteines are unmarked. (B): Propensity to 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<lots mark the aa that are indifferent. (C) Hydropathy plot of the deduced aa sequence of PDET. When the line is above the baseline, it represents a hydrophobia region, and when it is below the baseline, a hydrophilic region. Sing]c-leucr abbreviations for the aa are: A, AIa; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asp; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr,

kb

23 .1 - -

. 4 - -

. 6 - -

. 4 - -

1 2 3 4 5 6

I D t - - - i

Fig. 5. Autorediogram of a Southern blot of human-mouse somatic cell hybrid DNAs, DNA from the parental cell lines and somatic cell hybrids was prepared by published procedures (Linnenbach et el., 1980), digested with £coR! and electrophoresed in a !.0% agarose gel. DNAs were transblotted to nylon membranes which are prehybridized for 3-6 h at 65°C in a solution containing 7.0Yo SDS/0.$ M phosphate buffer pH 7.0/! mM EDTA/I% bovine serum albumin/human placental DNA. Hybridization was carried out at 65°C overnight in the same solution with the addition ofthe human PDEy probe labeled by random priming with [o~-32P]dCTP to a specific activity ofapproximately 6 x 109 cpm//Ag, Each lane has 6 #g of DNA. After hybridization the blots were washed at 37°C in 2 x SSC/0.1% SDS for 30rain. two times at 60°C in 2 × SSC/0.1~ SDS for 20min each time and two times at 60°C in 0.5 x SSC/0.1% SDS for 20 rain each time. After the washes the blots were exposed to X-ray film at -80°C for 6 h. The mouse control is in lane I and the human control is in lane 2. Three somatic cell hybrid DNAs representative of the original 15 are shown in lanes 3-S. Lane 6 shows a somatic cell hybrid DNA that lacks chromosome 17. The numbers on the left (in kb) mark the positions of ~ DNA fragments digested with HfndIll.

e e e e

e e Q • e • e e

e e e

17 Fig. 6. Regional chromosomal mapping of PDEyon human chromosome 17. in situ hybridization was performed according to Harper and Sannders (198 I) es modified by Zabel (1983). The human PDE? probe was labeled with a mixture of the four [3H]deoxynucleotides to a specific activity of approximately 5 × 10 a cpm/pg, Slides were exposed for seven days and silver grains were scored. The silver grain distribution on chromosome 17 with the major peak at 17q21.1 indicates this is probably the site for the human PDEy locus.

231

TABLE I

Correlation beewcen specific human chromosomes and the PDEG se- quence in 15 somatic cell hybrids

Human Number of hybrid clones" ~o Discordant b chromosome

+/+ - / - + t - - /+

I $ 0 10 0 66.7 2 5 0 10 0 66.7 3 10 0 5 0 33.3 4 !1 0 4 0 26.7 5 6 0 9 0 60.0 6 12 0 3 0 20.0 7 I I 0 4 0 26.7 8 12 0 3 0 20.0 9 0 0 15 0 100.0

I0 9 0 6 0 40.0 i I 6 0 9 0 60.0 12 10 0 5 0 33.3 13 8 0 7 0 46.6 14 12 0 3 iO 20.0 15 8 0 7 '~ 46.6 16 3 0 12 0 80.0 17 15 0 0 0 0.0 18 8 0 7 0 46.6 19 ? 0 8 0 53.3 2O 12 0 3 0 2O.O 21 8 0 7 0 46,6 22 8 0 7 0 46.6 X 2 0 13 0 86.7 Y 2 0 13 0 86.7

" PD£G sequence/chromosome retention. Human-mouse somatic cell hybrids were derived from the fusion of normal human fibroblasts (IMR 91) and a thymidine kinase (TK)-deficieut mouse cell line (GM 0347A, B82), both obtained from the Mutant Cell Repository (Camden, NJ). These cell hybrids preferentially, and in a random fashion, lose human chromosomes while retaining all mouse chromosomes. The chromosome content of each clone was determined on a minimum of 30 Q-banded photographed metaphases. Because these human.mouse hybrids con. sisteutly lose human chromosome 9, DNA from a Chinese hamster- human hybrid clone selectively retaining 9pter-9q34 by an X/9 trans- location was also analyzed (Mohandas etal., 1979). b % Discordant was calculated by dividing the total number of hybrids from the + / - and - / + columns by the total number of hybrids scored.

(g) Conclusions (1) We have isolated and characterized the eDNA

coding for the 7,-subunit of human cGMP-PDE. This 1012-bp eDNA has a coding region of 261 bp flanked by 5'- and 3'-untranslated regions of 101 bp and 650 bp, respec- tively.

(2) Human PDE~is a protein very well conserved during evolution which shows 97.7 % identity with the correspond- ing bovine protein and 96.6% identity with the V-subunit of the mouse enzyme.

(3) The mRNA encoding human PDE~ has the same size as the PDE~ mRNAs from mouse, dog and bovine retinas and is longer than the corresponding mRNA from

232

rat retina. The cone-dominant ground squirrel retina may have a different ?-subunit cGMP-PDE, since no cross hybridization of the cloned cDNA with ground squirrel mRNA could be detected.

(4) We have assigned the gene for PDET, PDEG, to human chromosome 17, in the q21.1 region.

All this information will be useful for the study of the DNA of individuals from families affected with different forms of RP.

ACKNOWLEDGEMENTS

This work was supported by NIH grants EY02651 and EY0331 (D.B.F.), by the National Retinitis Pigmentosa Foundation Fighting Blindness, Baltimore (D.B.F.), and by the George Gund Foundation (D.B.F. and R.S.S.). We want to thank Prof. Arturo Falaschi for allowing N.T. and R.T. to fmish the sequencing of the human PDEF clone at the International Center for Genetic Engineering and Biotechnology, Trieste, Italy.

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