GENOMICS 6,272-283 (1990)

Molecular Characterization of Human and Bovine Rod Photoreceptor cGMP Phosphodiesterase &-Subunit and

Chromosomal Localization of the Human Gene

STEVEN 1. PITTLER, **’ WOLFGANG BAEHR,t JOHN J. WASMUTH,* DAVID G. MCCONNELL,~ MARY 5. CHAMPAGNE, t PETER VANTUINEN, 11,2 DAVID LEDBETTER,” AND RONALD L. DAVIS*

Departments of *Cell Biology, t Ophthalmology, and “Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, SDepartment of Biological Chemistry, California College of Medicine, University of California, Irvine, California 92717; and

SDepartment of Biochemistry, Michigan State University, East Lansing, Michigan 48824

Received July 17, 1989, revised September 27, 1989

Defects in proteins that function in photoreceptor signal transduction are prime suspects as causes of some human hereditary retinal degenerations. We have characterized cDNA clones encoding the a-sub-

unit of human and bovine rod cell cGMP phosphodi- e&erase, a key phototransduction enzyme. Clones from both species contain an open reading frame capable of coding for an -lOO-kDa polypeptide of 859 amino acids, 94% of which are identical. Two or more tran- scripts were detected in both human and bovine retinal poly(A)+ RNA preparations, although the human transcripts ranging from 5.3 to 4.9 kb are significantly larger than the two bovine transcripts of 4.6 and 4.0 kb. The bovine and human genes appear to exist in single copy, with the bovine gene spanning more than 140 kb of genomic DNA. Somatic cell hybrids were used to map the human gene to the long arm of chro- mosome 5 (5q31.2 + q34). Finally, the use of the can- didate gene approach in the study of hereditary retinal dystrophies is discussed. o ISBO Academic PWB, IIIC.

INTRODUCTION

Bovine rod photoreceptor cGMP phosphodiesterase (cGMP PDE)3 is a heterotrimeric enzyme [(Y (88 kDa),

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. M26043 and M26061.

1 To whom correspondence should be addressed at present address: Department of Ophthalmology, Cullen Eye Institute, Baylor College of Medicine, Houston, TX 77030.

’ Present address: Department of Cellular and Structural Biology, University of Texas Health Science Center, San Antonio, TX 78284.

’ Abbreviations used: cGMP PDE, 3’,5’-cyclic guanosine mono- phosphate phosphodiesterase (EC 3.1.4.35); TMAC, tetramethyl ammonium chloride; TPCK, tosylphenylchloroketone; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin; TFA, trifluoroacetate; CN PDE, cyclic nu- cleotide phosphodiesterase; ADRP, autosomal dominant retinitis pigmentosa; PCR, polymerase chain reaction; Pipes, piperasine-iV,N’- bis-(2-ethanesulfonic acid); 1X SSC = 0.15 M NaCl, 0.015 M Na citrate.

,(3 (84 kDa), yz (10 kDa)] (Baehr et al., 1979; Deterre et al., 1988) that functions as a signal amplifier in pho- totransduction. In dark-adapted rods, the hydrolytic activity of the cGMP PDE is greatly reduced due to an inhibitory constraint imposed by the y-subunit. Transducin, a rod-specific G-protein, serves to couple the light activation of rhodopsin with a marked increase in cGMP hydrolysis. The sensitivity, speed, and am- plification of this signal transducing system are re- markable. A single photon is sufficient to trigger a re- sponse that leads to the hydrolysis of as many as lo6 cGMP molecules in less than 1 s. The reduction in cytoplasmic cGMP is coincident with the closure of cation channels in the plasma membrane (for review, see Stryer, 1986; Liebman et al., 1987; Hurley, 1987), which leads to a transient hyperpolarization of the plasma membrane.

A great deal of evidence that suggests a link between retinal degeneration and defects in phototransduction metabolism has been reported. Hollyfield and co- workers (Lolley et al., 1977; Ulshafer et al, 1980; Hol- lyfield et al, 1982, 1985) demonstrated that elevated levels of cGMP are toxic to photoreceptor cells in Xen- opus Levis eye rudiments and in the adult human retina maintained in culture. There is a marked specificity of the degeneration for rod cells. Several animal retinal degeneration mutants that exhibit very high levels of photoreceptor cGMP (Farber and Lolley, 1974; Aguirre et al., 1978; Woodford et al., 1982; Leon et al., 1988) prior to the onset of any morphological disruption have been identified. In addition, high cGMP levels were recently reported in rod photoreceptors of a 17-year- old male with ADRP (Farber et al, 1987). ADRP is one of a group of clinically related retinal dystrophies for which there is currently no treatment and no known cause (for review, see Heckenlively, 1988). Histological examination of the patient’s retina revealed that most retinal layers were intact, with the exception of the photoreceptor layer in which a reduction of rod cells

OS%7543/90 $3.oo Copyright 0 1990 by Academic Press, Inc. All risht. nfr.mmA..rtinn in mm., t-n-m ra.n...mA

272

ROD PHOTORECEPTOR cGMP PDE o-SUBUNIT 273

A

PDEl

PDE2

PDE3

PDE4

PDES

B cPDE1

cPDE2

cPDE3

cPbE4

cPDE5

FIG. 1.

12 3 4 5 6 7 8 9

Ser Gln Am Pro Leu Ala Lys

Gly Thr Asn Asn Leu Tyr

Phe Leu Asp Gln Asn

va1 Asp Ile Tyr 1le Leu - Gly Lys

Glu Ile Am Phe Tyr Lys

3'- G T ; T T ~GGIGAICGITT-5'

3'-CCITGITTgTT;GAIAT-5'

3’-AA: GAICT $GT ; T T - 5’

3'-CAICTtTAIATtTAIGA-5'

3’- C T ; TAITT ;AA;AT;TT-5’

Design of oligonucleotide probes. (A) Sequences of five

tryptic peptides from amino- to carboxyl-terminus. Peptide PDEP terminates with a tyrosine residue, presumably due to contaminating chymotryptic activity not quenched by TPCK. Peptide PDE3 is truncated due to an insufficient amount of sample. No PTH amino acid was observable for residue 7 in peptide PDE4. (B) Sequence of the oligonucleotides. Oligonucleotide mixtures containing deoxyino- sine (I) residues and reflecting some codon degeneracy were synthe- sized (see Materials and Methods).

was the most notable. In areas where rod cells were still present, a reduction in cGMP PDE specific activity was found. These studies provide sufficient impetus for intensely pursuing the study of defects in cGMP PDE as a potential causal factor in the etiology of certain hereditary retinal disorders.

We are employing the candidate gene approach to study retinitis pigmentosa. With this approach one studies a gene in which a defect is implicated as a caus- ative factor of a disease. This method is particularly amenable to the study of diseases that occur in well- studied, highly specialized tissues such as the retina. Here we report on the bovine cGMP PDE a-subunit primary structure, the characterization of cDNA clones encoding the human a-subunit, the preliminary anal- ysis of the bovine gene, and the chromosomal location of the human gene.

carboxymethylation (Gracy, 1977) and then digested by addition of 10 ~1 of TPCK-treated trypsin (100 pg/ ml). The reaction was allowed to proceed for 3 h at 37°C on a roller drum incubator, after which an ad- ditional 10 ~1 of trypsin solution was added and the incubation continued overnight. After digestion, the solution was dried in a Speed Vat concentrator and redissolved in 500 ~1 of water. A 20-4 aliquot was re- moved for SDS-PAGE analysis and the remainder dried and stored at -20°C. Fractionation of the trypsin- cleaved protein was achieved by elution from C4 or Cl8 reverse-phase columns using a linear gradient of O- 70% acetonitrile/0.075% TFA in H20/0.075% TFA. The gradient was generated over 60 min, and l-ml fractions were collected manually. Most of the fractions containing peptides were purified with two additional chromatography steps. First, the peptides were eluted a second time from a reverse-phase column using the same solvent system, but with a narrowed gradient range. Second, each sample was dried, resuspended, and rechromatographed using 25 mM ammonium ac- etate, pH 6.0 (buffer A), and 50 mM ammonium acetate, pH 6.0/60% acetonitrile (buffer B). Elution was achieved with a linear gradient of O-70% buffer B gen- erated over 60 min. Samples that appeared to be ho- mogeneous after the second chromatography step were not further purified. These were dried prior to sequence analysis. Peptides that were chromatographed in the ammonium acetate gradients were dried, redissolved in water, and dried again to remove excess buffer before

COOH

.A 1 3

- 5 2 4

: SPA-1 EPA-2

EPA-3

. . . ..-_ HPA-3

MATERIALS AND METHODS

Amino Acid Sequence Analysis of Purified Bovine cGMP PDE

Bovine eyes were obtained from Murco, Inc. (Plain- well, MI). Purified cGMP PDE was prepared from iso- lated rod outer segments according to Kohnken et al. (1981). Further purification to near homogeneity was achieved by rechromatography of the final product on Sephacryl S-200. The holoenzyme was modified by S-

H - HPAQ 200 bp - HPA-6

FIG. 2. Bovine and human cDNA clones. A schematic map of the full-length human cDNA clone is shown at the top. Wider rect- angles represent protein coding segments and more condensed rect- angles depict upstream or downstream untranslated regions. Included within the protein coding region is the “CN PDE HOMOLOGY” domain that defines a segment conserved in a number of CN PDEs (see text). Predicted ammo- and carboxy-termini are designated NH* and COOH, respectively. Bovine cDNA fragments labeled 1-5 and oligomers A-D were used as probes to isolate some of the cDNAs, for chromosomal localization, and for RNA blot analysis. Bovine cDNAs are designated BPA-1, BPA-2, and BPA-3 and human cDNA clones HPA-1 through HPA-6. Vertical lines mark the positions of internal EcoRI sites in the cDNA clones.

274 PITTLER ET AL.

1

121

241

361

481

601

121

041

1 11 21 t ~GEVTAEEVEKIFLDSN~VSFAKQ

A~~A~~~CCCTCACC~~~AT~A~~~TACC~~~M~~~~~~~~~~~~~CA~~A~~~~~A~~~~~~~C~~~~A~C~T~CACC~~C~~CAC 31 a ml 51 61

YYYLRYRAKVISDLLGPREAAVDF~SNYHALNSVEESElIF TACTACMC~CCCTAC~CCCCCCMCCTCATCTCACACCC

71 81 91 101 DLLRDFQDNLQAEKCVFNV~KKLCFLLQADRHSLFMYRAR

CACC~CC~CCCCAC~CACCMCCI‘CCACCCCACM~C~~~~~TCMGMG~C~C~CCTGCACGCCCACCGCATGACCCTA~CATCTACAC~CCCCC 111 121 131 141

NCIAELATRLFNVtiKDAVLEECLVAPDSEIVFPLD~CVVG MCCCCATCCCACAC~CCCCACC~C~~CMCCTCCA~~A~~CTCGAGCA~CCC~G~CCCC~CACTC~ACATCCT~CCCCCTCCACATCCCACTCCTCCCC

151 161 171 181 liVALSKKIVNVPNTEEDE~FCDFVDTLTEYQTKNILASPl

CAT~CCCT(TCCTAAAMCATCCTCM~TCCCCMCACACACCACCA~MCA~CTCTCAC~~CACACC~CAC~A~ACCA~CTMCMCATC~CCC~CTCCCATA 191 201 211 221

MNGKDVVAI I~VVNKVDGPHFTENDEEILLKYLNFANLltl ATCMTCCCM~TCTCCn;CCCATMTCATCC'PTCn;MTATCTMTCATC

231 241 251 261 KVFHLSYLIiNCETRRCQlLLUSGSKVFEELTDlERQFHKA

MCCTCTPCCACCTCAC~ACC~CACMCTCfCAGACACTCGCCCT~C~ATAC~~~C~CCACC~~C~MCAGC~ACACACA~ACACCCAC~CCAC~CCC 271 281 291 301

LYTVRAFLNCDRYSVGLLDblTKQKEFFDVUPVLltCEAPPY CTCTACACAGTCCCCGCCTTCCTCMCTCfCACACA7ACTCTCACACATACTCTC~CCG~C~ACACATCACC~CACMCGM TlTTTTGATCTCTCCCCACTCCTCATCCCCCACCCTCCACCCTAC

311 321 331 341 ACPRTPDCR~INFYKltVIDYILUCKjEDIKVlPNPPPDIlYAL

961 ~CTCCCAGCACTCCCCAn;CMCCCAMTCM~C~TCMC~ACMCCTCA~GA~ACATC~CCA~CC~CMCACATC~~CATCCCGMTCCTCCTCCACACCACTCGCC~A 351 361 371 381

V5'CLPTYVAQNCLICNIHNAPSEDFFAFQKEPLDESCW!lI 1081 CTCACCCCTCTCCCCACTTATCITGCCCC~~CCCTCA~CCMCA~ATGMT~CCCC~CAGA~A~ CATTCCACAAACACCCTCTCCATCAGTCTCCATCCATCAl-7

391 401 411 421 KNVLSMPIVNKKEEIVCVATFYNRKDCKPFDE~DETL~ES

1201 AAAMTCTCC~CTATCCCMTTCTGMCMCMCMCCACC~~C~CCCCTCGCCACG~ACMTCCC~CATGCC~CCC~GATG~TCCATCACACCCTCATCCACTCT 431 441 451 461

LTQFLGUSVLNPDTY ELHNKLENRKDIFQDHVKYHVKCDN 1321 1TCACCCAC'~CCTCCGCTCCTCTGTCTTAMTCCTGACT

- 471 481 491 501 EEIQTILKTREVYCKEPYECEEEELAE ILQCELPDADKYE

1441 CMCAMTCCACACMTC1TCACCAGACACACCTCTATCCCMGCACCCGTCCCMTCCCACCMCMCMC~CCTGACATCC~CMCCAGACCTCCCACA~CACAC~TATCM 511 521 531 541

lNKFHFSDLPLTELELVKCGlQtlYYELKVVDKFHlPQEAL 1561 A~MTAMTTCCACMCACCCACCTGCCCCCTCACCCMCTCGACCTCCTC~TCTCCM~CACATCTACTATCACCTC~CTCC~CAT~~CACA~CCTCACCACCCCCTC

551 561 571 581 VRFblYSLSKCYRRlTYHNYRtlCFNVCQTblFSLLVTCKLKR

1681 CTCCCC1"~CATGTA~~CCCTCACTMCCCATACCCCACCATCACCTACCACMCTCCCCCCACCCC~CMCCTCCCCCACACCAT~CTCC~CCTCCTCACCCCMACCTCMCCCA ---xi-- 601 611 621

YFTOLEALAWVTAAFCHDIDHR~CTYYL~Q~K~SQNPLAK~LH 1801 TA~ACACACCn;CACGCC7FCCCCAICGTCACCCCCCC~~CCA~ACA~ACCA~~~M~T~~ACCA~~~TCC~~CC~~CCMCCTCCAT

631 641 651 661 CSSILERHHLEFCKTLLRDESLNIFQNLNRRQilEHAIlf~~

1921 CtXTCCTCCATCTTCCAMGACACCAC3TCCACTlTGCT AAMCACTCITGACACA~AGACCCTAMTAT~CACMCC 671 681 691 701

DIAIIATDLALYFKKRTblFQKI VDQSKTYETQQEWTQYUH 2041 CACA~CGATCATTC~A~ACC~C~CTA~CMCA

711 721 731 741 LDQTRKElVblAbllfblTACDLSA ITKPYEVQSKVALLVAAEF

2161 ~ATCACACACCCMCCAMlXTCATCCCCATGATCATCATCACCGCCT~AT~~~CCCATCACCMCCC~~CAG~CACACCMG~C~C~~CC~~~CM~C 751 761 771 781

UEQCDLERTVLQQNP lPblblDRNKADELPKLQVGFIDFVCT 2281 ~CCMCMCCTCAC~ACCCCAC~~~CMCAGMTCCCA~CCCA~A~CACAC~CMCCC~ATCM~CC~MC~~CCTCCC~CATCCA~C~CACC

791 801 811 821 FVYKEFSRFHEEITPNLDGITNNRKEUKALADEYETKNKG

2401 mCTCTACMCCM~CTCCC~CCACGACCAGATCAGATCA~CCCA~~GATGGCA~ACCMCMCCGCMCCAC~G~CCG~C~A~A~A~A~ACCMGA~MG~C LEEEKQKQQAANQAAACSQilGCKQPCCGPASKS~CVQ* 831 I341 851 859

2521 ~ACCACGACMCCACAMCACCACCCACCCMCCMCCACCCCCACCM~CMCAC~CCCC~CCMCCTCG~CCCGCC~CATCCM~~~C~CCMTACCACACC

@Ii ICI 2641 GACCACCCACCCCCCACCCCCCCCCCCACCC~CACCCACCACACCACCCAGCCCAC~GC~CCATACGCCAC~~CGAGM~CA~~G~~~~CAGMT

193 2761 TWCllTACATCTTl'CCTACTCCCT'Il'CMCCCT~ CACCn;~ACMC~A~MIACMC~CA~AGC~CACACA~C~CA~A~CCCACA~~C~C

2881 IIACCT~MTCT171%1Y;I7CTTATACmCATmATACTAC

120

240

360

480

600

720

a40

960

1080

1200

1320

1440

1560

1680

1800

1920

2040

2160

2280

2400

2520

2640

2760

2880

29.58

FIG. 3. Nucleotide and deduced amino acid sequence of bovine cGMP PDE a-subunit. Nucleotide numbering is shown on each side of the sequence and amino acids are numbered above each tenth residue. Oligonucleotides synthesized for RNA blotting and DNA sequencing experiments are underlined, with the strand indicated by arrows. In-frame stop codons that define the open reading frame are denoted by asterisks. Peptide sequences obtained from cGMP PDE (Fig. 1A) are boxed. Minor discrepancies between peptide sequences PDE3 and PDE4 and the nucleic acid sequence may be due to errors in peptide sequence analysis. A solid box is present above residue Cys-, which is post- translationally modified in the mature subunit (see text).

ROD PHOTORECEPTOR cGMP PDE a-SUBUNIT 275

1 11 t NCEVTAEEVEKFLD

1 ~CCTC~~CAAMCCCCATTCCMGCCCTCCC~CCCA~CC~~ACAGACTACCCAC~CCA~CCCACCCATGCGCGAC~ACACCAC~CAG~CACM~C~C 21 31 41 51

SNICFAKQYYNLHYRAKLI SDLLCAKEAAVDFSNYHSPSS

121 TCCMTATTCC~CCC~CACTACTACTACMCCTCCA~ACCCCGCCMCCTCATCTCCCACCTCC~CCCCCCMCCACCCTCCC~CCAC~CACCM~ACCA~CCCCCACCACC 61 71 81 91

NEESEI IFDLLRDFQENLQTEKC IFNVNKKLCFLLQADRU

241 ATCCACCACACCCAMTCATCmCATCTCTCCTCCCCGAC~CACCACM~ACACACACAC~TCCATC~CMT~CA~MCMCCT~CC~C~C~CCACCCAGACCCCATC 101 111 121 131

SLFNYRTRNGIAELATRLFNVHKDAVLEDCLVUPDQE~VF 361 ACCCTC~CATCTACCCCACCCGCMTGGCATCCCATCCCACACCTCCCCACCACCCT~CMTCTCCACMCGATCC~TC~CCACCA~GCCTC~ATCCCCCACCMCACATC~C~C

141 151 161 171 PLDNCIVCHVAHSKKIANVPNTEEDEHFCDFVDILTEYKT

401 CC~CCACATCCCCATCCTCCCCCATCTCCCACACTCTMCMCA~C~MCCTCCCCMCACACACCACCATCAGCA~~~AC~~CCACATCCTCACACA~ACMCACC 181 191 201 211

KNILASPINNCKDVVAIINAVNKVDCSHFTKRDEEILLKY 601 MCMCATC~CCC~CCCCCATMTCAATCGCAACCCMCCATCTCCTCCCCATMTCATCCCTCTCMT~CTCCATGCATCCCAC~CACCMCACACA~MCACA~C~CTCM~AC

221 231 241 251 LNFANLIHKYYHLSYLHNCETRRCQI LLUSGSKVFEELTD

721 CTCM17TTCCAMTCTMTCATCMCTCCTACCACCTCAC~ACCTCCACMCTCTC~CTCCACGTCCCCACATA~CCTCTCCTCTCCCACC~CTC~CMCMC~ACCCAC 261 271 281 291

IERQFHKALYTVRAF LNCDRYSVGLLDUTKQKEFFDVYPV 841 ATCCMCCACAG~CCACAMCCCCTCTACACAGTCCC’TCC~rCCT~CCTCMCTCTCACACATACTCTGTGCCTCTC~ACACA~ACCMCCACMCCM~ ATCTCTGCCCCGTT

301 311 321 331 LNCEVPPYSCPRTPDCREINFYKVIDYILHGKEDIKVIPN

961 CTCATGCCTCMC~CCACCTTACTCTCTCCTCCCACCA~~CCCCATCCAACAC~~MC~ACMGGTCA~CA~ACATCCTGCATCGC~GAGGACATC~CTCATCCCCMT 341 351 361 371

PPPDHUALVSCLPTYVAQNCLICNInWAPSeDFFAFQKgP 1081 CCACCTCCTCACCA~CCCCmACTAACTMCCCCTCTCCCCACTTATG~CCCCACMTCCCCTCA~CCMCATCA~MCGCCCC~CACACCAC~CCA~CAC~CMC~

381 391 401 411 LDESCYUIKNVLSMPIVNKKEEIVCVATFYNRKDGKPFDE

1201 CTCCATCACTCTCCATCCATCATTAI\IUATCTCCmCAATCCCC~~CTCAACMCAACCMC~~C~CACTCGCCACA~ACMTCCT~CATCCGMCCCC~GATCM 421 431 441 451

UDETLUESLTQFLCUSVLNPDTYESNNKLENRKDIFQDIV 1321 ATCGATCACACCCTCATCCACTCT’TTCACTCA~~~~~~CCC~~C~~CT~~~~~TCCTCACACCTATCAGTCMTGMT~C~~TACCMCCATA~CCACCACATA~A

461 471 481 491 KYHVKCDNEEIQKI LKTREVYCKEPYECEEEELAEILQAE

1441 AMTATCATCTCMCTCTCACMTCMCAAA~CAC~T~~TC~CCACACAC~~CTATCCCMCCACCCATCCCACTCTCACCMCACCAGCTCCC~ACATCCTCCMCCCCAC

so1 511 521 531 LPDADKYEINKFHFSDLPLTELELVKCCIQNYYELKVVDK

1561 CTCCCACATCCACATAAATACCAAATTAATAMTTTCA~~TCA~~CA~~TACCCCTMCACMCTGCACCTGCT~TCTCCMTACACATGTA~ATCACCTC~GTCCTCCAT~ 541 551 561 571

FHIPQEALVRFNYSLSKCYRKITYHNURHCFNVCQTNFSL 1681 ~CACATTCCACAACACCCCCTCGTCCTCCC~~TCATCTAC’~CCCT~ACTMCCCCTACCGCAACATCACCTACCACAACTCCCCCCACCCC~CMCCTCCCCCACACCATCTTCTCCCTC

581 591 601 611 L V T C K L K R Y F T D 1. E A LAN V T A A F C H D 1 D H R C T N N L Y QN K S

1 I301 CT~~ACf:f:~AAAGCT~AA~~~~TA~~~T~A~l~~4~fTA~ACl:C~“I”I’~~~~ATl:~:T~A~I’~:(I’I’~~ITT~~~~~AT~A~A~~&~~4~A~A~~~AcCAATA&CCTCTACCA~AT~AA~TCC

621 b31 b41 651 QNPLAKLHCSSILERHHLEFCKTLLRDESLNIFQNLNRRQ

1921 CACMCCCACTCCCCMCCTCCATCGCTCCTCTAT~CC~CACACCAC~CCA~GC~CACTGCTCACACACCACAC~~~TAT~~~~~~~MT~~T~~A~A~

661 671 681 691 HEUAIHHNDIAIIATDLALYFKKRTUFQKIVDQSKTYRSR

2041 CATCACCA~CCATCCACATGATCCACA~CCMTCA~CCACACACCTCCCCCTCTA~CMCMCACCACCAT~CC~CATC~CATCACT~MCACATA~A~A~~

701 711 721 731 QEUTQYNNLEQTRKEIVNAUHNTACDLSAITKFNRVQSQV

2161 CACCACTCCACACACTACATA~CTCCACCACACACCCMCC~TCC~AT~CccA~ATCATCACCCC~TCATcTcTcACccATcAcc~~ccT~~~A~~~~A~A~~cA~~A

741 751 761 771 ALLVAAEFUEQCDLFRTVLQQNPIPNNDRNKADKLFKLQV

2281 CCT~C~CTCCCTCCTCM~CTCCGMCMC~CACCTCCACCCCACCCTCCTCCMCACMTCCCA~CCCATCATCCACAC~C~C~A~ATCM~~~~TM~~~~MCT~ 781 791 801 811

CFIDFVCTFVYKEFSRFHEEITPNLDCITNNRKEUKALAD 2401 GGCTTCA~XAC~~CCACCTTCCTCTACMCCM~CTCCCG~CCACCACCACATCACCCCMTC~CCACCCCATCAC~M~MTCC~MCCA~~MCC~C~~~~TCAT

821 831 841 851 EYDAKNKVQEEKKQKQQSAKSAAACNQPCCNQPRCATTSK

2521 GA~ACCATCCCMCATCMCCTCCACCACCACMCMCCACAMCACCACTCCGCCAACTCACCACCCCCACC~TCACCCCCCCCC~C~AC~~~ACCC~T~~~~A~ATC~MC SFCIQ+ 859

2641 TCCTC~CATCCACTMCACCACTCCCCATCTGCTCCCTCCACCCCACCACCCl”~CCTCCCMCACATCACTCMCCCA~TCCAACAccAcAcAc~A~M~AC~CACTCATAC

2761 CAmCAMCC~TTACACMmACC7TCCACCACCACT~~CMTC~rTCC~rTCCCTCCCCCACA~CCAACM~CTC~CCCTCACACAT~TACACTMCACTMTCATAC~

288 1 TICMCITA

PIG. 4. Nucleotide and deduced amino acid sequence of human cGMP PDE a-subunit. Nucleotide numbering is shown on the left of the

sequence and amino acids are numbered above each tenth residue. In-frame stop codons that define the open reading frame are denoted by asterisks. A putative nonconsensus polyadenylation signal is underlined. A solid box is present above Cyssr’s, a residue which is likely to be modified as shown for the bovine a-subunit (see text).

276 PITTLER ET AL.

analysis. Sequencing reactions were carried out on a Beckman System 890 M sequencer, and the resulting PTH amino acids were determined by two independent HPLC runs. All sequence analyses were carried out at the Macromolecular Facility of the Department of Biochemistry, Michigan State University under the direction of Dr. Y. M. Lee.

Design of Oligonucleotide Probes

A codon usage table was compiled from the deduced amino acid sequences of 12 bovine genes extracted from the GenBank nucleic acid database. From this table, it was found that leucine is encoded by CTX greater than 75% of the time. Therefore, the codon C T I was used for leucine, since preliminary evidence suggested that the hypoxanthine base of deoxyinosine acted in an inert fashion, neither contributing to nor destabi- lizing the DNA hybrid (Ohtsuka et aZ., 1985). For amino acids for which there were only two possible codons, both were included in the oligonucleotide mixture, and for any amino acid for which three or four codons were possible, a deoxyinosine was incorporated.

Isolation and Characterization of Bovine and Human cDNA Clones

The bovine and human retinal cDNA libraries were constructed in the bacteriophage X cloning vector XgtlO (Nathans and Hogness, 1983). The bovine library was screened according to a method developed by E. F. Fritsch (Genetics Institute, Boston, MA). Briefly, fil- ters containing -20,000 pfu were prehybridized at 65°C in a buffer consisting of 3.0 M TMAC, 50 mM sodium phosphate, pH 6.8, 1 mM EDTA, 0.5% SDS, 5~ Denhardt’s solution, and 100 pg/ml alkali-dena- tured salmon sperm DNA. After l-2 h, filters were rubbed vigorously with Kimwipes and placed into fresh prehybridization solution for 12-16 h at 65°C. Hy- bridization was carried out in a large volume (lo-15 ml per filter) of prehybridization solution with 0.1 pmol of end-labeled oligomer added per milliliter of solution. Filters were incubated at 35-45°C for at least 48 h, or 72 h for highly redundant probes. Washes were per- formed in a large volume of 2X SSC, 0.2% SDS at room temperature for 2 h with one change of solution. Filters were then air-dried and placed at -70°C for autora- diography. Hybridization with the N-terminal oligo- nucleotide was performed at 37°C in 50% formamide, 1 A4 NaCl, 0.5% SDS, 1X Denhardt’s, 0.1 M Tris, pH 8.0, and 200 pg/ml sheared, sonicated, denatured Escherichia coli DNA. Hybridizations with the cDNA probe were performed at 42’C in a buffer containing 50% formamide, 0.1 A4 Pipes (Ultrol), 0.8 M NaCl, 5~ Denhardt’s solution, 100 pg/ml denatured salmon sperm DNA, and 10% dextran sulfate as described (Pittler et aZ., 1987). Phage DNA was purified, and the inserts were excised and subcloned into pUC19 or

Bluescript vectors. Sequence analysis was performed with a sequencing kit and a modified protocol for dou- ble-stranded sequencing (Zhang et al., 1988). Ml3 uni- versal primers and sequence-specific oligomers were used to complete the sequence. Reverse transcription of bovine retinal RNA, with an oligo(dT) primer, was performed with a first-strand cDNA kit (Promega). The product was diluted fivefold and used directly for 25 PCR cycles (1’ at 92°C 2’ at 55”C, 2’ at 72°C) in an Ericomp thermal cycler.

RNA and DNA Blot Analysis

Retinal RNA was isolated by homogenization in guanidinium isothiocyanate as described (Chirgwin et al., 1979), and poly(A)+ RNA was twice selected by oligo(dT)-cellulose chromatography (Pittler et al., 1987). Genomic DNA from a single bovine brain was isolated by phenol extraction and selective precipita- tion according to Hogan et al. (1986). Electrophoresis, blotting, hybridization, and washings were performed as described (Pittler et al., 1987). RNA blot hybridiza- tion with oligonucleotide probes was performed by prehybridization in 12 ml of a buffer consisting of 40% formamide, 5X Denhardt’s, 5X SSC, 50 n&f sodium phosphate, pH 6.8, 0.1% SDS, and 250 pg/ml alkali- denatured salmon sperm DNA for 2-4 h at 42’C and hybridization in 8 ml prehybridization solution, 2 ml 50% dextran sulfate, and 100 ng unpurified end-labeled oligomer for 12-16 h at 42’C. The filters were washed for 1 h with three changes in 2X SSC at room tem- perature with agitation, 30 min in 0.5X SSC at 5O”C, and 5 min in 0.1X SSC at 50°C.

Chromosomal Localization

The initial chromosome location was determined using 14 human-rodent somatic cell hybrids (see Table 1). Construction and cytogenetic characterization of these hybrids have been previously described (Su et al., 1984; vanTuinen et al., 1987). Confirmation of the as- signment and regional localization was obtained from a panel of 11 human-hamster hybrids that contain various deletions of the short arm of human chromo- some 5 (Overhauser et al., 1986), and the assignment was further delimited using a hybrid panel with dele- tions of the long arm of chromosome 5 (see Fig. 7A). Deletion breakpoints on the q arm were determined by cytological analysis (J. Wasmuth, unpublished results). DNA blotting, hybridizations, and washings at high stringency were performed as described (Overhauser et al., 1986).

RESULTS AND DISCUSSION

Bovine cGMP PDE a-Subunit

Highly purified bovine retinal cGMP PDE (1 mg) was prepared to obtain partial amino acid sequences.

human bovine

human bovine

human bovine

human bovine

human bovine

humam bovine

human bovine

hum&n bovine

human bovine

human

bovine

human bovine

human bovine

human bovine

human bovine

human

bovine

ROD PHOTORECEPTOR cGMP PDE a-SUBUNIT

MGEVTAREVRKFLDSNIGFAKQYYNLK~ISDLLGAKEMVDFSNYRSPSSl4EESEI 60 vs R v PR ALNV

IFDLLRDFQENLQTEKCI -LLQADRMSLFMYRTRNGIAELAT~AV 120 D A V A

LFDCLVMPDQEIVFPLDMGIVGHVAHSKKIANVPNTEEDEHFCDFVDILTEYKTIWILAS 180 E A S V L v T Q

PIMNGKDWAIIMAVNKVDGSHFTKRDEEILLKYINFANLIMKWYHLSYLHNCETRRGQI 240 V PEN VF

LLWSGSKVFEELTDIERQFHKALYTVRAF LNCDRYSVGLLDhlTKQKF.FFDVWPVLMGEVP 300 A

PYSGPRTPDGREINFYKVIDYILHGKEDI~IPNPPPDHWALVSGLPTY+AQNGLICNIM 360 A

NAPSEDFFAFQKEPIDESGWMIlWnSMPIVNKKEEIVGVATFYNRXDGKPFDEMDETLM 420

ESLTQFLGWSVLNPDTYESMLENFUUJ IFQDIVKYHVKCDNEEIQKILKTREVYGKEPW 480 L M T

ECEEEELAEILQAELPDADKYEINKFHFSDLPLTELELVKC@QMYYELMDKFXIPQE 540 G

ALVRJ'MYSLSKGYRXIT YRNWRHGFNVGQTMFSLLVTGKLKRYFTD LlZXAWTAAFCHD 600 R

IDHRGTNNLYQMKSQNPIAKLRGSSILERHRLEFGKTLLRDESLNIFQNLNRRQHEwAIH 660

Mt4DIAIIATDLALYFKKRTMFQKIVDQSKTYESEQEWTQYMKLEQTRKEIVMAMMMTACD 720 TQ D

LSAITKPWEVQSQVALLVAAEFWEQGDLERTVLQQNP IPMMDRNKADELPKLQVGFIDFV] 780 K

CTFVYI~EFSRFHEEITPWGI TNNRKEWKALADEYDAKMKVQEEKKQKQQSAKSMAGN 8 4 0 ET GL E ANQ S

QPGGNQPRGATTSKSkIQ 859 H K GGPA V

277

FIG. 6. Comparison of human and bovine a-subunit primary structures. Amino acid alignment of the predicted primary structures of the bovine and human cGMP PDE o-subunits is shown. The bracketed region (522-780) roughly defines the region that is conserved in many CN PDEs (see text). CysW, which undergoes post-translational modification, is marked above by a solid box.

Following S-carboxymethylation and trypsinolysis, peptide fragments were purified to homogeneity by re- verse-phase HPLC. Five peptides were purified in suf- ficient yield to obtain amino acid sequence, from which oligonucleotide probes were designed (Fig. 1A).

One hundred thousand plaques from a bovine retinal cDNA library (Nathans and Hogness, 1983) were screened with each oligonucleotide (Fig. 1B) on dupli- cate filters. From the initial library screen, 226 hy- bridizing clones were identified on both the original and the duplicate filters. Twenty of these clones hy- bridized strongly to cPDE2 and cPDE5, and further characterization focused on 17 of these clones. DNA

samples from each clone were analyzed by restriction analysis (not shown). All of the clones share 662- and 144-bp EcoRI fragments and 6 of the clones contained more than two EcoRI fragments. One additional clone was isolated by rescreening the library with cDNA probe 1 (Fig. 2), and the clones were sequenced using a number of internal primers (underlined in Fig. 3).

Three clones (BPA-1, BPA-2, BPA-3, Fig. 2) overlap to define the sequence of the cGMP PDE a-subunit. The composite sequence of the cDNAs contains a 2577- nucleotide open reading frame which could encode a protein of 859 amino acids (Fig. 3). Although our en- zyme preparation contained both (Y- and &subunits of

278 PITTLER ET AL.

cGMP PDE, the sequence obtained from the cDNA clones is identified as CY for two reasons. First, the nu- cleotide sequence of this composite is nearly identical to a cDNA sequence reported as (Y in a preliminary analysis of cGMP PDE large subunits (Ovchinnikov et al., 1987). Discrepancies in the two sequences were identified at only 14 positions, 3 resulting in amino acid changes at positions 194 (Val to Ala), 424 (Thr to Ala), and 675 (Phe to Cys). These authors identified their cDNA sequence as encoding the a-subunit from the sequences of two peptides released from an a-sub- unit proteolytic hydrolysate that were absent in one from the P-subunit. In addition, the first 10 residues at the N-terminus were determined. Second, it has been shown that an antipeptide antibody directed against the first 15 amino acids of the sequence in Fig. 3 reacts only with the larger molecular weight PDE subunit (D. Takemoto, personal communication), confirming the identity of our cDNA clones. It is surprising that all five peptides obtained from the PDE hydrolysate (shown boxed in Fig. 3) are found in the a-subunit sequence, since the subunits should be present in roughly equimolar amounts. It is possible that some of the sequences are present in both subunits. Residue 4 in peptide PDE3 (Fig. 1A) differs from that predicted from the cDNA clones. This peptide may actually rep- resent the P-subunit sequence or an a-subunit variant. The assignment of the sequence in Fig. 3 as the a- subunit does not preclude the existence of several minor related proteins that migrate together with the a-sub- unit on SDS-PAGE. Indeed, we are currently analyzing

1

c 4.6- 4.0 -

FIG. 6. Analysis of human and bovine retinal RNA. Approxi- mately 2 pg retinal poly(A)+ RNA was fractionated on a 1.0% agarose, 2.2 M formaldehyde-containing gel and transferred to nitrocellulose (lane 1, human RNA; lanes 2-6, bovine RNA). The results of hy- bridization with radiolabeled human cDNA clone HPA-3 (Fig. 2) are shown in lane 1. A broad hybridizing band covering a size range from 5.3 to 4.9 kb is bracketed. Hybridization results with bovine cDNA probe 2 (Fig. 2) are shown in lane 2. Lanes 3-6 show the results of hybridization with oligonucleotide probes A-D (Fig. 2), respectively.

A

1 2 3

-27

- 9.4

- 2.3

- 2.0

- 1.3

- 1.0

-0.9

G

6

6 5 4 3 2

-6.2

FIG. 7. DNA blot analysis of the bovine o-subunit gene. (A) BamHI genomic DNA fragments identified with cDNA probes. Lanes are designated according to the probe used (see Fig. 2). Lines between lanes denote bands that represent identical size fragments due to overlap of probes. Sire markers are a mixture of X DNA digested with Hi&III and &X174 DNA digested with H&II. (B) Determi- nation of gene copy number. Genomic DNA (10 pg) and a X genomic clone were digested with HindI and transferred to a nylon mem- brane. Hybridizations were performed with probe 3. Lanes l-6 con- tain amounts of the X genomic clone DNA equivalent to one to six copies respectively of a 6200-nt fragment that would be present in 10 fig of genomic DNA (lane G).

cDNA clones with apparent heterogeneity to those in Fig. 2 that potentially represent a-subunit variants. The alternate forms, however, are thought to represent minor species since the major form reported here pre- dominated in the bovine cDNA clones isolated and was exclusively represented in the human and mouse (W. Baehr and S. J. Pittler, in preparation) cDNA clones.4

’ Some peptide sequences derived from Edman degradation of gel- purified and trypsinised ol-subunit do not match the deduced sequence in Fig. 3 (J. R. Leipprandt and D. G. McConnell, unpublished ob- servations). D.G.M. believes that the relative amounts of these variant peptides are too large to represent a minor form of the enzyme.

ROD PHOTORECEPTOR cGMP PDE (U-SUBUNIT 279

TABLE 1

Segregation of cGMP PDE n-Subunit Gene in Human-Rodent Somatic Cell Hybrids

Human chromosome

Hybrid” PDE,* 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

1 1.11 - +-+--+-----------f- 21.21 _ -----+++---+-++------~+- 3MR1.21- ------++-----------++-+- 41.4 _ --++-- ---- -----+--+-+-+- 5MR7.11- -e--------+-+----+--e++-

68.2 - -+++--++-+++-+++---+--f- 715.2 _ -------+------+-------+- 8SA-5 _ -e----+----+--‘-C--+---- 9 MH-7 - - ++ -- -- --- -- ----++--- - - -

10 MH-18 + - -+-+++++-+++++-----++---

11 P12 + + -+-+++---+-+-+++++++-+-

12 MH-74 + - ++-++++---++++-e++++++- 13 MH-41 + ++--+-+---------f-f-----

14 MH-82 - + -+--+++--+--+-+e+++++--

% Discordancy 21 29 36 43 0 29 36 43 29 36 43 36 14 36 21 43 21 36 21 36 21 36 64 29

n Hybrids l-7 are human-Chinese hamster hybrids and the rest are human-mouse hybrids. * cDNA probe 2 (Fig. 2) was used for all hybrids as described in the text. c t(15;17)(q15;p11.2), retains 17~11.2 + qter and 15q15 + qter. d t(9; 17)(q12;p11.2), retains 17~11.2 + qter and 9q12 + qter. ’ ring(l7)(pl3.3,q25.3), retains 17p13.3 * q25.3. ft(l7;19)(q23;p13), retains 17q23 + qter and 19p13 + qter.

We have confirmed that the sequence reconstructed from overlapping cDNA clones represents a contiguous retinal RNA molecule by the use of PCR. The cDNA product from a reverse transcription reaction with bo- vine retinal poly(A)+ RNA was amplified using a va- riety of primers (Fig. 3), including oligomer D and one complementary to oligomer A (Fig. 2). PCR products of the size expected were displayed after agarose elec- trophoresis (not shown), confirming that the primer sequences are linked at constant distances from one another on an RNA molecule.

Human cGMP PDE a-Subunit

A human retinal cDNA library (Nathans et al., 1986) was screened with a bovine a-subunit cDNA probe (probe 3, Fig. 2) representing about one-half of the protein coding region. Six cDNA clones, designated HPA-2 through HPA-6, that contained only partial sequence for the a-subunit were recovered (Fig. 2). In order to isolate a full-length cDNA clone, the library was rescreened with an oligonucleotide representing the first 12 amino acids (probe A, Fig. 2) of the bovine a-subunit sequence (Fig. 3). Twenty positive clones were recovered from a screen of 20,000 recombinants, one of which (designated HPA-1) contained the entire coding sequence, -100 bp 5’ flanking and -200 bp 3’ untranslated sequences. The nucleotide and deduced amino acid sequence of HPA-1 is shown in Fig. 4. Many of the oligonucleotide primers used to sequence the bo-

vine cDNAs were used successfully to obtain sequence from the human cDNA clones. No sequence differences were observed between HPA-1 and any of the truncated clones. A poly(A) sequence is present at the 3’ end of some of the cDNAs; however the AAUAAA sequence motif often found lo-30 bp upstream is absent. A number of other genes have been found to contain nonconsensus poly(A) sites (Ogawa et al., 1987).

Comparison of Human and Bovine cx-Subunits

Like the bovine cDNA sequence, the human cDNA sequence predicts a polypeptide chain of 859 residues, significantly larger than that predicted from its mo- bility on SDS-PAGE gels and from other measure- ments (Baehr et al., 1979; Kohnken et al., 1981; Hurwitz et al., 1984). The simplest interpretation is that the size inferred from direct determination differs from that deduced from the cDNA sequence due to post-trans- lational modifications.

A comparison of the predicted amino acid sequences for the human and bovine a-subunits is shown in Fig. 5. The human and bovine cDNA sequences are 88% identical over their matched lengths, and 94% identity was found at the protein level. The majority of the differences appear to be concentrated in the first -240 amino acids at the N-terminus and the last 43 amino acids at the C-terminus of the proteins. The region (bracketed in Fig. 5) identified as the conserved domain in several eukaryotic CN PDEs (Charbonneau et aZ.,

280 PITTLER ET AL.

13.3 13.2 13.1

:: 11.1 11.2

12

181 13.2 193

16

22

23.1 23.2 23.3

31.1 31.2 31.3

32 Sal

ii 34 36.1 36.2 36.3

5

FIG. 8. Regional localization of the cGMP PDE a-subunit gene. (A) The region of chromosome 5 retained in each hybrid cell line is shown. Hybrid cell line HHW105 contains an intact chromosome 5 as its only human chromosome. The hybridization results (B) are consistent with the regional location marked to the left of the idiogram (5q31.2 + q34). (B) Genomic DNA from each cell line was digested with HindHI, fractionated on an 0.8% agarose gel, and transferred to a nylon membrane. The blot was hybridized with radiolabeled cDNA insert from HPA- 3 (Fig. 2). Lanes designated HeLa and CHO are from control cell lines that contain only human or hamster chromosomes, respectively.

1986) is contained within the region that is the most highly conserved. The bovine and human sequences are 98% homologous within this region compared to about 30-45% homology with related CN PDEs in di- verse species. Identities at several positions appear to be conserved throughout eukaryotic phylogeny (re- viewed in Pittler and Baehr, 1989). No additional ho- mologies or similarities were found in a search of pro- tein or nucleic acid databases. Negative charges are in excess and appear to be uniformly distributed (calcu- lated p1,5.0). No apparent leader peptide was detected in the predicted amino acid sequence, consistent with the existence of the PDE a-subunit as an unsecreted, soluble protein.

The human and bovine PDE sequences vary signif- icantly near the C-terminal end (see Fig. 5). A cysteine residue, however, at position 856 is conserved in the human, bovine, and mouse (W. Baehr and S. J. Pittler, in preparation) sequences, and it has recently been shown in bovine that CysU ’ is lipidated following re- moval of the last three residues at the C-terminus (B. K.-K. Fung, personal communication). A number

of proteins that all share what has been termed a C- terminal four-amino-acid motif, CAAX (Magee and Hanley, 1988), are processed in a similar fashion. Re- cently, it was shown that all ras proteins examined are polyisoprenylated at the mature C-terminal cysteine, and only some are palmitoylated, and it was suggested that other proteins that also contain the CAAX motif may be similarly processed (Hancock et al., 1989). Post- translational lipidation of cysteines, particularly pal- mitoylation or addition of polyisoprenoids, has been observed in several proteins (Clarke et al., 1988; Mo- lenaar et al., 1988), including bovine rhodopsin (O’Brien and Zatz, 1984; Ovchinnikov et al., 1988). It has been suggested that methylation, previously shown to occur on the bovine a-subunit (Swanson and Ap- plebury, 1983), and lipidation may be coupled and serve an important role in membrane association (Clarke et al., 1988). This is consistent with the observation that limited trypsinolysis, which does not significantly alter the SDS-PAGE migration of the large subunits, abol- ishes the ability of the cGMP PDE to bind to the rod disk membrane (Wensel and Stryer, 1986; Whalen and

ROD PHOTORECEPTOR cGMP PDE a-SUBUNIT 281

Bitensky, 1989), suggesting that trypsin may cleave a small fragment from the N- or C-terminus. A potential cleavage site (Lysw) is present near Cys%. Palmitate addition to the &adrenergic receptor has recently been shown to be critical for normal receptor adenylate cy- clase coupling (O’Dowd et aZ., 1989).

Blot Analysis of Human and Bovine Retinal PolytAY RNA

To evaluate the transcription pattern of the gene in the adult human retina, we used a 1.4-kb human cDNA probe (HPA-3, Fig. 2) to hybridize to human poly(A)+ RNA. A broad band sized from 5.3 to 4.9 kb was iden- tified (Fig. 6). Several oligonucleotide probes specific to the a-subunit sequence and a bovine cDNA probe (probe 2, Fig. 2) hybridize predominantly to two rela- tively abundant bovine retinal RNAs of 4.6 and 4.0 kb (Fig. 6, lanes 2-6). At present, the significance of minor bands observed with these probes is unknown. Since the length of the open reading frame is identical in the two species, the larger size of the human transcripts must reflect larger 5’ and/or 3’ untranslated regions. The difference in the length of 3’ untranslated regions shown for the human and bovine RNAs (Figs. 2 and 3) suggests that alternative use of poly(A) attachment sites may account for at least some of the observed size heterogeneity.

DNA Blot Analysis of the Bovine cGMP PDE a-Subunit Gene

To assess the number of genes responsible for the bovine RNAs, we carried out several DNA blotting ex- periments. The intensity of hybridization of a 1.3-kb cDNA probe (probe 3, Fig. 2) to Hind111 fragments of genomic DNA compared to that of Hind111 fragments of varying amounts of an isolated genomic clone rep- resenting 1 to 6 genome equivalents suggests that the gene is present in single copy (Fig. 7B). Additionally, three different cDNA probes representative of N- and C-terminal sequences (probes 1,4,5, Fig. 2) hybridize to single genomic DNA fragments in EcoRI-, BumHI-, or PstI-digested DNA (only the BamHI digests are shown, Fig. 7A), consistent with the gene copy number data. Therefore, we conclude that a single bovine gene encodes the two predominant transcripts.

A minimum size for the gene was obtained by sum- ming the sizes of nonidentical-length BamHI fragments identified with representative cDNA probes (Fig. 7A). From this we estimate the bovine gene to be at least 140 kb, which is a minimum value since any restriction fragments that contain only intron sequence would not be detected, and the cDNA clones do not represent the entirety of the RNAs identified. However, we cannot formally rule out the possibility that some of the probes hybridized to fragments of closely related genes. The inability to detect overlapping fragments with probes

upstream or downstream of probe 4 is surprising, but reproducible. This suggests that the region represented by probe 4 is split by sizable introns in a manner that excludes hybridization with adjacent probes. The pres- ence of intron junctions close to each end of the probe 4 region is one possible arrangement that would explain this.

Chronwsornul Localizutiun of the Human cGMP PDE a-Subunit Gene

A 1.5-kb cDNA (probe 2, Fig. 2) was hybridized to a panel of 14 human-rodent somatic cell hybrid DNAs digested with EcoRI to determine the location of the human cGMP PDE a-subunit gene. The results from this analysis are summarized in Table 1. We observed 100% concordancy with chromosome 5 and at least two discordancies for all other chromosomes. In order to delimit the assignment further, we probed 11 hu- man-hamster hybrid DNAs (not shown) that contain various deletions of chromosome 5 (Overhauser et al., 1986). Two of these hybrids are deleted for the majority of 5q and the rest contain intact long arms and various deletions of 5p. Hybridization signals were observed in all of the hybrid DNAs except the two hybrids deleted for 5q. A more precise regional localization was deter- mined by analysis of a panel of hybrid DNAs containing various deletions of the long arm (Fig. 8A). Hybridiza- tion results from this panel using a 1.4-kb human cDNA probe (HPA-3, Fig. 2) are shown in Fig. 8B. The region 5q31.2 + q34 is the only portion of chromosome 5 retained intact in all of the hybrids. We therefore provisionally assign the gene location to the region 5q31.2 + 934, although we cannot completely rule out a 5q22 localization due to the inaccuracy inherent in the cytological analysis.

Since this is the first CN PDE gene to be localized, the locus designation CGPR-A is suggested. This des- ignation takes into account the classification system of Beavo (1988) for CN PDEs and is in agreement with the recommendations of the committee on human gene nomenclature (Shows et al., 1987). The locus name de- notes the human gene encoding the cGMP phospho- diesterase a-subunit of the rod photoreceptor cell.

The Candidate Gene Approach

The possibility that a defective cGMP PDE is the cause of certain forms of hereditary retinal degenera- tion makes the gene a good target for the candidate gene approach, in which the first step is to isolate and characterize a gene or a representative cDNA. The cDNA or gene fragment is then used to identify the chromosomal location for comparison to disease loci already known (McKusick, 1988). The second step is to analyze large kindreds with well-established pedi- grees for linkage to the disease of interest. This ap- proach complements more conventional linkage studies

282 PITTLER ET AL.

in which random probes are used in an attempt to nar- row the location of a disease gene. The candidate gene approach has the advantage of allowing relatively fast assessment of the potential involvement of a specific gene to a hereditary disease. Additionally, it guarantees that important information will be obtained from the analysis of a cDNA or gene, which is currently not true for conventional linkage analysis.

The completion of the first step for our candidate, the cGMP PDE a-subunit, is presented here. We have isolated a full-length cDNA and we have localized the gene to the long arm of chromosome 5 (5q31.2 + q34). We were, however, unable to find any retina-specific disorders that have been mapped to this region of 5q. The second step of the approach is currently underway.

ACKNOWLEDGMENTS

We thank Dr. Jeremy Nathans for the retinal cDNA libraries, Dr. Gregory I. Liou for human retinal RNA, and Dr. Thaddeus P. Dryja and Valerie Grondin for isolating the initial human cDNA clones. This work was supported in part by NIH Award GM29658 to J.J.W. NSF awards to R.L.D. and D.G.M., and an award from the National RP Foundation to W.B. W.B. is a Jules and Doris Stein Research to Prevent Blindness Professor.

1.

2.

3.

4.

5.

6.

REFERENCES

AGUIRRE, G., FARBER, D., LOLLEY, R., FLETCHER, R. T., AND CHADER, G. J. (1978). Rod cone dysplasia in Irish Setters: A defect in cyclic GMP metabolism in visual cells. Science 201: 1133-1134.

BAEHR, W., DEVLIN, M. J., AND APPLEBURY, M. L. (1979). Iso- lation and characterization of cGMP phosphodiesterase from bovine rod outer segments. J. Biol. Chem. 264: 11669-11677.

BEAVO, J. A. (1988). Multiple isozymes of cyclic nucleotide phosphodiesterase. Adv. Second Mess. Phosphoprot. Res. 22: l-38.

CHARBONNEAU, H., BEIER, N., WALSH, K. A., AND BEAVO, J. A. (1986). Identification of a conserved domain among cyclic nucleotide phosphodiesterases from diverse species. Proc. N&l. Acad. Sci. USA 83: 9308-9312.

CHIRGWIN, J. M., PRZYBLA, A. E., MACDONALD, R. J., AND RU?TER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294-5299.

CLARKE, S., VOGEL, J. P., DESCHENES, R. J., AND STOCK, J. (1988). Posttranslational modification of the Ha-ras oncogene protein: Evidence for a third class of protein carboxyl meth- yltransferases. Proc. Natl. Acad. Sci. USA 85: 4643-4647.

DETERRE, P., BIGAY, J., FORQUET, F., ROBERT, M., AND CHABRE, M. (1988). cGMP phosphodiesterase of retinal rods is regulated by two inhibitory subunits. Proc. Natl. Acad. Sci. USA 95: 2424-2428.

FARBER, D. B., AND LOLLEY, R. N. (1974). Cyclic guanosine monophosphate: Elevation in degenerating photoreceptor cells of the C3H mouse retina. Science 186: 449-451.

FARBER, D. B., FLANNERY, J. G., BIRD, A. C., SHUSTER, T., AND BOK, D. (1987). Histopathological and biochemical studies on donor eyes affected with retinitis pigmentosa. In “Degen- erative Retinal Disorders: Clinical and Laboratory Investiga- tions” (J. G. Hollyfield et al., Eds.), pp. 53-67, A. R. Liss, New York.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

GRACY, R. W. (1977). Two-dimensional thin layer methods. In “Methods in Enzymology” (C. H. W. Him and S. N. Timasheff, Eds.), Vol. 47, pp. 195-204. Academic Press, New York.

HANCOCK, J. F., MAGEE, A. I., CHILDS, J. E., AND MARSHALL,

C. J. (1989). All rus proteins are polyisoprenylated but only some are palmitoylated. Cell 67: 1167-1177.

HECKENLIVELY, J. (1988). In “Retinitis Pigmentosa” (J. Heck- enlively et al., Eds.), Lippincott, Philadelphia.

HOGAN

HOLLYFIELD, J. G., RAYBORN, M. E., FARBER, D. B., AND LOL- LEY, R. N. (1982). Selective photoreceptor degeneration during retinal development: The role of altered phosphodiesterase ac- tivity and increased levels of cyclic nucleotides. In “Structure of the Eye” (J. G. Hollyfield, Ed.), p. 97, Elsevier-North Holland, Amsterdam.

HOLLYFIELD, J. G., RAYBORN, M. E., ULSHAFER, R. J., AND FLIESLER, S. J. (1985). Simulation of an inherited disease in the normal retina: Involvement of cyclic nucleotides. In “Retinal Degeneration: Experimental and Clinical Studies” (M. M. LaVail et al., Eds.), pp. 341-348, A. R. Liss, New York.

HURLEY, J. B. (1987). Molecular properties of the cGMP cascade of vertebrate photoreceptors. Annu. Rev. Physiol. 49: 793-812.

HURWITZ, R. L., BUNT-MILAM, A. H., AND BEAVO, J. A. (1984). Immunologic characterization of the photoreceptor outer seg- ment cyclic GMP phosphodiesterase. J. Biol. Chem. 269: 8612- 8618.

KOHNKEN, R. E., EADIE, D. M., REVZIN, A., AND MCCONNELL, D. G. (1981). The light-activated GTP-dependent cyclic GMP phosphodiesterase complex of bovine retinal rod outer segments. J. Biol. Chem. 256: 12510-12516.

LEON, A., HUSSAIN, A. A., AND CURTIS, R. (1988). Rod-cone dysplasia in the Rdy cat: Electrophysiology and biochemistry. Invest. Ophthalmol. Vis. Sci. Suppl. 29: 143.

LIEBMAN, P. A., PARKER, K. R., AND DRATZ, E. A. (1987). The molecular mechanism of visual excitation and its relation to the structure and composition of the rod outer segment. Annu. Rev. Physiol. 49: 765-791.

LOLLEY

MAGEE, T., AND HANLEY, M. (1988). Sticky fingers and CAAX boxes. Nature (London) 336: 114-115.

MCKUSICK, V. A. (1988). “Mendellian Inheritance in Man,” Johns Hopkins Univ. Press, Baltimore, MD.

MOLENAAR, C. M. T., PRANGE, R., AND GALLWITZ, D. (1988). A carboxyl-terminal cysteine residue is required for palmitic acid binding and biological activity of the m-related yeast YPTl protein. EMBO J. 7: 971-976.

NATHANS, J., AND HOGNESS, D. (1983). Isolation, sequence analysis, and intron-exon arrangement of the gene encoding bovine rhodopsin. Cell 34: 807-814.

NATHANS, J., THOMAS, D., AND HOGNESS, D. (1988). Molecular genetics of human color vision: The genes encoding blue, green, and red pigments. Science 232: 193-202.

O’BRIEN, P. J., AND ZATZ, M. (1984). Acylation of bovine rho- dopsin by [sH]palmitic acid. J. Biol. Chem. 259: 505445057.

O’DOWD, B. F., HNATOWICH, M., CARON, M. G., LEFKOWITZ, R. J., AND BOUVIER, M. (1989). Palmitoylation of the human fix-adrenergic receptor. J. Biol. Chem. 264: 7564-7569.

OGAWA, H., TOMOHARU, G., MUECKLER, M. M., FUJIOKA, M., BACKLUND, P. S., JR., AKSAMIT, R. R., UNSON, C. G., AND CAN- TONI, G. L. (1987). Amino acid sequence of S-adenosyl-L-ho- mocysteine hydrolase from rat liver as derived from the cDNA sequence. Proc. Natl. Acad. Sci. USA 94: 719-723.

OHTSUKA, E., MATSUKI, S., IKEHARA, M., TAKAHASHI, Y., AND MATSUBARA, K. (1985). An alternative approach to deoxyoli-

ROD PHOTORECEPTOR cGMP PDE &SUBUNIT 283

31.

32.

33.

34.

35.

36.

gonucleotides as hybridization probes by insertion of deoxyino- sine at ambiguous codon positions. J. Biol. Chem. 260: 2605-

2608.

OVCHINNIKOV, Y. A., ABDULAEV, N. G., AND BOGACHUK, A. S. (1988). Two adjacent cysteine residues in the C-terminal cy- toplasmic fragment of bovine rhodopsin are palmitylated FEBS Z&t. 230: l-5.

OVCHINNIKOV, Yu. A., GUBANOV, N. V., KHRAMTSOV, K. A., ISCHENKO, V. E., ZAGRANICHNY, V. E., MURADOV, K. G., SHU- VAEVA, T. M., AND LIPKIN, V. M. (1987). Cyclic GMP phos- phodiesterase from bovine retina: Amino acid sequence of the d-subunit and nucleotide sequence of the corresponding cDNA. FEBS Z&t. 223: 169-173.

OVERHAUSER, J., BEAUDET, A. L., AND WASMUTH, J. J. (1986). A fine structure physical map of the short arm of chromosome 5. Amer. J. Hum. Genet. 39: 562-572.

PI’ITLER, S. J., SALZ, H. K., AND DAVIS, R. L. (1987). An in- terchromosomal gene conversion of the Drosophila dunce locus identified with restriction site polymorphisms: A potential in- volvement of transposable elements in gene conversion. Mol. Gen. Genet. 208: 315-324.

PIITLER, S. J., AND BAEHR, W. (1989). The molecular genetics of retinal photoreceptor proteins involved in cGMP metabolism. In “The Molecular Biology of the Retina: Basic and Clinically- Relevant Studies” (G. J. Chader and D. Farber, Eds.), A. R. Liss, New York, in press.

SHOWS, T. B., MCALPINE, P. J., BOUCHEIX, C., COLLINS, F. S., CONNEALLY, P. M., FRE~AL, J., GERSHOWITZ, H., GOODFELLOW, P. N., HALL, J. G., Issrrr, P., JONES, C. A., KNOWLES, B. B., LEWIS, M., MCKUSICK, V. A., MEISLER, M., MORTON, N. E., RUBINSTEIN, P., SCHANFIELD, M. S., SCHMICKEL, R. D., SKOL- NICK, M. H., SPENCE, M. A., SUTHERLAND, G. R., TRAVER, M.,

37.

38.

39.

40.

41.

42.

43.

44.

45.

VAN CONG, N., AND WILLARD, H. F. (1987). Guidelines for hu- man gene nomenclature. Cytogenet. Cell Gen-et. 46: 11-28.

STRYER, L. (1986). Cyclic GMP cascade of vision. Annu. Reu. Neurosci. 9: 87-119. Su, T.-S., NUSSBAUM, R. L., AIRHART, S., LEDBETTER, D. H., MOHANDAS, T., O’BRIEN, W. E., AND BEAUDET, A. L. (1984). Human chromosomai assignments for fourteen argininosuc- cinate synthetase pseudogenes: Cloned DNAs as reagents for cytogenetic analysis. Amer. J. Hum. Genet. 36: 954-964.

SWANSON, R. J., AND APPLEBURY, M. L. (1983). Methylation of proteins in photoreceptor rod outer segments. J. Biol. Chem. 258: 10599-10605. ULSHAFER, R., GARCIA, C., AND HOLLYFIELD, J. G. (1980). Sen- sitivity of photoreceptors to elevated levels of cGMP in the human retina. Znuest. Ophthalmol. Via Sci. 19: 1236-1241. VANTUINEN, P., RICH, D. C., SUMMERS, K. M., AND LEDBE~R, D. H. (1987). Regional mapping panel for human chromosome 17: Application to neurofibromatosis type 1. Gerwmics 1: 374- 381. WENSEL, T. G., AND STRYER, L. (1986). Reciprocal control of retinal rod cyclic GMP phosphodiesterase by its y subunit and transducin. Proteins: Struct. Funct. Genet. 1: 90-99. WHALEN, M. M., AND BITENSKY, M. W. (1989). Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments. B&hem. J. 259: 13-19. WOODFORD, B. J., LIU, Y., FLETCHER, R. T., CHADER, G. J., FARBER, D. B., SANTOS-ANDERSON, R., AND Tso, M. O.-M. (1982). Cyclic nucleotide metabolism in inherited retinopathy in collies: A biochemical and histochemical study. Exp. Eye Res. 34: 703-714. ZHANG, H., SCHOLL, R., BROWSE, J., AND SOMMERVILLE, C. (1988). Double stranded DNA sequencing as a choice for DNA sequencing. Nucleic Acids Res. 16: 1220.