GENOMICS 4, 215-217 (1989)

SHORT COMMUNICATION The Gene for the &ubunit of Retinal Rod

Mouse Chromosome 9 Transducin Is on

MICHAEL DArwGm,*,t CHRISTINE A. KOZAK,$ AND DEBORA B. FARBER**’

*Jules Stein Eye Institute, UCLA School of Medicine, Los Angeles, California 90024- 1777; tLoyola Marymount University, Los Angeles, California 90045; and *National Institute of Allergy and Infectious Disease,

National Institutes of Health, Bethesda, Maryland 20892

Received August 5. 1988; revised October 12, 1988

Mice carrying the autosomal recessive rd gene ex- perience total degeneration of the photoreceptor cells of the retina by 3 to 4 weeks of life. Biochemical stud- ies of the rd retina have demonstrated a lesion in cyclic guanosine monophosphate (cGMP) metabolism due to depressed rod-specific cGMP-phosphodiesterase (cGMP-PDE) activity. The depressed activity could result from, among other things, a lesion in the cGMP- PDE enzyme itself or in any of a number of proteins in the rod that regulate it. We have used a cDNA clone for the o-subunit of bovine rod transducin (Tel) to map the corresponding gene, Gnat-l, to mouse chro- mosome 9 with a panel of Chinese hamster-mouse so- matic cell hybrid DNAs. Transducin, a heterotrimeric G protein, is involved in the stimulation of cGMP-PDE when light hits the rod photoreceptors. Since the pri- mary defect in rd disease occurs in a gene(s) on mouse chromosome 5, our results suggest that Gnat-l is not the rd gene. 6’ 1989 Academic Press, Inc.

Retinal degeneration in mice carrying the rd mu- tation is an autosomal recessive disease which affects the photoreceptor cell layer. The disease is character- ized by a degenerative process which first appears in the rod photoreceptors at Postnatal Day 8 (Lasansky and De Robertis, 1960; Shiosi and Sonohara, 1969) and continues rapidly until 3-4 weeks of life when most of the photoreceptor cells have died (LaVail and Sidman, 1974). As early as Postnatal Day 6, well before any morphological pathology can be detected, the level of cGMP in the rd retina begins to rise above normal due to deficient cGMP-phosphodiesterase (cGMP-PDE) activity (Farber and Lolley, 1974,1976). The activation of cGMP-PDE is a complex process involving several different proteins (Farber and Shuster, 1986). There-

’ To whom reprint requests should be addressed at Jules Stein Eye Institute, B-237, UCLA School of Medicine, Los Angeles, CA 90024-1771.

fore, the mutation responsible for decreased activity may be in (among other things) the cGMP-PDE mol- ecule itself or any of the proteins involved directly or indirectly in its activation. One such protein is the het- erotrimer transducin, which is involved in the stimu- lation of cGMP-PDE when the rod photoreceptor is exposed to light. A lesion in this G protein could cause a deficiency in cGMP-PDE activity.

In this report we describe the mouse chromosome mapping of a cDNA for the c-r-subunit of bovine rod transducin (TLY~). The corresponding human gene has been designated Gnat-l at the Ninth International Workshop on Human Gene Mapping (1987).

A panel of 23 hamster-mouse somatic cell hybrid DNAs was analyzed for sequences homologous to TLV~ cDNA. The hybrids were derived from the fusion of E36 Chinese hamster cells with peritoneal or spleen cells from BALB/c or NFS.Aku-2 mice. The chromo- some content of most hybrids was determined by tryp- sin-Giemsa banding followed by staining with Hoechst 33258; all hybrids were typed for specific marker loci on (up to all 20 of) the mouse chromosomes (Kozak et al., 1975, 1977; Kozak and Rowe, 1979; Hoggan et al., 1988).

The Tal cDNA has 2190 bp with a 1050-bp coding region followed by a 1140-bp untranslated region at the 5’ end and was kindly provided by Dr. Melvin I. Simon, California Institute of Technology. This clone has been described previously (Tanabe et al., 1985). The probe was labeled with [a-32P]dCTP (3000 Ci/ mmol) by the random priming method (Feinberg and Vogelstein, 1983).

The Chinese hamster-mouse hybrid DNAs and pa- rentals were prepared by published procedures (Lin- nenbach et al., 1980), digested with EcoRI, and at 6 pg of DNA per lane were electrophoresed in 1.2% agarose gels. DNAs were transblotted to nylon membranes by the technique of Southern (1975). The blots were pre- hybridized for 3 to 6 h at 65’C in a solution containing 7.5% SDS (sodium dodecyl sulfate), 0.5 A4 phosphate

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216 SHORT COMMUNICATION

buffer at pH 7.0, 1 mM EDTA, and 1% bovine serum albumin. Hybridization was carried out at 60°C over- night in the same solution as that for prehybridization with the addition of radioactive probe. After hybrid- ization the blots were washed for 30 min at 37°C in 2X SSC (1X SSC is 150 mM NaCl and 15 mM sodium citrate, pH 7.0) + 0.1% SDS, two times for 20 min at 57°C in 2X SSC + 0.1% SDS, and two times for 20 min at 57°C in 0.4X SSC + 0.1% SDS. After washing, the blots were exposed to X-ray film with intensifier screens at -8O“C for 3 to 10 days.

The Chinese hamster parental DNA showed one band of 23 kb, while both of the mouse parental DNAs showed a single band of 5.8 kb (Fig. 1). Typical results of hybrid DNAs either positive or negative for the presence of Tal sequences are shown in lanes 3-5 of Fig. 1. Of 23 hybrids tested, 8 contained the 5.8-kb band. Table 1 shows no discordancies for the presence of T~yl on chromosome 9, and at least three discor- dancies for its presence on any other mouse chromo- some. To eliminate the possibility that sequences ho- mologous to Tcvl are also on mouse chromosome 11, which is not present in any of the hybrids (Kozak and Ruddle, 1977), we analyzed a rat-mouse hybrid whose only mouse chromosome is 11 (Killary and Fournier, 1984). The results showed no T~vl sequences in this hybrid. The same blot showed sequences in the hybrid

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FIG. 1. Autoradiogram of a Southern blot of hamster-mouse somatic cell hybrid DNAs digested with EcoRI and hybridized with the Tcvl cDNA probe; each lane has 6 gg of DNA. The Chinese hamster control is in lane 1 and one of the mouse controls, NFSAka- 2, is in lane 2. Three representative somatic cell hybrid DNAs are shown in lanes 3-5. The arrow points to the 5.8.kb mouse band which was used to score for the presence of T~vl sequences in the hybrids. The numbers on the left (in kb) mark the positions of X DNA fragments digested with HindIII.

TABLE 1

Correlation between Specific Mouse Chromosomes and the Gnat-1 Gene in 23 Somatic Cell Hybrids

Number of hybrid clones

Tal sequence/chromosome retention

Mouse chromosome +/+ -/- +/- -/+ % Discordant

1 5 9 2 6 36.4” 2 6 9 2 6 34.8 3 2 8 1 5 37.5 4 6 11 2 3 22.7 5 2 13 5 2 31.8 6 6 10 2 5 30.4 7 8 4 0 11 47.8 8 4 14 3 1 18.2 9 8 14 0 0 0.0

10 1 14 7 1 34.8 11 0 13 3 0 18.8 12 2 4 1 9 62.5 13 8 9 0 4 19.0 14 3 13 5 2 30.4 15 3 2 0 11 68.8 16 4 10 2 3 26.3 17 7 7 1 8 39.1 18 6 8 1 5 30.0 19 5 9 3 4 33.3 20 4 8 3 7 45.5

” Five hybrids have Tal sequences and also contain chromosome 1 (+/+l. Nine hybrids lack Tot1 sequences and also lack chromosome 1 t-/-). Two hybrids have TLV~ sequences, but lack chromosome 1 t+/-1. Six hybrids lack Tal sequences, but contain chromosome 1 c-/+j.

of another probe that had been mapped to mouse chro- mosome 11 (Danciger et al., 1988).

Our findings are in agreement with those of the lab- oratory of M. I. Simon, who mapped the same Tal cDNA to mouse chromosome 9 with restriction frag- ment length polymorphisms in recombinant inbred mice (Sparkes et al., 1987). Transducin is referred to there as guanine nucleotide-binding protein, alpha 1 (Gtal).

The assignment of Gnat-I to chromosome 9 dem- onstrates that this gene is not the site of the primary lesion in rd disease since linkage studies have assigned the rd gene to chromosome 5 (Sidman and Green, 1965). We are currently studying the cDNAs of several other proteins involved in cGMP metabolism to de- termine whether any of these map to mouse chromo- some 5.

ACKdOWLEDGMENTS

This research was supported by the National Retinitis Pigmentosa Foundation (Baltimore, MD), the George Gund Foundation, and NIH

SHORT COMMUNICATION 217

Grants EY 02651 and EY 00331. We thank Drs. Robert Sparkes and Bronwyn Bateman for helpful discussions and critical reading of the manuscript.

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