THE J O U R N A L OF BIOLOGICAL CHEMISTRY Vol. 266, No. 23, Issue of August 15, pp. 15505-15510,1991 Printed in U.S.A.

Expression of S-antigen in Retina, Pineal Gland, Lens, and Brain Is Directed by 5’-Flanking Sequences*

(Received for publication, April 3, 1991)

Martin L. BreitmanSB, Masahiko Tsuda, Jiro Usukuraq, Takanobu Kikuchi, Anthony ZucconiS, Wilson KhooS, and Toshimichi Shinoharan From the SDiuision of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Auenue, Toronto, Ontario M5G 1x5, Canada and Department of Molecular and Medical Genetics, University of Toronto, Toronto, Canada, TDepartment of Anatomy, Nagoya University School of Medicine, Tsurumai, Nagoya, Japan, and the Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892

S-antigen (S-Ag) is an abundant protein of the retina and pineal gland that elicits experimental autoimmune uveitis and pinealocytis in several animal species. To study the elements regulating the expression of S-Ag, we generated transgenic mice expressing the chlor- amphenicol acetyl transferase (CAT) gene under the control of a 1.3-kilobase pair 5”flanking segment of the mouse S-Ag gene. While all of the transgenic mice expressed CAT activity in the retina, in some animals CAT activity was also detected in the pineal gland, lens, and brain. Immunoblotting, polymerase chain re- action-mediated detection of RNA, and immunocyto- staining of transgenic tissues with antibodies to CAT and S-Ag established that the profile of expression of the transgene corresponded to that of S-Ag; both pro- teins were detectable in retinal photoreceptor cells, pinealocytes, lens fiber and epithelial cells, the cere- bellum, and the cerebral cortex. These results indicate that S-Ag is expressed in a wider spectrum of the cell types than previously recognized and that a 1.3-kilo- base pair S-Ag promoter segment contains sufficient information to direct appropriate tissue-specific gene expression in transgenic mice.

The outer segments of retinal photoreceptor cells contain several highly specialized proteins that mediate phototrans- duction, the receptor-mediated signal mechanism that con- verts light energy into neuronal impulses (Stryer, 1988; Chabre and Deterre, 1989). One of these proteins, S-Ag,’ is a hydrophilic polypeptide of approximately 45 kDa (Wacker et al., 1977; Kuhn et al., 1984). The precise mechanism of action of S-Ag is presently unknown, although it is generally believed to modulate the phototransduction cascade through its inter- action with either photoactivated, phosphorylated rhodopsin (Kuhn et al., 1984; Kuhn and Wilden, 1987; Bennett and Sitaramayya, 1988) or cGMP-dependent phosphodiesterase

* This work was supported by a grant from the Medical Research Council of Canada (to M. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

I A Medical Research Council Scientist. ( 1 TO whom correspondence should be addressed. Tel.: 301-496-

7799; Fax: 301-496-0823. ’ The abbreviations used are: S-Ag, S-antigen; CAT, chloramphen-

icol acetyltransferase; bp, base pair(s); SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; ROS, rod outer segment; RIS, rod inner segment.

(Zukerman and Cheasty, 1986), two additional components of the transduction mechanism.

In addition to its role in phototransduction, S-Ag has been studied extensively in relation to its ability to induce experi- mental autoimmune uveitis and pinealocytis, a T cell-me- diated disease resulting in severe inflammation of the retina and pineal gland (Wacker et al., 1977; Faure, 1980; Shinohara et al., 1988). The pineal gland is a secretory organ that is thought to have evolved from photoreceptor cells of lateral eyes (Collin et al., 1986; .Kappers, 1981). While mammalian pinealocytes, unlike those of teleosts, lack photoreceptor func- tion, they do contain various amounts of several photorecep- tor proteins, including S-Ag (Kalsow and Wacker, 1977; Korf et al., 1985). At present, the role of S-Ag in the mammalian pineal gland remains an enigma.

Recent studies suggested that S-Ag may be a member of a family of proteins that has been highly conserved during evolution. Thus, S-Ag shows sequence similarity to /3-arrestin of rat brain (Lohse et al., 1990) as well as to two S-Ag-like molecules isolated from the eyes of Drosophila melanogaster (Smith et al., 1990; Hyde et al., 1990; Yamada et al., 1990). While the function of the two fly proteins remains to be determined, @-arrestin is known to play an important regu- latory role within the @-adrenergic hormone transduction cascade (Lohse et al., 1990).

During the past few years, cDNAs and genomic clones for S-Ag have been isolated from several animal species (Yamaki et al., 1987; 1988; Shinohara et al., 1987; Tsuda et al., 1988; Abe et al., 1989; Abe and Shinohara, 1990). Detailed charac- terization of the mouse and human S-Ag genes showed that they span approximately 50 kilobase pairs and contain 16 exons, including a 5‘ noncoding exon (Yamaki et al., 1990; Tsuda et al., 1991). The 5’-flanking sequences of these genes, as well as those of bovine S-Ag,’ lack consensus TATA and CAAT boxes; however, they have been demonstrated to direct appropriate initiation of transcription in nuclear extracts derived from rat brain (Yamaki et al., 1990; Tsuda et al., 1991).

In this report, we reexamine the profile of expression of S- Ag and characterize the regulatory function of the 5“flanking sequences of the mouse S-Ag gene. We show that S-Ag is expressed not only in the retina and pineal gland, as previ- ously determined, but also in the epithelial and fiber cells of the lens, the cerebellum, and cerebral cortex. We also dem- onstrate that a 1.3-kilobase pair S-Ag promoter segment

a M. L. Breitman, M. Tsuda, J. Usukura, T. Kikuchi, A. Zucconi, W. Khoo, and T. Shinohara, unpublished results.

15505

15506 S-antigen Promoter Activity in Transgenic Mice

contains sufficient information to direct appropriate tissue- restricted gene expression in transgenic mice.

MATERIALS AND METHODS

Construction of the S-Ag-CAT Fusion Gene-An EcoRI-KpnI frag- ment spanning mouse S-Ag sequences -1283 to +163 was filled in with T4 DNA polymerase and ligated to HzndIII-digested pSVOCAT (Gorman et al., 1982). Recombinant plasmids were screened for the correct S-Ag-CAT orientation by DNA sequencing. The S-Ag-CAT fusion gene was retrieved from recombinant plasmid pS-Ag-CAT on an NdeI-BarnHI fragment containing 85 bp of pBR322 sequence upstream of the S-Ag promoter segment (see Fig. 1). The fusion gene fragment was eluted from an agarose gel, extracted with phenol/ chloroform (l:l), and precipitated with 70% ethanol.

Transgenic Mice-The purified NdeI-BarnHI fragment containing the S-Ag-CAT fusion gene was microinjected into one-cell CD-1 zygotes at a concentration of 5 pg/ml essentially as described by Hogan et al. (1986). To screen for transgenic founder animals, DNA was extracted from tail biopsies by the proteinase K/SDS method (Hogan et al., 1986) and analyzed for the presence of the transgene by Southern blot analysis as described previously (Southern, 1975; Breitman et al., 1987). Transgene copy number was estimated by comparing the band intensity of S-Ag-CAT hybridization with those of known internal standards.

CAT Assays-Organs dissected from S-Ag-CAT transgenic mice were snap-frozen in liquid nitrogen and stored at -70 "C. For enzyme determination, samples were thawed and homogenized in 0.1-0.2 ml of 250 mM Tris (pH 7.8), 10 mM EDTA. The homogenates were then spun in a microcentrifuge for 2 min and the supernatants heated at 65 "C for 10 min to inactivate endogenous deacetylases. CAT enzyme activity was assayed for 2 h at 37 "C with I4C-labeled chloramphenicol (Amersham Corp.) as described (Gorman et al., 1982). To quantitate the CAT activity in individual samples, signals on the autoradiograph corresponding to the acetylated forms of chloramphenicol were scraped from the silica-gel plate, emulsified in Optifluor (Packard), and counted by liquid scintillation spectroscopy.

Irnrnunocytostaining of CAT and S-Ag by Antibodies-Eyes were enuclated from anesthetized wild type or transgenic mice and soaked immediately in fixative consisting of 1% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer adjusted at pH 7.4. Retinas were isolated and dissected into small pieces (about 2 mmZ) in the same fixative. Excised retinas were washed briefly with the same phosphate buffer after 2 h of fixation. Subsequently, specimens were dehydrated through ascending series of ethanol up to 95% and embedded in Lowicryl K4M. For light microscope immunocytochem- istry, semi-thin sections were cut and mounted on the glass slide subbed with 2% aminopropyltriethoxysilane in dry acetone. Sections on the glass slide were covered with 4% bovine serum albumin in phosphate-buffered saline (PBS) for 10 min and then incubated overnight with polyclonal rabbit antibodies against highly purified (more than 95%) mouse S-Ag protein produced in a baculovirus vector and insect cell system or CAT purchased commercially from 3' prime-5' prime, Inc. (West Chester, PA). S-Ag and CAT antibodies were diluted 200- and 10-fold, respectively, in PBS containing 1% bovine serum albumin prior to using. After rinsing three times for 3 min in PBS, slides were incubated for 2 h with a 40-fold dilution of gold-conjugated anti-rabbit IgG (AuroProbe for E M, Amersham Corp.). Thereafter, slides were washed twice for 3 min with PBS followed by twice for 3 min with distilled water. Gold labeling was further enhanced for good visualization by using the silvery enhance- ment ki t (IntenSEM, Amersham). Specimens were observed with a confocal laser microscope (Bio-Rad) at reflection mode after counter- staining with toluidine blue. Reflection images from gold particles (antibody binding to the molecules) were superimposed on the trans- mission image while being color-inprinted by computer assisting (Usukura and Bok, 1987).

Analysis of rnRNA Using PCR-ampli/kd DNA-Poly(A)-contain- ing RNAs were extracted from mouse tissues using the Fast Track kit (Invitrogen, San Diego, CA). Approximately 10 pg of RNA were reverse-transcribed in a 10-pl volume with random primer and avian

Following cDNA synthesis, 100 pmol of each of a pair of specific myeloblastosis virus reverse transcriptase (Boehringer Mannheim).

oligomers corresponding to mouse S-Ag cDNA sense sequences 631- 657 and antisense sequences 1374-1400 (see Tsuda et al. (1991)) were added and the 50-pl reaction mixture was boiled for 3 min and quenched on ice. The remaining PCR reagents (Gilliland et al., 1990) were then added, and amplification was performed in 100 p1 of 50

mM KCI, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgC12,3 mM dithiothre- itol, 0.1 mg of bovine serum albumin, 200 p~ (each) dNTPs, and 2.5 units of Taq polymerase. Twenty cycles of PCR were carried out under the following conditions: annealing at 55 "C for 2 min, exten- sion at 72 "C for 3 min, denaturation at 94 "C for 1 min, and final extension for 20 min. The amplified DNA was analyzed on a 4.0% agarose gel.

Protein Blot Analysis-Cerebral cortex and cerebellum were dis- sected from the 14-day-old transgenic mouse. Bovine lenses were used instead of mouse lenses in order to obtain sufficient protein from the lens epithelium. The tissues (approximately 0.5 g) were homogenated in 0.5 ml of the ice-cold PBS buffer. The homogenates were centri- fuged (12,000 X g) for 20 min at 4 "C. The supernatants were mixed with 20 pl of polyclonal S-Ag antibody3 and kept at 4 "C for 72 h. After microcentrifugation, the precipitates were washed three times in PBS buffer, dissolved in 10 pl of SDS-PAGE sample buffer (Maizel, 1971), and heated for 3 min at 100 'C. The samples (1 pl) were fractionated by the SDS-PAGE and blotted to transfer membranes (Immobilon polyvinylidene difluoride) with Phastsystem (Pharmacia LKB Biotechnology Inc.). The membranes were incubated overnight with monoclonal S-Ag antibody (1/3,000 dilution) (Donoso et al., 1986) and then washed four times with 0.05% Tween 20,20 mM Tris- HCI, and 500 mM NaCl adjusted to pH 7.5. The filters were then incubated with alkaline phosphatase-conjugated goat anti-mouse an- tibody (1/3,000 dilution, Bio-Rad) for 90 min at room temperature and developed as recommended by the supplier. Gels were stained with the PhastGel silver kit (Pharmacia LKB Biotechnology Inc.).

RESULTS

Generation and Characterization of S-Ag-CAT Transgenic Mice-Since S-Ag is expressed in more than one cell type and photoreceptor cells are a terminally differentiated, post-mi- totic cell population that is not amenable to culture in uitro, it was necessary to take a transgenic approach to monitor the activity of the 5"flanking sequences of the mouse S-Ag gene. To this end, a 1.3-kilobase pair S-Ag 5'-flanking segment encompassing sequences -1283 to +163 was placed upstream of the cat reporter gene (Fig. LA) and microinjected into the male pronucleus of fertilized CD-1 mouse eggs to generate transgenic animals. Of 18 animals generated, Southern blot (Southern, 1975) analysis of tail DNA revealed that seven mice contained between 1 and 20 copies of the S-Ag-CAT transgene (Table I1 and data not shown). The profile of expression of the transgene in these animals was determined by assaying dissected organs for CAT activity. Two of the transgenic founders did not show detectable expression of the transgene. Of the remaining five S-Ag-CAT mice, two (H6 and 53) produced detectable CAT activity exclusively in the retina, while three (H4, J10, and D2) produced considerably higher levels of CAT activity in the retina and lower levels in the lens, pineal gland, cerebellum, and cerebral cortex (see Fig. 1B and Table I). Comparison of the activity profiles of the different animals revealed that there was no correlation between transgene copy number and the level or extent of expression of the S-Ag-CAT hybrid gene. In addition, whereas animals H4, J10, and D2 uniformly expressed the highest levels of CAT activity in the retina, no trend was observed with respect t o the relative levels of CAT activity produced by other tissues. For example, D2 expressed higher levels of CAT activity in the pineal gland than in the lens while the reverse was true for animal H4 (see Table 11). These differ- ences between founder animals could not ascribed to trans- genic mosaicism, since consistent quantitative trends in organ expression were also observed among F1 transgenic progeny (data not shown).

Co-localization of S-Ag and CAT Expression by Immunocy- tochernistry-The detection in S-Ag-CAT transgenic mice of

T. Seki, M. Tsuda, Y.-K. Ho, A. Sitaramayya, K. Yamaki, V. K. Singh, and T. Shinohara, submitted for publication.

S-antigen Promoter Activity in Transgenic Mice 15507

Hlndl l l /EcoRI A

Kvnl/Htndlll

Ndel CAP Site Barn HI

8

He

I -

FIG. 1. A, diagrammatic representation of the mouse S-Ag-CAT fusion gene injected to produce S-Ag-CAT transgenic mice. Indicated segments are 85 bp of pRR322 sequence derived from pSVOCAT (open bar), mouse S-Ag sequences -1283 to +163 (solid bar), cat gene (dotted bar), and SV40 processing signal (hatched bar). R, represent- ative profiles of CAT expression in S-Ag-CAT transgenic mice. Twenty micrograms of soluble protein from the indicated tissues of H6, D2, and H4 S-Ag-CAT F1 transgenic mice were assayed for CAT activity as described under "Materials and Methods." R, retina; L, lens; P, pineal gland; Ce, cerebellum; C, cerebral cortex; T, thymus; H , heart; Lu, lung; Li, liver; I , intestine; K , kidney; S, spleen; Te, testes; 0, ovary; M , muscle.

CAT activity in lens and brain, as well as in retina and pineal gland, raised the possibility that S-Ag might-be expressed in a wider spectrum of cell types than previously thought. As this would obviously have important implications for both the regulation and function of S-Ag, we compared the profile of

expression of S-Ag with that the transgene in D2 transgenic animals.

Using immunocytochemical methods and antibodies to CAT and S-Ag, we were able to detect cells expressing both proteins in the retina, pineal gland, and lens (Fig. 2). In the retina, positive staining with both CAT and S-Ag antibodies was observed for rod photoreceptor cells. Within these cells, CAT and S-Ag appeared to be localized preferentially to the rod outer segments (ROS), as these structures stained more intensely than both the rod inner segments (RIS) and nuclei (Fig. 2, B and C). No specific staining was observed for other cell types of the retina. Both antibodies produced relatively uniform staining of the pineal gland, suggesting that S-Ag and the transgene were co-expressed by most, if not all, pinealocytes (Fig. 2, E and F ) . In the lens, S-Ag and CAT were detected in epithelial cells as well as fiber cells. Fiber cell staining was localized to the bow region of the lens, with negligible staining observed for older, fully differentiated (i.e. enuclated) fiber cells displaced towards the lens nucleus (Fig. 2, H and I). Neither antibody showed specific staining of heart, kidney, muscle, thymus, spleen, ovary, and intestine (data not shown). We were also unable to detect immunostain- ing of S-Ag or CAT in the cerebellum and cerebral cortex, although CAT expression was detectable in these tissues by enzyme assay (see Fig. 2 and Tabe 11).

Detection of S-Ag Expression by Immunoblotting and PCR Analysis-Since we were able to detect CAT expression in the cerebellum and cerebral cortex by enzyme assay, but not by immunocytochemical methods, we reinvestigated the issue of whether S-Ag might be expressed in these tissues using im- munoblotting and highly sensitive PCR analysis. Moreover, as S-Ag belongs to a family of related proteins (Lohse et al., 1990; Smith et al., 1990; Hyde et al., 1990; Yamada et al., 1990), we also used immunoblotting analysis to confirm that the molecules we detected in the lens by immunocytostaining were indeed S-Ag.

In our initial studies, we found that we were unable o detect S-Ag by direct immunoblotting of electrophoretically fractionated tissue extracts. Therefore, to increase the sensi- tivity of detection, we first incubated tissue extracts with polyclonal antibody to S-Ag and then analyzed the resulting immunoprecipitates by protein blotting with monoclonal S- Ag antibody. Using this approach, we detected in extracts of cerebellum and cerebral cortex, as well as lens epithelial and fiber cells, a single immunoreactive species of 45 kDa that migrated at the same position as native bovine S-Ag (Fig. 3). Bovine lenses were used in this analysis in order to obtain sufficient material representative of both lens cell types. Com- parison of the band signal intensities with that generated by a known amount of purified bovine S-Ag suggested that the S-Ag content of the lens and brain was less than 0.001% of total protein. No immunoreactive species were detected in

TABLE I C A T activity in S-Ag-CAT transgenic mice

The scale for CAT activity values in pmol of '"C converted/pg protein/h is + (1-30), ++ (30-100), +++ (100- 200), ++++ (200-381). R, retina; L, lens; C, cerebral; Ce, cerebellum; P, pineal gland; T, thymus; H, heart; K, kidney; Lu, Lung; Li, liver; I, intestine; S, spleen; 0, ovary; Te, testes; M, muscle. ND, not dme; NA, not appropriate.

Transgenic mouse R I, C Ce P T H I,u I,i I S 0 Te M K

15508 S-antigen Promoter Activity in Transgenic Mice

TABLE I1 CAT activity from expressing tissues of S-Ag-CAT transgenic mice Expressed in pmol of ’ T converted/pg protein/h. Copy numbers

were determined by the Southern blot hybridization analysis. R, retina; L, lens; C, cerebral cortex; Ce, cerebellum; P, pineal gland; ND. not done.

Transgenic Transgene Tissue

mouse COPY number R L C C e P

H6 20 H4

4.1 10 320 81.1 4.8 1.2 10.2

53 <1 25.5 J10 <1 113 1.0 6.2 2.0 ND D2 5 384 8.3 2.1 2.2 15.0

parallel blots with nonimmune serum (data not shown). Unequivocal evidence for expression of S-Ag in the cere-

bellum and cerebral cortex was provided by PCR analysis. To this end, we synthesized two oligonucleotide primers specific for mouse S-Ag that in control reactions with mouse S-Ag cDNA produce a 769-bp segment that can be cleaved by NcoI into two fragments of 580 and 189 bp (see Fig. 4). Using these

FIG. 2. Localization of S-Ag and CAT in retina, pineal gland, and lens of D2 transgenic mice by im- munocytostaining. Sections ( 5 pm) were cut and reacted with antibodies of the S-Ag and CAT, then stained with second antibodies as described under “Materials and Methods.” Signal detec- tion was aided by computer-enhanced photography. Red corresponds to highest staining intensity and white to the lowest staining intensity. No staining of S-Ag and CAT was observed in the lens nu- cleus. A , retina with nonimmune control antibody (1/100 dilution); B, retina with S-Ag antibody (1/3,000 dilution); C, ret- ina with CAT antibody (1/20 dilution); D, pineal gland with control antibody; E , pineal gland with S-Ag antibody; F, pi- neal gland with CAT antibody; G, lens with control antibody; H, lens with S-Ag antibody; I, lens with CAT antibody. Scale bar corresponds to 20 pm in A , B, D, E , G, and H. C and I are one-half of magnification of the other panels.

two primers, we were able to generate a single cDNA band of 769 bp following reverse transcription and PCR amplification of RNA derived from the cerebellum and cerebral cortex, but not from liver or kidney (Fig. 4). As the amplified 769-bp segments could be cleaved by NcoI to yield the two diagnostic S-Ag fragments of 580 and 189 bp, we conclude that S-Ag is expressed in both the cerebellum and cerebral cortex.

DISCUSSION

We have demonstrated that a DNA fragment extending 1283 bp upstream and 163 bp downstream from the transcrip- tional start site of the mouse S-Ag gene contains cis-acting regulatory elements capable of directing expression of the CAT gene in the retina, pineal gland, lens, cerebral cortex, and cerebellum. All of the S-Ag-CAT transgenic mice we analyzed produced CAT activity in the retina, while those expressing relatively high levels in this organ also produced lower levels of activity in the pineal gland, lens, and brain. The correlation between high level expression in the retina and ability to detect CAT activity in other tissues suggests that any differences among animals with respect to the profile

S-antigen Promoter Activity in Transgenic Mice 15509

-c

Sa Le Lf Ce C

FIG. 3. Detection of the S-Ag in lens and brain by immu- noblotting. Approximately 500 pg of protein from the mouse cere- bellum, mouse cerebral cortex, and bovine lens fibers and 100 pg of protein from bovine epithelium were prepared and immunoprecipi- tated with 20 p1 (10 pI for lens epithelium) of a polyclonal antibody at 4 “C overnight. The precipitates were collected by microcentrifu- gation, dissolved in the sample buffer, and 1/10 of each sample was fractionated by SDS-PAGE. The proteins were then electrotrans- f‘erred to a filter and immunostained with monoclonal antibody. Arrou: indicates S-Ag. Sa, purified bovine S-Ag; k, lens epithelium; L f , lens fiber; Ce, cerebral cortex; C, cerebellum.

_ _ _ ~ ~ ~ ~ u c u C I I c u c u c M Ce C Li K Co

FIG. 4. Detection of S-Ag mRNA following reverse tran- scription and PCR amplification. Poly(A)-containing RNA was extracted from cerebral cortex, cerebellum, liver, and kidney of a month-old mouse. The mRNAs were reverse-transcribed and the resulting cDNAs amplified by PCR with two S-Ag cDNA-specific primers (see “Materials and Methods”). Control amplification (Co) of mouse S-Ag cDNA generates a single 769-bp band ( U ) , which is cleaved by NcoI into two fragments of‘ 580 and 189 bp (C). M, DNA size marker ($X174, replicative form DNAIHaeIII fragments, BRL); Ce, cerebellum; C, cerebral cortex; Li, liver; K, kidney. Arrows denote the predicted bands for mouse S-Ag cDNA in the amplified product.

of tissue expression are probably quantitative rather than qualitative. This view is consistent with our finding that, while we were unable to detect CAT activity in the pineal gland of founder animal H6 (Fig. l), we were able to detect expression of the transgene by immunocytostaining (data not shown).

Although position effects undoubtedly account for much of the quantitative variation in CAT expression among the transgenic animals, it is interesting that the CAT activity levels were uniformly highest in the retina, while no trend was observed with respect to the relative levels of activity produced by other tissues. This finding can be explained if a t least some of the tissue specificities of the S-Ag promoter are governed by distinct cis-acting regulatory elements that re- spond to position effects differentially or in a non-parallel manner. Characterization of the activity of the shorter pro-

moter segments should clarify the complexity of the regula- tory elements governing the various tissue specificity of the S-Ag promoter.

Using immunoblotting, PCR-mediated detection of RNA, and immunocytostaining of transgenic tissues with antibodies to CAT and S-Ag, we established that the profile of expression of the transgene corresponded to that of S-Ag. Specifically our immunocytostaining studies localized expression of S-Ag and the transgene to rod photoreceptor cells, virtually all pinealocytes, and both epithelial and fiber cells of the lens. Based on relative staining intensity, S-Ag and CAT appeared to accumulate a t higher levels in rod photoreceptor cells than in the other cell types. This could reflect a higher rate of transcription in rod cells or the virtual absence of proteolysis in these cells, which are highly specialized for phototransduc- tion and rely heavily upon phagocytic process of adjoining pigment cells for biosynthetic turnover (Young, 1976). Un- expectedly, CAT as well as S-Ag appeared to be localized preferentially to the photosensitive ROS. Protein synthesis in rod cells occurs on polysomes in the RIS. The nascent polypeptides are then transferred to the Golgi and either incorporated into the disk membrane or transported to the ROS through the ciliary connection (Young, 1976). The proc- ess of molecular transport is believed to be precise and selec- tive. The apparent preferential accumulation of CAT in the ROS is therefore intriguing, as it suggests that the bacterial enzyme, like S-Ag, is synthesized, processed, and transported from the perikaryon through the ciliary connection to the ROS by specific transport mechanisms. While the signals specifying this process are unknown, it is likely that they reside within the nascent polypeptide and not the transcript which, for cat, is predicted to contain 163 nucleotides of S-Ag 5”untranslated sequence a t its 5’ terminus.

Our immunocytostaining studies also revealed that while both CAT and S-Ag are expressed in lens epithelial cells, staining of these proteins is most pronounced in the bow region of the lens, where fiber cells elongation is initiated. Since no significant staining was observed for the older fiber cells comprising the lens nucleus, it would appear either that expression of S-Ag is developmentally restricted to cortical fiber cells or that S-Ag plays a transient role in lens fiber cell differentiation. Further developmental studies will be re- quired to distinguish between these possibilities.

Although we did detect S-Ag in the cerebellum and cerebral cortex by immunoblotting, we were unable to do so by im- munocytostaining. This discrepancy could indicate that expression of S-Ag in these tissues is restricted to a very small, cryptic population of cells. Expression in only a small number of cells could also account for our inability to detect expression of S-Ag and CAT by staining. In any event, PCR analysis established that the molecule we detected in the brain was indeed S-Ag and not the related protein, p-arrestin (Lohse et al., 1990; Benovic et al., 1987).

Our finding that S-Ag is not restricted to photoreceptor cells and pinealocytes is in keeping with recent reports that S-Ag is detectable by immunostaining in turkey and trout erythrocytes (Mirshahi et al., 1989) and choroid plexus (Faure and Mirshahi, 1990). While we cannot rule out the possibility that S-Ag may be expressed in additional mouse tissues that were not analyzed in our study, the presence of S-Ag in the lens and brain, as well as in the retina and pineal gland, raises fundamental questions about its function. Although the exact function of S-Ag in photoreceptor cells is unclear, it is gen- erally considered to play an important regulatory role within the phototransduction cascade. In this regard, S-Ag has been shown to bind both photoactivated, phosphorylated rhodopsin

15510 S-antigen Promoter Activity in Transgenic Mice

(Kuhn et al., 1984; Kuhn and Wilden, 1987; Bennett and Sitaramayya, 1988) and cGMP-dependent phosphodiesterase (Zuckerman and Cheasty, 1986; Fukuda et al., 1990) and to contain various activities, including Ca2+ binding (Huppertz et al., 1990), ATP binding (Gilscher and Ruppel, 1989) and an inhibition of phosphatase activity (Palczewski et al., 1989). I t is therefore possible that, in addition to its role in photo- transduction, S-Ag is utilized as an integral component of a number of different signal transduction processes that are characteristic of various different cell types. Finally, our dem- onstration that S-Ag is expressed in the lens and brain may be of potential significance with respect to the pathogenicity of S-Ag following induction of experimental autoimmune uveitis and pinealocytis. The relative involvement of the lens and brain in autoimmune inflammation is currently under investigation.

Acknowledgment-We thank Dr. L. A. Donoso for providing the monoclonal antibody to S-Ag.

REFERENCES

Abe, T., and Shinohara, T. (1990) Exp. Eye Res. 51, 111-112 Abe, T., Yamaki, K., Tsuda, M., Singh, V. K., Suzuki, S., McKinnon,

R., Klein, D. C., Donoso, L. A., and Shinohara, T. (1989) FEBS

Bennett, N., and Sitaramayya, A. (1988) Biochemistry 27,1710-1715 Benovic, J . L., Kuhn, H., Weyand, I., Codina, J., Caron, M. G. and

Lefkowitz, R. J. (1987) Proc. Natl. Acud. Sci. U. S. A. 8 4 , 8879- 8882

Breitman, M. L., Clapoff, S., Rossant, J., Tsui, L.-C., Glode, L. M., Maxwell, M. L., and Bernstein, A. (1987) Science 238, 1563-1565

Chabre, M., and Deterre, P. (1989) Eur. J. Biochem. 179, 255-266 Collin, J.-J., Brisson, P., Falcon, J., and Voisin, P. (1986) in Pineal

and Retina Relationships (Klein, D. C., O'Brien, P. J., eds) pp. 15- 32, Academic Press, New York

Donoso, L. A,, Merryman, C. F., Sery, T., Shinohara, T., Dietzschlod, B., Wistow, G., Craft, C., Morley, W. and Henry, R. T. (1986) Curr. Eye Res. 5,995-1004

Lett. 247,307-311

Faure, J. P. (1980) Curr. Top. Eye Res. 2, 215-302 Faure, J.-P., and Mirshahi, M. (1990) Curr. Eye Res. 9, 163-167 Fukada, Y., Yoshizawa, T., Saito, T., Ohguro, S., and Akino, T. (1990)

Gilliland, G., Perrin, S., and Bunn, F. (1990) in PCR Protocols: A Guide to Methods and Applications (1990) (Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. J., eds) pp. 60-69, Academic

FEBS Lett. 261,419-422

Press, San Diego Glitscher, W., and Ruppel, H. (1989) FEBS Lett. 256, 101-105

Gorman, c. M., Moffat, L. F., and Howard, B. H. (1982) Mol. Cell.

Hogan, B., Costatini, F., and Lacy, E. (1986) Manipulating of the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Labo- ratory, Cold Spring Harbor, NY

Huppertz, B., Weyand, I., and Bauer, P. (1990) J. Biol. Chem. 265, 9470-9475

Hyde, D. R., Mecklenburg, K. L., Pollock, J. A., Vihtelic, T. S., and Benzer, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1008-1012

Kalsow, C. M., and Wacker, W. B. (1977) Inuest. Ophthalmol. Vis. Sci. 16, 181-184

Kappers, J . A. (1981) in The Pineal Organ (Oksche, A., and Revet, P., eds) pp. 3-23, Elsevier/North-Holland Biomedical Press, Am- sterdam

Korf, H. W., Moller, M., Gery, I., Ziglar, S. J., and Klein, D. C. (1985) Cell Tissue Res. 239, 81-85

Kuhn, H., and Wilden, U. (1987) J. Recept. Res. 7, 283-298 Kuhn, H., Hall, S. W., and Wilden, U. (1984) FEBS Lett. 176 , 473-

Lohse, M. J., Benovic, J. L., Codina, M. G., and Lefkowitz, R. J .

Maizel, J . V., Jr. (1971) Methods Virol. 5, 179-246 Mirshahi, M., Borgese, F., Razaghi, A., Scheuring, U., Garcia-Romeu,

F., Faure, J.-P., and Motais, R. (1989) FEBS Lett. 258,240-243 Palczewski, K., McDowell, J. H., Jakes, S., Ingebritsen, T. S., and

Hargrave, P. A. (1989) J. Biol. Chem. 264 , 15770-15773 Shinohara, T., Dietzschold, B., Craft, C. M., Wistow, G., Early, J. J.,

Donoso, L. A., Horwitz, J., and Tao, R. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6975-6979

Shinohara, T., Donoso, L. A,, Tsuda, M., Yamaki, K., and Singh, V.

J., eds) Vol. 8, pp. 51-66, Pergamon Press, New York K. (1988) in Progress in Retinal Research (Osborne, N., and Chader,

Smith, D. P., Shieh, B.-H., and Zuker, C. S. (1990) Proc. Natl. Acad.

Southern, E. M. (1975) J. Mol. Biol. 98, 503-517 Stryer, L. (1988) Cold Spring Harbor Symp. Quant. Bwl. 53,283-294 Tsuda, M., Syed, M., Bugra, K., Whelan, J . P., McGinnis, J. F., and

Tsuda, M., Kikuchi, T., Yamaki, K., and Shinohara, T. (1991) Eur.

Usukura, J., and Bok, D. (1987) Exp. Eye Res. 45,501-515 Wacker, W. B., Donoso, L. A., Kalsow, C. M., Yankeelov, J. A., Jr.,

Yamada, T., Takeuchi, Y., Komori, N., Kobayashi, H., Sakai, Y.,

Yamaki, K., Takahashi, Y., Sakuragi, S., and Matsubara, K. (1987)

Yamaki, K., Tsuda, M., and Shinohara, T. (1988) FEBS Lett. 234,

Yamaki, K., Tsuda, M., Kikuchi, T., Chen, K.-H., Huang, K.-P., and

Young, R. (1976) Inuest. Ophthalmol. 15,700-725 Zuckerman, R., and Cheasty, J. E. (1986) FEBS Lett. 2 0 7 , 3 5 4 1

Biol. 2 , 1044-1051

4 78

(1990) Science 248, 1547-1550

Sei. U. S. A . 87, 1003-1007

Shinohara, T. (1988) Gene (Amst.) 73, 11-20

J . Bwchem., in press

and Organisciak, D. T. (1977) J. Immunol. 119, 1949-1958

Hotta, Y., and Matsumoto, H. (1990) Science 248,483-486

Bwchem. Biophys. Res. Commun. 142,904-910

39-43

Shinohara, T. (1990) J. Biol. Chem. 265, 20757-20762