Neuron,

Vol. 2, 469-476,

March,

1990, Copyright

0 1990 by Cell Press

Visinin: A Novel Calcium Binding Expressed in Retinal Cone Cells Kanato Yamagata,* Kaoru Goto,+ Che-Hui Hisatake Kondo,+ and Naomasa Miki* * Department of Pharmacology School of Medicine Osaka University Osaka

Kuo,*

I

Japan +Department of Anatomy School of Medicine Tohoku University Sendai

Japan *Department of Pharmacology Cancer Research Institute Kanazawa University Kanazawa

Japan Summary Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pl 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal hgtll cDNA library, using anti-visinin serum. The g-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a CaZ+ binding protein. Visinin protein expressed in E. coli exhibited CaZ+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca*+ binding protein and may be involved in phototransduction in the cone cells. Introduction Phototransduction is one of the most important functions of the retina. In the dark, ion channels of the outer segment plasma membrane are held open by cGMP, allowing both Na+ and Ca2+ to flow into the cell and thus keeping the membrane depolarized. On the other hand, a Na+lCa2+ exchanger in the outer segment and a Na+/K+ exchanger in the inner segment function to maintain the ionic balance. Phototransduction is initiated by light-induced conformational changes of opsin molecules in disk membranes. Sequential activations of GTP binding protein (transducin) and cGMP-dependent phosphodiesterase result in a rapid hydrolysis of cCMP, which causes the Na+/Ca2+ channels to close and the membrane to hyperpolarize (Stryer, 1986). Na+/Ca2+ exchange continues to work during the light response, so that the intracellular Ca2+ concentration [Ca2+], declines (Yau

Protein

and Nakatani, 1985). Several workers have postulated that this decline in [Ca2+], acts as a negative feedback, accelerating one or more of the restorative reactions of the cascade and possibly serving as the mechanism for adaptation (Matthews et al., 1988; Nakatani and Yau, 1988). Cone photoreceptors recover to a baseline after a flash much more rapidly than rod cells of the same species given the same degree of excitation. We have analyzed the soluble proteins of chick retina during development and found that a peptide of about 24 kd, designated visinin, appears in the retinas of embryos on the 14th day of development and gradually increases in concentration until hatching (Hatakenakaet al., 1983). It is not detected in the cerebrum, tectum, pigment epithelium, or vitreous body on SDSPAGE. We produced an antibody against visinin and demonstrated that this protein is located in the cone cells in various vertebrate retinas and is a good marker for these cells (Hatakenaka et al., 1985) and some pinealocytes (Goto et al., 1989). In the present paper, we isolated a cDNA encoding visinin from a chicken retinal cDNA library and determined its nucleotide sequence. Comparison of the deduced amino acid sequence with known protein sequences revealed that visinin IS a novel Ca*+ binding protein in retinal cone cells.

Results Molecular

Cloning

of Chicken

Visinin

cDNA

Since an antiserum raised against visinin was available, we tried to isolate cDNA clones by screening an expression library with the antiserum. However, the anti-visinin serum was found to also recognize calbindin, a vitamin D-dependent Ca2+ binding protein expressed in retinal cone and amacrine ceils (Pasteels et al., 1987). Therefore, before screening experiments, this antiserum was preadsorbed with rat renal extract, which contains a high concentration of calbindin. We constructed a cDNA library from chicken embryonic retinas (19-day-old) with the hgtll expression vector. When this library was screened with the visinin antiserum, four positive clones were obtained, all of which contained the same cDNA inserts. Figure 1 shows a Western blot analysis of the E. coli lysogen of one positive clone (h vis4) using the above antiserum. This clone produced a B-galactosidasevisinin fusion protein that was recognized by the antiserum. We also prepared a chicken retinal extension library by using a primer complementary to this cDNA. The h vis4 cDNA was used to screen the extension library and another chicken retinal hgtll library. Five clones (pBSvis-exl to 5), which had similar cDNA inserts, were isolated from an extension library. One clone (h vis25), which contained 5’ cDNA of visinin, was obtained from another retinal library (Figure 2).

NeLlrOn

470

A -+ B - +

tope-selected antibody specifically recognized a 24 kd protein from chicken retina but did not react with soluble proteins from brain, kidney, and intestine, which contained calbindin (Figure 3A). lmmunohistochemical studies with this antibody showed staining of the chicken retinal inner segment, outer nuclear layer, and some pinealocytes (Figure 38, a and d), but no reactions were observed with cerebellum and kidney (Figures 36, e and f). Unadsorbed antiserum stained retinal amacrine cells, cerebellar Purkinje cells, and renal tubular cells, all of which are reported to contain calbindin (Jande et al., 1981). Thus it was concluded that the epitope-selected antibody recognized visinin, but not calbindin. Chick retinas contain a small number of rods, and it is difficult to distinguish between rods and cones in this tissue. We used goldfish retinas for studies with this antibody (Figure 3B, c). It reacted with inner segments and the outer nuclear layer of cone photoreceptors of this species, but not with the rod photoreceptors. Rat photoreceptors also failed to react with this antibody because rat retinas are almost devoid of cones (Figure 3B, b). These results show that visinin is exclusively expressed in cone photoreceptors.

C

116,250+

66.200-c

Figure 1. Induction tein by IPTG

of the

B-Galactosidase-Visinin

Fusion

Pro-

Lysogens of Ivis4were induced with (+) or without (-) IPTG. After the induction, cells were collected and lysed. The cell lysates were subjected to 7.5% SDS-PAGE. The separated proteins were transferred to a nitrocellulose filter and reacted with anti-visinin serum. A single protein band was detected by anti-visinin serum. Numbers on the left side indicate daltons. (A) Coomassie (brilliant) blue stain. (B) Immunoblot. The arrows indicate the fusion proteins.

Identification of Isolated as Visinin cDNAs

Northern

cDNA Clones

Since no protein sequences were available for visinin, we tried to identify this clone as a visinin cDNA by the epitope selection method (Weinberger et al., 1985). The L vis4 phage was used to infect E. coli Y1090 on petri dishes and induce the expression of the B-galactosidase-visinin fusion protein with isopropyl B-o-galactopyranoside (If%). The expressed bacterial proteins were adsorbed in situ to nylon filters and incubated with the anti-visinin serum. The antibody adsorbed on the filters was eluted with acidic buffer and used to detect visinin by Western blot analyses. The epi-

-~

pl?s-exs-

localization of Visinin by In Situ Hybridization

---

lhb

Figure 2. Restriction k vis4, h vis25, and The solid regions, direction ment. All E, EcoRI;

Enzyme Maps pBSvis-ex5

and Sequencing

JatL

Strategy

Blot Analyses

mRNA in Chicken

Retina

The localization of visinin mRNA in chicken retina was determined by in situ hybridization with a nicktranslated probe of h vis4 cDNA. The positive cells were characterized by examining autoradiographs with phaseand dark-field optics (Figures 6A and 6B). Intense grains were observed over the inner segment and outer nuclear layer of the retina. No significant autoradiographic grains were observed in the remaining parts of this tissue. These results are compatible with the data of the immunohistochemical localization of visinin.

HE

L.

and Southern

Northern blot analysis of RNAs prepared from chicken embryonic retina, brain, liver, intestine, and kidney, using the h vis4 cDNA as a probe, showed a single band of about 1000 nucleotides (12s; Figure 4). The overlapping cDNA clones contained about 960 bases, suggesting that they covered the entire length of the mRNA, assuming that the poly(A)+ length is about 100 bases. In Southern blot analysis with the I. vis4 cDNA, each genomic DNA digested with BamHl or EcoRl showed a single band, suggesting that the visinin gene in the chicken genome is a single copy (Figure 5).

1 of

and open boxes indicate the coding and noncoding respectively. The arrows under the cDNA indicate the and extent of sequence determination for each fragnucleotide sequences were determined in both strands. H, Haelll; P, Pstl; S, Smal; X, Xhol.

Nucleotide of Visinin

and Deduced

Amino

Acid Sequences

Both strands of the h vis4, h vis25, and pBSvis-ex5 clones were sequenced by the method of Sanger et al. (1977). The entire nucleotide sequence of overlapping cDNA clones and the putative amino acid sequence are shown in Figure 7. The first ATG found at a position

i\ Calcium ‘ .71

Binding

Protein

in Retinal

Cones

12 345

Figure 3. Western Blot and chemical Characterization Selected Antibody

Immunohistowith Epitope-

(A) Western blot analysis with epitopeselected antibody to visinin. Total soluble proteins (about 50 pg) from various tissues were separated by electrophoresis on a 12.5% SDS-polyacrylamidegel and blotted to a nitrocellulose filter to which epitopeselected anti-visinin antibody was applied. Fixed antibody was visualized by reacting with anti-rabbit IgG-peroxidase and 3,3’diaminobenzidine. An arrow indicates a position of visinin in chicken retinal soluble protein. Chick brain (lane l), retina (lane 2), liver (lane 3), kidney (lane 4), and intestine (lane 5). (B) lmmunohistochemistry with epitopeselected visinin antibody. Chicken (a), rat (b), and goldfish (c) retinal sections and chicken pineal (d), cerebellar (e), and renal (f) sections were reacted with the epitope-selected visinin antibody. (a) Magnification 53x. The antibody binds to the inner segments and outer nuclear layer of the chicken photoreceptors. (b) Magnification 75x. No immunoreactive cells are seen in rat retinas. (c) Magnification 106x. A reactive protein is present within the majority of goldfish cone photoreceptors. (d) Magnification 106x. Some pinealocytes are immunostained. (e) Magnification 26x. No immunoreactive cells are seen. (f) Magnification 26x. No immunoreactive cells are observed. Abbreviations: PE, pigment epithelium; IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; CCL, ganglion cell layer; BL, bacillary layer.

123456

1 was

determined

codon,

since

-18s

be

a candidate

for

an

in a sequence

initiation fitting

the

codon (Kozak, 1984). This putative initiation codon is followed by an open reading frame of 576 bases and a TAA termination codon at position 574. The coding region is followed by 3’ unconsensus

-28s

to

it is embedded

Figure

for

4. Northern

an

initiation

Blot Analysis

Using

Yisinin

cDNA

as a Probe

Total RNAs isolated from the retina (lane I), brain (lane 2), liver (lane 3), heart (lane4), intestine (lane 5), kidney (lane 6) of 19-dayold chick embryos were resolved on a 1% agarose gel (about 60 vg RNA per lane) and transferred to a nylon filter. The filter was hybridized with a nick-translated EcoRl fragment (365 bp), washed at high stringency, and exposed to X-ray film. Positions of 285 and 18s ribosomal RNAs are shown on the right side as molecular size marker.

Neuron

472

12 -23.lkb

ligated with the transcription and translation signals of T7. The addition of IPTG induced the formation of a 24 kd protein band with the mobility of visinin (Figure 9A). The protein was transferred to nitrocellulose and reacted with the epitope-selected visinin antibody. As shown in Figure 9B, the protein synthesized in E. coli was visinin. The E. coli product was examined for its ability to bind Ca*+ by treating the nitrocellulose with 45Ca2+ and subjecting it to autoradiography. It is apparent from the data in Figure 9C that the visinin synthesized by E. coli possesses Ca2+ binding activity. Discussion

Figure 5. Southern Blot Analysis Using a Visinin cDNA as a Probe The genomic DNA isolated from chick liver was digested with BamHl (lane 1) and EcoRl (lane 2), applied to a 0.8% agarose gel (about 20 pg per lane), and transferred to a nylon filter. The filter was hybridized with a nick-translated EcoRl fragment (365 bp), washed with 2x SSC, and exposed to X-ray film. Reference markers are in kilobases.

sequences containing a poly(A)+ addition sequence (AATAAA) at position 710 and a poly(A)+ tail. of This open reading frame encodes a polypeptide 192 amino acids whose molecular weight and isoelectric point were calculated to be 22,514 and 5.1, respectively. These data agree with those determined by gel electrophoresis for visinin. The examination of hydrophobicity of visinin proved that visinin is a soluble protein. By using the NBRF protein database, it was found that visinin has three regions homologous with troponin C and calmodulin. These correspond to the Ca2+ binding sequences of the E-F hand structure (Figure 8). translated

Ca*+ Binding Properties of Visinin To determine whether visinin binds Ca2+, the retinal soluble proteins were subjected to SDS-PAGE and transferred to nitrocellulose. The sheet was incubated in a buffer containing %a*+ or subjected to Western blot analysis, using an epitope-selected visinin antibody. Autoradiography of the nitrocellulose membrane revealed the presence of a Ca2+ binding protein (data not shown), whose position was exactly the same as the immunoreactive visinin band of 24 kd. E. coli BLZl(DE3)pLysS was then transformed with a vector that included the entire visinin coding sequence

Visinin was originally shown to be a protein of 24 kd that appears in chick retina during embryogenesis (Hatakenaka et al., 1983). An antibody raised against the protein extracted from this SDS gel band stains cone cells as well as some amacrine cells in chick retina (Hatakenaka et al., 1985). Later it was found that this antibody also reacts with calbindin (28 kd), a vitamin D-dependent Ca *+ binding protein (Pasteels et al., 1987). We attempted to determine the amino acid sequence for visinin by cDNA cloning, but were unsuccessful because of its blocked N-terminus. We then proceeded to isolate visinin cDNA clones by using a visinin antibody adsorbed with calbindin. This permitted us to establish that the protein is not calbindin, but visinin, based on the following criteria. First, the molecular mass and isoelectric point of the deduced protein are 22.5 kd and 5.1, respectively, which are compatible with those of visinin. Second, visinin and its mRNA are only expressed in the photoreceptor cells of the retina and not in the calbindin-containing cells such as retinal amacrinecells, renal tubular cells, and cerebellar Purkinje cells. Finally, visinin exhibits homology with calmodulin and troponin C, but not with calbindin and calretinin (Rogers, 1987). These observations confirm that this novel photoreceptorspecific, Ca*+ binding protein is visinin. The investigation of the NBRF protein database revealed that visinin has three regions homologous to troponin C and calmodulin. These correspond to the Ca2+ binding sequences of the E-F hand structure, which consists of 29 amino acid residues arranged in a helix-loop-helix conformation. According to the theory of Kretsinger (1979), the consensus sequence of Ca2+ binding loops of an E-F hand consists of ‘!2 amino acids. However, the first Ca*+ binding domain of visinin shows some deviation from such a structure. It has an insertion of cysteine at position 39, which may diminish its Cal+ binding activity. However, we have demonstrated that the visinins extracted from chick retina and synthesized in E. coli have such activity. The role of visinin is unknown at present. Since it is found in the retinal cone cells and pineal photoreceptors, it may be involved in phototransduction

A Calcium 473

Binding

Protein

in Retinal

Cones

Figure 6 In Situ Hybridization ina with a Visinin cDNA Probe

of the Ret-

A frozen section of 3-day-old chick retina was hybridized to %-labeled visinin cDNA. After hybridization and washing, the section was dipped in NTB-2 emulsion and exposed for 2 weeks before development. (A) Giemsa staining under bright-field. (B) Darkfield of the same section. The radioactivity is concentrated in the inner segment and the outer nuclear layer. Abbreviations: IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; CCL, ganglion cell layer. Bar, 50 Pm.

Figure Visinin

Z Nucleotide

and

Deduced

Amino

Acid

Sequences

of

The sequence is numbered on the left side, beginning with the first residue of the ATG triplet encoding the initial methionine, and the nucleotides on the 5’ side of the sequence are shown by negative numbers. The numbers of deduced amino acid sequence are shown on the right side, beginning with the initiating methionine. The polyadenylation signal AATAAA is underlined, and *** indicates the stop codon.

mechanisms of cone and pineal cells. When newly hatched chicks were fed under continuous illumination for several days, the content of visinin in the pineal gland increased 4 to 8 times over that in animals maintained in the dark (Goto et al., unpublished data). When pineal bodies were cultured in the light, pineal visinin was induced as in vivo (Yamagata et al., unpublished data). These observations indicate, though not conclusively, that visinin may play a role in phototransduction mechanisms in retinal cone cells and pineal photoreceptors. Matthews et al. (1988) and Nakatani and Yau (1988) reported that [Ca*+]i in the rod outer segments is responsible for setting the sensitivity during light adaptation. Hodgkin and Nunn (1988) indicated that the decrease in [Ca2+], accelerates guanylate cyclase activity during the restoration phase. Koch and Stryer (1988) and Pepe et al. (1986) have reported that the guanylate cyclase activity in photoreceptor cells is highly sensitive to a low concentration of Ca2+, below 0.5 PM. Pugh and Altman (1988) proposed that a complex of Ca2+ with a hypothetical protein, designated M (a modulator protein having Ca2+ binding activity), inhibits

guanylate

develops

much

tion,

amphibian

cyclase

faster rods

activity.

in cones and

cones

than

Light

adaptation

in rods. both

exhibit

In addilight

NeUKNl 474

TC

EL--‘L--LD-0-OG-IO--EL--LL--L L--LO-O -OG-IO--EL--LL--L 32 YEGFQR~SDORIRCDEFERIYONFFPNSEPWYARHVFRSFDTNDWTLDPREYIIAL--HLT : : : :: : :I::::; :: : :I I :: : : 16 FSLFDRD-GDOCITTRELOTVnRSL-04NPTEAELODllVOEVDAWSOTIDFPEFLSLR-ARKn IIIIIIIt I :: ::: : : :: :: :: I: : ,,I,#, II 26 ~D~FDAD-GGGDISTKELOTVII-~L~NPTKEELDA~~EEVDRDD~OTIDFEEFLV~;IVRPIIK

\5

EL--LL--LD-0-OO-IO--EL--U--L 94 S-S-GKTHLKLE,,AFSLFDVDRWOBVSKSEVSKSEVLEIITAIF~IPEEERL

v!l IY

cc* ‘TC

: :I:

: ::

::

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77 R-D-SDSEEE;REAFRVFDKNOYISAAELR"hTNkLTDEEVD : I:; : :, I I.,. :: I : :: SS EDAKGKSEEELADCFRIFDKNAWFIDIEELQEILRAME"VTEED

adaptation, but only the cones of the primate retina show this phenomenon. A Cal+ binding modulator protein (M protein) may be more abundant in cones than in rods. Visinin, which is a Cal+ binding protein specific to cone cells, would be one of the candidates for the postulated M protein. The other possibility is that visinin may be related to the morphogenesis of cone photoreceptors by interacting with cytoskeletal proteins, as calmodulin does in the rod outer segments (Nagao et al., 1987). Experimental

Procedures

Preparation of cDNA libraries RNA was prepared from 19-day-pld chicken retinas by the guanidine thiocyanate method (Chirgwin et al., 1979). Poly(A)+ RNA was purified by chromatography on oligo(dT)-cellulose (Aviv and Leder, 1972). The retinal cDNAs were prepared using oligo(dT) primer as described by Cubler and Hoffman (1983). This cDNA was methylated with EcoRl methylase and then ligated with EcoRl linker. The linkers were then cleaved with EcoRI, and the cDNA was ligated with EcoRCdigested, phosphatase-treated

A 97,400

M12 k

66.200,

42,699

-

B

12

139 133 133

93 76 87

Figure& Binding

Homology Proteins

of Visinin

wtth

Cal’

The amino acids are shown as single letters, and those necessary for the Ca2+ binding activity in the three domains are marked above the sequences: 0, oxygencontaining residue; G, glycine; I, isoleutine; E, glutamate; L, hydrophobic amino acid. VS, chicken visinin; CM, chicken calmodulin (Putkey et al., 1983); TC, chicken troponin C (Wilkinson, 1976). The double dashes and dots represent the matching of the same and similar amino acids, respectively.

Igtll vector. Recombinant phage DNAs were packaged using extracts as described by Grosveld et al. (1981). Single-stranded cDNA of the extension library was synthesized using a unique primer (5’~CGTGCGTCITCCCGGACGAGGF3’), and double-stranded cDNA was synthesized in the same way. Double-stranded cDNAwas blunt-ended with T4 DNA polymerase and then ligated with a blunt-ended Bluescript KSII(+) vector. Isolation of cDNA Clones The chicken retina bgtll library was screened with polyclonal antiserum against visinin, as described elsewhere (Huynh et al., 1985). About 2 x IO5 plaques were screened, and positive ones were picked and plaque-purified. The inserts were sized by digestion with EcoRl and analysis on gels and then resubcloned into the plasmid vector pUC18. This insert DNA was labeled by nick translation and used to screen another hgtll library and a primer extension library by standard methods (Maniatis et al., 1982). Positive clones were isolated, and the inserts were analyzed by restriction enzyme digestion and Southern blotting. Preparation and Analysis of Lysogens h vis4 was lysogenized in E. coli Y1089 as described previously (Huynh et al., 1985). Lysogens were cultured at 30°C, induced for

C 12

Figure

9. Expression

of Visinin

In E. co11

Expression plasmids PET&-vis were introduced into E. coli, and the bacterial cell lysates were subjected to immunoblot analysis and Cal+ binding determination after SDS-PAGE. lane M, standard protein; lane 1, vector pET8c (control); lane 2, pET8c-vis containing an entire visinin open reading frame. (A) SDS-PAGE. An arrow shows the position of the visinin synthesized in E. coli. The gel was stained with Coomassie (brilliant) blue. (B) Western blot using epitope-selected antibody. An arrow indicates visinin. (C) Cal+ binding analysis. Note that synthesized visinin binds 45CaL+ (arrow).

A Calcium 475

Binding

Protein

in Retinal

Cones

20 min at 45”C, and then treated with IPTG for 30 min at 38OC. The cells were harvested by centrifugation and frozen. After two cycles of freezing and thawing, Triton X-100 (final concentration 1%) and lysozyme (final concentration 1 mg/ml) were added to the lysate, and the mixture was incubated at 4OC for 30 min. It was centrifuged, and the pellet was suspended in the loading buffer and electrophoresed on a 7.5% SDS-polyacrylamide gel. The proteins were transferred to a nitrocellulose filter and coated with 0.5% skim milk. The filter was then incubated with antivisinin serum. Bound antibodies were detected with peroxidaseconjugated anti-rabbit IgC and diaminobenzidine.

with 4sCa2+ and al. (1984).

Epitope Selection E. coli strain Y1090 was infected with k vis4 (10,800 pfu) at 42°C for 6 hr on petri dishes and induced for the expression of fusion protein by IPTG. For epitope selection, 20 ~rl of crude antiserum was diluted with a solution of 0.5% skim milk in TBS (50 mM Tris [pH 8.0],150 mM NaCl) and incubated with the nitrocellulose filter for 1 hr. The filter was washed three times with a solution of 0.5% Triton X-100 in TBS. Antibody was eluted from the filter with three 1 min washes in 5 mM glycine-HCI (pH 2.3) containing 150 mM NaCl and 0.5% Triton X-100. The combined washes were immediately neutralized with 1 M Tris to pH 7.5. This antibody preparation was used for Western blot analyses and immunohistochemistry. Northern and Southern Blot Hybridization For Northern blot analysis, total cellular RNA was isolated from chicken retina, brain, liver, kidney, and intestine (19-day-old embryo). After glyoxal and DMSO treatment, 60 pg of each RNA was electrophoresed on a 1% agarose gel containing 10 mM sodium phosphate buffer (pH 7.0) and transferred to a nylon filter. Prehybridization, hybridization, and washing conditions were as described previously (Kuo et al., 1986). For Southern blot analysis, DNA was prepared from chicks and digested with BamHl and EcoRI. Twenty microgram samples were run on 0.8% agarose gels and transferred to a nylon filter. The filter was hybridized with nick-translated probe and then washed at 65OC in 2x SSC and 0.1% Sarkosyl (Southern, 1975). In Situ Hybridization In situ hybridization probe was performed

experiment as described

employing by lseki

a visinin cDNA et al. (1989).

Expression of Visinin in E. coli Procedures for expressing visinin cDNA were essentially as previously described (Studier and Moffatt, 1986). For the addition of new restriction enzyme sites (Ncol and BamHI, respectively) to the Sand 3’ends of the coding sequence, visinin cDNA was amplified with primers (S-CCATGGCGAACAGCCCCAGCAGCGCT-3 and 5’-GGATCCATLATTTCTTGGGTTCG-TAT-3’) by polymerase chain reactions. The Ncol- and BamHI-digested cDNA was ligated with pEF8c. To direct expression of visinin, the recombinant plasmid was introduced into E. coli BL21(DE3JpLysS. The resulting strain was grown with shaking at 37°C in M9ZB medium supplemented with ampicillin. The lac UV5 promoter was induced by adding IPTG to a final concentration of 0.4 mM when the cultures reached ODm = 0.6. After an additional 2 hr of shaking at 3PC, the cells were harvested by centrifugation and the lysate was subjected to SDS-PAGE on a 12.5% gel. Following electrophoresis, the proteins were visualized by staining with Coomassie (brilliant) blue or subjected to immunoblot or Ca*+ binding analyses. Ca*+ Binding Properties of Visinin About 50 ug of the soluble proteins rated on SDS-PAGE and transferred brane. Ca*+ binding proteins were

from E. coli lysate was sepato a nitrocellulose memdetected by the incubation

as described

by Maruyama

et

Acknowledgments We wish to thank Dr. W. F. Studier for the T7 plasmids and strains. Dr. Y. Fukada for the chick retinal cDNA library, Drs. S. Hatakenaka, M. Nonaka, H. Sato, T. Kuno, and K. Maruyama for helpful advice, and Dr. H. F. Deutsh and Mrs. A. Hino for reading and typing this manuscript. Received

Nucleotide Sequences Nucleic acid sequence was determined by the chain termination method of Sanger et al. (1977). In most cases, overlapping sequence data were obtained for both strands.

autoradiography

October

20, 1989;

revised

January

19, 1990

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up Nucl.

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Miki, Mol.

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J. (1988).

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Visinin: a novel calcium binding protein expressed in retinal cone cells.

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick...
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