β-crystallin mRNAs: Differential distribution in the developing chicken lens

DEVELOPMENTAL

BIOLOGY 86.403-408

(1981)

,&Crystallin mRNAs: Differential Distribution in the Developing Chicken Lens HARRY OSTRER,*DAVID C. BEEBE,?AND JORAMPIATIGORSKY* *Section 012Cellular D$ferentiatim, Laboratory of Molecular Genetics, National Institute of Child Health and Human Lkvelqpment, National Institutes of Health, Bethesda, Maryland 20205; and ~Departnwnt of Anatomy, Uniformed .%-vices University of the Health Sciences, Bethesda, Ma&and 20014 Received November

27, 1980; accepted in revised

fm March 25, 1981

The synthesis of the p-crystallin polypeptides has been studied in different regions of the embryonic chicken lens. Seven j%crystallin polypeptides ranging in molecular weight from approximately 19,000 (19K) to 35,000 (35K) daltons were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Each polypeptide was synthesized in a rabbit reticulocyte cell-free system supplemented with RNA from the embryonic lens fiber cells suggesting that each is encoded by a separate mRNA. Analysis of the cell-free translation products of the RNAs from 6, 15-, and Is-dayold embryonic chicken lens fibers demonstrated that all seven polypeptides are translated at each of the stages and that the proportion of @-crystallin mRNAs increases as the chicken embryo matures. Fingerprints of methioninecontaining tryptic peptides indicated that the three predominant @-crystallin polypeptides synthesized in the reticulocyte lysate @OK, 26K, and 35K) have related but distinct primary structures. Surprisingly, both the 35K @-crystallin polypeptide and its mRNA were selectively absent from the cells in the central region of the epithelium. Synthesis of this polypeptide from extracted RNAs was detected in the elongating cells of the equatorial region of the epithelium and from the fiber cells. In contrast to the 35K polypeptide, the six lower-molecular-weight j3-crystallin polypeptides were synthesized in a reticulocyte lysate directed by RNAs extracted from all three regions of the lens. These data indicate that lens cell elongation and fiber cell differentiation in the embryonic chicken are accompanied by the appearance of the mRNA for the 35K polypeptide. INTRODUCTION

Development of the chicken lens is characterized by the sequential synthesis of crystallins, which constitute about 90% of the soluble protein of the lens (GenisGalvez et al., 1968; Zwaan and Ikeda, 1968; Clayton, 1970). d-Crystallin is the first to appear during development (Zwaan and Ikeda, 1968) and is the principal crystallin synthesized in the embryonic lens (Yoshida and Katoh, 1971; Piatigorsky et al., 1972). @-Crystallin has been first detected at 3% days of development (Zwaan and Ikeda, 1968) and accumulates in the lens following hatching (Genis-Galvez et al., 1968). The 8crystallins consist of several polypeptides of heterogeneous size and charge (Clayton, 1969; Truman and Clayton, 1974; Zigler and Sidbury, 1976; Beebe and Piatigorsky, 1976; Ostrer and Piatigorsky, 1980). @-Crystallin polypeptides have partially overlapping tyrosinecontaining tryptic-peptide maps (Ostrer and Piatigorsky, 1980) and share antigenic determinants (Rana and Maisel, 1970; Clayton and Truman, 1974), indicating that they form a family of related polypeptides. In lens extracts, the /3-crystallins exist as aggregates which are composed of different combinations of the polypeptides (Clayton, 1969; Rana and Maisel, 1970; Truman and

Clayton, 1974; Zigler and Sidbury, 1976; Ostrer and Piatigorsky, 1980). In the present study, we have examined the synthesis of the @-crystallin polypeptides in the embryonic chicken lens by labeling lens explants in vitro and by translating the lens mRNA in a heterologous cell-free system. The 35K /3-crystallin polypeptide was found to be a specific marker for lens cell elongation; the appearance of this polypeptide correlated with the appearance of its mRNA in the elongating cells. MATERIALS

AND METHODS

Obtaining and dissecting lenses. Lenses were excised from 37-week-old adult or from 6-, 15-, or 19-day-old embryonic White Leghorn chickens (purchased from Truslow Farms, Inc., Chestertown, Md.). Usually, before analysis of proteins or extraction of RNA from the embryonic lenses, the epithelia were separated from the fiber cells, and the central regions of the epithelia were dissected from the equatorial regions as described elsewhere (Piatigorsky, 1975). The central epithelial cells are cuboidal while the equatorial epithelial cells are columnar. Preparation of lens RNA. Poly(A+) RNA was ex-

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DEVELOPMENTALBIOLOGY

tracted from whole embryonic lenses as described previously (Zelenka and Piatigorsky, 1974), with the exception that the RNA bound to oligo(dT)-cellulose (PL Biochemicals, Inc., Milwaukee, Wise.) was eluted in a single step with 0.01 MTris-HCl, pH 7.5. For extraction of total RNA from the central epithelial cells, the equatorial epithelial cells and the fiber cells, the tissues were thawed in 0.1 M NaCl, 0.01 M Tris-HCl, pH 7.5, 0.001 M EDTA, 1% SDS, and vortexed to disrupt the cell membranes. Insoluble material was pelleted by centrifugation at 10,OOOgfor 15 min at 4°C. The supernatant fraction was extracted twice with an equal volume of phenol-chloroform-isoamyl alcohol (50:50:1), each time for 10 min at room temperature. The aqueous phase was adjusted to 2% potassium acetate. Nucleic acids were precipitated at -20°C for at least 2 hr by the addition of 2% vol of absolute ethanol and centrifuged at 10,OOOgfor 15 min at -20°C. The precipitates were redissolved in 25 ~1 sterile water. Aliquots were taken for in vitro translation. In vitro translation. A micrococcal nuclease-treated, rabbit reticulocyte, cell-free, translation system was purchased from New England Nuclear Corporation, Boston, Massachusetts. Reactions were carried out in 25-~1 volumes in the presence of 50 &i [35S]methionine (New England Nuclear, 800-1000 Ci/mmole) and of 100 ng to 1 pg of RNA. Substitution of potassium acetate (KOAc) and magnesium acetate (MgOAc) for potassium chloride and magnesium chloride substantially enhanced translation of the 8-crystallin mRNAs (unpublished results) as has been reported previously for other eukaryotic mRNAs (Weber et al., 1977; Shinohara and Piatigorsky, 1980). Mid-optimal range concentrations of KOAc (80 mM) and MgOAc (1 m&f) were routinely used. The reaction mixture was incubated at 37°C for 60 min followed by chilling on ice. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Electrophoresis was carried out in discontinuous 0.1% SDS, 10% polyacrylamide gels as described previously (Reszelbach et al., 1977), except that urea was omitted from the gels. Immunoprecipitation. Chicken /?-crystallin was purified by isoelectric focusing twice in a sucrose-density gradient as described previously (Ostrer and Piatigorsky, 1980). The &&, high-isoelectric-point fractions were pooled, dialyzed exhaustively against water, and then lyophilized. Two milligrams of antigen were mixed with Freund’s complete adjuvant and used to immunize an adult sheep twice over a 6-week period. Four weeks following the second immunization, the sheep serum was found to have a high titer against the test antigen. The antibody fraction was partially purified by ammonium sulfate precipitation as described elsewhere (Garvey et al., 1977).

VOLUME86,1981

Immunoprecipitation was performed following the method of Jones et al. (1981). Aliquots of 6 ~1 from the in vitro translation reaction were mixed with 5 ~1 of column-purified, @crystallin (1.5 Az,/ml), 50 ~1 partially purified antiserum, 39 ~1 HzO, and 50 ~1 2~ immunoprecipitation buffer (0.2 1MTris-HCl, pH 7.5, 0.02 M EDTA, 0.1 M NaCl, 0.05 M L-methionine, 2% Triton X-100,1% deoxycholate, 0.5% BSA). The samples were kept at 4°C overnight; the precipitates were washed three times with immunoprecipitation buffer. The resulting immune complexes were redissolved in 5% glycerol, 50 mlM Tris-HCl, pH 6.6, 10 mM P-mercaptoethanol, 0.1% SDS, and 0.1% phenol red by boiling for 3 min, then examined by SDS-polyacrylamide gel electrophoresis. Tryptic-peptide mapping. [35S]Methionine-labeled pcrystallin polypeptides were prepared by in vitro translation and were purified by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Two-dimensional, tryptic-peptide maps were prepared of these polypeptides following the procedure described by Shinohara et al. (1980). Labeling lens epithelia. Six lens central epithelia were labeled immediately after explantation in 250 ~1 Ham’s FlO (Grand Island Biological Company (GIBCO), Grand Island, N. Y.) medium supplemented with 250 PCi [?S]methionine and 15% fetal calf serum (GIBCO). The epithelia were incubated at 37°C in the presence of 5% COz for an hour, washed once with saline G (GIBCO), and frozen on dry ice. RESULTS

Identification

of &Crystallin

Polypeptides

We have reported previously the resolution by SDSurea-polyacrylamide gel electrophoresis of six p-crystallin polypeptides ranging in molecular weight from about 19,000 (19K) to 35,000 (35K) daltons (Ostrer and Piatigorsky, 1980). In the present investigation, we have omitted urea from the gel and resolved the P-crystallin polypeptides into seven, more distinct bands (Fig. 1A). The newly observed polypeptide migrated between the 26K and 29K polypeptides and was designated 27K. Sephadex G-200-purified a-crystallin comigrated with the 19K polypeptide under these conditions. Translation of @Crystallin mRNAs in a Cell-Free System To examine whether these polypeptides are primary translation products, poly(A+) RNA was isolated from 19-day-old embryonic lenses and translated in the rabbit reticulocyte lysate (Fig. 1B). All seven polypeptides were synthesized. Their identity was confirmed by im-

OSTRER, BEEBE, AND PIATICORSKY

35K P-CRYSTALLIN

/3-CRYSTALLINS

ah26K 20KC

FIG. 1. SDS-polyacrylamide gel electrophoresis of &crystallin polypeptides of the chicken lens. (A) fl-Crystallins purified from the lenses of 37-week-old chickens by Sephadex G-290 chromatography as given elsewhere (Ostrer and Piatigorsky, 1930); (B) in vitro translation of 19-day-old embryonic lens mRNAs in a rabbit reticulocyte cell-free system; (C) immunoprecipitation of B using partially purified sheep anti-chicken @-crystallin antiserum. A is a stained gel; B and C are autoradiograms.

munoprecipitation of the in vitro translation products with anti-chicken /3-crystallin antiserum (Fig. 1C). In vitro translation was dependent upon the quantity of mRNA added to the system. As little as 100 ng of mRNA (as judged by ultraviolet absorption at 260 nm) yielded resolvable /3-crystallin polypeptides on 24-hr autoradiograms. To examine age-dependent differences in the accumulation of 8-crystallin mRNAs in embryonic chicken lens fibers, equivalent amounts of total RNA from 6-, 15-, and 19-day-old embryonic fibers were translated in vitro (Fig. 2). All seven polypeptides were identified at each of the stages and their identity was confirmed by immunoprecipitation (data not shown). Over the course of development of the embryo, the fraction of mRNA coding for 8-crystallins increases. Fingerprints Peptides

of Methionine-Containing

Tmptic

The three major @crystallin polypeptides which were synthesized in vitro were compared by tryptic-peptide s-

15 DAY

19DAY

/3-Crgstallin mRNAs

405

mapping (Fig. 3). In each case, there were three major peptides which appeared to migrate to similar regions in the fingerprint of the 20K, 26K, and 35K polypeptides (Fig. 3, solid arrows). Peptide “a” consistently resolved into two spots in the map of the 26K polypeptide and was quite faint in the map of the 35K polypeptide. For the 20K and 26K polypeptides, there were additional peptides which were unique to those maps (Fig. 3, dotted arrows). Other minor spots were visible, especially in the map of the 20K polypeptide. These may be due to incomplete trypsin digestion or, more likely, contamination with other polypeptides. As these peptides were formed by in vitro translation in a reticulocyte lysate it is unlikely that they represent post-translational modifications. Similar results were obtained in three separate experiments. The data suggest that these three related @-crystallin polypeptides have differences in their primary structures, which is consistent with their being encoded by separate mRNAs. Wferential Accumulation of P-Crystallin mRNAs in the Lens Epithelial and Fiber Cells Labeling of 6- and 15-day-old embryonic lens central epithelia with [%]methionine suggested that the 35K polypeptide was not synthesized in these cells (Fig. 4). To examine whether the mRNA for this polypeptide was present in these cells, total RNA was extracted from the central and equatorial epithelia and the fibers of 19-day-old embryonic lenses and translated in vitro (Fig. 5). Synthesis of the 35K polypeptide was not found from the central epithelial RNA, confirming that within the limits of detection of this technique, this mRNA is absent in these cells. The 35K polypeptide was a minor in vitro translation product of the equatorial cell RNA and a major product of the fiber cell RNA. In addition, this polypeptide was not translated from RNA isolated from 6-day-old embryonic central epithelia. Thus, the 35K polypeptide appears to be a marker for elongating cells in the developing lens. DISCUSSION

--35K

/3crystallin

FIG. 2. Autoradiograms of SDS-polyacrylamide gel electrophoresis of in vitro translation products of mRNA isolated from 6-, 15-, and 19-day-old embryonic chicken lens fiber cells.

These experiments demonstrate that at least seven @-crystallin polypeptides resolvable by SDS-polyacrylamide gel electrophoresis may be synthesized in a heterologous cell-free system supplemented with mRNA from the embryonic chicken lens, suggesting that each of the polypeptides is encoded by a separate messenger RNA. The methionine-containing tryptic-peptide maps of the three most abundant P-crystallin polypeptides present in the embryonic lens are consistent with our earlier maps of radioiodinated tryptic peptides indicating that these polypeptides belong to a closely related family (Ostrer and Piatigorsky, 1980). The fin-

406

DEVELOPMENTAL BIOLOGY

+ t ORIGIN

VOLUME 86,1981

t ORIGIN

-

t ORIGIN

ELECTROPHORESIS FIG. 3. Fingerprints of the methionine-containing peptides of j%crystallin polypeptides synthesized in vitro. The origins are on the lower left of each photograph as indicated. Electrophoresis was in the horizontal direction and chromatography in the vertical direction. Solid arrows indicate the spots which may he shared. Dashed arrows indicate spots unique to that map.

gerprints of the methionine-containing tryptic peptides also suggest differences in their primary sequences and thus in their mRNA coding sequences. Furthermore, the increase in P-crystallin accumulation in the lens that occurs during development (Genis-Galvez et al., 1968) is correlated with the accumulation of B-crystallin mRNAs. It is not known whether further modification of the chicken p-crystallin polypeptides occurs in the lens. In contrast to these findings, the calf fi-crystallin Blb cannot be synthesized from mRNA in a reticulocyte cell-free system (Vermorken et al., 1977) or in Xenoms oocytes (Asselbers et al., 1979) and is assumed, therefore to be derived by post-translational modification of a precursor polypeptide. The present experiments indicate that the 35K Pcrystallin polypeptide is a specific marker for lens cell elongation in the chicken. As this polypeptide was resolved by molecular weight it is not known whether it has heterogeneous charge forms. The synthesis of the

35K /3-crystallin polypeptide is directed by an mRNA which appears in the equatorial region of the lens epithelium. Our results do not exclude the possibility that a few 35K P-crystallin mRNAs are present in the central lens epithelial cells, which may be detected by a more sensitive technique such as molecular hybridization. Inspection of published data from another investigation is consistent with our finding that the mRNA for the 35K p-crystallin polypeptide accumulates in the lens fiber cells of 15-day-old chicken embryos (Fig. 4, Thomson et al. (1978)). In addition to the appearance of the 35K /3-crystallin mRNA, there is a marked accumulation of mRNAs for the other /3-crystallins during development of the embryonic lens fiber cells. This correlates well with the increase in the accumulation of &crystallins during embryogenesis (Genis-Galvez et al., 1968). A 37K chicken fl-crystallin polypeptide, which we A

&DAY

B

C

l&DAY

d-crysmllin-

35K pcfY&llinlow MW &c~stallins fmnt-

I P-CRYSTALLINS FRONT

FIG. 4. Autoradiograms of SDS-polyacrylamide gel electrophoresis of in vitro [%]methionine-labeled soluble proteins from the central epithelia of 6- and 15-day-old chicken embryonic lenses, A standard for the crystallins was run in an adjacent lane of the gel. The arrow indicates that the 35K polypeptide was not detectable.

-

FIG. 5. Autoradiograms of SDS-polyacrylamide gels of in vitro translation products of mRNA isolated from (A) central epithelia, (B) equatorial epithelia, and (C) fiber cells of 19-day-old embryonic chicken lenses.

OSTRER, BEEBE, AND PIATIGORSKY

assume is the same as our 35K polypeptide, described as anodal /3-crystallin, has been reported previously (Maisel et al., 1976). Immunofluorescence studies on chicken embryos with anodal /3-crystallin have given different results with respect to the localization of this protein within the developing lens. In one investigation the anodal B-crystallin was confined to the lens fibers until 18 days of incubation, at which time positive fluorescence was observed in the epithelium (Waggoner et aZ., 1975); in another investigation immunofluorescence for anodal P-crystallin was observed in the lens epithelium in 3-day-old chicken embryos (McDevitt and Clayton, 1979). One possible explanation for these data is that the anodal p-crystallin is not composed solely of the 35K polypeptide but may be associated with other /3-crystallin polypeptides. This is suggested by our previous experiments concerning the molecular weight and acidic isoelectric point of the native /3-crystallin containing the 35K polypeptide (Ostrer and Piatigorsky, 1980). It is also possible that the sensitivity or specificity of the antisera used in the different experiments was not the same. Our data based on SDS-polyacrylamide gel electrophoresis and in vitro translation of mRNA indicate that at least the 35K polypeptide component of anodal /3-crystallin occurs only in the elongating lens cells. In other organisms different fiber-cell-specific crystallins have been reported. These include y-crystallin in bovine lenses (Papaconstantinou, 1965) and p- and y-crystallins in rat lenses (McAvoy, 1978). Amphibians are interesting since only y-crystallin is specific for fiber cells in regenerating lenses (Takata et al., 1965), while both (Y-and y-crystallins are confined to the fiber cells during normal development (McDevitt and Brahma, 1981). In cultured calf lens epithelia it has been shown recently that P-crystallins appear only in the epithelial cells which are undergoing a transition from epithelial to fiber-like cells (Vermorken et al., 1978). Synthesis of the 35K P-crystallin polypeptide in elongating lens cells and differentiating fiber cells in the chicken is of special interest since it represents selective regulation within a class of related crystallin polypeptides. The authors would like to express their appreciation to Douglas Feagans for his assistance with dissecting lenses and labeling lens epithelia. REFERENCES ASSELBERGS,F. A. M., KOOPMANS,M., VANVENROOIJ,W. J., and BLOEMENDAL,H. (1979). /3-Crystallin synthesis in Xenqpzls oocytes. Exp. Eye Res. 28,475-482. BEEBE, D. C., and PIATIGORSKY,J. (1976). Differential synthesis of crystallin and non-crystallin polypeptides during lens fiber cell differentiation in vitro. Exp. Eve Res. 22, 23’7-249. CLAYTON,R. M., and TRUMAN,D. E. S. (1974). The antigenic structure of chick fl-crystallin subunits. Exp. Eye Res. 18.495-506.

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mRNAs

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CLAYTON,R. M. (1969). Properties of the crystallins in the chick in terms of their subunit composition. Exp. Eye Res. 8, 326-339. CLAYTON,R. M. (1970). Problems of differentiation in the vertebrate lens. In “Current Topics in Developmental Biology” (A. A. Moscona, and A. Monroy, eds.), Vol. 5, pp. 115-180. Academic Press, New York. GARVEY,J. S., CREMER,N. E., and SUSSDORF,D. H. (1977). “Methods in Immunology: A Laboratory Text for Instruction and Research.” W. A. Benjamin, Reading, Mass. GENIS-GALVEZ,J. M., MAISEL, H., and CASTRO,J. (1968). Changes in chick lens proteins with aging. Exp. Eye Res. 7.593-602. JONES,R. E., DEFEO. D., and PIATIGORSKY,J. (1981). Initial studies on cultured embryonic chick lens epithelial cells infected with a temperature-sensitive Rous-sarcoma virus. Vision Res. 21.5-10. MCAVOY, J. W. (1978). Cell division, cell elongation and distribution of a-, j3-, and y-crystallins in the rat lens. J. Embryol. Exp. Mvrphol. 44,149-165. MCDEVI?T, D. S., and BRAHMA, S. K. (1981). Ontogeny and localization of the a-, ,%, and y-crystallins in newt eye lens development. Develop. Biol., in press. MCDEVI’IT, D. S., and CLAYTON,R. M. (1979). Ontogeny and localization of the crystallins during lens development in normal and Hy1 (hyperplatic lens epithelium) chick embryos. J. Embrgol. Exp. Morph& 50,31-45. MAISEL, H., ALCALA, J., and LIESKA, N. (1976). The protein structure of chick lens fiber cell membranes and intracellular matrix. Dot. Ophthdmol. 8, 121-133. OSTRER,H., and PIATIGORSKY,J. (1980). &Crystallins of the adult chicken lens: Relatedness of the polypeptides and their aggregates. Exp. Eve Res. 30.679-689. PAPACONSTANTINOU, J. (1965). Biochemistry of bovine lens proteins. II. The y-crystallins of adult bovine, calf and embryonic lenses. B&him. Biophys. Acta 107, 81-90. PIATIGORSKY,J. (1975). Lens cell elongation in vitro and microtubules. Ann N. K Acad. Sci. 253,333-347. PIATIGORSKY,J., WEBSTER,H. DEF., and CRAIG, S. (1972). Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in viwo and in vitro. Develup. Biol. 27, 176-189. RANA, M. W., and MAISEL, H. (1970). The subunit structure of chick lens beta-crystallin. Ophthalmol. Res. 1.156-165. RESZELBACH,R., SHINOHARA,T., and PIATIGORSKY,J. (1977). Resolution of two distinct embryonic chick d-crystallin bands by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea. Exp. Eye Res. 25, 533-593. SHINOHARA,T., and PIATIGORSKY,J. (1980). Anion and cation effects on y-crystallin synthesis in the cultured embryonic chick lens and in a reticulocyte lysate. Exp. Eye Res. 30, 351-360. SHINOHARA, T., RESZELBACH,R., and PIATIGORSKY,J. (1980). TWO tryptic peptide differences among the subunits of &crystallin of the embryonic chick lens. Exp. Eye Res. 30,361-370. TAKATA, C., ALBRIGHT, J. F., and YAMADA, T. (1965). Lens fiber differentiation and gamma crystallins: Immunofluorescent study of Wolffian regeneration. Science 147,1299-1301. THOMSON,I., WILKINSON, C. E., JACKSON,J. F., DE POMERAI, D. I., CLAYTON, R. M., TRUMAN, D. E. S., and WILLIAMSON, R. (1978). Isolation and cell-free translation of chick lens crystallin mRNA during normal development and transdifferentiation of neural retina. Develop. Biol. 65, 372-332. TRUMAN, D. E. S., and CLAYTON,R. M. (1974). The subunit structure of chick &crystallins. Exp. Eye Res. 18, 485-494. VERMORKEN,F. J. M., HERBRINK, P., and BLOEMENDAL,H. (1977). Synthesis of lens proteins in vitro: Formation of j3-crystallin. Eur. J. Biochem. 78,617-622. VERMORKEN,A. J. M., HILDERINK, J. M. H. C., VANDEVEN, W. J. M.,

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and BLOEMENDAL,H. (1978). Lens differentiation. Crystallin synthesis in isolated epithelia from calf lenses. J. CeU Biol. 76, 175183. WAGGONER,P. R., LIESKA, N., ALCALA, J., and MAISEL, H. (1976). Ontogeny of chick lens fi-crystallin polypeptides by immunofluorescence. Ophthalmol. Res. 8,292-301. WATANABE,H., and KAWAKAMI, I. (1973). Fractionation of the soluble proteins of the chick lens on Sephadex column. Exp. Eye Res. 17, 205-207. WEBER,L. A., HICKEY, E. D., MARONEY,P. A., and BAGLIONI,C. (1977). Inhibition of protein synthesis by Cl. J. Biol. Chem. 252,4007-4010. YOSHIDA, K., and KATOH, A. (1971). Crystallin synthesis by chick lens.

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II. Changes in synthetic activities of epithelial and fiber cells during embryonic development. Ezp. Eye Res. 11,184-194. ZELENKA, P., and PIATIGORSKY,J. (1974). Isolation and in vitro translation of &crystallin mRNA from embryonic chick lens fibers. Proc. Nat. Acad. Sci. USA 71, 1896-1900. ZELENKA, P., and PIATIGORSKY,J. (1976). Molecular weight and sequence complexity of 6-crystallin mRNA. Exp. Eye Res. 22, 115124. ZIGLER,J. S., JR., and SIDBURY,J. B., JR. (1976). A comparative study of the /3-crystallins of four sub-mammalian species. Comp. Biochem. Physiol. 55B, 19-24. ZWAAN, J., and IKEDA, A. (1968). Macromolecular events during differentiation of the chicken lens. Exp. Eye Res. 7,301-311.