δ-Crystallin mRNA in chick lens cells: mRNA accumulates during differential stimulation of δ-crystallin synthesis in cultured cells

DEVELOPMENTAL

6-Crystallin Differential

(1976)

mRNA in Chick Lens Cells: mRNA Accumulates during Stimulation of &Crystallin Synthesis in Cultured Cells

LEONARD Laboratory

48, 197-204

BIOLOGY

M.

MILSTONE,’

PEGGY

ZELENKA,

AND

of Molecular

JORAM

Genetics, National Institute of Child Health National Institutes of Health, Bethesda, Maryland Accepted

September

PIATIGORSKY

and Human 20014

Development,

4, 1975

Increasing specialization for 6-crystallin synthesis is a prominent feature of the differentiation of chick lens epithelial cells into lens fiber cells and can be studied in cultured embryonic lens epithelia. Quantitation of &crystallin mRNA by molecular hybridizaton to a r3H]DNA complementary to S-crystallin mRNA demonstrates that differentiation, both in ouo and in tissue culture, is associated with the accumulation of 6-crystallin mRNA. In the cultures, there is an overall stimulation of protein synthesis, including &crystallin mRNA during the first 5 hr in uitro. Between 5 and 24 hr in vitro there is a differential stimulation of &crystallin synthesis and an accumulation of S-crystallin mRNA that can quantitatively account for this stimulation.

pott and Coulombre, 1965) and this in vitro The developing lens is one of many tis- system is particularly well-suited to quansues in which differentiating cells become titative studies. The central region of the increasingly specialized for the synthesis epithelium contains a population of simiof tissue-specific protein. The central ques- ‘lar cells attached to collagenous capsule and contains no blood vessels or connective tion raised by this observation is whether increasing specialization, revealed by a dif- tissue. All cells normally accumulate 6ferential or selective increase in the syn- crystallin as judged by immunofluoresthesis of the tissue-specific protein, is cence (Zwaan and Ikeda, 1968). In uitro, caused by an increase in the amount of the the rate of &crystallin synthesis per cell is differentially stimulated during the first corresponding mRNA or by more effective 24 hr in culture (Milstone and Piatigorsky, utilization of a fixed pool of that mRNA. We have chosen to study this problem by 19751, the cells uniformly elongate, and the tissue demonstrates other biochemical quantitative comparison of b-crystallin and morphological features that conform synthesis and the amount of &crystallin mRNA in cultured, embryonic chick lens to the in uiuo patterns of lens cell differenepithelia. tiation (Piatigorsky et al., 1973). The recent purification of the &crystalIn lenses of g-day-old chick embryos, the anterior epithelial cells, which are already lin mRNA (Zelenka and Piatigorsky, 1974) has allowed us to synthesize a radioactive specialized for the synthesis of the chick lens protein, &crystallin, further differenDNA ([3H]cDNA) complementary to a-crystiate into lens fiber cells that are even tallin mRNA. Using this [3H]cDNA in a more highly specialized for &crystallin syn- hybridization assay that relies on the specific annealing of the 13HlcDNA to &crysthesis (Genis-Galvez et al., 1968; Piatigorsky et al., 1972a; Katoh and Yoshida, tallin mRNA and the ability of the S, nu1973). Lens fiber cell differentiation can be clease from Aspergillus oryzae to digest studied in explanted lens epithelia cul- single-stranded DNA but not DNA:RNA tured in medium containing serum (Philhybrids (Sutton, 19711, we have quantitated &crystallin mRNA in differentiating I Present address: Department of Dermatology, Yale Medical School, New Haven, Connecticut. lens tissue in vitro and in ouo. INTRODUCTION

197 Copyright All rights

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

198

DEVELOPMENTAL

MATERIALS

Preparation

AND

and Culture

BIOLOGY

Sucrose

METHODS

ofLens

Epithelia

Lenses were removed from eyes of white Leghorn chick embryos (obtained from Truslow Farms, Inc., Chestertown, Md.) at the end of the sixth day of development at 37°C. The central regions (approximately 1 mm21 of the lens epithelia were isolated and cultured in Ham’s F-10 medium (Ham, 1963) supplemented with 15% (v/v) fetal calf serum (Grand Island Biological Co.) as described previously (Milstone and Piatigorsky, 1975). Six explants per dish were cultured for 0,5, or 24 hr at 37°C in 5% CO%-95% air. Extraction

VOLUME

of RNA

Groups of 20 epithelia were transferred to conical centrifuge tubes, washed with Saline G (Puck et al., 19581, and allowed to swell for 5 min at 4°C in 1.0 ml of 10 m&f Tris pH 7.5, 0.14 M NaCl, and 1% Triton X-100 (Packard). The plasma membranes were ruptured by vortex mixing for 5 set and nuclei were removed by centrifugation for 10 min at 750g. RNA was isolated from cytoplasm that had been made 10% in sodium dodecyl sulfate and extracted with 1.0 ml of buffer-saturated-phenol: chloroform:isoamyl alcohol (50:50:1) for 10 min at room temperature. The aqueous phase was then made 2% in potassium acetate, pH 5.5, and extracted with chloroform:isoamyl alcohol (5O:l). Unwashed nuclei from these preparations or washed nuclei, isolated from epithelia that had been homogenized in buffer containing 0.24 M sucrose rather than Triton X-100, were extracted as above, digested for 25 min at 37°C with 35 pg DNAse (Worthington Biochemical Corp.) in 0.9 ml of 10 mM Tris pH 7.5, 0.14 M NaCl, 50 mM MgC12, and extracted again as for cytoplasmic RNA. RNA was precipitated from the aqueous phase by the addition of 2.5 volumes of ethanol, washed with ethanol-ethyl ether (50:50), and then dried from ethyl ether.

48,

Density

1976

Gradient

Centrifugation

Sucrose density gradients of 5 to 20% (w/v) sucrose were made in cellulose nitrate tubes in 10 mM Tris-HCl, pH 7.5, containing 1 n-&f EDTA and used for mRNA purification. The mRNA was dissolved in the same buffer, heated to 85°C for 10 min and “quick-cooled” in an ice bath before layering onto the gradients. Centrifugation was for 4.5 hr at 2°C and 50,000 rpm in a Spinco SW56 rotor. Alkaline sucrose density gradients of 5% sucrose-O.1 N NaOH to 20% sucrose-O.3 N NaOH were made in 0.9 M NaCl containing 1 mM EDTA and used in the preparation of the cDNA. Centrifugation was for 19.5 hr at 2°C and 40,500 rpm in a Spinco SW41 rotor. Synthesis

of L3HlcDNA

r3H]cDNA was synthesized by reverse transcriptase from avian myeloblastosis virus (Kacian et al., 1972; Ross et al., 1972; Verma et al., 1972) in O.l-ml reaction mixtures containing 0.2 mM dATP, dCTP, and and mM [3HldGTP dTTP, 0.025 (SchwarzNann, sp act 7 Ci/mmole) as described elsewhere (Zelenka and Piatigorsky, 1975). Each reaction mixture contained 3 pg of b-crystallin mRNA, isolated from lens fibers of 15-day-old chick embryos and purified by oligo(dT)-cellulose chromatography (Zelenka and Piatigorsky, 1974) and sucrose density gradient centrifugation. The reaction mixtures were incubated for 1 hr at 37”C, made 1% in sodium dodecyl sulfate, and extracted with an equal volume of phenol:chloroform:isoamyl alcohol (1:1:0.04). The nucleic acids were incubated 1 hr at 37°C in 0.5 M NaOH to hydrolyze the RNA and then dialyzed against 0.01 M Tris-HCl, pH 7.5, containing 0.1 M NaCl and 1 m&f EDTA. The cDNA was centrifuged in an alkaline sucrose density gradient and fractions of limited size distribution were pooled and dialyzed.

MILSTONE,

Hybridization RNA

ZELENKA

AND

of Wrystallin

PIATIGORSKY

cDNA

to

Lens RNA or purified Gcrystallin mRNA was hybridized to 500 cpm of 6crystallin 13HlcDNA essentially as described by Honjo et al. (1974) for hybridization of an immunoglobulin mRNA to its L3HlcDNA. The RNA and r3HlcDNA were incubated at 75°C in 20 ~1, oil-sealed, reaction mixtures containing 10 mM TrisHCl, pH 7.5, 0.6 NaCl, 0.2 mM Na2EDTA, and 0.5 pg single-stranded salmon sperm DNA (Sigma Chemical Co.). Hybridization was stopped by adding 2.2 ml of 30 mM NaCH&OOH, pH 4.5, containing 40 m&f NaCl, 0.125 mM ZnSO,, and 10 pg/ml of single-stranded DNA. Each sample was divided into two l-ml aliquots; one received 5 pg of S, nuclease from Aspergillus oryzae (kindly provided by P. Leder). Both aliquots were incubated for 1 hr at 45°C. Undigested 13HlcDNA was precipitated with 5% Cl,CCOOH at 4°C and collected on Whatman GF/C glass fiber filters in preparation for scintillation counting in a toluene solution of Biosolv (Beckman) and Spectrafluor (AmershamSearle). Percent hybridization was the radioactivity in the nuclease treated aliquot divided by the radioactivity in the aliquot not treated with nuclease. The background subtracted from each determination was the amount of undigestible material in hybridization mixtures not allowed to anneal at 75°C and was never more than the equivalent of 4% hybridization.

GCrystnllin

mRNA

in Chick

Lens

199

Cells

&crystallin, and trypsin digests of all the protein made in vitro contained only 8 crystallin peptides (Zelenka and Piatigorsky, 1974). The poly(A)-containing RNA was further fractionated by sucrose density gradient centrifugation and a peak of RNA, estimated to be approximately 17 S by comparison with rRNA markers in parallel gradients, was isolated (Fig. 1). This material produced a single band in formamide-polyacrylamide gels that was readily distinguishable from 18 S rRNA, and caused the synthesis of 8crystallin in a rabbit reticulocyte lysate (Zelenka and Piatigorsky, 1975). The 17 S RNA from the sucrose gradient was used as a template for the synthesis of a L3HlcDNA by the reverse transcriptase from avian myeloblastosis virus. The reaction was strongly dependent upon the addition of the enzyme, &crystallin mRNA, and deoxyribonucleoside triphosphates. The 13HJcDNA was heterogeneous in size when examined by alkaline sucrose density gradient centrifugation (Fig. 2). All the 13H]cDNA greater than 400 nucleotides in length hybridized to &crystallin mRNA, but for the experiments reported here, only molecules with 950-1300 nucleo-

RESULTS

Properties cDNA

of GCrystallin

mRNA

and its

RNA was isolated from lens fibers of 15day-old chick embryos and poly(A)-containing RNA was obtained by adsorption to oligo(dT)-cellulose as previously described (Zelenka and Piatigorsky, 1974). This RNA stimulated protein synthesis in a Krebs ascites in vitro protein synthesizing system: 70% of the new protein made was

wFRACTION Eonom

NUMBER 1
FIG. 1. Sucrose density gradient centrifugation of embryonic chick lens fiber mRNA. 72 pg of mRNA, twice chromatographed on oligo(dT! cellulose, was centrifuged through a 5 to 20% sucrose density gradient as described under Methods. Fractions of 0.17 ml were collected from the bottom of the tube and fractions 12-16 were poole,d for reverse transcription.

200

DEVELOPMENTAL

c

5

Bottom

10

15

20

FRACTION

950-1300

25

30

BIOLOGY

N T

35

40

NUMBER TOP

FIG. 2. Alkaline sucrose density gradient centrifugation of d-crystallin PH]cDNA synthesized from the 17 S RNA isolated from the sucrose gradient shown in Fig. 1. t3HlcDNA was dialyzed and centrifuged through an alkaline 5 to 20% sucrose gradient as described under Methods. Fractions (0.3 ml) were collected from the bottom of the tube. Aliquots (5 ~1) were neutralized with acetic acid and assayed for radioactivity. The positions of DNA markers of known size, centrifuged in a parallel gradient, are indicated by arrows.

tides were used. The cDNA of this size was used because it was a large transcript with sufficient total radioactivity to perform a complete series of experiments. A complete description of the synthesis and physical properties of &crystallin L3HlcDNA and its hybridization to purified Ccrystallin mRNA will be published elsewhere (Zelenka and Piatigorsky, 1975). The r3H]cDNA was complementary to purified &crystallin mRNA as demonstrated by the hybridization curve in Fig. 3. Less than 10% of the cDNA hybridized to RNA extracted from headless bodies of chick embryos, even when the amount of RNA added to the reaction was 100 times more than the amount of RNA from lens epithelia needed to give 50% hybridization. This demonstrates the specificity of the hybridization reaction. Quantitation of Wrystallin mRNA in Cultured Lens Epithelia RNA was extracted from cultured lens epithelia and hybridized to 6-crystallin

VOLUME

48, 1976

r3HlcDNA (Fig. 4). With increasing time in culture, RNA from fewer epithelia was required to obtain the same amount of hybridization to the r3H]cDNA under identical conditions. The data in Fig. 4 thus show that Ccrystallin mRNA accumulates in the epithelia during the 24 hr of culture. The amounts of &crystallin mRNA in the tissue extracts can be estimated by reference to a standard curve if the amount of cDNA hybridized is related solely to the concentration of tissue extract in the hybridization reaction mixtures. Figure 4 shows that for RNA concentrations (expressed as number of epithelia per reaction mixture) giving 25-70% hybridization of a fixed amount of 13H]cDNA in a fixed amount of time, there was an approximately linear relationship between log [RNA] x (time) and percentage hybridization. Similar curves were obtained if the same amount of cDNA and the lowest amount of RNA used in Fig. 4 were hybridized for increasing amounts of time. These data demonstrate that the amount of hybridization was proportional to the amount of tissue extract and that sufficient excess of RNA was present so that the reactions were not limited by nearly complete hybridization of all available mRNA. Since the concentration-dependent hybridization curve (for which the ratio of mRNA/cDNA varied fourfold between tubes giving 25 and 70% hybridization) and the time-dependent hybridization curve (for which the ratio of mRNA/cDNA was constant) were indistinguishable, we concluded that this fourfold difference in the ratio of mRNA/cDNA did not measurably affect hybridization. Nonetheless, it has been reported that hybridization is affected by the ratio of mRNA/cDNA (Young et al., 1974). To minimize any effect of different ratios of mRNA/cDNA, the standard curve used for quantitating S-crystallin RNA was generated by hybridizing different amounts of purified &crystallin mRNA to the same amount of cDNA for the same amount of time in the same reaction volumes as used

MILSTONE.

ZELENKA

AND

PIATICORSKY

GCrystallin

mRNA

FIG. 3. Hybridization of YH]cDNA to &crystalhn mRNA. Increasing lin mRNA were hybridized to 500 cpm of [3H]cDNA (specific activity 10’ under Methods R$ is the product of initial RNA concentration (moles (seconds); moles per liter x seconds is numerically equal to A 260 per hybridized was determined by resistance to S, nuclease digestion.

60

0

1

3 (NUMBER

5

OF EPITHELIA

10

20

1 x IHRS.)

FIG. 4. Increasing concentrations of RNA, extracted from the central region of lens epithelia, were hybridized to 500 cpm of [3H]cDNA for 3 hr at 75°C in 20-&l reaction mixtures as described under Methods. The abscissa is the number of epithelia per reaction mixture multiplied by the hours (3) of hybridization. Percentage of L3H]cDNA hybridization was determined by resistance to S, nuclease digestion. Epithelia were cultured 0 (A), 5 (B), or 24 hr (0).

for tissue samples (Fig. 3). In this way the mRNA/cDNA ratios for the standard curve were the same as those in the tissue hybridization at a given Rd. The amount of &crystallin mRNA in the cultured lens epithelia was determined

in Chick

Lens

Cdls

201

concentrations of purified h-crystalcpm/Fg) for 3 hr at 75°C as described of ribonucleotide per liter) and time ml12 x hr. Percentage of ?HlcDNA

from the standard curve and was divided by the number of cells in the epthelia to give the E-crystallin mRNA content per cell (Table 1). During the first 5 hr of culture, the amount of &crystallin mRNA per cell did not change, In contrast, after 24 hr in vitro the amount of Ccrystallin mRNA per cell had increased by a factor of 2.59. This increased amount of 6-crystallin mRNA at 24 hr was not derived from a preexisting nuclear pool of Ccrystallin mRNA or from a single-stranded precursor of Ccrystallin mRNA, since very little hybridizable RNA was found in the nuclear fraction of cells at the earlier times (Table 1). A control experiment showed that the DNAse treatment used in the preparation of the nuclear RNA had no effect on the hybridizability of purified Ccrystallin mRNA. Comparison of Rates of Mrystallin Synthesis with Amounts of Krystallin mRNA Relative amounts of cytoplasmic 6-crystallin mRNA were compared with previously reported rates of Scrystallin synthesis (Milstone and Piatigorsky, 1975) in similarly treated explants of lens epithelia (Ta-

202

DEVELOPMENTAL TABLE

1

HYBRIDIZABLE XRYSTALLIN mRNA CULTURED LENS EPITHELIA~ Time in vitro

(hr)

Cell number

Gcrystallin

mRNA cules/cell)

IN

(mole-

VOLUME

48, 1976

in the amount of 8-crystallin mRNA, a corresponding differential stimulation of Gcrystallin synthesis, and the result is a cell more highly specialized for the synthesis of &crystallin.

(CellS/eX-

plant) 0 5 24

BIOLOGY

23,750 28,500 32,900

Cytoplasm* 6,080 5,990 15,750

2 1,370 + 1,290 + 1,610

Nucleus’ 60 31 218

” The central region of lens epithelia from 6-dayold embryonic chicks were explanted and cultured as in the text. Triplicate samples of RNA from three epithelia for the 0- and 5-hr cultures and one epithelium for the 24-hr cultures were hybridized to 500 cpm of Gcrystallin [3H]cDNA for 3 hr at 75°C in 20-&l reaction mixtures as described under Methods. These dilutions of the tissue extracts hybridized to approximately 50% of the indicated amount of 13HlcDNA in 3 hr. Percentage hybridization was converted to molecules of mRNA per cell using the standardization curve as described in the text, and separately determined cell counts (Milstone and Piatigorsky, 19751 as follows: molecules mRNA per cell = [(Rot x 2 x ml react. mix)/(hr of hybridization) x (40 x 1O-6 g mRNA)/(A,,,) x Nl/(mol wt mRNA x cell number); where, R,t x 2 = Azso per ml x hr; mol wt mRNA = 6.8 x lo5 (Zelenka and Piatigorsky, 1975); N = Avogadro’s number. b Average of three experiments 2 1 SEM. c Average of two experiments.

ble 2). During the first 5 hr in vitro, there was no change in the amount of &crystallin mRNA and the stimulation of protein synthesis equally affected Gcrystallin and the bulk of other proteins. Two additional observations are pertinent. First, the ratio of polysomes to single ribosomes visible in electron micrographs of these tissues is increased after culture (Piatigorsky et al., 1972b; compare Figs. 5a and Fig. 5b therein). Second, the total amount of ribosomal RNA per cell does not change appreciably during culture (Milstone, unpublished). Taken together these data suggest that the increased capacity of these cells to translate many different mRNAs after 5 hr in culture is related to a functional rather than a quantitative change in the protein synthesizing machinery. Between 5 and 24 hr in culture, there is an increase

GCrystallin mRNA in Lens Cells of Whole Epithelia and Fiber Masses To test whether the accumulation of 6crystallin mRNA in vitro is a fair representation of lens fiber differentiation in ouo, the amount of Gcrystallin mRNA was measured in isolated whole epithelia and fiber massesfrom g-day-old chick embryos. The hybridization results (Table 3) showed that the fiber cells contained an average of four times as much b-crystallin mRNA as the epithelial cells. Since epithelial cells from this stage of embryonic development normally differentiate into fiber cells that are more highly specialized for S-crystallin synthesis (Piatigorsky et al., 1972a; Katoh and Yoshida, 19731, this result is entirely consistent with the result obtained in the in vitro system. DISCUSSION

The data we have presented show that 6crystallin mRNA normally accumulates in differentiating chick lens in ouo and in TABLE RELATIVE mRNA

Time in (hr)

uitro

0 5 24

2

CHANGES IN CYTOPLASMIC 6-CRYSTALLIN AND RATES OF PROTEIN SYNTHESIS IN CULTURED LENS EPITHELIA” Gcrystallin mRNA” 1 0.98 2.59

Total protein synthesis’ 1 1.63 1.73

Gcrystallin synthesi@ 1 1.68 2.80

a Data are expressed relative to the amount of RNA per cell or rates of protein synthesis per cell at time zero. b Data for Gcrystallin mRNA from Table 1. c Data for rates of protein synthesis, measured by [3H]valine incorporation into total protein and into immunoprecipitable Gcrystallin, with allowances made for the specific activity of the precursor pool of valine, the turnover of the proteins, and the number of cells, comes from Milstone and Piatigorsky (1975).

MILSTONE,

ZELENKA

AND PIATIGORSKY

vitro. Comparisons of mRNA accumulation and rates of Bcrystallin synthesis in the cultured lens epithelia support the hypothesis that increasing specialization for Gcrystallin synthesis is controlled by the amount of cytoplasmic 6-crystallin mRNA. This conclusion is similar to that reached by other investigators who have studied increasing specialization for the synthesis of globin (Ross et al., 1972; Tereda et al., 1972; Chan et al., 1974; Harrison et al., 1974), ovalbumin and avidin (Chan et al., 1973), actin (Paterson et al., 1974), and tryptophan oxygenase (Schutz et al., 1975). We see no reason to invoke specific translational control mechanisms as the cause of increasing specialization. Nonetheless, the stimulation during the first 5 hr in uitro, which occurs in the absence of a change in the amount of &crystallin mRNA, might be a useful system in which to study regulated translation of a specific mRNA. Our data do not answer the important question of how &crystallin mRNA accumulates in differentiating lens cells. Possible mechanisms are: increasing rates of transcription, decreasing rates of degradation, decreasing intranuclear degradation coupled with an increasing rate of transport out of the nucleus, or a constant rate of overall turnover coupled with a decreasing rate of cell division. Whatever the mechanism, if &crystallin mRNA accumulation in the intact lens occurs at the same rate as it does during fiber cell differentiation in the cultured epithelia, a nondividing fiber cell would require about 4 days to accumulate the observed level of mRNA.2 This is approximately the amount of time that has elapsed between the first detection of Gcrystallin at 2 days of develop ment (Zwaan and Ikeda, 1968; Katoh and Yoshida, 1973) and the present estimates of 8-crystallin mRNA in fiber cells at 6 days of development. * The in vitro “rate” of accumulation the time averaged difference between after 24 and 5 hr in uitro.

referred to is the amounts

GCrystallin

mRNA

XRYSTALLIN

in Chick

TABLE 3 mRNA IN LENSES CHICK

Tissue

Whole Whole

epithelium fiber mass

Lens

203

Cells

OF ~-DAY-OLD

EMBRYOS” Cell number rcell/tissueY

Gcrystallin mRNA (molecules/ ce111r

64,000 51,000

12,210 48,780

0 RNA from the equivalent of 0.1 lens fiber mass or 0.5 lens whole epithelium was hybridized to 500 cpm of LRH]cDNA for 3 hr at 75°C in 20-~1 reaction mixtures as described under Methods. Percentage hybridization was converted to molecules of mRNA per cell as described in Table 1. * Cell numbers from unpublished data of A. Coulombre. c Average of two experiments We thank Dr. Philip Leder for his gifts of AMV reverse transcriptase, A. oryzae S, nuclease, and the DNA molecular weight markers. We are indebted to Dr. A. J. Coulombre for allowing us to use his unpublished cell number data. We are grateful to Ms. Catherine Kunkle for typing the manuscript. REFERENCES CHAN, L., MEANS, A. R., and O’MALLEY, B. W. (19731. Rates of induction of specific translatable messenger RNAs for ovalbumin and avidin by steroid hormones. Proc. Nut. Acad. Sri. USA 70, 1870-1874. CHAN, L.-N. L. WIEDMANN, M., and INGRAM, V. M. (19741. Regulation of specific gene expression during embryonic development: synthesis of globin messenger RNA during red cell formation in chick embryos. Develop. Biol. 40. 174-185. GENIS-GALVEZ, J. M., MAIZEL, H., and CASTRO, J. (1968). Changes in chick lens proteins with ageing. Exptl. Eye Res. 7. 593-602. HAM, R. G. (1963). An improved nutrient solution for diploid Chinese hamster and human cell lines. Exptl. Cell Res. 29, 515-526. HARRISON, P. R., CONKIE, D., AFFARA, N., and PAUL, J. (1974). In situ localization of globin messenger RNA formation. I. During mouse fetal liver development. J. Cell Biol. 63, 402-413. HONJO, T., PACKMAN, S., SWAN, D., NAU, M., and LEDER, P. (19741. Organization of immunoglobulin genes: reiteration frequency of the mouse K chain constant region gene. Proc. Nat. Acad. Ser. USA 71, 3659-3663. KACIAN, D. L., SPIEGELMAN, S., BANK, A., TERADA, M., METAFORA, S.. Dow, L., and MARKS, P. A. (19721. In vitro synthesis of DNA components of human genes for globins. Nature New Biol. 235, 167-169.

204

DEVELOPMENTAL

BIOLOGY

KATOH, A., and YOSHIDA, K. (1973). Delta crystallin synthesis during chick lens cell differentiation. Exptl. Eye Res. 15, 353-360. MILSTONE, L. M., and PIATIGORSKY, J. (1975). Rates ofprotein synthesis in explanted chick lens epithelia: differential stimulation of S-crystallin synthesis. Deuelop. Biol. 43, 91-100. PATERSON, B. M., ROBERTS, B. E., and YAFFE, D. (1974). Determination of actin messenger RNA in cultures of differentiating embryonic chick skeletal muscle. Proc. Nat. Acad. Sci. USA 71, 44674471. PHILPOTT, G. W., and COULOMBRE, A. J. (1965). Lens development. II. The differentiation of chick lens epithelial cells in vitro and in vivo. Exptl. Cell Res. 38, 635-644. PIATIGORSKY, J., ROTHSCHILD, S. S., and MILSTONE, L. M. (1973). Differentiation of lens fibers in explanted chick lens epithelia. Deuelop. Biol. 34, 334-345. PIATIGORSKY, J., WEBSTER, H. DEF., and CRAIG, S. P. (1972a). Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in vivo and in vitro. Develop. Biol. 27, 176189. PIATIGORSKY, J., WEBSTER, H. DEF., and WOLLBERG, M. (1972b). Cell elongation in the cultured embryonic chick lens epithelium with and without protein synthesis. J. Cell Biol. 55, 82-92. PUCK, T. T., CIECIURA, S. J., and ROBINSON, A. (1958). Genetics of mammalian cells. III. Longterm cultivation of euploid cells from human and animal subjects. J. Exptl. Med. 108, 945-956. Ross, J., IKAWA, Y., and LEDER, P. (1972). Globin

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messenger-RNA induction during erythroid differentiation of cultured leukemia cells. Proc. Nat. Acad. Sci. USA 69, 3620-3623. SCHUTZ, G., KILLEWICH, L., &EN, G., and FEIGEG SON, P. (1975). Control of the mRNA for hepatic tryptophan oxygenase during hormonal and substrate induction. Proc. Nat. Acad. Sci. USA 72, 1017-1020. SUTTON, W. D. (1971). The crude nuclease preparation suitable for use in DNA reassociation experiments. Biochim. Biophys. Acta 240, 522-531. TERADA, M., CANTOR, L., METAFORA, S., RIFKIND, R. A., BANK, A., and MARKS, P. A. (1972). Globin messenger RNA activity in erythropoietin. Proc. Nat. Acad. Sci. USA 69, 3575-3579. VERMA, I. M., TEMPLE, G. F., FAN, H., and BALTIMORE, D. (1972). In vitro synthesis of DNA complementary to rabbit reticulocyte 10s RNA. Nature New Biol. 235, 163-167. YOUNG, B. D., HARRISON, P. R., GILMAN, R. S., BIRNIE, G. D., HELL, A., HUMPHRIES, S., and PAUL, J. (1974). Kinetic studies of gene frequency. J. Molec. Biol. 84, 555-568. ZELENKA, P., and PIATIGORSKY, J. (1974). Isolation and in vitro translation of 8-crystallin mRNA from embryonic chick lens fibers. Proc. Nat. Acad. Sci. USA 71, 1896-1900. ZELENKA, P., and PIATIGORSKY, J. (1975). Molecular weight and sequence complexity of 6-crystallin mRNA. Exptl. Eye Res., in press. ZWAAN, J., and IKEDA, A. (1968). Macromolecular events during the differentiation of the chick lens. Exptl. Eye Res. 7, 301-311.