THE JOURNAL OF BIOLOGICAL CHEM~~RY

Vol 253, No 10, Issue of May 25, pp 3494-3503, 1978

Prmted tn U S A

The Action of Estrogen and Progesterone on the Expression of the Transferrin Gene A COMPARISON OF THE RESPONSE IN CHICK LIVER AND OVIDUCT*

(Received for publication, November 21, 1977)

DAVID C. LEE, G. STANLEY MCKNIGHT,$ AND RICHARD D. PALMITER§

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195

We present evidence that conalbumin and transferrin, synthesized in chick oviduct and liver, respectively, are products of the same gene, but the gene is regulated differ- ently in the two tissues. Conalbumin mRNA was purified 500-fold from hen oviduct by a combination of immunopre- cipitation of polysomes, oligo(dT) chromatography, and sucrose gradient centrifugation. A cDNA complementary to conalbumin mRNA was synthesized and used to demon- strate that by size and hybridization properties, conalbumin mRNA in the oviduct is identical with transferrin mRNA in the liver. Polyacrylamide gel analysis suggests that both mRNAs have an apparent molecular weight of 1 x 10”. In addition, hybridization of conalbumin cDNA to chick DNA indicates there are 1 to 2 gene copieslhaploid genome.

The effects of estrogen and progesterone on the expression of this gene in chick oviduct and liver were examined. Both steroids induced a marked stimulation of conalbumin syn- thesis and after 96 h of hormone, the rate was elevated 6- to S-fold with estrogen and 5- to &fold with progesterone. In both cases, the response could be accounted for by a rapid increase in conalbumin mRNA sequences. Although estro- gen also stimulated transferrin synthesis, the effect was much less pronounced with only a 1.5- to Z-fold stimulation after 96 h of hormone, and there was a lag in the appearance of transferrin mRNA. Progesterone had no effect on trans- ferrin synthesis. The cell-specific regulation of this gene in the liver and oviduct is particularly interesting because both are estrogen-responsive tissues induced by the hormone to synthesize egg yolk and egg white proteins, respectively. This system may provide a model for exploring the differ- ential programming of a specific gene during development and differentiation.

Current investigations into the mechanisms regulating gene expression in eukaryotes are conducted largely within the context of a single tissue or cell type, and often the gene

* This work was supported by Grant HD-9172 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebv marked “advertisement” in accordance with 18 U.S.C. Section 173i solely to indicate this fact.

$ Associate Investigator of the Howard Hughes Medical Institute. 8 Investigator of the Howard Hughes Medical Institute.

studied (e.g. globin, ovalbumin, immunoglobulin, vitello- genin) is virtually inactive in other tissues. In some cases such as the actin (1) or myosin (2) genes, the mRNAs induced during differentiation derive from a different gene than that transcribed constitutively. If the same gene is expressed in multiple tissues, however, various control mechanisms acting at the transcriptional or translational levels might be invoked, depending on the ultimate role of the gene product in those tissues. Comparative analysis of the tissue-specific regulation of such a gene could provide further insight into the mecha- nism of differentiation and the control of gene expression in higher organisms. We provide evidence here to suggest that regulation of the avian transferrin gene constitutes such an example.

Transferrin, the major serum iron-binding protein, is a glycoprotein with a molecular weight of approximately 77,000 (3). It transports iron from intestinal adsorption sites to storage areas in the liver, spleen, and bone marrow and subsequently to reticulocytes for the biosynthesis of hemoglo- bin (4). Although liver hepatocytes are the principal site of synthesis of serum transferrin in rats (5-7), the protein is produced in a number of tissues including spleen, kidney, lung, brain, and bone marrow (5, 8).

Conalbumin is a major egg white protein constituting roughly 10 to 15% of the egg white. Although its function is uncertain, it is presumed to be a nutritional source and may function as an antimicrobial agent because of its iron-binding capacity. Conalbumin has a molecular weight similar to that of transferrin, and in fact, the two proteins appear to differ only in their carbohydrate constituents; conalbumin contains only mannose and glucosamine, whereas transferrin contains, in addition, 2 galactose and 1 or 2 sialic acid residues/molecule of protein (9). Various criteria including immunoelectropho- resis, analysis of iron-binding properties, amino acid composi- tion, and peptide patterns indicate that the protein compo- nents are identical (10, 11).

The fact that conalbumin and transferrin appear to be identical polypeptides suggests that the same gene codes for both proteins. This is supported by the findings of Williams (lo), that for a given individual, polymorphic forms of trans- ferrin are accompanied by the same polymorphisms of conal- bumin. In addition, hens displaying an electrophoretic variant of transferrin show the same variant of conalbumin. If the hypothesis postulating a single genetic locus is correct, this

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Regulation of the Transferrin Gene 3495

raises interesting questions about the control of expression of this gene in the liver and in the oviduct.l

Synthesis of conalbumin in the chick oviduct is induced by steroid hormones, rising from approximately 1% of total protein synthesis in the absence of exogenous steroids to a maximum of 10 to 12% in an estrogen-stimulated chick or laying hen (12, 13). Furthermore, the increase in conalbumin synthesis is paralleled by an increase in the cellular levels of conalbumin mRNA (14).

Morgan (5) has studied the synthesis of transferrin in rat liver and has demonstrated a 2.5-fold increase in synthesis in response to hemorrhagic iron deficiency anemia. Serum trans- ferrin is elevated during pregnancy in a variety of animals (E-171, possibly because of iron deficiency incurred during gestation. Alternatively, changing hormone levels during pregnancy might stimulate transferrin synthesis. Horne and Ferguson (15) reported a 1.5 to 2-fold increase in serum transferrin values following administration of estrogen (but not progesterone) to rats, and the increase is quantitatively similar to that observed during pregnancy. In addition, a variety of other hormones including cortisol (18), thyroxine (191, and glucagon (20) have been shown to increase transfer- rin synthesis in the liver. The level at which any of these factors act is uncertain.

We have further characterized the regulation of transferrin synthesis in the liver in order to ascertain whether the expression of this gene is controlled differently in the oviduct and liver. In this paper, we provide evidence that conalbumin and transferrin are coded for by the same gene, and compare the effects of estrogen and progesterone on the synthesis of transferrin and conalbumin in the chick liver and oviduct, respectively.

EXPERIMENTAL PROCEDURES

Animals - White Leghorn chicks (3 to 4 days old) were given 10 to 12 days of primary stimulation with 15-mg hexestrol pellets and then withdrawn for 10 days. Chicks were restimulated with daily subcutaneous injections of 17P-estradiol benzoate or progesterone (1 mglchick) dissolved in corn oil.

Preparation of Antigens and Antibodies- Conalbumin was ex- tracted from egg white (21) and further purified by DEAE-cellulose chromatography essentially as described (22). The protein prepared in this manner was shown to be pure by sodium dodecyl sulfate- acrylamide gel electrophoresis. Chicken serum albumin, Fraction V (Sigma) was fractionated by DEAE-Sephadex A-50 chromatography (23). Grade V ovalbumin (Sigma) was purified further on DEAE- cellulose as described before (13). Rabbit IgG2 was prepared by precipitation with ammonium sulfate at 40% saturation and chro- matography on DEAE- and CM-cellulose (12).

Purification of Antibodies - Goat anti-rabbit IgG was purified and freed of RNase as before (12). Rabbit anti-conalbumin IgG was purified by immunoabsorption. Crude IgG was prepared by (NH&SO, precipitation and dialyzed against PBS. It was then applied to a conalbumin/Sepharose column prepared essentially as described (24) with purified conalbumin. The column was washed with sterile PBS and the absorbed antibody was then eluted with sterile 0.1 M glycine adjusted to pH 2.8 with HCl. The eluant was neutralized and dialyzed against 10 mM sodium phosphate (pH 7.2) containing 15 mM NaCl and then made ribonuclease-free as de- scribed above.

I This genetic locus has sometimes been called the ovotransferrin locus in reference to the oviduct protein. Although it may be inappropriate to use different names for the same protein in two tissues, we will retain the use of separate names in order to avoid confusion in the ensuing comparisons.

* The abbreviations used are: IgG, immunoglobulin G; PBS, phos- phate-buffered saline (0.15 M NaCl, 0.015 M NaH,PO,, pH 7.2); Hepes, 4-(2-hydroxyethyl-1-piperazineethanesulfonic acid; SDS, so- dium dodecyl sulfate.

Preparation of Conalbumzn-synthesizing Polysomes - Frozen ovi- duct from laying hens was disrupted with a Polytron (Tekmar) followed by Dounce homogenization, and polysomes were isolated by magnesium precipitation as before (251, except that a 5% homoge- nate was prepared in 12.5 rnM Tris (pH 7.51, 12.5 rnM NaCl, 2.5 rnM MgCl,, 10 PM cycloheximide, and the heparin concentration was increased to 1.5 mg/ml. Examination of polysome profiles indicated that these changes tended to minimize degradation. Following Mg’+ precipitation, polysomes were resuspended in 20 rnM Hepes (pH 7.5) and adjusted to 0.15 M NaCl, 0.5% Triton X-100, 5 rnM MgCl,, and 500 yglml of heparin. The polysome suspension was centrifuged for 20 min at 18,000 x g,,, to remove aggregates; the concentration of polysomes remaining in the supernatant was adjusted to 10 to 20 A,,,lml. Polysomes were used immediately for immunoprecipitation.

Conalbumin-synthesizing polysomes were enriched essentially as described before for ovalbumin-synthesizing polysomes (26). Poly- somes were incubated with 30 yg of rabbit anti-conalbumin/A,,,, of polysomes for 60 min at 4”C, followed by a 50-fold weight excess of RNase-free goat anti-rabbit IgG (twice the immunological equiva- lence point) for an additional 60 min at 4°C. The polysome. antibody complex was isolated by centrifugation for 15 min at 18,000 x g,,, through a 0.5 M sucrose pad layered over a 1.0 M sucrose pad, both containing 25 rnM Tris-Cl (pH 7.51, 0.15 M NaCl, 0.5% Triton X-100, and 500 yglml of heparin. The pellet was resuspended in the same buffer and recentrifuged through sucrose again. The sides of the tube were washed carefully with sterile H,O and the pellet resus- pended in an amount of SDS sufficient to yield an excess of approximately 0.75% (assuming that 1 g of protein binds 1.5 g of SDS) and proteinase K was added to 200 fig/ml. The sample was incubated for 60 min at 41°C and the RNA was extracted as described below. A typical preparation started with 6 g of tissue and yielded 800 A%,,, units of polysomes, of which 30 A,,, units were immunopre- cipitated.

Purification of Conalbumin mRNA -Ethanol-precipitated RNA extracted from conalbumin polysomes was collected by centrifuga- tion and resuspended in 10 rnM Tris-Cl (pH 8.01, 4 rnM EDTA, 500 rnM NaCl, and 1% SDS. The RNA was heated at 68°C for 3 min and applied to an oligo(dT)-cellulose column (Collaborative Research) equilibrated with the same buffer. The column was washed and the poly(A)-containing RNA was then eluted with sterile H,O. The RNA was lyophilized, resuspended, and ethanol-precipitated. RNA was resuspended in SET buffer (10 rnM Tris (pH 7.6). 5 rnM EDTA, 1% SDS), heated for 3 min at 65”C, and layered on a 12-ml, 5 to 20% sucrose gradient in SET buffer. Centrifugation was at 280,000 x g for 8 h at 25°C in a Beckman SW 41 rotor. Fractions (0.5 ml) were collected and the absorbance at 260 nm measured.

Translation of Conalbumin mRNA - RNA fractions were assayed for conalbumin mRNA activity using a rabbit reticulocyte transla- tion system essentially as described (27) except for the following modifications. 1) The lysate was treated with staphylococcal nu- clease prior to use in order to diminish endogenous globin mRNA activity (28) and then chromatographed on a Sephadex G-50 column to reduce the endogenous amino acid pool. These procedures im- proved the incorporation of isotope into conalbumin and lowered the background significantly. 2) Affinity-purified, RNase-free rabbit anti-conalbumin was added to the reaction at a final concentration of 80 pg/ml; this enhanced recovery of 13Hlconalbumin up to 3-fold in the subsequent immunoprecipitation. 3) 13HlTyrosine (35 &i/ml) was used as the label.

Purification of Oualbumin mRNA-Oviduct RNA was enriched for ovalbumin mRNA activity by two passages over oligo(dT) and sucrose gradient centrifugation essentially as described (29). Oval- bumin mRNA was further purified by preparative gel electrophore- sis according to the method of Hagen and Young (30).

Preparation of Total RNA and DNA-Total nucleic acid was extracted from chick liver or oviduct by preparing a 2.5% homoge- nate in SET buffer containing proteinase K at 25 yglml. The sample was incubated for 2 h at 41”C, adjusted to 0.1 M NaCl, and extracted with an equal volume of phenol/chloroform (1:l). The resulting nucleic acid was precipitated with 2 volumes of ethanol, washed with 67% ethanol, 0.04 M NaCl, and resuspended in 0.1% SDS. High molecular weight RNA was prepared from total nucleic acid by precipitation with 2 volumes of 3 M NaOAc, 5 rnM EDTA (pH 7.0). DNA was isolated from chicken erythrocytes in a manner similar to that described for total nucleic acid. RNA was eliminated by base hydrolysis.

Synthesis of 13HlcDNA -Reaction mixtures contained the follow- ing components in a total volume of 20 ~1: 50 rnM Tris-Cl (pH 8.31, 5

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3496 Regulation of the Transferrin Gene

rn~ MgCl,, 10 rn~ dithiothreitol, 0.33 rn~ dATP, dGTP, and TTP, 0.125 rn~ 13HldCTP (specific radioactivity, 20 Ci/mmol), 5 pg/ml of oligo(dT), 100 pg/ml of RNase inhibitor (Searle), 1 pg of conalbumin or ovalbumin mRNA (purified as described above), and 0.8 units of avian myeloblastosis virus reverse transcriptase (a generous gift from Dr. J. W. Beard, Viral Cancer Program, National Cancer Institute). Incubations were carried out for 30 min at 45°C. The reaction was terminated by adding NaOH to a final concentration of 0.3 N followed by heating for 5 min at 65°C. Samples were neutralized with HCl, adjusted to single strength SET buffer and chromato- graphed on a Sephadex G-75 column equilibrated with SET buffer. The peak fractions of radioactivity were pooled and ethanol-precipi- tated in the presence of carrier DNA. The DNA was dissolved in 0.1 N NaOH and 0.9 M NaCl, layered on 12-ml, 5 to 20% sucrose gradients in 0.1 N NaOH, 0.9 M NaCl, and centrifuged at 280,000 x g for 24 h at 4°C. Gradients were fractionated, radioactivity deter- mined, and the high molecular weight material precipitated with ethanol. Based on markers of known molecular weight, the number average size of conalbumin and ovalbumin cDNAs were 750 and 1260 nucleotides, respectively.

maximum of 9% of labeled polysomes could be precipitated using 30 to 50 pg of rabbit anti-conalbumin/Az,,, unit of polysomes. This is consistent with the finding that conalbumin constitutes about 10% of total protein synthesis in a fully stimulated oviduct (13). Nonspecific adsorption of polysomes was measured using rabbit anti-bovine serum albumin as the

Hybridization of cDNA to RNA-Hybridization reactions and quantitation of hybrids using S, nuclease were carried out essen- tially as described (31). S, enzyme was purified from Aspergillus oryzae according to the method of Vogt (32).

Measurement of Relative Rates of Protein Synthesis in Oviduct and Liner- The rate of protein synthesis in the liver was determined by administering 100 PCi of [3H]leucine (intraperitoneally) 15 min before the chicks were killed. At the time of death, livers were excised and rapidly frozen in liquid nitrogen. Oviduct explants were labeled with [‘*Clleucine at 1.5 &i/ml as described (14). Incorpora- tion of leucine into total and specific proteins was determined as before (13). Immunoprecipitations were generally carried out at several antigen concentrations to ensure antibody excess.

RESULTS

Purification of Conalbumin mRNA- Immunoprecipitation of polysomes was carried out using the indirect immunoprecip- itation procedure of Shapiro et al. (26). Magnesium-precipi- tated polysomes were first reacted with affinity-purified rabbit anti-conalbumin followed by an excess of goat anti-rabbit IgG. In order to optimize the immunoprecipitation of conalbumin- synthesizing polysomes and minimize nonspecific adsorption, the reaction conditions were defined using oviduct polysomes containing labeled nascent chains. As shown in Fig. 1, a

Antibody ( pg 1

FIG. 1. Effect of antibody concentration on the indirect immuno- precipitation of [3”S]methionine-labeled oviduct polysomes. Oviduct polysomes (1 A,,, unit; 11,000 cpm of 13‘Slmethionine-labeled nascent chains) were incubated for 1 h at 4°C in 0.1 ml with the indicated amounts of purified rabbit anti-conalbumin (0-O) or rabbit anti- bovine serum albumin (O-O). Polysomes were then incubated for 1 h at 4°C with a 50-fold weight excess of goat anti-rabbit and sedimented once through a discontinuous sucrose gradient as de- scribed under “Experimental Procedures.” Immunoprecipitates were dissolved in 1% SDS and counted in Aquasol (New England Nu- clear).

first antibody; less than 1.0% of the radioactive nascent chains were precipitated (Fig. 1). This background could be reduced further by washing the immunoprecipitate (data not shown).

During the course of these investigations, it became appar- ent that the important factor in obtaining a quantitative immunoprecipitate was related to there being an adequate amount of the first antibody to form a precipitate of sufficient size with the goat anti-rabbit IgG. Thus, the amount of specific rabbit antibody could be reduced if a corresponding amount of nonspecific IgG was added. Hence, it is not neces- sary to affinity-purify the first antibody, and doing so does not necessarily improve the specificity of the immunoprecipita- tion. It is vital, however, to ensure that all antibodies are RNase-free.

Based on the above findings, conalbumin polysomes were immunoprecipitated with 30 pg of anti-conalbumin/A,,,,. The immunoprecipitate was resuspended in SDS and digested with proteinase K. The RNA was extracted with phenol/chloroform and chromatographed on oligo(dT) cellulose. Poly(A)-contain- ing RNA was eluted and sedimented on a 5 to 20% sucrose gradient in 1.0% SDS. Purification was assessed by monitor- ing RNA fractions at each step for conalbumin mRNA activity in a messenger-dependent translation system derived from rabbit reticulocytes. The translation data are presented in Fig. 2, and summarized in Table I. Since increasing amounts of RNA may result in inhibition of translation, each fraction was assayed over a lo-fold range of RNA concentration and the amount of conalbumin mRNA activity, expressed as radioactive conalbumin synthesized per pg of RNA, deter- mined from the initial slopes. As shown in Table I, the final purification for the peak fraction of conalbumin mRNA on the sucrose gradient was approximately 500-fold. This value is based on the conalbumin mRNA activity in magnesium-pre- cipitated polysomes (which include free ribosomes and sub-

RNA (vg)

FIG. 2. Comparison of the amount of conalbumin synthesis di- rected by various oviduct RNA fractions. RNA extracted from magnesium-precipitated polysomes (O-O), immunoprecipitated polysomes (A-A), poly(A)-containing RNA (O-O), and RNA from sucrose gradient fractions containing the peak of conalbumin mRNA activity (m-m) was added to messenger-dependent retic- ulocyte cell-free translation assays at various concentrations. Radio- activity incorporated into conalbumin was determined immunologi- cally as described under “Experimental Procedures.”

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Regulation of the Transferrin Gene 3497

TABLE I

Purification of conalbumin messenger RNA Oviduct polysomes were subjected to immunoprecipitation and

the immunoprecipitated RNA was then purified by oligo(dT) chro- matography and sucrose gradient centrifugation as described under “Experimental Procedures.” The total activity is the product of the amount of RNA (micrograms) and the specific activity of the RNA. Fold purification and yield were calculated from the specific activity and total activity, respectively. Reticulocyte lysate assays were carried out at five RNA concentrations over a lo-fold range.

Fraction RNA Specific Total ac- Purifica- activity tivity tion Yield

w cpmilrg cpm x lo-" -fold %

Polysomal RNA 38.2 150 5,730 1 100 (Mg”+-precipi- tated)

Immunoprecipi- 1.46 833 1,220 5.6 21 tated polysomes

Poly(A1 RNA 0.052 15,400 801 103 14 Sucrose gradient 0.0044 74,100 326 494 6

(peak fraction only)

units) as the starting material. The specific activity of conal- bumin mRNA in total nucleic acid and the magnesium-precip- itated RNA was virtually identical. A maximum theoretical purification of 700-fold can be calculated based on the assump- tion that conalbumin mRNA represents 0.14% of the total RNA present in magnesium-precipitated polysomes.”

Purification obtained by immunoprecipitation was approxi- mately 6-fold. Assuming that conalbumin polysomes represent 10% of total polysomes (131, the theoretical maximum at this step is lo-fold. Oligo(dT)-cellulose chromatography yielded another 20-fold purification followed by a &fold enrichment on the sucrose gradient. The sucrose gradient profile of the oligo(dT)-purified RNA revealed an absorbance peak co-mi- grating with conalbumin mRNA activity slightly ahead of the 18 S rRNA (Fig. 3). The poly(A) RNA contained some residual 18 S and 28 S rRNA as expected after only one passage over oligo(dT)-cellulose.

The yield of conalbumin mRNA activity after immunopre- cipitation was less than anticipated and suggested that some degradation of the mRNA may have occurred. This was confirmed by examination of the sucrose gradient profile of purified conalbumin mRNA by translation and by hybridiza- tion with cDNA (Fig. 3B). Translation of the gradient frac- tions gave a sharp peak of activity centered on Fraction 7. Although there was a peak of conalbumin mRNA sequences detected by hybridization in Fractions 7 and 8, a significant proportion of the total sequences was distributed through the lighter side of the gradient, suggesting that the degraded fragments are not translated. Hybridization of cDNA to gra- dient fractions of total Mg’+-precipitated RNA did not reveal these lower molecular weight components (Fig. 3A), suggest- ing that they arise during the purification procedure.

Gradient fractions were also examined for contamination by ovalbumin mRNA sequences by translation and by hybridiza- tion using cDNA prepared from purified ovalbumin mRNA. Fig. 3 shows that the peak of ovalbumin mRNA occurred in

3 Ovalbumin mRNA constitutes 1% of polysomal RNA (33). Since rates of synthesis indicate that conalbumin is synthesized at one- fifth the rate of ovalbumin, we presume that conalbumin mRNA is 0.2% of polysomal RNA. This value is corrected for the fact that magnesium precipitates not only polysomes but also free monosomes which account for approximately 30% of total ribosomes in hen oviduct (25).

Fraction 12. Comparison with sucrose gradient profiles of total polysomal RNA (Fig. 3A) revealed that the ratio of conalbu- min to ovalbumin mRNA sequences had been increased 6-fold. Furthermore, in the peak conalbumin mRNA fraction, the contamination by ovalbumin mRNA was less than 1%.

We have examined the purity of the peak conalbumin mRNA fraction by acrylamide gel electrophoresis of its in vitro translation products. An autoradiogram of the total translation products obtained revealed a major band in the position of authentic conalbumin (Fig. 4). Several minor bands migrating faster than conalbumin were also observed; how- ever, greater than 95% of total radioactivity was present in the major band and the other bands may represent premature termination products from conalbumin mRNA. In contrast, when the products obtained from translation of total oviduct polysomal RNA were analyzed, the major band was associated with ovalbumin, and conalbumin was present as a minor band. Analytical acrylamide gels of this same RNA revealed a single band by optical density with an apparent molecular weight of 1,033,OOO (see below). We conclude that the conal- bumin mRNA is >95% pure in terms of messenger activity and hence is an appropriate template for preparing conalbu- min cDNA. The preparation does, however, contain some other RNA sequences (most likely rRNA degradation prod- ucts), and we estimate that intact conalbumin mRNA consti- tutes about 50% of the total RNA sequences present.

Comparison of Conalbumin and Transferrin mRNA Se- quences- We used the mRNA which sediments at approxi- mately 20 S and has peak conalbumin translational activity as a primer for cDNA synthesis. The cDNA synthesized in these reactions is about 1000 nucleotides and hence represents only the 3’terminal 30% of the conalbumin mRNA molecule. Various attempts to make a full length conalbumin cDNA, involving several different lots of reverse transcriptase, were unsuccessful despite the fact that it was possible to make full length ovalbumin and globin cDNAs.

We have used this conalbumin cDNA to compare conalbu- min and transferrin mRNA sequences. Fig. 5 shows the hybridization of conalbumin cDNA to total hen liver and oviduct RNA. The extent of hybridization was identical in both cases with 85 to 90% of the probe protected. In addition, the curves were superimposable, suggesting that the cDNA was hybridizing to the same species in both tissues. This is supported by studies of the thermal denaturation of the hybrids (Fig. 6), in which duplexes of conalbumin cDNA and oviduct or liver RNA showed identical melting curves at both low (48 mM NaCl) and high (300 mM NaCl) salt. These findings indicate complete homology in the 3’ ends of the two mRNAs.

In order to compare the size of conalbumin and transferrin mRNAs, hen liver and oviduct RNA were subjected to electro- phoresis on 2.5% acrylamide gels. The mRNA sequences were localized by slicing the gels, eluting the RNA, and hybridizing it with conalbumin cDNA; Fig. 7 shows that the size of conalbumin and transferrin mRNAs are the same and that both have an apparent molecular weight of 1,033,000, corre- sponding to approximately 3200 nucleotides. This value is consistent with the sedimentation value on sucrose gradients. These data suggest that within the limits of our analysis, these mRNAs are identical and that conalbumin cDNA can be used to quantitate the number of transferrin genes and to measure transferrin mRNA sequences in the liver.

Determination of the Number of Transferrin Genes-We have examined the hybridization of conalbumin cDNA to

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3498 Regulation of the Transferrin Gene

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Fraction FIG. 3 (left). Sedimentation of total polysomal RNA and poly(A)-

containing RNA in sucrose gradients. A, approximately 150 pg of Mg2+-precipitated polysomal RNA in 200 ~1 of 25 mM Tris-Cl (pH 7.5), 25 mM NaCl, 500 pg/ml of heparin, and 1% SDS was heated for 1 min at 65”C, and sedimented on a 12-ml, 5 to 20% sucrose gradient as described under “Experimental Procedures;” B, poly(A)-contain- ing RNA (42 pg) from immunoprecipitated oviduct polysomes was dissolved in 200 ~1 of 10 mM Tris, pH 7.6, 5 mM EDTA, and 1% SDS, heated for 1 min at 65”C, and sedimented as above. Fractions (0.5 ml) were collected and assayed directly for conalbumin (A-A) and ovalbumin (0-O) mRNA sequences using appropriate cDNAs. A portion of each fraction was ethanol-precipitated and assayed for conalbumin mRNA activity (C-M) in a messenger- dependent reticulocyte lysate system. The absorbance of each frac-

,, I

0 200 400 600 1400

RNA hg)

FIG. 5. Comparison of the hybridization of liver and oviduct RNA to cDNA prepared from purified conalbumin mRNA. RNA was isolated from hen liver (0-O) and oviduct (0-O) and the indicated amounts hybridized to 500 cpm of conalbumin cDNA for 3 days at 68°C. The extent of hybridization was measured by resistance to S, nuclease as described under “Experimental Procedures.” The . . upper abscwa mdlcates the amount of oviduct RNA and the lower abscissa indicates the amount of liver RNA.

,: -con ‘I. r;.

top-

tion (0-O) was measured at 260 nm. FIG. 4 (right). Sodium dodecyl sulfate-polyacrylamide gel elec-

trophoresis of the reaction products from mRNA-dependent rabbit reticulocyte lysates primed with exogenous RNA. Translations were carried out in 20 ~1 using [3H]proline as the label. Slot 1 shows the result with no exogenous RNA added (8,500 cpm); Slot 2 shows the reaction products obtained from 1.7 yg of total hen polysomal RNA (120,000 cpm) and Slot 3 contains the products derived from 57 ng of the peak of purified conalbumin mRNA activity (100,000 cpm). The gel, containing 12.5% acrylamide with a 5% stacking gel, was prepared according to Laemmli (34); it was developed by fluorogra- phy using the method of Bonner and Lasky (35) with a l-day exposure of preflashed film.

Temperature PC)

FIG. 6. A comparison of the melting curves for hybrids of conal- bumin cDNA and liver or oviduct RNA. Excess liver (0, A) or oviduct (0, A) RNA was hybridized to conalbumin cDNA for 22 h at 68°C in 40 ~1. Samples were then diluted to 500 yl with 20 mM Tris (pH 7.5) containing 0.1% SDS and an amount of sodium chloride such that the tinal salt concentration was either 48 mM (circles) or 300 mM (triangles). Samples were then divided into 50-111 aliquots, overlayed with paraffin oil, and equilibrated for 10 min at the indicated temperatures. The extent of denaturation was then mea- sured by adding 1 ml of buffer containing S, nuclease as described under “Experimental Procedures.”

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Regulation of the Transferrin Gene 3499

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FIG. 7. A comparison of the size of conalbumin and transferrin mRNA. Liver RNA (24 pg) and hen oviduct polysomal RNA (13 pg) were heated for 5 min at 65°C and electrophoresed on 2.5% acrylam- ide gels for 3 h at 110 V according to the method of Mirault and Scherrer (36). Gels were sliced and the RNA eluted with 20 ~1 of 10 mM Tris (pH 7.6), 5 mM EDTA, and 1% SDS by repeated freezing and thawing followed by centrifugation. Conalbumin cDNA was hybridized to 2~1 aliquots of the oviduct RNA fractions and lo-p1 aliquots of the liver RNA fractions for 20 h at 68°C. The extent of hybridization was measured as described under “Experimental Pro- cedures.” Gels were scanned at 260 nm in a Gilford spectrophotome- ter equipped with a linear transport device; only the profile of the liver RNA is shown. The position of the 18 S and 28 S ribosomal RNAs were used to align the two gels.

chick DNA in an attempt to determine whether the conalbu- min gene is reiterated or unique. Both biochemical (37) and genetic evidence (38) have been reported, suggesting that the major egg white gene, ovalbumin, is a single copy gene and we have utilized ovalbumin cDNA to provide a direct compar- ison in the experiments shown in Table II. Chick erythrocyte DNA was hybridized to an excess of conalbumin and ovalbu- min cDNA for times sufficient to achieve saturation and the percentage of hybridization observed as a function of DNA in the hybridization reaction is shown in Table II. From these data and from standard curves of hybridization between polysomal RNA and cDNA, we have calculated the gene copy number in two ways. Method A relies on assumptions of the specific activity of cDNA and its size (given in Table II) to give a direct determination of between 1.6 and 2.9 genes/haploid genome for conalbumin, and 1.4 and 2.2 genes/haploid genome for ovalbumin. Method B relies on a comparison of cDNA- DNA hybridization to cDNA-mRNA hybridization and is based on the observed molecular weights for ovalbumin and conalbumin mRNA and their relative abundance in polysomal RNA. The number of genes per haploid genome calculated by Method B for conalbumin is 1.1 to 1.6, and for ovalbumin is 1.6 to 2.2. By both methods, the number of genes for conalbu- min is very close to that determined for ovalbumin. Uncertain- ties in the assumed values given in Table II do not allow an unequivocal conclusion that both genes are single copy, but the resuts strongly suggest that there are the same number of copies of each gene and that there are no more than two

copies/haploid genome for either ovalbumin or conalbumin. Effects of Steroid Hormones on Conalbumin and Transfer-

rin Synthesis- We have examined the effects of two steroid hormones, estrogen and progesterone, on conalbumin and transferrin synthesis. Fig. 8A shows the induction of conalbu- min synthesis in the oviduct during the first 96 h of secondary stimulation with an optimal dose of estrogen. For each time point, the magnum portion of three oviducts were divided into two parts. One sample was incubated for 30 min in vitro with [YJleucine and the fraction of total labeled protein repre- sented by conalbumin determined by immunoprecipitation. Total RNA was isolated from the remaining tissue and as- sayed for conalbumin mRNA sequences by hybridization with cDNA. Fig. 8A shows an &fold increase in conalbumin synthe- sis following the administration of estrogen, with conalbumin rising from 1.5 to 1.7% of total protein synthesis in withdrawn chicks to greater than 12% by 96 h. The stimulation of synthesis occurred in two phases, with an initial, rapid phase that plateaued at 6 to 7% followed by a slower rate of increase. The initial component we attribute to the induction of conal- bumin in pre-existing tubular gland cells; the subsequent slower rise is probably the result of new tubular gland cells entering the population through cell division (41). Fig. SA also shows that the early increase in conalbumin synthesis was paralleled by an accumulation of its mRNA, and that both occurred rapidly with little or no lag after the administration of estrogen (Fig. 8A, inset). For the data shown in the inset of Fig. 8A, we have corrected the number of conalbumin mRNA

TABLE II Determination ofgene copy numbers for ovalbumin and conalbumin

Ovalbumin and conalbumin cDNAs were hybridized to the indi- cated amounts of chick reticulocyte DNA isolated as described under “Experimental Procedures.” Hybridizations were carried out in 20 ~1 for 3 days at 68”C, and the extent of hybridization measured by S, digestion.

Method A” Method B”

cDNA DNA Hybridi- zation cDNA hl- hcaep;ETi Eqt2- Gene?’

bridize haplold mxxmle mRNA eenome

Pg !4 % molecules x 10~”

Ovalbumin 30 1.48 5.9 2.6

30 2.96 9.4 4.3

30 7.4 19.1 9.9 30 14.8 29 17.1

Conalbumin 30 1.48 5.5 3.5

30 2.96 10 6.8

30 7.4 16.8 12.2

30 14.8 24 19.2

P&Y

2.2 2.8 2.2

1.8 4.8 1.9

1.7 10.5 1.7 1.4 20 1.6

2.9 2.9 1.4

2.9 5.6 1.4 2.1 11.5 1.1 1.6 20.5 1.0

’ These values are calculated based on the following set of assumptions: 1) average cDNA length determined by alkaline-su- crose gradient sedimentation: ovalbumin cDNA = 1260 nucleotides, conalbumin cDNA = 750 nucleotides; 2) specific activity of cDNAs = 17 cpmipg based on using 13HldCTP at 20 Ci/mmol and assuming an equal distribution of bases; 3) 13HlcDNA and the DNA strand identical with cDNA compete equally in the hybridization; and 4) the haploid chick genome contains 1.25 pg of DNA (39).

b This set of values relies on assumptions 3 and 4 above and the following: 1) length of mRNAs: ovalbumin mRNA = 1930 nucleotides (40), conalbumin mRNA = 3200 nucleotides; and 2) standard curves relating percentage of hybridization to picograms of mRNA using polysomal RNA and assuming mRN&> = 50% of mRNA and mRN&,,, = 10% of mRNA. This value for ovalbumin has been checked by hybridization to pure mRN& (33).

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3500 Regulation of the Transferrin Gene

Hours of secondary stimulotion

i 0’ 0 20 40 60 80 100

Hours of secondary stimulation

molecules per cell for the fraction of tubular gland cells assuming that only tubular gland cells synthesize conalbumin in the oviduct (42) and that 15% of the cells in the withdrawn oviduct are tubular gland cells (41). Thus, the corrected number of conalbumin mRNA molecules per tubular gland cell in a withdrawn oviduct is about 430. After 6 h of estrogen stimulation, the number had increased to 3600 molecules.

Fig. 3I? shows the simultaneous effects of estrogen on transferrin synthesis in the liver. To measure transfer-r-in synthesis, chicks were injected with [3H]leucine 15 min before they were killed, and at the times indicated, the livers were removed and divided into two portions. The rate of transfer-i-in synthesis and the number of transferrin mRNA molecules per cell were determined as described for the oviduct samples. We have measured transfer-i-in synthesis in uiuo since labeling of liver minces in vitro gave very low levels of isotope incorpora- tion and the fractional rate of transferrin synthesis was lo- fold lower than in viuo. Fig. 6B shows only a small increase in transferrin synthesis in response to estrogen; it rose from an average of 1.7% of total protein synthesis to a plateau of about 2.5%. The increase in protein synthesis occurred rapidly with the maximum value attained by 5 h. Although we have observed variability in the actual values for withdrawn and stimulated rates of synthesis, we have consistently found a 1.5- to 2-fold stimulation of transferrin synthesis upon admin- istration of estrogen. Fig. &B also shows a quantitatively similar increase in the number of transferrin mRNA mole- cules per cell but the accumulation of mRNA molecules lagged significantly behind the stimulation of transferrin synthesis. These results suggest that estrogen may act initially by enhancing the translational efficiency of pre-existing transfer- rin mRNA molecules and, secondarily, by increasing the cellular concentration of transfer-i-in mRNA. The latter rose from 700 molecules to approximately 1600 molecules/cell after 96 h of estrogen.

As a comparison, we have examined the effect of estrogen on the synthesis of serum albumin since this protein is synthesized by the same cells that produce transferrin (6, 43, 44). The relative synthesis of serum albumin (as a percentage of total) declined from approximately 11% to less than 7% after 96 h of hormone as shown in Fig. 8B. This is consistent with previous findings that the amount of circulating serum albu- min falls in chicks given estrogen (45). It is important to point

I

IO 20

FIG. 8. Estrogen induction of conal- bumin synthesis and mRNA in the oviduct (A) and transferrin synthesis and mRNA in the liver (B). 17P-Estra- diol benzoate (1 mg) was administered daily to withdrawn chicks, and at the times indicated, the chicks were killed. Oviducts (A) and livers (B) were la- beled with 114Clleucine and 13Hlleucine, respectively, as described under “Ex- perimental Procedures.” Part of each tissue was used to determine the per- centage of total protein synthesis that was specifically immunoprecipitable and part was used for the isolation of RNA. Samples of RNA were hybridized to conalbumin cDNA as described un- der “Experimental Procedures.” Note that in the inset of Fig. 8A, the number of conalbumin mRNA molecules per cell has been corrected for the fraction of tubular gland cells.

60 :4d

40

20

0 l!!iif IO 20

0 IO 20

Fraction

FIG. 9. Electrophoresis of pulse-labeled proteins and immunopre- cipitates isolated from livers of hormone-stimulated (B, D) and withdrawn (A, C) chicks. Withdrawn chicks and chicks given estrogen (1 mg) for 4 days (E4d) were injected with 100 yCi of L3Hlleucine and killed 15 min later. Liver homogenates were pre- pared as described under “Experimental Procedures.” Total protein was precipitated and washed with 90% acetone and aliquots of total protein (30,000 cpm) from withdrawn or estrogen-treated chicks were immunoprecipitated with anti-conalbumin or anti-chick serum albumin. Samples were resuspended in 0.1 M Tris (pH 7.51, 2% SDS, 25% glycerol, and 0.1 M dithiothreitol and subjected to electrophore- sis on 5% acrylamide gels with 0.5 M urea for 7 h at 2 mA/gel. Gels were sliced, dissolved in Soluene-350 (Packard) and counted in toluene-based scintillant. A and B, total protein from hormone- withdrawn (A) and estrogen-treated chicks @I). C and D, immuno- precipitates prepared with anti-conalbumin (tf, (0) and anti-chick serum albumin (alb) (0) from hormone-withdrawn (C) and estro- gen-treated chicks CD).

out that the liver is an estrogen-responsive tissue and that a number of prominent changes occur in response to the hor- mone. These include changes in cell morphology, increased cell division, and the induction of yolk protein synthesis (45, 46). We have examined the changing pattern of protein synthesis in the liver in response to estrogen. Pulse-labeled (15 min) total protein was prepared from livers of hormone-

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3501 Regulation of the Transferrin Gene

TABLE III Effects of estrogen and progesterone on conalbumin and transferrin synthesis and mRNA

17@Estradiol benzoate (1 mg) and progesterone (1 mg) were given to withdrawn chicks as indicated and fractional rates of synthesis and mRNA concentrations for conalbumin and transferrin were determined as described in Fig. 8.

Tissue Treatment Conalbumin

Transferrin synthesis

Conalbumin mRNA Transferrin

% total mgig wet weight of ouiduct

Oviduct None Progesterone (96 h) Estrogen (96 h) Progesterone + estrogen (96 h)

Liver None Progesterone (96 h) Estrogen (96 h) Progesterone + estrogen (96 h)

1.7

9.5 12.2

12.0

1.7

2.1 2.5

2.8

100 (660)” 0.56

1500 4900 (5760)” 15.7

6700 (7900)”

mgiml of serum

700 1.1

800 1.2 1600 2.4

1500 2.5

’ Corrected for the fraction of tubular gland cells.

withdrawn chicks, as well as chicks stimulated with estrogen for 96 h. Fig. 9, A and B show the molecular weight profiles of protein synthesized before and after estrogen. The serum albumin peak clearly drops after estrogen, confirming the results obtained by immunoprecipitation. The prominent peak that appears at the top of the gel after the administration of hormone is presumed to be vitellogenin, the precursor of yolk proteins (-250,000 daltons). Fig. 9, C andD show the products of immunoprecipitation and demonstrate the specificity of the reactions. Aside from a low, dispersed background, the major- ity of the radioactivity was present as a single peak in each case.

We have also compared the effects of progesterone alone, and in combination with estrogen, on the synthesis of conal- bumin and transferrin. The results are shown in Table III. Progesterone was chosen because, although it is an active steroid in the oviduct when given in secondary stimulation (13), it is, nevertheless, nonmitogenic in that tissue and it does not produce the gross changes in the liver characteristic of the estrogen response. As shown in Table III, an optimal dose of progesterone caused a marked stimulation of conalbu- min synthesis, as well as an increase in the number of conalbumin mRNA molecules per cell. In terms of both parameters, the response evoked by progesterone was not as great as that of estrogen and, in this experiment, the response to a combination of optimal doses of estrogen and progesterone was similar to that caused by estrogen alone. In other experi- ments, we have found the combination of hormones is more effective than either steroid alone. In contrast, 96 h of proges- terone treatment caused only a slight increase in transferrin synthesis and there was no change in either the number of transferrin mRNA molecules per cell or the serum transferrin concentration. In addition, progesterone does not affect the fractional rate of serum albumin synthesis (data not shown). The response obtained in the liver with a combination of estrogen and progesterone was similar to that evoked by estrogen alone.

DISCUSSION

The avian transferrin gene is a particularly interesting model system both for exploring steroid regulation of gene expression within a single tissue, as well as for studying cell- specific programming of gene function. We report here that as a steroid-inducible model, conalbumin mRNA synthesis in the oviduct responds dramatically to estrogen with little or no lag following the administration of hormone in secondary stimu-

lation. The onset of the induction of conalbumin mRNA sequences corresponds to the movement of estrogen receptors into the nucleus and the response is directly proportional to their nuclear concentration (47). This contrasts with the accumulation of ovalbumin mRNA which shows a lag of 3 to 4 h, and requires a higher concentration of nuclear estrogen receptors. It is possible, therefore, that the conalbumin re- sponse is a primary effect of hormone, whereas the stimulation of ovalbumin expression is a secondary response.

A crucial question is whether the accumulation of conalbu- min mRNA sequences under the influence of estrogen is caused by an increased rate of synthesis or rather by stabili- zation of the mRNA against degradation. We cannot yet distinguish between these possibilities. However, the purifi- cation of conalbumin mRNA and the preparation of cDNA should now permit the development of methods for measuring the rates of transcription of the gene in the presence and absence of hormone. Another feature of the conalbumin re- sponse which will aid in its study is that it can be reproduced with almost complete fidelity in uitro.4

The transferrin gene may provide a model for investigating tissue-specific programming of gene function during differen- tiation and development. Previous findings demonstrated the identity of conalbumin and transferrin (lo), and we have shown that both proteins are synthesized as precursors and that the NH,-terminal leader sequences are identical.: Here we show that, in addition, the mRNAs for conalbumin and transferrin appear to have identical sequences at their 3’ ends and that both have an apparent molecular weight on poly- acrylamide gels of 1 x 10” (-3200 nucleotides). This identity of proteins and mRNAs suggest that conalbumin and transferrin are coded for by the same gene. We made direct measurements of the gene copy number for transferrin (conalbumin) and ovalbumin and conclude that there are no more than two copies/haploid genome for either gene. Although this data cannot rule out a gene duplication, the lack of RNA or protein sequence divergence and the genetic evidence for both oval- bumin (38) and transferrin (10) argue that these genes are both single copy.

If the one-gene hypothesis is correct, then this raises inter- esting questions concerning the regulation of the gene in the two tissues. We have initiated an investigation of these questions by comparing the effects of estrogen and progester-

4 G. S. McKnight (1978) Cell, in press. 5 S. N. Thibodeau, D. C. Lee, and R. D. Palmiter (1978) J. Biol.

C/tern., in press.

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3502 Regulation of the Transferrin Gene

one on conalbumin and transferrin synthesis in the oviduct and liver, respectively. We conclude that estrogen stimulates both conalbumin and transferrin synthesis but that the re- sponse differs both qualitatively and quantitatively in the two tissues. While conalbumin mRNA levels increase approxi- mately lo-fold after 96 h of estrogen, transferrin mRNA increases only 2-fold. Furthermore, in contrast to immediate induction of conalbumin mRNA, there is a lag of at least 6 h prior to the increase in transferrin mRNA. Progesterone, on the other hand, causes an increase in conalbumin mRNA sequences similar to that of estrogen, but has no effect on transferrin mRNA levels. The differential response evoked by estrogen in the liver is particularly significant in view of the fact that it is an estrogen-responsive tissue, contains recep- tors, and is induced by the hormone to synthesize vitellogenin, the precursor for egg yolk proteins, as well as a variety of lipids. A number of dramatic biochemical and morphological changes occur in the liver in response to estrogen, raising the possibility that the stimulation of transferrin mRNA synthesis is, in fact, an indirect response resulting from these other changes. Morgan (48) found that liver iron is mobilized under the influence of estrogen and shifted from transferrin to the egg yolk protein, phosvitin. In addition, we have recently observed that iron deficiency increases the rate of transferrin synthesis in chick liver.” These data suggest the possibility that the transferrin gene is primarily responding to iron levels in the liver and that the observed response to estrogen represents a long term adaptation to the increased demand for iron resulting from vitellogenin biosynthesis.

internal sequences may be removed from primary mRNA transcripts (X-531, imply a greater repertoire of regulatory mechanisms than previously imagined. Further study of the transferrin gene may yield clues as to the mechanisms by which tissue-specific regulation of genes is established during differentiation and development.

Acknowledgments -We are grateful to R. Moen for the purification of conalbumin, to D. Hemmaplardh for the mea- surement of serum transferrin, and R. Eisenman for gel analysis of translation products. We thank R. Moen and S. Sandmeyer for helpful discussions and the Viral Cancer F’ro- gram, National Cancer Institute (Dr. Beard) for gifts of avian myeloblastosis virus reverse transcriptase.

1. Storti, R. V., and Rich, A. (1976)Proc. N&l. Acad. Sci. U. S. A. 73, 2346-2350

2. Chi, J. C., Fellini, S. A., and Holtzer, H. (19’75) Proc. N&Z. Acad. Sci. U. S. A. 72, 4999-5003

3. Greene, F. C., and Feeney, R. E. (1968) Biochemistry 7, 1366- 1371

4. Hemmaplardh, D., and Morgan, E. H. (1974) Biochim. Biophys. Acta 373,84-99

5. Moraan, E. H. (1969) J. Biol. Chem. 244,4193-4199 6. Jeejeebhoy, K. N., Ho, J., Greenberg, G. R., Phillips, M. J.,

Bruce-Robertson, A., and Sodtke, U. (1975) Biochem. J. 146, 141-155

7. Lane, R. S. (1968) Br. J. Haenatol. 15, 355-364 8. Thorbecke. G. J.. Liem. H. H.. Knight, S.. Cox, K., and Muller-

A related question concerns the basal level of conalbumin and transferrin expression. In a hormone-withdrawn chick, there are approximately 500 to 700 molecules of conalbumin mRNA/tubular gland cell and conalbumin represents 1 to 2% of total protein synthesis. In the liver, we find about 700 molecules of transferrin mRNA/cell in the absence of exoge- nous steroids and the fractional rate of synthesis is similar to that of conalbumin in the oviduct. Whether this basal rate of synthesis is maintained by endogenous hormone levels or represents a constitutive level of synthesis that is steroid- independent, is not known.

9. 10. 11. 12.

13. 14.

Eberhard, U. (1973) J. Clin. In&. 52, 725-731 Graham, I., and Williams, J. (1975) Biochem. J. 145, 263-279 Williams. J. (1962) Biochem. J. 83. 355-364 Gafni, A.: and Steinberg, I. Z. (1974) Biochemistry 13, BOO-803 Palacios, R., Palmiter, R. D., and Schimke, R. T. (1972)J. Biol.

Chem. 247, 2316-2321 Palmiter. R. D. (1972)5. Biol. Chem. 247, 6450-6461 Palmiter; R. D., Moore, P. B., Mulvihill, E. R., and Emtage, S.

(1976) Cell 8, 557-572 15. 16. 17.

In summary, we have presented evidence that suggests that the transfer-i-in gene is responding differentially to the same hormones in two tissues, namely chick liver and oviduct. In comparison with the response in the oviduct, estrogen induc- tion of the transferrin gene in the liver is relatively small and, as discussed above, we suspect that the effect in this tissue may, in fact, be an indirect response to the hormone. This differential response could be explained by the presence of two identical genes, one of which is linked to iron regulation in the liver and the other coupled to steroid-responsive elements in the oviduct. According to this scheme, the weak stimulation of transferrin mRNA synthesis by estrogen could represent a low level induction from the hormone-responsive gene in the liver. An alternative explanation is that the same gene(s) is linked to either iron or steroid regulation elements in the liver and oviduct, respectively. This possibility raises fundamental questions about the architecture of the gene in the two tissues. The differential response may be the result of differences in chromatin proteins associated with the gene or it may be the result of differences in controlling DNA sequences in or around the gene. Recent findings suggesting that DNA se- quences may be rearranged in the genome (49, 50) and that

18.

19.

20.

21.

Home, C. H., and Ferguson, J. (197215. Endocrinol. 54, 47-53 Morgan, E. H. (1972)Aust. J. Ezp. Biol. Med. Sci. 50, 777-779 Cartei, G., Meani, A., Okalicsanyi, L., and Naccarato, R. (1970)

Folia Endocrinol. 23, 579-592 Jeejeebhoy, K. N., Bruce-Roberston, A., Ho, J., and Sodtke, U.

(1972) Biochem. J. 130, 533-538 Jeejeebhoy, K. N., Bruce-Robertson, A., Ho, J., and Sodtke, U.

(1972) CIBA Found. %‘mD. 9. 217-247 Tavill, A. S., East, A.” G:, Black, E. G., Nadkami, D., and

Hoffenbere. R. (1972) CZBA Found. Symp. 9, 155-179 Azari, P., and Baugh, R. F. (1967)Arch. giochem. Biophys. 118,

138-144 22. 23.

24. 25. 26.

27. 28.

29.

30.

31.

32. 33.

Mandeles, S. (1959) J. Chromatog. 3, 256-264 Janatova, J., Fuller, J. K., and Hunter, M. J. (1968) J. Biol.

Chem. 243,3612-3622 Cuatrecasas, P. (1970) J. Biol. Chem. 245, 3059-3065 Palmiter. R. D. (1974) Biochemistry 13, 3606-3615 Shapiro, ‘D. J., Taylor, J. M., McKnight, G. S., Palacios, R.,

Gonzalez, C., Kiely, M. L., and Schimke, R. T. (1974) J. Biol. Chem. 249, 3665-3671

Palmiter, R. D. (1973) J. Biol. Chem. 248, 2095-2106 Pelham, H. R., and Jackson, R. J. (1976) Eur. J. Biochem. 67,

247-256 Haines, M. E., Carey, N. H., and Palmiter, R. D. (1974) Eur. J.

Biochem. 43, 549-560 Hagen, F. S., and Young, E. T. (1974) Biochemistry 13, 3394-

3400 McKnight, G. S., and Schimke, R. T. (1974) Proc. N&Z. Acod.

Sci. U. S. A. 71,4327-4331 Vogt, V. M. (1973) Eur. J. Biochem. 33, 192-200 Shapiro, D. J., and Schimke, R. T. (1975) J. Biol. Chem. 250,

1759-1764 34. Laemmli, U. K. (1970) Nature (Land.) 227, 680-685 35. Banner, W. M.. and Laskv, R. A. (1974) Eur. J. Biochem. 46,83-

’ G. S. McKnight and D. C. Lee, unpublished observations. 88

REFERENCES

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

36.

37.

Mirault, M. E., and Scherrer, K. (1971) Eur. J. Biochem. 23, 372-386

Sullivan, D., Palacios, R., Stavnezer, J., Taylor, J. M., Faras, A. J., Kiely, M. L., Summers, N. M., Bishop, J. M., and Schimke. R. T. (1973) J. Biol. Chem. 248. 7530-7539

Lush, I. E.‘(1964) Genet. Res. 5, 257-268 38. 39. Palmiter. R. D.. Catlin, G. H.. and Cox. R. F. (1973) Cell Differ.

40.

41. 42.

43.

44.

2, 163-170 .,

Humphries, P., Cachet, M., Krust, A., Gerlinger, P., Kourilsky, P., and Chambon, P. (1977) Nucleic Acids Res. 4, 2389-2406

Palmiter, R. D. (1973) J. Biol. Chem. 248, 8260-8270 Palmiter, R. D., and Gutman, G. A. (1972) J. Biol. Chem. 247,

6459-6461 Kaighn, M. E., and Prince, A. M. (1971) Proc. N&l. Acad. Sci. 52.

U. S. A. 68, 2396-2400 53. Szpirer, J., and Szpirer, C. (1975) Cell 6, 53-60

Regulation of the Transferrin Gene 3503

45.

46. 47.

48.

49. 50.

51. Chow. L. T.. Gelinas. R. E.. Broker. T. R.. and Roberts. R. J. (1977) Celi12, l-8

Klessig, D. F. (1977) Cell 12, 9-21 Dunn, A. R., and Hassell, J. A. (1977) Cell 12, 23-26

Schjeide, D. A., and Lai, G. G. B. (1970) in Cell Differentiation (Schieide. D. A., and DeVellis. J.. edsl DD. 447-475, Van Nosirand’Reinhold Co., New York - -

Jost, J.-P., and Pehling, G. (1976)Eur. J. Biochem. 62, 299-306 Mulvihill, E. R., and Palmiter, R. D. (1977) J. Biol. Chem. 252,

2060-2068 Morgan, E. H. (1975) Q. J. Enp. Physiol. Cogn. Med. Sci. 60,

233-247 Peterson, P. A. (1976) Genetics 84,469-483 Hozumi, N., and Tonegawa, S. (1976) Proc. Natl. Acad. Sci. U.

S. A. 73, 3628-3632

by guest on June 18, 2018http://w

ww

.jbc.org/D

ownloaded from

D C Lee, G S McKnight and R D Palmitergene. A comparison of the response in chick liver and oviduct.

The action of estrogen and progesterone on the expression of the transferrin

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