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[CANCER RESEARCH 38, 2694-2699, September 1978] 0008-5472/78/0038-0000$02.00 Differential Regulation of a-Lactalbumin and Casein Messenger RNA's in Mammary Tissu& Nicholas J. Nardacci, Jimmy W. C. Lee, and William L. McGuire2 Department of Medicine, The University of Texas Health Science Center, San Antonia, Texas 78284 ABSTRACT Gateway Immunosera Co., Cahogia, ill. Rat caseinswere characterizedwith respectto molecu lare weight and carbohydrate and amino acid content. MessengerRNA was extractedfrom rat mammaryglands at 5, 10, 15, and 20 days of pregnancyand at 2, 8, and 15 days of lactationand translated in a wheat germ cell-free system. Nascent [3H]caseinand a-[3H]lactaIbuminwere precipitatedseparatelywith specificantibodiesand iden tified by their mobilities on sodium dodecyl sulfate:acryl amide gels and by competition with unlabeled casein and a-lactalbumin, respectively.Casein messengerrose continuouslyfrom Day 5 of pregnancyto Day 15 of lacta tion. In contrast, a-Iactalbumin messenger RNA levels remainedlow duringpregnancybut rose markedlyduring lactation. INTRODUCTION a-Lactalbumin is the regulatory subunit of lactose synthe AnimalsandTissues.Thoracicandabdominalmammary glands were removed from female Sprague-Dawley rats (150 to 200 g), weighed, and immediately frozen in liquid nitrogen until use. For development studies Day 0 of preg nancy was the morning of a sperm-positive test by micro scopic examination of vaginal smears. Casein Isolation, Characterization,and Antibody Pro duction. Milk was collected from Sprague-Dawley rats 8 to 15 days after partunition by the suction method of Young and Nelstrop (19). Casein was isolated by a modification of the method of McMeekinet a!. (8). After the second isoelec tnic precipitation at pH 4.6 and neutralization of the resus pended precipitate, 3 volumes of ethanol were added and the pH was adjusted with HCI until maximal precipitation occurred (pH 6.0 to 6.4). The precipitate was then collected on Whatman No. 1 filters, dried in a vacuum desiccator, and dissolved in 0.1 M imidizole-HCI buffer, at pH 7.0, containing 3.3 M urea, 1% 2-mercaptoethanol, and 1 mM tase (UDP-galactose:D-glucose 1-galactosyltransfenase; EC 2.4.1 .22), the enzyme that catalyzes the rate-limiting step in the biosynthesis of lactose (1). Casein is the major secretory protein synthesized in mammary gland. Both proteins are unique to mammary tissue and are under the control of prolactin (6, 16). Casein mANA has been isolated from bound polysomes of the lactating mammary glands of ewe and rabbit (2, 5) as well as from total RNA extracts from mammary glands of mouse and rat (13, 14). In the rat mammary gland a selective induction of casein mRNA has been reported during preg nancy and lactation (11—13). We recently reported that in experimental breast cancer disodium tissue both casein and a-iactalbumin mRNA'swere present, Beckman Model 118 automatic amino acid analyzer (Beck man instruments, Palo Alto, Calif.). Dialyzed, lyophilized protein samples were hydrolyzed in 6 N HCI at 110°for 18 but only casein mRNA was consistently stimulated by pro lactin (10). The purpose of this study is to examine the regulation of an a-Iactalbumin mRNA in normal rat mam mary tissue to determine whether this message can be detected during pregnancy and lactation and, furthermore, whether its levels are regulated similarly to those of casein mANA. MATERIALS AND METHODS Chemicals. L-[4,5-3H]Isoleucine, 65 Ci/mmol, was from New England Nuclear Corp. Boston, Mass. L-[2,3-3H]valifle, 17 Ci/mmol, L-[4,5-3H]ieucine, 38 Ci/mmol, and Na'25l, 100 Ci/mI, were obtained from Amersham/Seanle, Arlington Heights, III. Goat anti-rabbit ‘y-globulinwas produced by 1 Supported in part by NIH Grant CA 11378 should be and by the Robert W. Welch EDTA. Individual caseins were separated on DEAE-cellulose by the method of Thompson et a!. (15). Casein fractions were dissolved in 3% SDS,3subjected to 10% SDS:polyacrylamide gel electrophoresis, and stained with Coomassie blue as described previously (9). Carbohy drate measurements were carried out by directly staining polyacrylamide gels with periodic acid-Schiff reagent with the use of the quantitative method for glycoproteins of Matthieu and Quanles(7). Gels were scanned in a Gilford 240 spectrophotometer fitted with a 2410-5 linear transport mechanism and a 6050 recorder (Gilford Laboratories, Inc., Oberlin, Ohio). Preliminary amino acid analysis was performed on a hn, under vacuum prior to analysis. Antibodies were raised in New Zealand White rabbits by the method of Vaitukaitus et al. (17), with the use of 300 pg protein for primary injection followed by 1 mg as a booster injection 45 days later. Preliminary studies were carried out with casein antibodies provided by Dr. J. M. Rosen; purified rat a-Iactaibumin was a gift of Dr. W. F. Manzluff. RNA Extraction. Total RNA was extracted from whole mammary glands by a modification of the method of Rosen et a!. (13). Whole frozen tissues were pulverized in a Thenmovacpulverizer and homogenized in a VirTis homog enizer (5 mm) in 5 volumes of 88% phenol and 5 volumes of 0.5% SDS:25 nM disodium EDTA:75 mM NaCI, pH 8.0. The aqueous phase was reextracted with the use of 88% phenol:chloroform (1:1) with several strokes in a Dounce Foundation. 2 To whom requests for reprints addressed. Received December 12, 1977; accepted May 17, 1978. 2694 3 The abbreviation used is: SDS, sodium dodecyl sulfate. CANCERRESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. cr-LaCta!bUmin and Casein mRNA's homogenizer. Crystalline NaCI was added to the aqueous layer to 0.2 M followed by 1 volume of cold ethanol. DNA was removed by “spooling out― on glass rods followed by filtration through glass wool to remove remaining strands. The RNA was then precipitated with an additional volume of ethanol for 18 hr at 20°.The precipitate was collected by centnifugation at low speed, washed twice in absolute ethanol, dissolved in sterile demineralized water, and stored at —70° on used directly in the translation assay. Concentrations of RNA-containing fractions were deter mined by absorbance at 260 pm on the assumption that 1 mg/mi = 20 A2@:A280 ratio for these fractions was approxi mately 2.0. 0 In In Wheat Germ TranslocationSystem. Wheat germ trans lation assays, precipitation with specific antibodies, and analysis on SDS:polyacrylamide gels were described previ ously (10) except for the following changes. Two hundred pl assays were used routinely. After incubation for 60 mm at 25°,polynibosomes were removed at 105,000 x g for 1 hr. Specific rabbit antiserum (3 pI) was added to the superna tant in the presence of 2% Triton X-100, 0.14M NaCI, and 10 mM unlabeled leucine, isoleucine, and valine. [3HjCasein and a-[3H]lactalbumin were separately precipitated for 2 hr at 30°,then for 18 hr at 4°.Goat anti-rabbit y-globulin was added to equivalence for 2 hr at 30°to increase the mass of the precipitates. The antibody precipitates were centrifuged through a 1.0-mI layer of 1 M sucrose containing 1% sodium deoxycholate, 1% Triton X-100, and 10 mM leucine, isoleu cine, and valine in a Microfuge B (Beckman Instruments Co.) for 10 mm at maximum speed to minimize nonspecific precipitation. The precipitates were then dissolvedin 3% SDS:0.1 M sodium phosphate buffer, pH 7.8, containing 2% 2-mercaptoethanol at 95°for 3 mm, and a-[3H]lactalbumin and [3H]casemn were identified and quantitated on 10% SDS:polyacryIamide gels (9). The amount of newly synthesized protein was propor tional to the amount of added RNA. Equal quantities of mammary gland RNA were assayed from the linear portions of the translation curve to compare the activity of the various RNA extracts. 1251-labeleda-lactalbumin and 125l-casein were prepared from purified samples of rat a-lactalbumin and casein (Protein C) by the chlonamine-T procedure of Hales and 0 I 234567 MIGRATiON (cm) Chart 1. SDS:acrylamidegel electrophoresisof rat caseinsisolatedfrom whole milk. Rat milk was fractionated by isoelectric and ethanol precipitation as described in “Materials and Methods.―Aliquots of each fraction were dissolved In 3% SDS:sodium phosphate buffer, pH 7.8, and subjected to 10% SDS:acrylamidegel electrophoresis(9).Coomassieblue-stainedgels of (la) whole milk, (lb) skim milk, (ic) first isoelectric precipitate, and (le) ethanol precipitate were scanned at 550 nm. Direction of migration is from left to right. beled A, B, and C were then separated on DEAE-cellulose in the presence of 3.3 M urea. Rechromatography of the separated caseins produced 3 electrophoretically homoge neous proteins with apparent molecular weights of 38,000, 24,000, and 22,000 (Chart 2). Amino acid analysis by acid hydrolysis suggests that distinct protein species are pres ent, although all 3 are highly acidic and particularly rich in glutamic acid (Chart 3). Differences in the proportions of isoleucine, leucine, lysine, and arginine are especially prominent. Periodic acid-Schiff stains of a mixture of Ca seins A, B, and C also revealed an unequal distribution of carbohydrate (Chart 4); the ratio of periodic acid-Schiff and Coomassie blue staining for Caseins A and B was approxi mately 3:1 , whereas carbohydrate was not detectable in Randle (4). A calibration curve of log molecular weight CaseinC. Antibodiesto Casein and a-Lactalbumin.Since Rosen and Osbonn(18)with the use of proteins of known molecu et a!. (13) had shown that cell-free synthesis of casein in the versus mobility was determined by the method of Weber Iansize. RESULTS Purificationand Characterizationof Rat Caseins. Since isolation and characterization of rat casein as well as antibody production was required to study casein mRNA synthesis, we purified to homogeneity the 3 principle rat caseins and determined their molecular weights, amino wheat germ system yielded primarily low molecular weight casein, we used our purified casein C to prepare specific antibody. Also since Casein C does not contain attached carbohydrate (Chart 4), which has been shown to alter the migration of other glycoproteins synthesized in cell-free heterogeneous systems, its identification on SDS:acrylam ide gels should be more definitive. Approximately 0.4 pI of serum obtained 45 days after primary injections of casein antigen precipitated 60%of ‘25l-casein (Fraction C) at equiv acid distribution, and carbohydrate content. The isolation of the 3 rat caseins by selective precipitation from rat milk was monitored on SDS:polyacrylamide gels (Chart 1). The alence (Chart 5A). Similar low-affinity antibodies for casein have been reported (5, 13). Booster injections of 1 mg s.c. effectively whey protein from the caseins and to solubilize lipids associated with the caseins. Individual caseins Ia A small quantity of antiserum (1 p1) obtained 45 days after primary injections of a-Iactalbumin was sufficient to precip incomplete Freund's adjuvant did not markedly increase ethanol precipitation step was included to separate more titers or alter antibody affinities. SEPTEMBER1978 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. 2695 N. J. Nardacci et a!. TOTAL PROTEINS It) 4 @ ,,,II 0 It) IL, 4 I 0 (Si It) 4 MiGRATiON (cm) Chart 2. SDS:acrylamide gel electrophoresis of rat caseins separated on DEAE-cellulose columns. Caseins isolated from whole milk were separated on DEAE-cellulose columns, dissolved in 3% SDS:sodium phosphate buffer, pH 7.8, and applied to 10% SDS:acrylamlde gels. The migrations of protein markers of known molecular size are (left to right): 68K, bovine serum albumin; 50K, y-globulln (heavy chain); 24K, ‘y-globulln (light chain); 14K, pancreatic RNase; 6K, insulin. 25@ A MIGRA11ON(cm) 15 5 Chart4. Carbohydratecontent of rat caseins. Rat caseins isolated by isoelectricand ethanol precipitationwere subjectedto SDS:acrylamidegel AALAA@J@ 0 electrophoresis and stained with Coomassle blue for total proteins (top) or with periodic acid-Schiff (7) for glycoproteins (bottom). @20 @J1LIl@AthI@ 0 0 Ui 5 @ : 0 i i @. • _ CysAsp71w Ser GkiPro Gly liii. Met lie LauTyrPhe H@ LysArg AMINOACIDS C-, Ui Chart3. Amino acid content of rat caseins. Rat caseins purified to homogeneity by chromatography on DEAE-cellulose were lyophilized, hydro lyzed, and analyzed for amino acid content as described in “Materials and Methods.― A, B, and C, mol percentageof aminoacids in CaseinsA, B, and Ui C-, C, respectively. itate 75% of ‘25i-a-lactalbumin(5 ng) as shown in Chart 5B. Similarly, booster injections of 1 mg did not further increase antibody titers on affinities. Analysis of (3H]Caseinand a-[3H]LactalbuminSynthe sized in the Wheat Germ System. Translationof total mRNA from lactating rat mammary glands resulted in a large population of newly synthesized proteins of various sizes (Chart 6A). When antibodies against Casein C were added to the incubation mixture, a major [3H]-labeledpro tein was precipitated that migrated similarly to authentic l'25-labeled Casein C (Chart 6, A and B). The peak of 2696 0 0.2 @4 Q6 iii ANTISERA Q8 1.0 0 02 Q4 06 ph AN11SERA 0.8 1.0 Chart5. Immunopreclpitationof ‘2I-casein and ‘25l-a-lactalbumin (125l@ aLA). Rat casein (Protein C) purified to homogeneity on DEAE-cellulose and a-Iactalbumin were iodinated with Na'25l by means of the chloramine-T procedure (4). Increasing quantities of specific antiserum raised in rabbits were added to 3.8 ng of 125I-casein,227,000 cpm (A), and to 5.3 ng of ‘25I..@@ lactalbumin, 145,000 cpm (B), in 200 pi of 50 m@sodium phosphate buffer, pH 7.2, containing bovine serum albumin (40 mg/mI) and 2% Triton X-100. Normal rabbit serum was added to make the final ‘y-globuhin concentration equal in all tubes. Antibody precipitation was carried out as described in “Materials and Methods.―Precipitates were washed 3 times by centrifuga tion at low speed in phosphate buffer, to remove unbound “5l-Iabeled protein, and counted In a gamma counter. CANCERRESEARCH VOL. 38 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. a-LaCtalbumin and Casein mRNA's $5 .5 Q Q a. C-) Chart 6. Immunoprecipitation and identification of (3Hjcasein and a-[3H]Iactalbumin.Aliquots of translation mixtures pro grammed with 15-day lactating gland RNA were precipitated with either 5% trichloroacetic acid or with specific antibodies as described in “Materials and Methods.―The SDS:gel migration profiles show: (A) trichloroacetic acid precipitate (0) and casein antibody precipitate (•); (B) ‘2l-Iabeledcasein; (C), a-lactalbumin (aLA) antibody precipitate; (D) “l-labeleda-lactalbumin; (E) $5 Q Q5 caseinimmunoprecipitates in presenceor absenceof 25 @g of casein (Protein C) competitor; and (F) a-lactalbumin immunopre cipitates in the absence or presence of 25 @g of a-iactaibumin competitor. 0 Q 10 20 30 40 51D 1.0 Jo ) N Q Q a. C-) I .5 GELSUCE lOOK [3Hjcasemncould be eliminated by adding 25 pg of nonra dioactive authentic Casein C to the antibody incubation mixture, which further demonstrates the specificity of our @ @ assay (Chart 6E). The low-molecular-weight 3H-labeled ma terials, migrating near the casein peak in Chart 6, A and E (Gel Slices 20 to 25), probably represent casein fragments since they are competed by unlabeled casein. Such frag ments may arise by premature release of nascent peptide chains from polynibosomes, by mild proteolysis, or by “nicked― casein mRNA molecules that produce short casein segments. When antibodies against a-lactalbumin were added to the newly synthesized protein mixture, a major [3H]-Iabeled pro tein was precipitated that migrated identically with authen tic ‘251-lactalbumin (Chart 6, C and D). Twenty-five pg of unlabeled authentic a-lactalbumin, added prior to specific 40K CHAIN I-. I l@ 10K C-) Ui -j 0 probably represents high-molecular-weight proteins that are nonspecifically trapped in the antibody precipitate. The molecular weight of the newly synthesized casein was 22,000(Chart 7), which is similar to the 25,000that Rosenet a!. (13) reported for their low-molecular-weight casein. 4K @; 0 antibodies, eliminated 95% of the a-[3H]IactaIbumin peak (Chart 6F) which showed specificity. The 3H-labeled mate nial remaining near the origin (Gel Slices 1 to 6) was not competed by nonradioactive a-Iactalbumin and therefore RNAau Ui 10 MIGRATiON (cm) Chart 7. Molecular weight of [2H]casein and a-[@H]Iactalbumln (f3HJaLA) synthesized in the translation assay. [3H]Casein and a-rH]Iactalbumin im munoprecipitated from the translation assays were dissolved In 3% SDS: 0.1 N sodium phosphate buffer, pH 7.8, and run on 10% SDS-acrylamide gels along with proteins of known molecular size. The positions of the protein markers were determined from scans of Coomassie blue-stained gels. The migrations of (3H]casein and a-[3H]Iactalbumln were determined by counting gel slices digested with protosol (10). y-G, y-globulin; BSA, bovine serum albumin. SEPTEMBER 1978 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. 2697 N. J. Nardacci et a!. is undergoing intense stimulation during both pregnancy and lactation. Casein mRNA levels in the glands rose continuously, increasing 20-fold from Day 5 of pregnancy a-Lactalbuminand Casein mRNA in Pregnantand Lac to Day 15 of lactation (Chart 9A). The important of hormonal tating Glands.Total mRNAwasisolatedfrom rat mammary control of casein mRNA is suggested by removal of the gland on various days of pregnancy and lactation. Chart 8 pups during lactation; casein messenger levels are dimin shows total protein synthesized as a function of the pg of ished (Chart 9A). When a-lactalbumin mRNA activity was measured at RNA added to the translation system. It is apparent from the increase in total mRNA synthesis that the mammary gland various times during pregnancy and lactation, it remained low during pregnancy, unlike that of casein, but rose sharply during lactation (Chart 9B). When the animals were prematurely weaned on Day 2 of pregnancy, the rise other wise seen by Day 8 was prevented. Nascent a-[3H]lactalbumin had a molecular weight of 15,000 (Chart 7), which agreed with the molecular weight reported for native a-lactalbumin for other species (3). DISCUSSION IL, We isolated and purified 3 separate rat caseins and prepared specific antibodies to the low-molecular-weight species. This low-molecular-weight protein (Casein C) was b ‘C ci@ shown to be distinct from the other species of casein by its C-) amino acid composition and absence of attached carbohy drate. Analysis of Casein C mRNA in the mammary gland of the rat during pregnancy and lactation revealed progressive increases in casein mRNA activity beginning in early preg nancy despite the absence of true lactational activity until partunition. Levels of cr-Iactalbumin mRNA, on the other hand, were barely detectable during pregnancy and nose only during lactation. The failure of the 2 mRNA's to increase in parallel during pregnancy is of considerable interest. Evidently, the con trols oven a-lactalbumin are at least in part distinct from those of casein. These findings in normal mammary tissue are similar to those recently reported in experimental breast cancer tissue (9) where casein but not a-lactalbumin mRNA was enhanced following prolactin stimulation . Simultane ous study of the regulation of these 2 mRNA's in both nor mal and neoplastic breast tissue should provide additional insight into hormonal control mechanisms in breast cancer. z Ui a: a-J 0 20 40 60 80 100 120 pgRNA Chart 8. Total 3H-Iabeled proteins synthesized in the translation assay with increasing quantities of RNA extracted from glands at various stages of pregnancy and lactation. Fifty-@tlaliquots of [email protected] assays programmed with RNA from glands during pregnancy and lactation were pipetted into 1 ml of 5% trichloroacetic acid containing 0.1% unlabeled Ieucine, isoleucine, and valine. The precipitates were heated to 90-95°for 10 mm to deacylate tRNA, washed several times in the 5% trichloroacetic acid solution by centrifuga tion at low speed, and dissolved in 0.5 ml of protosol (55°for 1 hr) before being counted in an Omnifluor-toluene cocktail (10). Equal quantities of mammary tissue from 3 to 5 rats were pooled for each time point. d, days. ACKNOWLEDGMENTS We thank Dr. J. M. Rosen for providing anti-rat casein antibody, Dr. B 1.0 Q In 0 ‘C @ a. 0.5 C-) I ,6) 0 @5 PREGNANC( LACTATION lö 15 PREGNANCY 2@@è I5@S LACTATION Chart 9. Mammary gland casein and a-lactalbumin (aLA) mRNA during pregnancy and lactation. [‘H]Casein and a-[3H]lactalbumin were immunoprecipi tated from translation assays programmed with RNA from pregnant and lactating glands as described in Chart 8. Allquots containing equal RNA concentrations from the linear portions of the translation curves were used. Weaned, removal of pups on Day 2 of lactation and assay for specific mRNA's on day 8 of lactation. 2698 CANCER RESEARCHVOL. 38 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. a-Lacta!bumin and Casein mRNA's Marzluff for a-lactalbumin antigen and antibodies, Dr. Robert E. Olson for his gift of active wheat germ, and Dr. B. P. Yu for assistance with the amino acid analysis. REFERENCES 1. Brew, K., Vanaman, T. C., and Hill, R. L. The Role of a-Lactalbumin and the A Protein in Lactose Synthetase: A Unique Mechanism for the Control of a Biological Reaction. Proc. NatI. Aced. Sd. U. S., 59: 491495, 1968. 2. Gaye, P. , and Houdebine, L. M. Isolation and Characterization of Casein mRNAs from Lactating Ewe Mammary Glands. Nucleic Acids Rae., 2: 707-722,1975. 3. Gaye, P., Viennot, N., and Denamur, R. In Vitro Synthesis of a-Lactal bumin and @-LactoglobuIinby Microsomes and Bound Polyribosomes from the Mammary Gland of Lactating Sheep. Blochim. Biophys. Acts, 262:371-380,1972. 4. Hales, C. N., and Randle, P. J. Immunoassay of Insulin with Insulin Antibody Precipitate. Biochem. J., 88: 137-146, 1963. 5. Houdebine, L. M., and Gaye, P. Regulation of Casein Synthesis in the Rabbit Mammary Gland. Titration of mRNA Activity for Casein Under Prolactin and Progesterone Treatments. Mol. Cellular Endocrinol., 3: 37-55, 1975. 6. Juergens, W. G., Stockdale, F. E., Topper, V. J., and Elias, J. J. Hormone-Dependent Differentiation of Mammary Gland In Vitro. Proc. NatI. Acad. Sci. U. S., 54: 629-634, 1965. 7. Matthieu, J. M., and Quarles, R. H. Quantitative Scanning of Glycopro teins on Polyacrylamide Gels Stained with Periodic Acid-Schiff Reagent (PAS). Anal. Biochem., 55: 313-316, 1973. 8. McMeekln, T. L., Hipp, N. J., and Groves, M. L. The Separation of the Components of a-Casein. I. The Preparation of a,-Casein. Arch. Bio chem. Biophys., 83: 35-43, 1959. 9. Nardacci, N. J., Jones, J. P., HaIl, A. L., and Olson, R. E. Synthesis of Nascent Prothrombin and Albumin at a Heterologous System Using Rat Liver Messenger RNA Purified on Oligo (dT)-Cellulose. Biochem. Bio phys. Rca. Commun., 64: 51-58, 1975. 10. Nardacci, N. J., and McGuire, W. L. Casein and a-Lactalbumin Messen ger RNA in Experimental Breast Cancer. Cancer Res., 37: 1186-1190, 1977. 11. Rosen, J. M. Isolation and Characterization of Purified Rat Casein Messenger Ribonucleic Acids. Biochemistry, 15: 5263-5271 , 1976. 12. Rosen, J. M., and Barker, S. W. Quantitation of Casein Messenger Ribonucleic Acid Sequences Using a Specific Complementary DNA Hybridization Probe. Biochemistry, 15: 5272-5280, 1976. 13. Rosen, J. M., Woo, S. L. C., and Comstock, J. P. Regulation of Casein Messenger RNA During the Development of the Rat Mammary Gland. Biochemistry, 14: 2895-2903, 1975. 14. Terry, P. M., Ganguly, R., Ball, E. M., and Banerjee, M. R. Murine Mammary Gland RNA Directed Synthesis of Casein in a Heterologous Cell-Free Protein Synthesis System. Cell Differentiation, 4: 113-122, 1975. 15. Thompson, M. P. DEAE-Cellulose-Urea Chromatography of Casein in the Presence of 2-Mercaptoethanol. J. Dairy Sci., 49: 792-795, 1966. 16. Turkington, R. w., Brew, K., Vanaman, T. C., and Hill, A. L. The Hormonal Control of Lactose Synthetase in the Developing Mouse Mammary Gland. J. Blol. Chem., 243: 3382-3387, 1968. 17. Vaitukaitis, J., Robbins, J. B., Nieschlag, E., and Ross, G. T. A Method for ProducingSpecificAntiserawith Small Dosesof Immunogen.Clin. Endocrinol., 33: 988-991, 1971. 18. Weber, K., and Osbom, M. The Reliability of Molecular Weight Determi nations by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis. J. Biol. Chem., 244: 4406—4412, 1969. 19. Young, S., and Nelstrop, A. E. The Detection by Immunofluroescence of Casein in Rat Mammary Glands. Brit. J. Exptl. Pathol., 51: 28-33, 1970. SEPTEMBER1978 Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research. 2699 Differential Regulation of α-Lactalbumin and Casein Messenger RNA's in Mammary Tissue Nicholas J. Nardacci, Jimmy W. C. Lee and William L. McGuire Cancer Res 1978;38:2694-2699. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/38/9/2694 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 11, 2017. © 1978 American Association for Cancer Research.