Download Differential Regulation of a-Lactalbumin and

[CANCER RESEARCH 38, 2694-2699, September 1978]
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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
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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
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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
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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
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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
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chem. Biophys., 83: 35-43, 1959.
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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
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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
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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.
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