potter hudson - Europe PMC

REGULATION OF MOUSE MAJOR URINARY PROTEIN PRODUCTION BY THE MUP-A GENE P. R. SZOKA

AND

K. PAIGEN

Molecular Bio!ogy Deparimeni, Roswell Park Memorial Instiiute, 666 Elm Sireei, Buffalo, New York 14263 Manuscript received January 31, 1978 Revised copy received May 12, 1978 ABSTRACT

A method was developed to quantitate the daily excretion of the three major urinary proteins (mups) to test which parameters of the mup phenotype are controlled by the the Mup-a gene. Electrophoretic separation of the mup proteins, followed by staining and spectrophotometric scanning was used to characterize the phenotypes of various inbred strains. The mup phenotype of a strain proved to have two components: the absolute levels and the relative proportions of the mups present in the urine. Testosterone treatment alters both components of the mup phenotype, increasing mup excretion and altering their relative proportions. The induced proteins are the same as the basal proteins as judged by electrophoretic mobility, molecular weight, and reactivity with antibody. All strains excrete all three mups when induced. The Mup-a gene appears to be a single, codominantly expressed regulatory locus that controls the induced proportions of the three proteins. However, other genes in addition to Mup-a participate i n controlling the basal mup proportions, as well as individual and total mup levels before and after testosterone treatment.

HE major urinary proteins (mups) of inbred mice are a family of three Tandrogen-inducible proteins that are synthesized in the liver and ultimately POTTER and RUNNER1963; RUMKEand excreted into the urine (FINLAYSON, THUNG 1964; FINLAYSON et al. 1965). The three proteins are defined by their electrophoretic mobilities, and inbred strains of mice exhibit variation in their urinary mup electrophoretic patterns. This variation is controlled by the Mup-a locus on chromosome 4 (HUDSON, FINLAYSON and POTTER 1967; FINLAYSON, HUDSON and ARMSTRONG 1969). All mice excrete mup 3, but strains carrying the Mup-a' allele appear to excrete mup 1, but not mup 2; conversely strains carrying the Mup-az allele appear to excrete mup 2, but not mup 1. The mup system provides an excellent model for studying the hormonal regulation of specific gene expression. First and most significant, there is definable genetic variation in the production of the mups in response to androgenic hormones; consequently, this system may ultimately offer insight into the role of specific DNA sequences in the stimulation of gene expression by steroid hormones. Second, when induced by androgens, mice excrete milligram quantities of the mups daily. Therefore, mup synthesis must constitute a considerable portion of the daily liver protein synthesis. This large response, combined with Genetics 90: 597-612 November, 1978.

598

P. R . SZOKA A N D K. P A I G E N

the small molecular weight of the mups, should facilitate development of techniques to study mup gene expression at the level of messenger RNA. Third, protein turnover should not be a complicating factor in studying mup induction by testosterone, since the mups are rapidly excreted rather than degraded. Although untreated female homozygotes excrete either mup 1 or mup 2, depending on the Mup-a allele carried by the strain, Mup-d/Mup-a2 heterozygotes excrete both mup 1 and mup 2. This suggests that mup 1 and mup 2 represent the allelic products of a structural polymorphism at the Mup-a locus (HUDSON, FINLAYSON and POTTER 1967). This hypothesis was supported by biochemical studies demonstrating that mup 1 and mup 2 are nearly identical proteins; they have identical amino acid sequences for the first 36 N-terminal residues (FINLAYSON et a1 1974), and differ by only a single tryptic peptide (FINLAYSON et aZ. 1968). The exact structural difference between mup 1 and mup 2 is still unknown. Although mup 3 is related to the other two proteins, it is distinct, since it differs in amino acid sequence from mup 1 and mup 2 (FINLAYSON et al. 1974). Our current finding that all three mups may be present after induction of homozygous animals suggests that this interpretation of the Mup-a locus as a structural polymorphism may be overly simplified. The genetics of the mups is also uncertain in the sense that it is not known whether genes other than Mup-a participate in the regulation of the mup production. I n order to clarify these questions we have developed a method for quantitating the urinary mup phenotypes of mice and, using this method, asked what parameters of the phenotype are under the control of the Mup-a locus. The phenotype proved to be complex. Testosterone not only induces an increase in the absolute levels, but also alters the relative proportions of the mups in urine. A comparison of inbred strains makes it clear that the Mup-a gene does not define a structural polymorphism as was originally suggested, since all strains excrete all three mups when induced. Rather, Mup-a appears to be a regulatory locus that determines the relative proportions of the three mups, when induced. However, by itself, Mup-a does not control the basal proportions of the mups, nor the levels of mup excretion. The determination of these phenotypes includes additional genetic factors. MATERIALS A N D METHODS

Animals: Female inbred mice were obtained from the Jackson Laboratory, Bar Harbor, Maine. All mice were between two and four months old. Whenever possible, within an experiment mice of each strain were age matched. Only female mice were used to avoid the problem of variation in endogenous androgen levels found among males. Urine collection and testosterone induction: For urine collections, animals were maintained on a schedule of 12 hr light alternated with 12 h r darkness and housed two to four mice per cage in stainless steel metabolism cages equipped with food and water. Food was presented as a paste of water and ground Rockland Mouse Breeder Diet (Teklab, Inc., Monmouth, Illinois) containing 17% minimum crude protein, 10% minimum crude fat, and 2.5% maximum crude fiber. Urine was collected over 24 h r periods into 12 ml conical test tubes containing mineral oil, but no preservative. Urine samples were clarified at 1,000 x g for 10 min and stored at -20". The same mice were used for both basal and testosterone-induced urine collections. 30 mg testos-

REGULATION O F M U P PRODUCTION

599

terone pellets (Schering Corp. or Department of Pharmaceutics, University of Tennessee College of Pharmacy) were implanted subcutaneously and maintained throughout the induction period. Induced urine samples were collected on days 17, 18, and 19 after implantation of testosterone pellets, unless otherwise noted. EZectrophoresis: Urine samples were dialyzed at 4" overnight against 100 volumes of 0.01 M tris, 0.01 M acetate, 0.25 M sucrose pI-1 5.4, then assayed for protein content by the microbiuret procedure of GOA(1953). Samples of 5 pg protein in a volume of 5 or 10 c1 were electrophoresed at 200 V for 90 min through a 10 cm x 1.5 m m 5% polyacrylamide slab gel containing 0.2% bisacrylamide and buffered with 0.01 M tris, 0.01 M acetate p H 5.5 (final concentrations). The electrophoresis buffer was 0.01 M tris, 0.01 M acetate p H 5.4. After electrophoresis gels were stained with either Coomassie brilliant blue G or Amido Schwartz. For Coomassie brilliant blue G staining, the gels were first fixed for 10 min i n 12.5% TCA and then stained for two hr with stirring with 20:l (v/v) of 12.5% TCA: 0.25% Coomassie brilliant blue G (Sigma). The stain was further developed by incubating the gel overnight in 5% acetic acid. For Amido Schwartz staining, the gels were stained for 30 min with 1% Buffalo Black NBR (Allied Chemical) in 7.5% acetic acid with stirring and then destained with 7% acetic acid. Gels were scanned using a Corning 750 densitometer using a setting of 1 O.D., a slit of 0.2 x 3 mm, and wavelengths of 650 nm for Coomassie brilliant blue G or 625 nm for Amido Schwartz. By comparison of the peak areas of urine mup bands with that of a purified mup 3 standard, pg mup protein/band was determined. The p H 5.5 polyacrylamide gel system for separating the mfips was used in a modification of the method of FERGUSON (1964) that related molecular size with the dependence of electrophoretic mobility on acrylamide gel concentration. The concentration of acrylamide was varied appropriately. The gels were stained with the Coomassie brilliant blue G stain. The SDS polyacrylamide gel electrophoresis was done by the method of LAEMMLI(1970) using 4 to 6 pg of each standard protein. Gels were stained for 30 min with 0.25% Coomassie brilliant blue R (Sigma) in 5 parts methanol: 5 parts water: 1 part acetic acid, destained with 50% methanol, 7% acetic acid, followed by treatment for one hr with 5% methanol, 7% acetic acid. Gels were stored i n 7% acetic acid. Purification of the mups: The mups were purified from urine of testosterone-treated C57BL/6J female mice, since this strain excretes considerable quantities of each mup when induced. The procedure used was a modification of that of FINLAYSON et a2. (1968). Approximately 120 mg of urinary protein in 0.1 M tris, 0.1 M acetate p H 5.5 was applied to a 1.5 x 81 cm column of Sephadex GI00 (Pharmacia) and eluted with the same buffer at a flow rate of 5 ml/hr. The peak containing the mups was dialyzed against distilled water, lyophilized, and reconstituted with 0.05 M tris, 0.05 M acetate p H 5.5; 22 mg of this protein was applied to a 1 X 25 cm column of DEAE cellulose (Whatman DE52, microgranular, preswollen) preequilibrated with the same buffer. After washing the column with the same buffer containing 0.05 M NaCl elution was performed by applying 400 ml of a linear 0.05 t o 0.10 M NaCl gradient i n the column buffer; this gradient resulted in elution of mup 1, mup 2, and mup 3 in separate peaks. Application of another 400 ml linear gradient containing 0.10 to 0 . 1 5 ~NaCl in the same buffer to the column eluted a second fraction containing mup 3. All of the peak fractions were dialyzed against distilled water, lyophilized, reconstituted in 0.02 M imidazole, 0.25 M sucrose p H 7.4, and stored a t -20". Throughout the purification procedure, protein concentration were determined by the method of LOWRY et a2. (1951) and fractions containing the mups were identified by the pH 5.5 polyacrylamide gel electrophoresis. Preparation of antibody: Equal quantities of each purified mup in 0.02 M imidazole, 0.25 M sucrose p H 7.4 were combined as a pooled antigen. Rabbits were immunized with 90 pg of antigen mixed with a n equal volume of Freund's complete adjuvant. Injections were given intradermally at multiple sites in the back and were administered every two weeks for a total of six injections; then blood was collected. The serum prepared from the blood was stored a t -20".

600

P. R. SZOKA A N D K. PAIGEN

Double diffusion analysis: The Ouchterlony technique was performed by the method of OUCHTERLONY (1958). Wells with a capacity of 15 pl were cut in plates containing 1% agarose (Fisher) dissolved in 0.85% saline, 0.09 M tris HCl pH 7.5, with 0.1% Na azide added as a preservative. After addition of the appropriate antiserum and antigens to the wells, the plates were allowed to develop overnight a t room temperature. RESULTS

M u p induction Untreated mice carrying the Mup-a' allele excrete the mup 1 protein, but not mup 2, while untreated mice carrying the Mup-a* allele have the reciprocal phenotype and excrete mup 2, but not mup 1. All strains excrete mup 3, although often the levels are quite low. Although before induction most strains excrete either mup 1 or mup 2, but not both, after testosterone treatment all strains appear to excrete both mup 1 and mup 2, as well as mup 2, albeit to varying levels (Figure 1) . Strains carrying the Mup-a' allele, which excrete mup 1 before induction, excrete much more mup 1 than mup 2 after induction. Strains carrying the M u p d allele which excrete mup 2 before induction, excrete more mup 2 than mup 1 after induction. Mup 3 is present in the urine of all strains, and induced levels of mup 3 vary independently of the Mup-a genotype. )r

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FIGURE 1.-Electrophoresis and protein staining for the major urinary proteins of inbred mice. Aliquots of urine from testosterone treated mice containing 5 fig protein were separated electrophoretically on p H 5.5 polyacrylamide gels and stained with Coomassie brilliant blue G, as stated in MATEnxArs AND METHODS.

REGULATION O F MUP PRODUCTION

601

Thus, induction causes a change in the relative amounts of each mup present, as well as an increase in total mup production. Furthermore, all strains of mice excrete all three mups when induced by testosterone, regardless of the Mup-a allele carried by the strain.

Comparison of basal and induced mups

If all three mups are present after induction in all strains regardless of the Mup-a genotype, and if the induced mups are the same species of proteins as the uninduced mups, then the Mup-a locus cannot determine a structural polymorphism. Instead it would represent a regulatory locus modulating mup production. For this reason we have accumulated additional data confirming the identity of the basal and induced proteins. Zmmunological properties: Rabbit antiserum was prepared against a mixture of purified mup 1, mup 2, and mup 3 and used in Ouchterlony double-diffusion analysis. Each purified mup gave a line of identity when tested against the other two (Figure 2a). Hence, all three mups share a common set of antigenic determinants, and none of the mups contains an antigenic site lacking in another mup. Given that the three induced mups are antigenically identical, we have asked whether are basal and induced mups also show antigenic identity (Figure 2b). Pretreatment of urine with anti-mup antiserum removes the mups from the pH 5.5 polyacrylamide gels. This is true for the mups from both basal and testosterone induced Mup-a' and Mup-a2 homozygotes (Figure 2b, lanes 1, 3, 5, and 7). The antiserum is specific for the mups since it does not remove albumin from the gel (Figure 2b, lane 9). Hence, the same mups are expressed before and after induction, irrespective of the Mup-a genotype, since the antiserum crossreacts with both basal and induced mups. Physical properties: Ferguson plot analysis of urinary protein from basal and testosterone treated Mup-a1I2mice shows that mup 1, mup 2 and mup 3 differ from each other by charge and not by molecular weight, and that each mup from urine of untreated mice exhibits the same mobility characteristics as the corresponding mup from urine of testosterone-treated mice (Figure 3). Identical results were obtained with the urinary mups from untreated and testosteronetreated Mup-a' and Mup-az homozygotes (data not shown). Thus, the three mups excreted after induction have the same molecular weight and electrical charge as the corresponding proteins made before induction. The molecular weight was not calculated from the Ferguson plot data because not many proteins migrate anodally at pH 5.5, and it is difficult to calibrate the system. Therefore, we used SDS polyacrylamide gel electrophoresis to determine the molecular weight of the mups. We find that the three mups have molecular weights of approximately 15,000 (Figure 4). Therefore, we conclude that the same mups are present in urine of various inbred strains of mice before and after induction, as judged by electrophoretic mobility, molecular weight, and reactivity with antibody. Carbohydrate analysis: Because secreted proteins are often glycosylated, we asked whether the charge difference between the mup proteins was due to car-

602

P. R. SZOKA A N D K. P A I G E N

a

1

4

b

FIGURE 2.-Ouchterlony immunodiffusion studies with purified and urinary mup components. The center well contains rabbit antiserum prepared against a mixture of the three mups, which had first been isolated separate from each other. (a) wells 1 and 4 contain purified mup 1; wells 2 and 6 contain purified mup 2; wells 3 and 5 contain purified mup 3. (b) The major urinary proteins and bovine serum albumin were electrophoresed on pH 5.5 polyacrylamide gels and stained for protein as described in MATERIALS AN METHODS. After collection of the basal urine samples, increased mup production was induced by testosterone. Before electrophoresis, the samples in the odd-numbered lanes were preincubated with anti-mup antiserum at 37" for 30 min.Lanes 1 and 2: BALB/cBy (Mup-a') without testosterone; lanes 3 and 4: BALB/cBy (Mupa') with testosterone; lanes 5 and 6: C57BL/6By ( M u p - d ) without testosterone; lanes 7 and 8: C57BL/6By (Mup-a2) with testosterone; lanes 9 and 10: bovine serum albumin.

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% Acrylamide FIGURE 3.-Ferguson plot analysis of mups from (A/J x C57BL/6J) F, mice. The mice are heterozygous for the Mup-a gene; p H 5.5 polyacrylamide slab gel electrophoresis and Coomassie brilliant blue G staining were performed as stated in MATERIALS AND METHODS. Open symbols represent mups from the urine of untreated mice; closed symbols represent mups from the urine ) mup 3. of testosterone induced mice. (0,0 ) mup 1; (A, A) mup 2; (0,.

bohydrate residues. We were unable to demonstrate the presence of covalently attached carbohydrate on any of the mups. Neuraminidase treatment to remove any sialic acid residues did not affect the mobility of urinary mup 1, mup 2 or mup 3 on pH 5.5 polyacrylamide gels. The neuraminidase used was active, since treatment of fetuin, a known sialoprotein, with the enzyme did decrease the mobility of fetuin on pH 5.5 polyacrylamide gels. PAS straining of the purified mups also failed to detect any carbohydrate, even when the sensitivity of the test was sufficient to detect a single sugar residue per molecule of protein. Therefore, the observed charge difference between the mups does not seem to be due to the presence of carbohydrate residues. Quantitation of indiuidual mup excretion: Because the Mup-al/Mup-aLdifference appears to be due to genetic regulation of mup production, rather than resulting from a structural polymorphism, a quantitative assay for each of the mups was developed in order to define the phenotypes more precisely. The mups

604

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Cm migrated FIGURE 4.--SDS polyacrylamide gel molecular weight analysis of the purified mups. Four to six p g of each standard and of each purified mup were electrophoresed through 12% polyacrylamide disc gels containing 0.1% SDS and stained with Coomassie brilliant blue R as described in MATERIALS AND METHODS. Mup 1, mup 2 and mup 3 all exhibit the same mobility and, hence, the same molecular weight.

were separated electrophoretically from 24 hr urine samples collected from groups of two to four mice, and the resulting gels were stained for protein and scanned with a densitometer. Representative scans for Mup-a' and Mup-ae homozygotes are shown in Figure 5. By comparing the peak areas to that of a purified mup standard, and knowing the 24 hr urine volume per mouse, the mg of each mup excreted/mouse/day can be calculated. Each of the three mups reacts equivalently with the Coomassie brilliant blue G strain used and the assay is linear for each mup up to at least 4 pg/band (Figure 6). Both observations are also true with the less sensitive Amido Schwartz strain (data not

605

R E G U L A T I O N O F MUP P R O D U C T I O N

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FIGURE 5.-Densitometry traces of electrophoretic separated mups from urine of untreated (- - -) and testosterone-treated (-) female SWR/J (Mup-a') and C57BL/6J (Mup-as) mice. The mups were electrophoresed, stained and scanned as described in MATERIALS AND METHODS.

shown). For convenience we have used a purified mup 3 preparation as a standard. The intensity of each stained mup band is proportional to the volume of urine applied to the gel, for both basal and induced samples, for Mup-a' and Mup-a* homozygotes and for Mup-a'l" heterozygotes (Figure 7 a and b). In addition, assay of a given urine sample is reproducible (Table I), and separate groups of animals from the same strain give quite consistent results (Tables 2 and 3 ) . Thus, the procedure appears to give a valid measure of the amount of each mup excreted, both before and after induction. The mup phenotype: When characterized by quantitation of the mups, phenotype proved to have two components: the absolute level of mups excreted and

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pg Protein /band FIGURE 6.-Standard curve of stain intensity us. pg mup/band. Varying amounts of each purified mup were separated on pH 5.5 polyacrylamide slab gels and stained with Coomassie brilliant blue G as described in MATERIALS AND METHODS. Stain intensity as measured by peak area/band was determined by spectrophotometricscanning at 650 nm. ( 0 )mup 1, (A)mup 2, mup 3.

(m)

their relative proportions. The absolute levels of the mups vary considerably between strains, both before and after administration of testosterone, and these are not correlated in any obvious way with the Mup-a genotype. The sole exception to this is the qualitative identity of the mups present in the urine of uninduced mice (Table 2). Testosterone treatment leads to a 20- to 40-fold induction of total mup excretion (Table 3). When mup excretion is expressed as the relative proportions of each mup present, the strains fall into discrete groups (Tables 4 and 5 ) . The relative proTABLE 1 Reproducibility of the mup quantitntion assay ( m g mup/mouse/day) Strain

BALB/cBy C57BL/6J

Testosterone

-

+ +

Mup 1 Expt. 1 Expt. 2

0.42 12.3 N.D. 1.06

0.39 14.0 N.D. 0.81


N.D. 0.88 0.40

3.01

N.D. 0.96 0.44 2.97


0.17 3.0 0.08 3.50

0.13 3.2 0.10 3.26

Urinary mups were separated on pH 5.5 polyacrylamide slab gels and stained with CBB-G. From peak area/mup band as determined by spectrophotometricscanning, daily excrdon of each mup was calculated. Each experiment represents analysis of the same sample performed on different days. N.D. = not detected.

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FIGURE 7.-Standard curve of stain intensity us. urine volume/well. Increasing volumes of urine protein were electrophoresed through pH 5.5 polyacrylamide slab gels and stained with Coomassie brilliant blue G. Stain intensity as measured by peak area/band was determined by spectrophotometric scanning at 650 nm. (a) urine from untreated BALB/CByJ mice (Mup-a'; mup 1 and mup 3) and from untreated C57BL/6J mice (Mup-a"; mup 2); (b) urine from testosterone-treated (A/J x C57BL/6J) F, mice (Mup-a'/Z); ( 0 ) mup 1; (A)mup 2; (m) mup 3. TABLE 2

Major urinary protein excretion by various inbred strains of mice before induction

Strain

SWR/J BALB/cBy DBA/2J AKR/J C3H/HeJ C57L/J C57BL/6J C57BL/6By

Mup-a Allele

1 1 I I

I 2 2 2

Mup 1

Mup 2

Mup 3

Total Mups

0.28 f 0.08 0.28 f 0.09 0.16 0.04 0.05 f 0.00 0.14 f 0.01 N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D. 0.17 _+ 0.02 0.33 f 0.06 0.62 0.10

0.26 f 0.04 0.09 2 0.02 0.12 f 0.01 0.18 f 0.00 trace trace 0.07 f 0.04 0.13 0.03

0.54 f 0.06 0.30 f 0.04 0.28 f 0.04 0.24 k 0.01 0.14 f 0.02 0.17 f 0.03 0.39 f 0.04 0.75 f 0.01

*

*

For each strain, three consecutive 24 hr urine samples were collected from two groups of untreated mice, housed three per metabolism cage. Then 5pg of urine protein/sample were separated on pH 5.5 polyacrylamide gels and stained with Coomassie briIliant blue G. From peak area/mup band as determined by spectrophotometric scanning, the daily excretion of each mup was calculated. Data presented as the mean of the mg/mouse/day of the two cages/strain 2 half the range. N.D. = not detected.

608


TABLE 3

Major urinary protein excretion by uarious strains of inbred mice after induction Strain

SWR/J BALB/cByJ DBA/2J AKR/J C3H/HeJ C57L/J C57BL/6J C57BL/6By

Mup-U Allele

Mup 1

I I I I I 2 2 2

12.8 -C 0.4 8.1 t 0.1 10.0 f 0.4 9.7 t 1.0 4.9 f 0.1 1.0 f 0.1 1.2 f 0.3 4.6 f 0.2

n1up 2

Mup 3

Total Mups

0.50 f 0.02 0.50 t 0.05 0.46 t 0.04 0.39 f 0.04 1.4 0.2 3.6 t 0.4 5.9 f 0.2

3.8 t 0.1 2.1 f 0.1 3.1 t 0.1 2.8 t 0.2 0.54 f 0.2 1.3 t 0.2 4.3 f 0.7 7.9 f 0.3

17.3 f 0.4 10.4 f 0.1 13.4 t 0.6 12.9 f 1.2

______ 0.70 f 0.08

*

5.9 f 0.2 3.6 f 0.5 9.1 f 1.4 19.8 f 0.0

After induction by testosterone, urine samples were collected and analyzed as described in the legend to Table 2.

portions of the mups present in untreated mice are not entirely determined by the Mup-a locus. Among uninduced animals with the same Mup-a genotype, there is considerable variation in the proportion of mup 3. However, once induced, the strains fall into two discrete groups with respect to relative mup proportions (Table 5 ) . These are correlated with the Mup-a allele carried by the strain, suggesting that the Mup-a locus is a regulatory gene controlling the induced mup proportions. The regulation of mup production by genes in addition to Mup-a is supported by analysis of the mup phenotypes of Mup-a*/eheterozygotes. Heterozygotes TABLE 4

Relatiue amounts of each mup before induction Total Strain


Mup-u Allele

Mup i

Mup 2

Mup 3

0.49 t 0.02 0.70 f 0.01 0.52 t 0.02 0.21 f 0.01 1.o N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D.

0.51 f 0.2 0.30 f 0.01 0.48 t 0.02 0.79 f 0.01 trace trace 0.20 f 0.01 0.11 i. 0.02

1.o 0.80 f 0.01 0.89 f 0.02

mup excretion (mg/mouse/day)

Urine samples were collected and analyzed as described in the legend of Table 2. Data presented as the mean of the two cages/strain f one-half the range. N.D. = not detected. mg(mup 1, mup 2, or mup 3)/mouse/day Relative amounts of each mup = ____----mg(mup I mup 2 mup 3)/mouse/day

+

+

0.54 0.30 0.28 0.24 0.14 0.17 0.39 0.75

609


TABLE 5

Relative amount of each mup after induction

Strain


Mup-a Allele

Mup 1

Mup 2

Mup 3

Total mup excretion (mg/mouse/da y)

I I I I I 2 2 2

0.75 k 0.01 0.76 f 0.01 0.74 f 0.01 0.75 f 0.01 0.85 f 0.01 0.27 f 0.01 0.24 k 0.01 0.12 f 0.01

0.04 2 0.01 0.05 k 0.01 0.03 -+ 0.01 0.04 t 0.01 0.07 t 0.01 0.37 k 0.01 0.32 -+ 0.01 0.41 I .0.01

0.21 2 0.01 0.19 k 0.01 0.23 f 0.01 0.21 k 0.01 0.08 k 0.01 0.35 f 0.01 0.44 f 0.01 0.47 f 0.01

17.3 10.4 13.4 12.9 5.9 3.6 9.1 19.8

Urine samples were collected and analyzed as described in the legend of Table 2. Data presented as the mean of the two cages/strain k one-half the range. N.D. = not detected. mg(mup 1, mup 2, or mup 3)/mouse/day Relative amounts of each mup = mg (mup 1 mup 2 mup 3)/mouse/day '

+

+

excrete higher levels of the three mups than either homozygous parent (Table 6), suggesting complementation between genes determining the absolute amounts of the mups produced. Similarly, the basal proportions of the mups in heterozygotes are intermediate for mup 1 and mup 2, but not for mup 3 (Table 7 ) . Thus, heterozygotes provide additional evidence that the Mup-a gene is probably not the only genetic factor controlling the relative amounts of the mups present in the urine of untreated mice. However, once induced, the heterozygotes have proportions for each mup that are intermediate between those of the TABLE 6

Major urinary protein excretion by Mup-a1 and Mup-a2 homozygotes and b y Mup-al/Mup-az heterozygotes (mg/mouse/day) Strain

A/J (A) B6A F, C57BL/6J (B6) BALB/cByJ(C) (CXB) F, C57BL/6By (B)

Mup-a Genotype

1/1 1/2 2/2 1/1 1/2 2/2

Mup 1

0.06+-0.00 0.3220.01 N.D. 0.2320.05 0.85t0.10 N.D.

-Testosterone Mup 2

Mup 3

Mup 1

N.D. 0.08k0.01 6.1 k 0 . 2 0.71t0.02 0.23t0.01 6.5 k 0 . 2 0.3220.06 0.07k0.04 0.42f0.02 N.D. 0.0920.03 13.8 k0.9 1.25f0.05 0.74f0.04 16.8 21.0 0.6720.12 0.1620.04 4.3 k0.4 ~~~

+Testosterone Mup 2

Mup 3

0.79f0.04 3.5 20.1 2.4 a0.05 trace 6.0 k0.3 8.1 k0.4

1.2k0.05 4.020.1 2.1a0.05 2.9k0.3 9.2k0.6 8.9k0.6

~

For A/J, C57BL/6J, and B6A F, mice, three 24 hr urine samples were collected from two groups of mice housed three per metabolism cages. For BALB/cByJ, C57BL/SBy and (CXB F, mice, 24 hr urine samples were collected from four groups of mice housed three per metabolism cage. Urine samples were collected and analyzed as described in the legend to Table 2. Data presented as cage means f one-half the range for A/J, C57BL/6J and BGAF, and as cage means f s.e. for BALB/cByJ, C57BL/6By, and (CXB) F,.

610


TABLE 7 Relative amount of each mup in Mup-al/Mup-az heterozygotes before induction Strain

A/J (A) B6A F, C57BL/6J (B6) BALB/cByJ (C) (CXB) F, C57BL/6By (B)

Mup-a Genotype

Mup 1

Mup 2

0.45 +- 0.010 0.26 f 0.002 N.D. 0.70 f 0.007 0.28 i: 0.02 N.D.

1/1 1/2 2/2

1/1 1/2 2/2

N.D. 0.56 f 0.0 0.89 f 0.02 N.D. 0.45 f 0.01 0.80 f 0.02

Mup 3

0.55 f 0.01 0.18 f 0.002 0.11 f 0.02 0.30 f 0.01 0.27 i.0.01 0.20 0.02

*

Urine samples were collected and analyzed as described in the legend of Table 2. Data presented as cage means +- one-half the range for A/J, C57BL/6J, and BGAF,, and as cage means f se. for BALBjcByJ, C57BL/6By, and (CXB) F,. N.D. = not detected. mg(mup 1, mup 2, o r mup 3)/mouse/day _________Relative amounts of each mup = __ mg(mup 1 mup 2 mup 3) /mouse/day

+

+

homozygous parents (Table S ) , indicating that the Mup-a gene is the major determinant of the relative proportions of the mups after induction, and establishing that Mup-a alleles show additive inheritance. DISCUSSION

All the inbred strains of mice tested excrete three mups when induced by testosterone; the differences between them lie in the proportions of the mups. Consequently, the Mup-a locus represents a regulatory gene and not a structural POTTER gene. Ostensibly, this contradicts the original observations of FINLAYSON, and RUNNER (1963), who implied that Mup-a defined a structural polymorphism for mup 1 and mup 2. The source of the difference between the two sets of results lies in the electrophoretic methods used. The polyacrymalide gel TABLE 8 Relative amount of each mup in Mup-al/Mup-a2 heterozygotes after induction Strain

A/J B6A F, C57BL/6J BALB/cByJ (C) (CXW F, C57BL/6By (B)

Mup-a Genotype

Mup 2

Mus 1

1/1

0.74 +- 0.005

1/2

0.46 +- 0.002 0.08 t 0.003 0.78 i. 0.04 0.52 f 0.004 0.20 i. 0.01

2/2 1/1 1/2 2/2 ~

~

0.10 i: 0.002 0.24 +- 0.002 0.48 +- 0.003 0.05 f 0.01 0.19 i: 0.003 0.37 f 0.01 ~

~~~~~~

Mup 3

0.16 f 0.002 0.28 f 0.002 0.43 +- 0.002 0.17 f 0.006 0.29 f 0.004 0.43 f 0.008 ~

Urine samples were collected and analyzed as described in the legend of Table 2. Data presented as cage means f one-half the range for A/J, C57BL/6J, and BGAF,, and as cage means f s.e. for BALB/cByJ, C57BL/6By, and (CXB) F,. N.D. = not detected. mg(mup 1, mup 2, or mup 3)/mouse/day ______~___ Relative amounts of each mup = ___ mg (mup 1 mup 2 mup 3) /mouse/day

+

+


611

system used here resolves the three mups into distinct bands, while with the agarose gel electrophoresis used by FINLAYSON, POTTER and RUNNER (1963) considerable overlapping occurs, obscuring the true phenotype. Later experiments by FINLAYSON and coworkers when employing polyacrylamide gel electrophoresis are in agreement with the present results. They find that male mice excrete all three mups regardless of their Mup-a genotype, and that induced females exhibit urinary mup electrophoretic patterns identical with those of male mice of the same strain (FINLAYSON, POTTER and RUNNER 1963; HUDSON, FINLAYSON and POTTER 1967). The three mups are antigenically identical, and all strains of mice have the same mups regardless of their Mup-a genotypes. In addition, the same mups are present in the urine both before and after testosterone treatment, as determined by their electrophoretic mobilities, molecular weights and antigenic similarity. Testosterone treatment alters both components of the mup phenotype, increasing the levels of the mups and changing the relative proportions of the mups found in the urine. All strains of mice fall into one of two groups with respect to induced mup proportions, and these are correlated with the Mup-a allele each strain carries. Furthermore, the induced proportions of the mups in mice heterozygous at the Mup-a locus are intermediate between those of their homozygous parents. Therefore, Mup-a appears to be a single, co-dominantly expressed regulatory locus that controls the induced mup proportions. We have confirmed this by examining a set of recombinant inbred lines derived from crossing progenitor inbred strains carrying the Mup-a' and Mup-as alleles ( SZOKA and PAIGEN, in preparation). However, the basal proportions and total levels of the mups appear to be determined by one or more additional genes. Strains carrying the same Mup-a allele, and having the same proportions of mups after induction, may differ considerably in the basal proportions and in the total amounts of mup excreted daily. Similarly Mup-al/Mup-as heterozygotes arising from different parental crosses show considerable differences in both basal mup proportions and total mup production, despite their identical Mup-a genotype. In addition, in both the basal and induced states, heterozygotes excrete as much or more mup than either homozygous parent. Such elevated levels of mup production in Mup-a heterozygotes presumably reflects complementation among several genes whose identities are presently unknown. W e thank LARRY P u s s for technical assistance on some experiments. This work was supported by Public Health Service grant GM-19521 from the National Institute of General Medical Sciences. P. R. SZOKA was a predoctoral trainee supported by the same grant. LITERATURE CITED

FERGUSON, K. A., 1964 Starch-gel electrophoresis-application to the classification of pituitary proteins and polypeptides. Metabolism (Clin. Exp.) 13 : 985-1002.

J. S., R. ASOFSRY, M. POTTER and C. C. RUNNER,1965 Major urinary protein comFINLAYSON, plex of normal mice origin. Science 149: 981-982.

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