P. R. Szoka1 and K. PAIGEN2 Manuscript Received July 13, 1978 Revised Copy Received June 13, 1979

GENETIC REGULATION OF MUP PRODUCTION IN RECOMBINANT INBRED MICE

P. R. szoKA1 AND K. PAIGEN2 Manuscript received July 13, 1978 Revised copy received June 13, 1979

ABSTRACT Inbred strains of mice excrete all three major urinary proteins (mups) when induced by testosterone, but differ as to the relative proportions and total levels of each mup present. We have now determined the urinary mup phenotypes before and after testosterone treatment of seven recombinant inbred strains derived from progenitor strains exhibiting different mup phenotypes. The results codirm previous observations indicating that total control of mup protein production is a multigenic process. One locus, Mup-a on chromosome 4, determines the relative mup protein proportions after induction by testosterone. Mup-a, together with other genetic sites, determines the basal mup proportions. Genes other than Mup-~determine the kinetics of mup induction and total mup excretion.

HE major urinary proteins (mups) of the inbred mouse are three electro- "p h oret'cally 1 separable proteins that are biochemically related. They exhibit identical molecular weights, have similar amino acid sequences, and cross-react antigenically (FINLAYSONet al. 1974; SZOKAand PAIGEN1978). Increased mup synthesis in the liver and excretion in the urine are induced by androgen (RUMKEand THUNG1964; FINLAYSONet al. 1965; OSAWAand TOMINO1977; SZOKAand PAIGEN1978). The mups represent an excellent model system for studying control of specific gene expression by steroid hormones since (1) the magnitude of the induction by androgens is large, and (2) inbred strains exhibit genetic variation in the relative amounts of each mup present after induction (FINLAYSON,POTTER and RUNNER1963; SZOKAand PAIGEN1978). Our previous results indicated that the induced mup protein proportions are determined by the Mup-a gene on chromosome 4. Mup-a also contributes to the determination of basal mup proportions, but is probably not the only locus determining this aspect of the mup phenotype (SZOKAand PAIGEN1978). Total mup levels before and after induction are also likely to be determined by genes other that Mup-a. To examine the roles of Mup-a and other loci participating in the expression of mup phenotype, we have determined the mup phenotypes of recombinant inbred (RI) strains derived from progenitor strains differing at the Mup-a locus. RI strains are produced by crossing two genetically different progenitor inbred

1 Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge Massa- chusetts 02139. 3 Molecular Biology Department, Roswell Park Memorial Institute, 666 Elm Street. Buffalo, New York 14263.

Genetics 93: 173-181 September, 1979. 174 P. R. SZOKA AND K. PAIGEN strains, and then inbreeding pairs of progeny to produce new sets of inbred strains. After a number of generations of brother-sister mating, the resulting recombinant strains are homozygous at nearly every locus, carrying alleles of one or the other progenitor strains (BAILEY1971). RI strains are useful for determining the number of genes involved in the regulation of a biochemical trait if that trait differs between the progenitor strains. The appearance of new phenotypes among the RI strains indicates that more than one gene determines that trait and may provide evidence concerning their respective functions (SWANK and BAILEY1973). The seven RI strains used here were developed from a cross of BALB/cByJ X CS 7BL/SBy (BAILEY1971 ) . The mup electrophoretic phenotypes of these lines were determined by POTTERet al. (1973) using untreated female mice. These investigators postulated that the illup-a locus defines a structural polymorphism controlling the presence or absence of mup 1 and mup 2. However. our previous study showed that Mup-a cannot define a structural polymorphism since genetically homozygous inbred mice excrete all three mups when treated with testosterone. Instead, Mup-a appears to act as a regulatory locus controlling the relative amounts of mup excretion after testosterone treatment In addition the total mup phenotype is complex and most likely determined by genes in addition to Mup-a (SZOKAand PAIGEN1978). Therefore, to test the validity of our previous results, we re-examined the mup phenotypes of the same RI strains used by POTTERd al. (1973). The results confirm our previous observations that Mup-a is a regulatory gene determining the induced proportions of the mups; Mup-a and additional genes are involved in controlling the relative proportions of the mup proteins before testosterone treatment. Genes other than Mup-a determine total mup levels before and after testosterone treatment and the rate of increase in mup excretion after testosterone treatment.

MATERIALS AND METHODS

Animdsr Female inbred mice used were between two and four months of age and were obtained from the Jackson Laboratory, Bar Harbor, Maine. Only female mice were used to avoid the problem of variation in endogenous androgen levels found among male mice. Urine col2ection and testosterone induction. Collection of urine samples and induction of mup excretion by subcutaneous implantation of 30 mg testosterone pellets were done as described previously (SZOKA and PAIGEN1978). Urine samples were collected at Whr intervals from at least six mice per strain housed in groups of two to four in metabolism cages. The same mice were used before and after testosterone treatment. Ebctrophoresis and densiiomelry: Quantitation of mup protein excretion by electrophoresis and densitometric scanning of the mups was done as described previously (SZOKA and PAIGEN 1978). The mup proteins were separated on polyacrylamide gels at pH 5.5. Under these condi- tions the mups are the only urinary proteins that migrate anodally.

RESULTS Quantitative studies of mup excretion by various inbred strains of mice have shown that the mup phenotype has two components: the relative proportions of the three mups excreted in the basal and induced states, and the total amounts REGULATION OF MUP PRODUCTlON 175 of the mups excreted ( SZOKAand PAIGEN1978). It is important to determine if only one, or both, of these components of the phenotype is controlled by the Mup-a gene on chromosome 4. Therefore, we determined the mup phenotypes of the seven recombinant inbred strains derived from progenitor strains BALB/ cByJ (C) and C57BL/6By (B), which are homozygous for Mup-al and Mup-az, respectively, before and during testosterone induction. Mup-a controls the induced mup proportions and electrophoretic patterns: After testosterone treatment, the relative proportions of the three mups are in- herited together as a unit phenotype (Table 1, Figure 1). All strains excrete mup 3. Four of the RI strains (CXBE, CXBG, CXBH and CXBJ) excrete much more mup 1 than mup 2 and had relative proportions similar to progenitor strain BALB/cByJ; they appear to carry the Mup-al allele. Mup 2 is excreted in trace amounts by these strains and consequently is difficult to quantitate. The three remaining RI strains (CXBD, CXBI, and CXBK) excrete more mup 2 than mup 1 and have relative proportions very much like the C57BL/ 6By progcnitor strain; they appear to carry the Mup-a2allele. That the RI strains exhibit only progenitor-like phenotypes suggests that a single gene, Mup-a, determines the relative proportions of the mups following induction. After induction, the strains carrying the Mup-az allele (C57BL/6By, D, I and K) also excreted very small amounts of another protein that migrates more slowly than mup 1 (Figure 1). Strains carrying the Mup-a' allele excreted this protein in variable amounts. Because it represents less than 5% of the total protein, we were unable to quantify its level of excretion with any accuracy. Mup-a plus other loci control the basal mup proportions: Before induction, the presence or absence of mup 1 and mup 2 is correlated with Mup-a (Table 2). The four recombinant inbred strains, E,H,G and J, and the progenitor strain BALB/cByJ, which all carry the Mup-a' allele, excrete mup 1, but not mup 2.

TABLE 1 Relative amounfs of each mup after induction*

Mup-a Strain Mup 1 Mup 2 Mup 3 assignment C 0.83 2 0.01 trace 0.17 t 0.01 E 0.82 f 0.02 trace 0.18 f 0.02 G 0.83 f 0.01 trace 0.17 f 0.01 H 0.76 +- 0.01 trace 0.M f 0.01 J 0.76 f 0.005 trace 0.24 t 0.005 B 0.20 t 0.01 0.37 f 0.01 0.43 f 0.008 D 0.16 t 0.008 0.4.2 +- 0.01 0.42 -C 0.008 I 0.15 f 0.01 0.44 f 0.02 0.39 t 0.OW K 0.11 t 0.006 0.36 f 0.004 0.53 f 0.008

Urine samples were collected on days ten and 12 after implantation of testosterone pellets, and daily mup excretion was quantitated as described in the MATERIALS AND METHODS section. Data presented as the mean of four cages +- s.e. for the progenitors BALB/cByJ (C) and C57BL/SBy (B) and as the mean of two cages t range for the seven RI lines. mg(mup I, mup 2, or mup 3) /mouse/day * Relative amounts of each mup = a mg(mup 1 4- mup 2 4- mup 3)/mouse/day 176 P. R. SZOKA AND K. PAIGEN

FIGURE1.-Electrophoresis and protein staining for the major urinary proteins from testosterone treated, female, (C x B) recombinant inbred mice. Aliquots containing 5 ccg protein from 24-hr urine collections were separated electrophoretically on pH 5.5 polyacrylamide gels and stained with Coomassie brilliant blue G.

The progenitor strain C57BL/6By and the two recombinant inbred strains, D and I, which all carry the Mup-az allele, excrete mup 2 but not mup 1. However recombinant inbred strain K, which also carries the Mup-a' allele, excretes both mup 1 and mup 2 before induction. This suggests that other genes in addition to Mup-a control the basal proportions of the mup proteins.

TABLE 2 Mup excretion before induction

Urine samples were collected and daily mup excretion quantitated as described in the MATERIAIS AND METHODS section. Data presented as the mean of four cages +- s.e. for the nitors BALB/cByJ (C) and C57BL/SBy (B) and as the mean of two cages -C l/2 range &% seven RI lines. N.D.= not detected. mg(mup 1, mup 2, or mup3)/mome/day Relative amounts of each mup = ' mg(mup I + mup 2 + mup 3)/mouse/day REGULATION OF MUP PRODUCTION 177 Determinations of the relative amounts of excreted mup 3 support this conclusion. For example, in both progenitor strains, mup 3 represented 20% of the total mup excreted. However, the two recombinant inbred strains, G and D, produced only trace amounts of mup 3, while nearly half of the total excreted mup in recombinant inbred strain J was mup 3 (Table 2). The observation that several of the recombinant inbred strains have basal mup proportions different from those of the progenitor strains implies that genes in addition to Mup-a control this aspect of the mup phenotype. Genes other than Mup-a control total mup Lxcretion: Before induction, the total levels of the mups excreted are not correlated with the Mup-a genotype of the strains (Table 2). For example, among the RI strains carrying the Mup-a' allele, urinary mup 1 levels varied over a five-fold range, from 0.11 to 0.56 mg per mouse per day. Among strains carrying the Mup-ae allele, the levels of mup 2 excreted varied over a three-fold range from 0.5 to 1.5 mg per mouse per day. Nor were levels of mup 3 or total levels of all three mups correlated with the Mup-a genotype. SWANKand BAILEY(1973) demonstrated that RI strains can be used to ana- lyze the induction kinetics of a protein in response to a hormonal stimulus. To determine whether Mup-a controls the rate of increase of mup excretion after induction of mup synthesis by testosterone, we measured the kinetics of mup induction in the progenitor and RI strains (Figures 2-5). Two strains, J and

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o024681012 246 8012 24 6 81012 Time (days) FIGURE2.-Induction of mup 1 excretion: After implantation of the testosterone pellets at day zero, urine samples were collected and analyzed as described in the legend to FIGUREI. (a) Induction kinetics of the progenitor strains C57BL/SBy (B; Mup-h) and BALB/cByJ (C; Mup-a'); (b) induction kinetics of RI strains carrying Mup-a'; (c) induction kinetics of RI strains carrying Mup-a&;the shaded gray area represents the range of mup 1 excretion for the progenitor strains. 178 P. R. SZOKA AND K. PAIGEN

Time (days)

FIGURE3.-Induction of mup 2 excretion: After implantation of the testosterone pellets at day zero, urine samples were collected and analyzed as described in the legend to Figure 1. (a) Induction kinetics of the progenitor strains C57BL/SBy (B; Mup-as) and BALB/cByJ (c; Mup-al); (b) induction kinetics of RI strains carrying Mup-as (closed symbols) and Mup-~’ (open symbols) ; the shaded gray area represents the range of mup 2 excretion for the C57BL/6By progenitor.

E, had faster rates of induction of mup 1 and mup 3 excretion compared to either progenitor strains (Figures 2 and 4). In addition, strain K had a faster rate of induction of both mup 2 and mup 3 excretion compared to the progenitor strains (Figures 3 and 4). Since three of the seven RI strains had induction curves different from either progenitor strain, we conclude that genes other than Mup-a control the kinetics of mup induction.

Time (days) FIGURE4.-Induction of mup 3 excretion: After implantation of the testosteron pellets at day zero, urine samples were collected and analyzed as described in the legend to Figure 1. (a) induction kinetics of the progenitor strains C57BL/SBy (B; Mup-us) and BALB/cBy (C; Mup-a’); (b) induction kinetics of RI strains carrying Mup-a’; (c) induction kinetics of RI strains carrying Mup-a2; shaded gray area represents the range of mup 3 excretion for the progenitor strains. REGULATION OF MUP PRODUCTION 179 t"

2468DE 24681012 ri(days) FIGUIRE5.-Induction of total mup excretion: After implantation of the testosterone pellets at day zero, urine samples were collected and analyzed as described in the legend to Figure 1. (a) Induction kinetics of the progenitor strains C57BL/6By (B; Mup-us) and BALB/cByJ (C; Mup-al); (b) induction kinetics of RI strains carrying Mup-U'; (c) induction kinetics of RI strains carrying Mup-us; shaded gray area represents the range of total mup excretion for the progenitor strains.

Fully induced levels of mup excretion are also not controlled by the Mup-a gene, since the absolute amounts of the mups excreted by several of the RI strains differed markedly from those of either progenitor strain. For example, strain J excreted more mup 1 and mup 3 (Figures 2 and 4); strain K excreted more mup 2 and mup 3 (Figures 3 and 4) ; and strain G excreted less of the mups than either progenitor strain (Figures 2,4 and 5).Thus, like basal levels, the induced levels of mup excretion are not controlled by the Mup-a locus. It seems likely that the genes determining the time course of mup induction are different from those determining the final absoluate levels of mup excretion, since two of the RI strains, E and K, which induce faster than the progenitor strains, nevertheless excrete the same total mup levels as the progenitor strains (Figure 5). Since there are several new phonotypes among the RI strains involving the rate of induction by testosterone, as well as total mup excretion, both basal and induced, it is not possible to determine the number of genes controlling these aspects of the phenotype and whether they are linked to Mup-a.

DISCUSSION Analysis of the phenotypes of a set of RI strains can reveal whether a trait is determined by a set of alleles at one locus or by a number of different genes. If the trait is determined by a set of alleles, then only the two phenotypes of the progenitor strains will be observed among the RI strains. However, appearance of new phenotypes among the strains indicates the involvement of more than one gene. If the number of new phenotypes is limited, it may allow determination of the number of genes controlling that phenotype (SWANKand BAILEY 1973). 180 P. R. SZOKA 4ND K. PAIGEN After testosterone treatment, the Mup-a gene alone appears to determine the relative mup proportions, since there are only progenitor-like phenotypes for mup proportions observed among the RI strains. Although the number of RI strains examined here was small, the chance that a second unlinked gene determining the induced mup proportions co-segregates with Mup-a is (1/2)' or 1/128. We feel this is unlikely since Mup-al/zheterozygotes express intermedi- ate mup proportions after testosterone treatment (SZOHAand PAIGEN1978). The additive expression of Mup-a by heterozygoes combined with the expression of only parental mup phenotypes among the RI strains together suggest that Mup-a acts as a single regulatory locus controlling the induced mup proportions. Our assignment of the Mup-a genotypes among these RI strains based on the induced mup proportions agrees with those previously published (POTTERet al. 1973). In addition, Mup-a influences the basal phenotype since segregation at this locus is correlated with the qualitative phenotype of the presence or absence of mup 1 and mup 2. However, several of the RI strains exhibit nonprogenitor phenotypes for basal mup proportions and absolute levels. Therefore, it is likely that genes in addition to Mup-a participate in the regulation of mup production before testosterone treatment. After testosterone treatment, genes other than Mup-a control the rate of increase in mup excretion and the final urinary mup levels, since several of the RI strains have mup induction curves different from those of the progenitor strains. In sum, the studies with RI strains support our original conclusion that Mup-a is a regulatory locus determining the relative proportions of the three mups after induction, and demonstrate that additional loci participate in determining other aspects of mup regulation. Thus, the mup system is more complex than originally anticipated. They also demonstrate the power of RI strains in the analysis of complex phenotypes. Special thanks to F. BERGERfor helpful discussions. This work was supported by Public Health Services Grant GM-19521. P. R. SZOKAwas a predoctoral trainee supported by this same grant.

LITERATURE CITED

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