Proc. Nati. Acad. Sci. USA Vol. 75, No. 11, pp. 5623-5626, November 1978 Genetics

Location on 1 of Rnr, a gene that regulates renin in the submaxillary gland of the mouse (androgen action/hypertension/recombinant inbred strains/regulatory genes) CAROL M. WILSON*t, ERVIN G. ERDoS*t, JEAN D. WILSONtI, AND BENJAMIN A. TAYLOR§ Departments of *Pharmacology and tInternal Medicine, and tthe Eugene McDermott Center for Growth and Development, The University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, Texas 75235; and §The Jackson Laboratory, Bar Harbor, Maine 04609 Communicated by P. Kusch, August 21, 1978

ABSTRACT Renin activity (EC 3.4.99.19) was measured in MATERIALS AND METHODS submaxillary gland extracts from four sets of recombinant inbred mouse strains. Recombination between Rnr, a gene that Animals. Inbred strains of mice were obtained from the mediates the susceptibility of submaxillary gland renin to in- Jackson Laboratory. BXD recombinant inbred strains were duction by androgen, and Dip-i, a chromosome 1 marker, was derived by brother-sister inbreeding, beginning with the F2 found in only 1 of 51 recombinant inbred strains, indicating that generation from the cross of C57BL/6J and DBA/2J inbred the two genes are closely linked on chromosome 1. Renin ac- mice (13). AKXL recombinant inbred strains were similarly tivity in androgen-treated female mice of all recombinant inbred derived from the cross of AKR/J and C57L/J (14), and SWXL strains resembled that of one or the other progenitor strains, as recombinant inbred strains the cross expected if a single gene is responsible. Documentation that a fyom of SWR/J and single gene can have major effects on renin in the submaxillary C57L/J (15) inbred mice. HP/Ei is a single recombinant inbred gland of the mouse implies that single gene differences might strain derived from AKR/J and C57BL/6J inbred mice (16). explain known variations in renin in other species. Sample Preparation and Assays. Eight-week-old female mice were treated with 2 mg of dihydrotestosterone in 0.2 ml Renin is a proteolytic (EC 3.4.99.19) that cleaves a of sesame oil (induced) or sesame oil alone (control) every other plasma protein to release angiotensin I. Angiotensin day for one week. Submaxillary glands were removed, ho- I is subsequently converted to angiotensin II, a potent vaso- mogenized, and centrifuged for 60 min at 104,000 X g as pre- pressor agent. Regulation of plasma renin activity plays an viously described (11). Renin enzymatic activity in the super- important role in the maintenance of normal blood pressure natant fraction was assayed essentially as previously described under conditions such as sodium deprivation (1); moreover, (11). The final incubation mixture (63 ,ul) contained supernatant elevated renin levels in plasma constitute an important cause (0.02-50.ug protein), 4 mM EDTA, 1 mM o-phenanthroline, of hypertension in humans (2). The is the primary source 1 mM diisopropylfluorophosphate, 13 mM 2,3-dimercap- of plasma renin. However, in some strains of mice, submaxillary topropanol, 50 mM Tris-HCl, and partially purified hog an- glands contain large amounts of renin, exceeding that of the giotensinogen containing the equivalent of 0.3 nmol of angio- kidney (3, 4). Submaxillary gland is tensin I (final pH 7.8). The supernatants had been preincubated renin present in higher at a protein concentration of 0.6-2 mg/ml for 15 min at 37'C concentration in males than in females and can be induced in in the presence of the o-phenanthroline/diisopropylfluoro- females by treatment with androgen (5-7). The submaxillary phosphate/2,3-dimercaptopropanol/Tris mixture to inhibit gland enzyme has been purified to homogeneity (8) and appears angiotensin I converting enzyme and angiotensinases. The re- indistinguishable from mouse kidney renin (9, 10). Using two action was initiated by adding aliquots of preincubated su- inbred strains of mice that differ widely in submaxillary gland pernatant to the substrate. After incubation of the final mixture renin activity, we have previously shown by segregation analysis at 37°C for 10 min, the reaction was stopped by freezing at that the susceptibility of submaxillary gland renin to induction -50°C, and the amount of angiotensin I generated was deter- by androgen is apparently mediated by a single gene (11). Al- mined by radioimmunoassay. Results are expressed as nmol though the possibility of variation in renin structure has not angiotensin I produced per min at 37°C. In some experiments, been excluded, this gene seems to regulate the synthesis of renin. samples from two different strains were mixed, either before We therefore propose to designate the gene renin regulator and or after preincubation, and assayed as described. the gene symbol Rnr.1 Rnrs and Rnrb are designated as symbols The supernatant fraction was analyzed for high or low renin for alleles carried by SWR/J and C57BL/10J, the high and low concentration by double diffusion versus antiserum to mouse reference strains, respectively. submaxillary gland renin as previously described (11), except In the current study we analyzed submaxillary gland extracts that 25-,ul samples from control animals were used. Epidermal from control and androgen-treated female mice of 24 additional growth factor was also estimated by immunodiffusion using inbred strains for renin. Four informative sets of recombinant 1O-.sl samples from either control or induced animals. Protein inbred mouse strains were identified and tested to confirm that was measured by the method of Lowry et al. (17) the induction of renin by androgen is determined by a single Reagents. Standard angiotensin I was obtained from Beck- gene and to locate this gene on the mouse linkage map. man. Antiserum to angiotensin I was the gift of T. F. Good- We have symbolized the gene that controls the regulation of renin The publication costs of this article were defrayed in part by page activity Rnr, in accordance with the terminology used by Paigen et charge payment. This article must therefore be hereby marked "ad- al. (12) for genes that are involved in the realization of f3-glucuron- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate idase activity in mice. With this terminology, the structural gene for this fact. renin would be symbolized Rns. 5623 Downloaded by guest on September 29, 2021 5624 Genetics: Wilson et al. Proc. Natl. Acad. Sci. USA 75 (1978)

friend, University of Wisconsin. Antisera to mouse submaxillary creasingly powerful tools for linkage testing. To map a newly gland renin and epidermal growth factor were the gift of T. discovered polymorphism it is necessary only to type the Inagami and S. Cohen, Vanderbilt University. Angiotensinogen recombinant inbred strains for that locus; the strain distribution was prepared from hog serum by the method of Green (18) pattern thus obtained is compared with previously defined followed by chromatography on a preparative Sephadex G-150 patterns to evaluate linkage relationships (19, 21). In addition column or was purchased from Miles Laboratories. to detecting linkage, in favorable cases it is also possible to infer the correct gene order and to estimate map distances (13- 15). RESULTS Fifty-one recombinant inbred strains derived from four All inbred strains of mice tested fall into two distinct classes. original crosses between high and low renin progenitor strains SWR/J, AKR/J, AU/SsJ, BDP/J, BUB/BnJ, CBA/J, DBA/1J, were available-24 BXD strains (13) derived from DBA/J DBA/2J, NZB/B1NJ, P/J, PL/J, RF/J, SJL/J, and SM/J pro- (high) and C57BL/6J (low); 19 AKXL strains (14) derived from duced immunodiffusion bands with antiserum to renin both AER/J (high) and C57L/J (low); 7 SWXL strains (15) derived before and after treatment with androgen. These strains are from SWR/J (high) and C57L/J (low); and HP/Ei (16), a single presumed to carry the Rnrs allele. C57BL/10J, A/J, BALB/cJ, recombinant inbred strain derived from AKR/J (high) and CBA/CaJ, CE/J, C3H/HeJ, C57BL/6J, C57BR/cdJ, C57L/J, C57BL/6J (low). The recombinant inbred strains could be LP/J, SEA/GnJ, and SEC/lReJ produced no detectable im- classified unambiguously as either low renin (Rnrb) or high munodiffusion bands even when extracts from androgen- renin (Rnrs) strains by testing either control or androgen-treated treated animals were examined. These strains are presumed to mice, indicating that genetic fixation had occurred in all 51 carry the Rnrb allele. strains. Segregation at the Rnr locus was concordant with Dip-i, We have employed recombinant inbred strains (19) derived a gene that determines electrophoretic variation in the enzyme from crosses between high and low renin inbred strains to locate -1 (22, 23), in 50 of the 51 recombinant inbred the Rnr locus on the mouse linkage map (20). Recombinant strains (Table 1), indicating close linkage (P < 0.001). The inbred strains are derived by continuous inbreeding from the Dip-la allele is found in C57BL/6J and C57L/J, and Dip-lb F2 generation of a cross between two unlike highly inbred is found in AKR/J, SWR/J, and DBA/2J (14, 24). The single progenitor strains. Unlinked pairs of genes reassort indepen- instance of recombination between Rnr and Dip-i is found in dently in the F2 generation. Therefore each recombinant inbred the HP/Ei strain, which is fixed for the Rnrb allele from strain is equally likely to become fixed for parental or recom- C57BL/6J and the Dip-lb allele from AKR/J. The Dip-l locus binant genotypes. Linked pairs of genes tend to be transmitted is on chromosome 1 in the mouse. Linkage to two other chro- together during the inbreeding process and are therefore more mosome 1 markers, leaden coat color (In) and adrenal lipid likely to become fixed in parental combinations. The recom- depletion (ald) (14) is documented in Table 1. These findings binant inbred strains are typed for numerous polymorphic loci indicate that Rnr is also located on chromosome 1. that distinguish the progenitor strains. As the number of typed Segregation of Dip-1, Rnr, and aid in the AKXL and HP/Ei loci increases, the recombinant inbred strains become in- strains favors the gene order: Dip-1-Rnr-ald. The ald locus has

Table 1. Segregation of Rnr and chromosome 1 markers in BXD, AKXL, SWXL, and HP/Ei recombinant inbred strains of mice BXD Number Locus 1 2 5 6 8 9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 27 28 29 80 Crossover of strains Dip-1 B B D D B B D B D B D B B B D B B D D B D DD D Dip-l-Rnr 0 Rnr B B D D B B D B D B D B B B D B B D D B DD D D AKXL Number Locus 2 5 6 7 8 9 12 13 14 16 17 19 21 24 25 28 29 37 38 Crossover of strains In *L L A A A L A A L A L L A A L A L L X X X X X X X X 1n-(Dip-l,Rnr) 8 Dip-1 AAA L A A A L L L A LL A A A A L A Dip-1-Rnr Rnr A A A- L -A A A L LL A L L A A A A L A 0 X X X X (Dip-1,Rnr)-ald 4 ald A A A A A A L L L L A LL L A L A L A SWXL Number Number Locus 4 7 12 14 15 16 17 Crossover of strains Locus HP/Ei Crossover of strains In L S L S L L L Dip-l A X X 1n-(Dip-1,Rnr) 2 X Dip-i-Rnr 1 Dip-i L S L S L SS Rnr B Dip-1-Rnr 0 Rnr-ald 0 Rnr L S L S L S S ald B Chromosome 1 markers are: dipeptidase-1 electrophoretic variant (Dip-i), leaden coat color (In), and adrenal lipid depletion (ald). A, B, D, L, and S are used as generic symbols for alleles inherited at each locus from progenitor strains AKR/J, C57BL/6J, DBA/2J, C57L/J, and SWR/J, respectively. The AKR/J genotype is + Dip-lb Rnrs ald/+ Dip-lb Rnrs ald. The C57BL/6J genotype is + Dip-la Rnrb +/+ Dip-la Rnrb +. The DBA/2J genotype is + Dip-lb Rnrs +/+ Dip-1b Rnrs +. The C57L/J genotype is In Dip-1a Rnrb +/in Dip-1a Rnrb +. The SWR/J genotype is + Dip-lb Rnr' +/+ Dip-lb Rnrs +. Regions where crossovers have resulted are denoted by an X. * Genotype not determined. Downloaded by guest on September 29, 2021 Genetics: Wilson et al. Proc. Natl. Acad. Sci. USA 75 (1978) 5625 recombined with Rnr and Dip-l in AKXL-7, AKXL-12, Quantitative estimates of renin activity in the recombinant AKXL-24 and AKXL-28, while Dip-i has recombined with Rnr inbred strains and in the progenitor strains from which these and aid in the HP/Ei strain. An alternative gene order, Rnr- were derived are presented in Fig. 1. Because the enzyme ac- Dip-l-ald, would require two recombination events within a tivity in each recombinant inbred strain after treatment with relatively short distance in the HP/Ei strain. androgen is either like that of the low renin progenitor or the The single recombinant between'Rnr and Dip-i indicates high renin progenitor, we conclude that variation at a single that the two genes are not identical and provides a basis for the locus is responsible for the difference in activity between inbred estimation of map distance. Estimation of recombination fre- strains after androgen treatment (21). The separation between quency from these data must account for the fact that there are high and low strains is less clear cut in mice that have not been multiple opportunities for recombination in the process of de- treated with androgen. The occurrence of some recombinant veloping a recombinant inbred strain. As previously described inbred strains with control submaxillary renin levels interme- (14), the probability of fixation of a recombinant genotype (R) diate between those found in the progenitor strains suggests, with respect to two loci that recombine with a frequency r is as did our previous study (11), that while inducibility is deter- 4r/(1 + 6r). In the present case R is estimated to be 1/51 and mined by a single gene, additional genes as yet unidentified the estimated recombination frequency (r) between Rnr and influence the basal level of renin activity. Dip-l is 0.0050 + 0.0052. The frequency of recombination A/J, CBA/CaJ, CE/J, C3H/HeJ, and HP/Ei samples were between Rnr and aid is estimated to be 0.071 + 0.046 on the tested for the presence of a renin inhibitor by mixing each with basis of 4 recombinants among 20 recombinant inbred strains. a high renin (SWR/J) sample prior to the quantitative deter- (Only the AKXL and the HP/Ei strains are informative in the mination of renin activity. Additive values were obtained in latter case because aid is a recessive gene that has been found each case, indicating that no inhibition of the high renin or only in the AKR strain and its descendants.) activation of the low renin samples has resulted (data not pre- There is apparent linkage disequilibrium for Rnr and Dip-i sented). among the inbred strains tested. All 14 high renin strains and Control and androgen-treated females of all inbred strains four of the low renin strains (A/J, CBA/CaJ, CE/J, and and of the recombinant inbred strain, HP/Ei, were tested by C3H/HeJ) carry the Dip-lb allele, the remaining eight low immunodiffusion (11) for epidermal growth factor, a peptide renin strains carry the Dip-la allele (24, 25; T. Roderick, per- of the submaxillary gland that is regulated by androgen (26). sonal communication). The two CBA substrains differ with In each instance epidermal growth factor was undetectable in respect to the Rnr locus. These substrains were separated in control animals and easily detectable after androgen treatment. 1932 and are known to differ at several polymorphic loci, in- Thus, these strains retain a similar capacity for response to an- cluding the Mls locus, which maps 17 recombination units distal drogen in regard to epidermal growth factor despite the fact to Dip-i on chromosome 1 (13). The CBA strain was evidently that they differ markedly in the inducibility of renin by the heterogeneous with respect to several chromosomal segments same treatment. It may be inferred that the renin regulatory at the time the substrains were established. gene affects only a specific androgen response.

1 10 1 10 20 30 4o Renin activity, nmol angiotensin I/min per mg protein FIG. 1. Submaxillary gland renin activity in 5 progenitor strains of mice and 51 strains of mice from 4 recombinant inbred (RI) sets. The range of values for progenitor strains is indicated by arrows, and the number of animals tested for each progenitor strain is shown in parentheses. Recombinant inbred strains classified as low in renin activity according to the immunodiffusion test are represented by open circles, and those classified as high in renin are represented by filled circles. Each circle represents an average value from two animals. Control values for two recombinant inbred strains (AKXL-28 and AKXL-29) were not available. Downloaded by guest on September 29, 2021 5626 Genetics: Wilson et al. Proc. Natl. Acad. Sci. USA 75 (1978) DISCUSSION 5. Oliver, W. J. & Gross, F. (1967) Am. J. Physiol. 213,341-346. 6. Bhoola, K. D., Dorey, G. & Jones, C. W. (1973) J. Physiol. 235, Results obtained with (3-glucuronidase and f3-galactosidase, two 503-522. other that have been studied in great detail in the 7. Gescse, A., Wilson, C. M. & Erdos, E. G. (1976) Biochem. Phar- mouse, indicate that the mammalian genome may contain macol. 25, 763-768. many functional units in which genes that control the regulatory 8. Cohen, S., Taylor, J. M., Murakami, K., Michelakis, A. M. & In- and developmental information for a protein are located near agami, T. (1972) Biochemistry 11, 4286-4292. the gene that codes for the structure of the protein (12, 27). 9. Michelakis, A. M., Yoshida, H., Menzie, J., Murakami, K. & In- Individuals heterozygous for these linked regulatory elements agami, T. (1974) Endocrinology 94, 1101-1105. are intermediate with respect to enzyme activity, an observation 10. Malling, C. & Poulsen, K. (1977) Biochim. Biophys. Acta 491, consistent with cis expression. The fact that heterozygotes at 542-550. J. F. & J. D. the Rnr locus are also strictly intermediate in renin expression 11. Wilson, C. M., Erdos, E. G., Dunn, Wilson, (1977) renin Proc. Natl. Acad. Sci. USA 74, 1185-1189. (11) is compatible with the possibility that the structural 12. Paigen, K., Swank, R. T., Tomino, S. & Ganschow, R. E. (1975) gene is also near the Rnr locus. Because no difference has yet J. Cell Physiol. 85, 379-392. been found between submaxillary renin and kidney renin from 13. Festenstein, H., Bishop, C. & Taylor, B. A. (1977) Immuno- the same mouse strain, the chromosomal location of a gene that genetics 5, 357-361. controls submaxillary renin is of particular interest. The human 14. Taylor, B. A. & Meier, H. (1976) Genet. Res. Camb. 26, 307- homologue of Dip-i is the structural gene for peptidase-C 312. (PEPC) (28), which has been assigned to the long arm of 15. Taylor, B. A. (1976) Genetics 83,373-377. chromosome 1 of man by somatic cell hybridization (29, 30). 16. Taylor, B. A., Bedigian, H. G. & Meier, H. (1977) J. Virol. 23, If the mouse renin structural gene is located near the Rnr locus, 106-109. it is possible that the human renin structural gene is located near 17. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. PEPC. The confirmation that a single gene has a major effect (1951) J. Biol. Chem. 193, 265-275. 18. A. A. in 2, 135-139. in influencing submaxillary gland renin in the mouse implies Green, (1955) Methods Enzymol. 19. Bailey, D. W. (1971) Transplantation 11, 325-327. that differences in renin activity among individuals of other 20. Womack, J. E. (1977) Mouse News Lett. 57,6. species may also be controlled by single-gene effects. 21. Swank, R. T. & Bailey, D. W. (1973) Science 181, 1249-1252. 22. Lewis, W. H. P. & Truslove, G. M. (1969) Biochem. Genet. 3, The technical assistance of Tess A. Stewart and Jacqueline L. Uhr 493-498. is acknowledged. This work was supported by Grants AM03892, 23. Chapman, V. M., Ruddle, F. H. & Roderick, T. H. (1971) Bio- HL20594, GM 18684, and 5-S07-RR05426 from the National Institutes chem. Genet. 5, 101-110. a Affiliate of the American Heart of Health and grant from the Texas 24. T. H., F. H., Chapman, V. M. & Shows, T. B. Association awarded to C.M.W. The Jackson Laboratory is fully ac- Roderick, Ruddle, credited by the American Association for the Accreditation of Labo- (1971) Biochem. Genet. 5, 457-466. ratory Animal Care. 25. Taylor, B. A. (1972) J. Hered. 63,83-86. 26. Byyny, R. L., Orth, D. N., Cohen, S. & Doyne, E. S. (1974) En- 1. Samuels, A. I., Miller, E. D., Jr., Fray, J. C. S., Haber, E. & Barger, docrinology 95, 776-782. A. C. (1976) Fed. Proc. Fed. Am. Soc. Exp. Biol. 35, 2512- 27. Breen, G. A. M., Lusis, A. J. & Paigen, K. (1977) Genetics 85, 2520. 73-84. 2. Laragh, J. H. & Sealey, J. E. (1977) Cardiovasc. Med. 2, 1053- 28. Rapley, S., Lewis, W. H. P. & Harris, H. (1971) Ann. Hum. 1075. Genet. 34, 307-320. 3. Werle, E., Vogel, R. & Goldel, L. F. (1957) Arch. Exp. Path. 29. Ruddle, F., Ricciuti, F., McMorris, F. A., Tischfield, J., Creagan, Pharmakol. 230, 236-244. R., Darlington, G. & Chen, T. (1972) Science 176, 1429-1431. 4. Bing, J. & Poulsen, K. (1971) Acta Path. Microbiol. Scand, Sect. 30. Burgerhout, W., van Someren, H. & Bootsma, D. (1973) Hu- A 79, 134-138. mangenetik 20, 159-162. Downloaded by guest on September 29, 2021