Proc. Natl. Acad. Sci. USA

Vol. 76, No. 12, pp. 6539-6541, December 1979 Genetics

Regulation of mitochondrial malic enzyme synthesis in mouse brain (regulatory genes/rate of enzyme synthesis/gene dosage)

EDWARD G. BERNSTINE, CHONGKUN KOH, AND CAROLYN CAIN LOVELACE Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

Communicated by William L. Russell, September 21, 1979

ABSTRACT In a previous study [Bernstine, E. G. (1979)1. Biol. Chem. 254, 8387] it was shown that inbred strains of mice fall into two classes based on the specific activity of mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.401 in brain. In this report we demonstrate differences between high- and low-activity strains in the development of enzyme activity levels in adult mice and show that the rate of enzyme synthesis quantitatively accounts for the inherited level of the brain enzyme. Genetic analysis has established that the locus controling the amount of enzyme in brain (Mdr-1) is located on chromosome 7. Its linkage to Hbb and c places it in the same region of the chromosome as Mod-2, the structural gene for mitochondrial malic enzyme. By making use of deletions and a duplication that include Mod-2, evidence for cis action of Mdr-1 was obtained.

Mitochondrial malic enzyme [L-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40] expression varies with the number of copies of its structural gene (Mod-2) present in a given animal (1-4). For animals carrying three copies of Mod-2 , resulting from an unbalanced X-autosome translocation [T(X;7)Ctl ] in males (3), and for a large series of deletions involving the adjacent c-locus (4), the relationship between gene dosage and enzyme activity is clearly linear. The linear relationship between gene dosage and enzyme activity in all of these systems indicates that no regulation of the level of mitochondrial malic enzyme occurs to compensate for the presence of either a smaller or greater number of copies of the Mod-2 gene than is normally present in diploid animals. A recent survey of 10 inbred strains has shown that whereas the specific activity of the enzyme in heart is invariant, the strains fall into two classes on the basis of enzyme-specific activity in extracts of brain mitochondria (5). The enzyme has been extensively purified from mouse heart and brain, and antibody against it has been produced in rabbits (5). By use of this antibody it was shown that the difference in the specific activity of the enzyme in brain mitochondria between C3H and C57BL/10 mice reflected the presence of more enzyme molecules in C3H individual and that the difference segregated as a single genetic locus designated Mdr-1 (5). In this report we have examined the postnatal development pattern of enzyme activity in brain and have genetically mapped Mdr-1. In addition, we have shown that Mdr-1 regulates the rate of synthesis of mitochondrial malic enzyme in brain. Data obtained from gene dosage studies are consistent with a cis mode of action of Mdr-L. MATERIALS AND METHODS Malic enzyme activity was determined spectrophotometrically at 260C. Each reaction mixture contained 40 mM triethanolamine-HCl (pH 7.5), 4 mM MnCl2, 0.23 mM NADP+, and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate

this fact.

Age, days

FIG. 1. Postnatal development of mitochondrial malic enzyme activity in brain. Mitochondrial pellets were prepared (4) from brains of individual animals of the ages indicated and were assayed for enzyme activity and protein content (7). &, C3H; 0, (C3H X C57BL/ 10)FI; 0, C57BL/10.

5 mM potassium malate (pH 7.6). One unit of enzyme reduces 1 pmol of NADP+ per min at 260C. Specific activities are given as units/mg of protein. Protein was determined by the method of Lowry et al. (6). Mitochondrial pellets were prepared and lysed as described (4). The rate of synthesis of mitochondrial malic enzyme was determined by pulse labeling with either [3H]leucine or [35S]methionine injected intracranially. Labeled extracts of mitochondria were heated at 500C for 8 min, centrifuged, and applied to columns of NADP+-agarose. After the column was washed, the enzyme was eluted with 500 Ag of NADP+ per ml. Carrier enzyme and sufficient antibody were added to the eluate, which was incubated at 370C for 60 min and then overnight on ice. Precipitates were collected and washed by centrifugation, dissolved in sodium dodecyl sulfate (NaDodSO4) sample buffer (7), and run in 10% NaDodSO4/polyacrylamide gels at 5 mA per gel. Gels were frozen and then sliced. The radioactivity in gel slices was measured by liquid scintillation spectrometry after oxidation with H202 at 650 C. Hemoglobin was analyzed on starch gels by the method of Jacobson and Vaughan (8). RESULTS The development of mitochondrial malic enzyme activity as a function of time after birth in C3H/RI and C57BL/1ORl mice and their F1 hybrid is shown in Fig. 1. The adult levels of activity were reached in 8 weeks. C3H is readily distinguished from C57BL/10 by both its higher plateau value of enzyme specific activity and its rate of increase during development. Adult brain enzyme activity of the F1 animals is clearly intermediate to and distinguishable from both parents. Chromatography on DEAE-cellulose, which resolves the mitochondrial enzyme from the cytoplasmic form (4, 5), has shown that the Abbreviation: NaDodSO4, sodium dodecyl sulfate.

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Genetics: Bernstine et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Backcross progeny of (C57BL)/1ORl X C3H/Rl)Fl X C57BL/1ORl* Genotype No. Mdr-1B Hbbs Mdr-JB Hbbs

20

Mdr-JA Hbbd Mdr-1B Hbbs

25

Mdr-JB Hbbs Mdr-1B Hbbd Mdr-JA Hbbs 1 Mdr-1B Hbbs * Mdr-1 occurs in two allelic forms: high mitochondrial malic enzyme activity in brain (Mdr-1A) and low activity (Mdr-1B). C57BL/1ORl is Mdr-JB Hbbs/Mdr JB Hbbs; C3H/Rl is Mdr-JA Hbbd/Mdr-

JA Hbbd.

measured increase in activity during the first 8 weeks after birth reflects the appearance of the mitochondrial enzyme only (unpublished results of E.G.B.). Similar developmental studies of this enzyme activity in heart showed no difference between C3H and C57BL/10 animals; the developmental patterns resembled that of the enzyme in C57BL/10 brain. The genetic segregation of mitochondrial malic enzyme activity in brain was shown to be consistent with control by a single genetic determinant (5). As shown in Table 1, this determinant (Mdr-1) is linked to Hbb, the f3-globin structural gene, on chromosome 7. Forty-seven (C57BL/10 X C3H)F1 X C57BL/10 backeross progeny were screened for Hbb type and enzyme specific activity in brain mitochondria. Only two recombinants between Mdr-1 and Hbb were detected. In order to obtain a more precise genetic localization of the determinant of the tissue-specific difference in enzyme development, we set up a three-point cross in which alleles at c, Mdr-1, and Hbb were segregating by using (SEC/RI X 101/RI)F1 males backcrossed to SEC/RI females. All backcross progeny could be rapidly screened for crossovers between c and Hbb (6 cM) by coat color and electrophoresis of hemoglobin without killing the animals. Thirteen such animals, out of 240 progeny, were identified (Table 2). If Mdr-1 were distal to Hbb, then these two loci would not be expected to segregate among these recombinants. As shown in Table 3, however, Mdr-1 is clearly separated from Hbb whereas it is very tightly linked to c. Because recombinants between c and Hbb were selected, the order is c-Mdr-l-Hbb, with extremely close linkage of c and Mdr-1. Table 2. Backcross progeny of (SEC/Rl X 101/Rl)Fj X SEC/Rl* No. Genotype + Hbbd cch

Hbb8

122

cch Hbbs

cch Hbb8

105

+ Hbbs

cch Hbbs

6

cch Hbbd 7 cch Hbbs *(SEC X 101)F, animals are genotypically + Hbbd (Mdr-1A)/ cch Hbbs (Mdr-1B); SEC animals are cch Hbbs (Mdr-1B)/ cch Hbbs

(Mdr-jB).

Table 3. Analysis of offspring showing recombination between c and Hbb from the backcross (SEC X 101)Fl X SEC Alleles Specific Alleles present at activity at c locus Animal Hbb locus of enzyme* Mdr-1 1 +/cch s/s 0.045 A/B 2 +/Cch s/s 0.041 A/B cch/cch 3 sld B/B 0.025 cch/cch 4 sld 0.024 B/B 5 cch/cch sld 0.025 B/B 6 cch/cch sld 0.023 B/B 7 +/cch s/s 0.042 A/B 8 +/Cch s/s 0.049 A/B 9 cch/cch s/d 0.028 B/B 10 cch/cch s/d 0.024 B/B 11 +/c ch s/s 0.048 A/B 12 cch/cch s/d 0.028 B/B 13 +/cch s/s 0.028 B/Bt * Mitochondrial malic enzyme in brain, units per mg of protein. t This is the only animal in which recombination occurred between c and Mdr-1.

As a check of our methods, 50 animals that were scored as nonrecombinants between c and Hbb were killed to obtain their brain enzyme levels. All animals of type +c Hbbd/cch Hbbs were Mdr-1 A/Mdr-J B (i.e., intermediate specific activity), whereas all animals of type cch Hbbs/cch Hbbs were Mdr1 B/Mdr-J B (i.e., low specific activity). Although the detailed mechanism of action of Mdr-1 remains to be elucidated, we have shown that the overall difference between brain enzyme activities in C3H and C57BL/10 is quantitatively accounted for by a higher rate of enzyme synthesis in the former strain (Table 4). In these experiments, the amount of radioactivity incorporated into immunoprecipitable enzyme in a 2-hr pulse-labeling period was normalized to the total incorporation into brain for both strains. . Identification of the mitochondrial malic enzyme subunit (5) in the immunoprecipitate was made on NaDodSO4/polyacrylamide gels (Fig. 2). Animals used were 8-10 weeks old and, thus, the enzymes had attained plateau values of activity. The results given in Table 4 are those of a typical experiment. In four such experiments with both [3H]leucine and [a5S]methionine, the fraction of total cpm in the enzyme in C3H mice was 5.25 X 10-4 + 1.70 whereas in C57BL/10 mice it was 2.02 X 10-4 ± 0.77. These numbers are significantly different from one another, P < 0.01. Their ratio is 2.6, which accounts very well for the inherited difference in the level of enzyme activity in brain. Table 4. Rate of synthesis of mitochondrial malic enzyme in brains of C3H/Rl and C57BL/lORl mice* C3H/Rl C57BL/AORl 1.75 X 106 2.08 X 106 Total acid-precipitable cpm 700 266 cpm in mitochondrial malic enzyme 3.37 X 10-4 1.52 X 10-4 cpm in enzyme/total cpm Ratio of rates of synthesis, 2.2 C3H:C57BL/10 * Three mice of each strain were injected with (133 ,gCi). After 3 hr, total isotope incorporation into trichloroacetic acid-insoluble material was measured, and mitochondria were prepared from brains from each strain. Partially purified enzyme was precipitated and analyzed on NaDodSO4Jpolyacrylamide gels.

L-[3mS]methionine

Genetics: Bernstine et al.

Proc. Natl. Acad. Sci. USA 76 (1979)

E

2/ 0

0~~~~* 0

10

20

30

40

50

Sl ice

FIG. 2. NaDodSO4polyacrylamide gel electrophoresis of immune precipitate of mitochondrial malic enzyme. Brains were labeled in vivo and the enzyme was partially purified from mitochondrial lysates, immunoprecipitated, and prepared for electrophoresis. The molecular weight of the material in the peak was identical to that of a standard that was stained for protein.

The fact that FI animals of Mdr-1 A X Mdr-1 B crosses exhibited exactly intermediate levels of mitochondrial malic enzyme in brain (Fig. 1) suggested codominant expression of these alleles, which is most easily understood if each allele acts cis. The availability of deletions (2, 4) and a duplication (9) including the region c-Mod-2 allowed us to examine genedosage effects on the level of enzyme expression in brain by constructing strains with varying numbers of Mod-2 loci and various combinations of the Mdr-1 A and Mdr-1 B alleles. As shown in Table 5, each combination gives a level of enzyme activity that is the sum of contributions by the Mdr-1 A and Mdr-1 B alleles present. Thus, each allele acts completely autonomously. The results suggest that Mdr-1 acts cis although other, more complex, explanations are not excluded by the data. Table 5. Mitochondrial malic enzyme activity as a function of gene dosage of Mdr-1 A and Mdr-1 B alleles Specific activity of mitochondrial malic enzymea Dosage Mdr - 1A Mdr - jB Heart Brain 2b -c 0 (3) 0.027 i 0.001 id 0 c (3) 0.014 i 0.001 3e 0 (3) 0.030 (3) 0.050 + 0.002 0 2f (3) 0.019 (3) 0.032 b 0.001 Og 2 (1) 0.017 (2) 0.073 + 0.003 oh 1 (2) 0.008 + 0.001 (2) 0.036 ± 0.001 1i 1 (2) 0.018 I 0.002 (2) 0.045 + 0.001 1 2i ND (2) 0.067 + 0.002 a Units per mg of protein. Numbers in parentheses represent number of animals assayed. ND, not done. b Stock 65K Cch/Cch animals; see ref. 4. c See ref. 4 for data. d Stock 65K Cch/C* animals, where c* is a deficiency covering cMod-2. e Combination of a duplication, extending from at least c to Hbb, with the cch chromosome of 65K. Progeny were genetically identified by crosses to c/c animals. f Combination of the above duplication with the c* chromosome of 65K. Progeny were genetically identified as above. g Stock 4 animals (c Mdr-1 A/c Mdr-1 A). h c/c * offspring of stock 4 X 65K crosses. i cch/c* offspring of stock 4 X 65K crosses. A duplication chromosome, in combination with a stock 4 chromosome 7.

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DISCUSSION The results presented here have established the postnatal developmental pattern of mitochondrial malic enzyme in brain in animals possessing different alleles of a locus that regulates the expression of Mod-2. The relevant locus, Mdr-1, has been located on chromosome 7, where it shows very close linkage to c and lies between c and Hbb. The question of the positional relationships of Mdr-1 to Mod-2 is of extreme importance in this system. No one has succeeded in separating, by recombination, a structural gene from a "closely linked" regulatory locus in the mouse (10-13). In this connection, the relatively fine genetic analysis possible near the c locus will be used in order to resolve this question. We now have strains that will allow us to detect visually all crossovers in about 4 cM of chromosome 7 that includes the Mod-2 locus and Mdr-1. Thus, screening for recombinants between Mdr-1 and Mod-2 will be greatly facilitated. Some data suggest that Mdr-1 is a locus distinct from Mod-2. The brain and heart enzymes are identical in all biochemical properties studied (5), and the electrophoretic variant of the enzyme found in strain SM/J (14) is found in both brain and heart (unpublished results). Thus, it is difficult to explain simply the tissue-specific action of Mdr-1 if it is founded in the coding sequences of the structural gene (Mod-2). It should also be noted that the Mdr-l A allele is not associated with a particular electrophoretic form of the enzyme. We have established that Mdr-1 determines the rate of synthesis of Mdr-1 the enzyme, but the detailed mechanism of its action remains to be elucidated. The availability of deletions covering this part of chromosome 7 (2, 4) offers unique possibilities for pursuing the mechanism of Mdr-1 action at the nucleic acid level. Gels for Hbb typing were run by Ms. C. Cornett. We especially thank Dr. Liane B. Russell for her encouragement of our work, many helpful comments on the genetics, and development of the genetic analysis that underlies this system. This research was sponsored by the Office of Health and Environmental Research, U.S. Department of Energy,

under Contract W-7405-eng-26 with the Union Carbide Corp. 1. Diamond, R. P. & Erickson, R. L. (1974) Nature (London) 248, 418-419. 2. DeHamer, D. L. (1975) Dissertation (Univ. of Tennessee,

Knoxville, TN). 3. Eicher, E. M. & Coleman, D. L. (1977) Genetics 85,647-658. 4. Bernstine, E. G., Russell, L. B. & Cain, C. S. (1978) Nature (London) 271, 748-750. 5. Bernstine, E. G. (1979) J. Biol. Chem. 254, 83-87. 6. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 7. Laemmli, U. K. (1970) Nature (London) 227,680-685. 8. Jacobson, K. B. & Vaughan, C. (1977) Anal. Biochem. 78, 295-301. 9. Russell, L. B., Russell, W. L., Popp, R. A., Vaughan, C. M. & Jacobson, K. B. (1976) Proc. Natl. Acad. Sci. USA 73, 28432846. 10. Paigen, K., Swank, R., Tomino, S. & Ganschow, R. E. (1975) J.

Cell Physiol. 85, 379-392. 11. Doyle, D. & Schimke, R. T. (1969) J. Biol. Chem. 244, 54495459. 12. Paigen, K., Meisler, M., Felton, J. & Chapman, V. (1976) Cell 9, 533-539. 13. Kozak, L. P. (1972) Proc. Natl. Acad. Sci. USA 69, 3170-

3174. 14. Shows, T. B., Chapman, V. M. & Ruddle, F. H. (1970) Biochem.

Genet. 4, 707-718.

Regulation of mitochondrial malic enzyme synthesis in mouse brain.

Proc. Natl. Acad. Sci. USA Vol. 76, No. 12, pp. 6539-6541, December 1979 Genetics Regulation of mitochondrial malic enzyme synthesis in mouse brain...
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