Tne JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 252, No. 3, Issue of February 10, pp. 930-834, 1977

Panted in U.S.A.

Transmission of the Cytochrome c Structural Gene in Horse- Donkey Crosses*

(Received for publication, September 9, 1976)

OTTO F. WALASEK AND E. MARGOLIASH

From the Biochemistry Laboratories of the Experimental Biology Division, Abbott Laboratories, North Chicago, Illinois 60064, and the Department ofBiochemistry and Molecular Biology, Northwestern University, Evanston, Illinois 60201

Donkey cytochrome c was shown to differ from horse cytochrome c by having a serine in position 47 rather than a threonine- The rest of the amino acid sequences are identi- cal. Mules and hinnies, both males and females, carry equal amounts of horse and donkey cytochromes c. The same ratio is found in hinnies in preparations from heart tissue and from skeletal muscle. These results demonstrate that cyto- chrome c is transmitted in horse-donkey crosses as a simple Mendelian character which is neither sex-linked nor shows dominance. The cytochrome c gene is therefore located in the nuclear genome, as earlier shown to be the case for Saccharomyces iso-1-cytochrome c.

During a routine survey of the chymotryptic peptide maps of a variety of cytochromes c, it was noted that the horse and donkey proteins differ by a single spot. As the amino acid compositions of the proteins and of the isolated chymotryptic peptides of donkey cytochrome c confirmed that the two pro- teins vary by a single residue, an experimentally simple op- portunity was afforded to examine whether in mammalian species the structural gene for cytochrome c is part of the nuclear Mendelian genome or part of the mitochondrial ge- nome. Preparations were examined from individual donkeys, mules (female horse-male donkey cross) and hinnies (male horse-female donkey cross), both males and females. It was determined that all mules and hinnies carry a cytochrome c complement consisting of half horse protein and half donkey protein, demonstrating that cytochrome c behaves as a simple Mendelian character in these mammalian species, shows nei- ther sex linkage nor dominance and must be coded for by a chromosomal gene. The only other examination of the mode of inheritance of cytochrome c has been carried out in bakers’ yeast, employing a structural mutant of the protein (11, and here again, it is clear that the protein is transmitted as a simple Mendelian character.

EXPERIMENTAL PROCEDURES

Cytochrome c Preparations - Cytochrome c was prepared essen- tially according to Margoliash and Walasek (2) from two individual hearts of horses of unrecorded sex, three hearts from mules of unre- corded sex, five male mule hearts, four female mule hearts, one male

* This work was supported in part by Grant GM 19121 from the National Institutes of Health (to E. M.).

hinny heart and skeletal muscle from the same animal, one female hinny heart and skeletal muscle of the same animal, two hearts of donkeys of unrecorded sex, and a pool of four hearts of donkeys of unrecorded sex. The yield of protein for these 22 separate prepara- tions varied from 121 to 252 mg/kg of tissue, with an average of 182 mg/kg for the hearts, and were 45 and 72 mg/kg of tissue for the two skeletal muscle samples. The iron content (3) averaged 0.456% and the ratio of the absorbance at 550 nm in the ferrous form to that at 280 nm in the ferric form averaged 1.24, typical of the value for eukaryotic cytochromes c containing a single tryptophan/molecule. In no preparation was any detectable carbon monoxide complex of cytochrome c observed in the ferrous form at 1 atm pressure of the gas (4, 5). Spectra were determined with a Zeiss PMQII spectropho- tometer.

Amino Acid Sequence Determination -The cytochrome c samples were digested with chymotrypsin (6% by weight) (Worthington, 3x crystallized) in 0.05 M ammonium bicarbonate, at 37” for 10 to 18 h. Peptide maps were obtained on Whatman 3MM paper and stained with ninhydrin and specific residue reagents as previously described (6, 7). Amino acid compositions of the proteins and purified peptides were determined, following acid hydrolysis in U~CUO in 6 N HCl, with a model 120C Beckman amino acid analyzer equipped with a model CRS 10-A Infotronics digital integrator. Peptides were hydrolyzed for 20 h, proteins for 20, 40, and 80 h. The chymotryptic peptides of donkey cytochrome c (196 mg) were separated by chromatography on a column of Dowex 50-X2 developed under a linear gradient of pyridine acetate buffers as previously described (6, 7), and the col- umn chromatographic fractions were further purified by preparative paper chromatography (6, 7) or free flow electrophoresis, or both, in the pyridine acetate buffer (pH 6.5) used for peptide mapping (Brink- man model FF, continuous flow electrophoretic separator; retention time 70 min, 2000 V). The chymotryptic heme peptide which re- mained at the top of the Dowex 50 column was eluted in 1 N ammo- nium hydroxide, taken to dryness, and purified by gel filtration on a column (0.9 x 150 cm) of Sephadex G-25 (Pharmacia) in 0.1 M ammonium bicarbonate. The Ser-Tyr and Thr-Tyr peptides of mule (197 mg) and hinny (150 mg) cytochromes c were isolated by passing the chymotryptic digests of the proteins through a column (3.6 x 250 cm) of Sephadex G-10 in 1.0 N acetic acid, locating the required tyrosine-containing (Pauly positive) electrophoretically neutral pep- tides by peptide mapping of the appropriate column fractions, and purifying them by free flow electrophoresis and paper chromatogra- phy, as given above. Edman degradation of these peptides was carried out as previously described (7), identifying which residue had been removed from the amino acid composition of the residual mate- rial (8).

RESULTS

Primary Structure of Donkey Cytochrome c-The amino acid composition of a preparation of donkey cytochrome c from an individual heart given in Table I, demonstrates that it is identical to that of horse cytochrome c (Table I) (91, except for 1

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Cytochromes c of Donkey, Mule, and Hinny-Gene Transmission

TABLE I

Amino acid compositions of horse, donkey, mule, and hinny cytochromes c

All protein preparations were hydrolyzed in duplicate for 20, 40, and 80 h. The values reported are the average values or the values extrapolated to zero time. Cysteine and tryptophan were not deter- mined and assumed to be 2 and 1, respectively. Separate prepara- tions from two horse hearts, two donkey hearts, and a pool of four donkey hearts, four hearts from mules of undetermined sex, two hearts from male mules, one heart from a female mule, a heart and skeletal muscle tissue from a male hinny, and a heart and skeletal muscle tissue from a female hinny, were each analyzed (a total of 16 separate cytochrome c preparations). The amino acid compositions of the two horse preparations were indistinguishable, as were those of the three donkey proteins, and also those of the seven mule and four hinny cytochrome c preparations. The analyses listed are average values. The numbers in parentheses represent the number of resi- dues obtained from the amino acid sequences. The bold face type indicates those values which showed significant differences among the proteins of the four groups of animals examined.

Amino acid Residues per molecule of protein

Horse Donkey Mule Hinny

Aspartic acid 8.31 (8) 8.22 (8) 8.24 (8) 8.38 (8) Threonine 9.83 (10) 8.95 (9) 9.57 (9.5) 9.54 (9.5) Serine 0.07 (0) 1.06 (1) 0.56 (0.5) 0.57 (0.5) Glutamic acid 12.23 (12) 12.27 (12) 12.50 (12) 12.68 (12) Proline 4.32 (4) 4.44 (4) 4.21 (4) 4.20 (4) Glycine 12.25 (12) 12.12 (12) 12.38 (12) 12.28 (12) Alanine 6.15 (6) 6.29 (6) 6.17 (6) 6.27 (6) Valine 2.90 (31 2.99 (3) 3.01 (3) 3.05 (3) Methionine 1.60 (2) 1.87 (2) 1.84 (2) 1.27 (2) Isoleucine 5.84 (6) 5.93 (6) 5.94 (6) 5.87 (6) Leucine 6.05 (6) 6.09 (6) 6.05 (6) 6.08 (6) Tyrosine 3.77 (4) 3.84 (4) 3.87 (4) 3.70 (4) Phenylalanine 3.99 (4) 3.91 (4) 3.95 (4) 3.81 (4) Lysine 19.30 (19) 19.21 (19) 19.17 (19) 19.53 (19) Histidine 2.96 (3) 2.85 (3) 2.97 (3) 2.89 (3) Arginine 1.77 (2) 1.95 (2) 1.88 (2) 1.80 (2)

less residue of threonine and the appearance of 1 residue of serine, an amino acid which is totally lacking in the horse protein. Peptide maps of chymotryptic digests of the donkey protein (Fig. 1) showed that all peptide spots were indistin- guishable from those yielded by horse cytochrome c, except for one neutral peptide. This peptide from the horse protein is Thr-Tyr (residues 47 and 48) (6, 10). It is absent in the donkey protein digest, being replaced by a neutral tyrosine-containing peptide of slightly lower chromatographic mobility. That this was the only peptide map difference between the chymotryptic digests of the horse and donkey proteins was verified by map- ping a mixture of equal amounts of the digests of the two proteins and demonstrating that no new peptide spots could be detected with ninhydrin or any of the specific residue re- agents. In fact, the peptide maps of such mixtures were indis- tinguishable from the peptide maps yielded by the mule and hinny proteins. It should be noted that the amino acid compo- sitions and peptide maps from two other donkey cytochrome c preparations, one from a second individual heart, and one from a pool of four small hearts, were indistinguishable from those given in Table I and Fig. 1.

From the amino acid compositions and the peptide maps it could be concluded that donkey cytochrome c most probably differs from the horse protein merely by having a serine replace the threonine in position 47, the corresponding chymo- tryptic peptide, Ser-Tyr, having the slightly lower chromato- graphic mobility expected from the relative mobilities of ser-

FIG. 1. Photographs of peptide maps of chymotryptic digests of horse (A), donkey (B), mule (C), and hinny (D) cytochromes c. In each case the origin is shown by the inked circle; - indicates the cathode; + , the anode. The standard conditions for peptide mapping are: Whatman 3MM paper; electrophoresis (pyridine acetate buffer, pH 6.5) for 90 min at 30 V/cm; descending chromatography (butanol- l/pyridine/acetic acid/water, 1200/800/240/960, v/v) for 15 h (see Ref. 7). The Thr-Tyr and Ser-Tyr dipeptides are indicated.

ine and threonine in the solvent employed. This conclusion was further substantiated when the isolated and purified chy- motryptic peptides of donkey cytochrome c were shown to have amino acid compositions identical to those of the correspond- ing peptides from horse cytochrome c (see supplementary ma- terial).’ The only exceptions were the peptides containing residues 41 and 48 and residues 47 to 54, both of which show a serine instead of the threonine of the corresponding horse cytochrome c peptides and encompass the variant position 47.

Structures of Mule and Hinny Cytochromes c-The amino acid compositions of mule heart cytochrome c (seven prepara- tions from individual hearts of which two were known to be from male mules and one from a female mule) and of hinny heart and skeletal muscle cytochrome c (one preparation from each tissue from a male and a female hinny) were indistin- guishable (Table I). They differed from those of both the horse and donkey proteins, appearing to lack 0.5 residue of threo- nine and to have instead 0.5 residue of serine. The peptide

* Details of the amino acid compositions and properties of chymo- trypic peptides of donkey cytochrome c are presented as a miniprint supplement immediately following this paper (p. 834). For the convenience of those who prefer to obtain these data in the form of eight pages of full size photocopies, it is available as JBC Document Number 76M-1280. Orders should specify the title, authors, and reference to this paper, the JBC Document Number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014, and must be accompanied by a remittance to the order of the Journal in the amount of $1.20 per set of photocopies.

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832 Cytochromes c of Donkey, Mule, and Hinny-Gene Transmission

maps of chymotryptic digests of mule and hinny cytochrome c preparations were the same as those given by the horse and donkey proteins, except that both the Thr-Tyr and Ser-Tyr dipeptides were present, and as far as could be judged from the ninhydrin coloration, in approximately equal amounts (Fig. 1).

To establish that mule and hinny cytochromes c were indeed mixtures of the horse and donkey proteins, gel filtration on columns of Sephadex G-10 followed by free flow electrophoresis and paper chromatography were employed to isolate the Ser- Tyr and Thr-Tyr dipeptides from chymotryptic digests of both proteins. Amino acid compositions and Edman degradation showed their structures to be the expected ones (Table II). The relative proportions of the two peptides and the amino acid compositions of the eleven mule and hinny cytochrome c prep- arations demonstrated that, within the error of the methods employed, mules and hinnies whether male or female carried equal complements of horse and donkey cytochrome c.

DISCUSSION

The fact that donkey cytochrome c has the same structure as horse cytochrome, except for the replacement of threonine 47 by a serine, made it straightforward to determine what pro- portion of the two proteins was present in the hybrids of these species, mules and hinnies. Indeed, horse cytochrome c con- tains no serine at all, so that the fraction of a residue of serine present in acid hydrolysates of mule and hinny cytochrome c represents the proportion of donkey protein they carry. How- ever, the analytical problem is not very simple, since both serine and threonine are relatively labile to acid hydrolysis. Furthermore, the two cysteines in any cytochrome c, including the horse protein, that bind the heme prosthetic group to the polypeptide chain by thioether bonds, appear to decompose fractionally to serine on acid hydrolysis (9). Acid hydrolysates of horse cytochrome c have been observed with as much as a full residue of serine,2 the usual amount being between 0.03 to 0.20 residue. The conditions which minimize or maximize the yield of serine from thioether-bonded cysteines have not been defined and the yields vary erratically.

Notwithstanding these difficulties the analytical results presented in Table I clearly indicate that the horse-donkey hybrids have approximately half their cytochrome c derived from each parent. For the seven individual mule proteins analyzed the serine content varied from 0.50 to 0.64 residue/ molecule, with an average of 0.56, while for the four hinny protein preparations the serine content varied from 0.55 to 0.63 residue/molecule, with an average of 0.57. When the common yield of serine from the thioether-bonded cysteines is sub- tracted, the values are as near 0.5 residue/molecule as amino acid analyses are capable of yielding. Threonine is also some- what acid-labile. It occurs ten times in horse cytochrome c and nine times in the donkey protein and is therefore analytically less favorable than serine for the estimation of the proportion of the two proteins in the hybrids. Even so, the threonine content of the seven mule cytochromes c varied from 9.40 to 9.73 residues/molecule, with an average of 9.57, while that of the four hinny preparations varied 9.38 to 9.75 residuesimole- cule, with an average of 9.54, in excellent concordance with the serine results.

The above conclusion was further bolstered by the peptide maps, which when prepared from the proteins of the hybrids showed approximately equal amounts of the Thr-Tyr and Ser-

2 E. Margoliash, unpublished observation.

TABLE II

Identification of variant dipeptides from chymotryptic digests of mule and hinny cytochromes c

Source Mule Hinny

Peptide designation LA LB 89A 89B Amino acid composi- Ser, Tyr Thr, Tyr Ser, Tyr Thr, Tyr

tion Yield (%I Edman degradation

69 80 78 80

Phenylthiohydan- Ser Thr Ser Thr toin derivative

Residual amino TY~ ‘br ‘br Tyr aeid

Tyr dipeptides. In contrast, peptide maps of horse cytochrome c showed no trace of Ser-Tyr, while in peptide maps of donkey cytochrome c no trace of Thr-Tyr was ever observed, even when the maps were overloaded to make it easier to detect smaller quantities of peptides. Finally, the two dipeptides were isolated from mule and hinny cytochrome c chymotryptic digests in approximately equal amounts and in high yields in both cases, as shown in Table II above. This again confirms that equal amounts of both proteins were present in both hybrids.

Thus, the conclusion that mules and hinnies, males and females, all carry half of their cytochrome c as the horse protein, and the other half as the donkey protein is well established. It is therefore evident that cytochrome c is inher- ited as a normal Mendelian character in horse-donkey crosses and that the structural gene for the protein is part of the nuclear genome, not the mitochondrial genome. Furthermore, the structural genes for cytochrome c in horses and donkeys are not sex-linked and do not exhibit dominance. Mules and hinnies are heterozygous in their cytochrome c locus, one allele deriving from the horse (whether mother or father) and one from the donkey (whether mother or father). Both alleles express themselves independently and equally. This contrasts with the apparently purely maternal inheritance of the mito- chondrial genome in horse-donkey crosses, as evidenced by the electrophoretic separation patterns of DNA fragments pro- duced by restriction endonucleases from the mitochondrial DNA of the hybrid, maternal, and paternal species (11).

The only other demonstration of the mode of inheritance of cytochrome c was by Sherman et al. (1) who showed that in bakers’ yeast, the iso-l-cytochrome c gene segregates as a normal nuclear Mendelian character. It was therefore consid- ered important to investigate whether the same situation prevails in a higher organism. The interest in the genetics of cytochrome c stems from the protein being a typical compo- nent of mitochondria. Since mitochondria are commonly sup- posed to have derived in the course of evolution of eukaryotic cells from a prokaryote symbiont (see Refs. 12-14), it is impor- tant for our understanding of this major evolutionary event to find out which of the mitochondrial proteins is under control of the nuclear genome and therefore possibly derived from the original cell, and which are products of the mitochondrial genome and thus descended from the hypothetical symbiont. Moreover, a much more extensive knowledge than now availa- ble of which mitochondrial proteins derive from the nuclear genome and which are autonomous mitochondrial products in different groups of species is likely to clarify how mitochon- drial activities and biogenesis are controlled by and integrated with nuclear activities and to what extent gene redistributions between nuclear and organelle genomes have occurred in the

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Cytochromes c of Donkey, Mule, and Hinny-Gene Transmission a33

course of evolution. Other respiratory chain proteins for which some information is available are cytochrome oxidase in fungi (bakers’ yeast and Neurosporu) and the F, ATPase (15-M). The present evidence indicates that in these cases some of the polypeptides which constitute the enzymes are synthesized in mitochondria, while the others are made by the cytoplasmic ribosomal system. The locations of the structural genes are so far unknown.

1504 2. Margoliash, E., and Walasek, 0. F. (1967)Method.s Enzymol. 10,

339-348 3. Cameron, B. F. (1965) Anal. Biochem. 11, 164-169 4. Tsou. C. L. (1951) Biochem. J. 49. 362-367 5. Mariohash, E. (1962) Brookhaven Symp. Biol. 15, 266-281 6. Margoliash. E.. and Smith, E. L. (196215. Biol. Chem. 237,2151-

2160 Nolan, C., and Margoliash, E. (1966) J. Biol. Chem. 241, 1049-

1059 Finally, the fact that the structural gene for cytochrome c is

in the nuclear genome does not necessarily mean that the protein is made in the cytoplasm and not intramitochondri- ally. However, whatever evidence is available appears to indi- cate that cytochrome c is indeed synthesized in full by the cytoplasmic ribosomal system, including the attachment of the heme prosthetic group (18).

7.

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13. 14. 15. 16.

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18.

Hirs, C. H. W., Moore, S., and Stein, W. H. (196O)J. Biol. Chem. 235, 633-647

The fact that donkey cytochrome c differs from the horse protein by a single residue is not unexpected since the two species are in the same perissodactyl genus, Equus. It should also be noted that after this work was completed, zebra cyto- chrome c was shown to be identical to donkey cytochrome c (19).

Margoliash, E., Kimmel, J. R., Hill, R. L., and Schmidt, W. R. (1962) J. Biol. Chem. 237, 2148-2150

Margoliash, E. (1962) J. Biol. Chem. 237, 2161-2174 Hutchison, C. A., Newbold, J. E., Potter, S. S., and Edgell, H.

H. (1974) Nature 251, 537-538 Margulis, L. (1970) Origin of Eukaryotic Cells, Yale University

Press, New Haven Bogorad, L. (1975) Science 188, 891-898 Uzzell, T., and Spolsky, C. (1974) Am. Sci. 62, 334-343 Poyton, R. O., and Schatz, G. (1975) J. Biol. Chem. 250, 752-761 Sebold, W., Machleidt, W., and Otto, J. (1973) Eur. J. Biochem.

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REFERENCES

1. Sherman, F., Stewart, J. F., Margoliash, E., Parker, J., and Campbell, W. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 1498-

Tzagoloff, A., Rubin, M. S., and Sierra, M. F. (1973) Biochim. Biophys. Acta 301, 71-104

Nicholls, P., and Elliott, W. B. (1974) in Iron in Biochemistry and Medicine (Jacobs, A., ed) pp. 221-227, Academic Press, New York

19. Giirtler, L., and Horstmann, H. J. (1971)FEBSLett. 18,106-108

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834 Cytochromes c of Donkey, Mule, and Hinny-Gene Transmission

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O F Walasek and E MargoliashTransmission of the cytochrome c structural gene in horse-donkey crosses.

1977, 252:830-834.J. Biol. Chem. 

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