Pigment 0 1 1 Research Suppl. 2: 57-60 (1992)
The Shift from Physiological Genetics to Molecular Genetics in the Study of Mouse Tyrosinase WALTER C. QUEVEDO, Jr AND THOMAS J. HOLSTEIN Division of Biology and Medicine, Brown University, Providence RI 02912 and Department of Biology, Roger Williams College, Bristol, RI 02809
In general, molecular genetics has focused on the central dogma pathway of DNA --> mRNA --> Protein. The much older field of physiological genetics has examined the path from protein (the initial phenotype) to the derived phenotype (a trait expressed at some higher level of developmental complexity). In the case of mouse tyrosinase, for more than 40 years, physiological geneticists have examined the enzymatic properties of intracellular and extracted tyrosinase emphasizing genetic influences on its contribution to the generation of ocular and integumentary melanin pigmentation. They could not penetrate more closely to the tyrosinase gene for the required methodology was not available. In recent years, this deficiency has been corrected largely due to brilliant analytical and technological advances in biochemistry, molecular and cell biology. Molecular geneticists have centend their attention on the isolation of the tyrosinase gene, determination of its structure, transcription of its information into mRNA, translation of the mRNAencoded message into tyrosinase protein, post-translational modification of nascent tyrosinase, and the structure, kinetics and stability of the enzyme.
This paper provides a very brief, selective account of how physiological genetic studies on the pigmentationof the mouse helped to establish the agenda for molecular genetic studies of tyrosinase, some of the accomplishments thus far, and important questions that remain unanswered. A major task confronting pigment cell biologists is that of assembling the many fragmentary insights provided by molecular and physiological genetics into an uninterrupted epigenetic pathway stretching from its origin in the tyrosinase gene to its termination in ocular and integumentarypigmentation. In the mouse, this entails linking the numerous c-locus alleles to the heritable coat color variations which some time ago revealed their existence. Among the first questions asked about the physiological genetics of mice were "What influence do coat color genes have on tyrosinase activity?" and "Does tyrosinase catalyze early steps in the synthesis of eumelanin and pheomelanin?" In the late 1940s, E. S. Russell(1-3), examined the quantitative and qualitative properties of melanin granules (melanosomes) in the hair of a great variety of mouse coat
color mutants. The properties evaluated included melanosome color, size, shape, volume and distribution along the hair shaft. E. S. Russell's study established the foundation upon which all subsequent studies of mouse coat color genetics have been based. In 1948, L. B. Russell and W. L. Russell(4) started building on this foundation by examining histochemically the DOPA-oxidasehyrosinase activity (tyrosinase had not as yet gained universal recognition as the catalyst in the initial stages of melanogenesis) in hair bulb melanocytes. They found that only the c-series of alleles significantly modified the intensity of the DOPA-reaction within melanocytes. No DOPAmelanin was deposited in albino WE) melanocytes. The DOPA-reaction was approximately equal in otherwise corresponding da (eumelanic) and AYla (pheomelanic) genotypes. Surprisingly, the intensity of the DOPA-reaction correlated better with gene action on the amount of pheomelanin in the yellow hair coats of the AY/a (pheomelanic) gene series than it did with eumelanin in coats of the da (eumelanic) series. Foster[ 19511 used manometric techniquesto measure tyrosinase activity as a function of oxygen consumption when tyrosine was the principal melanogenic substrate(5). He demonstratedthat % consumption (tyrosinase activity) varied with coat color (e.g., black IB] > brown [hl > yellow BY] > albino Very little tyrosinase activity was found in yellow or albino skin specimens. Based on a variety of experiments, Foster concluded that eumelanin synthesis was catalyzed by tyrosinase whereas phmmelanogenesisrequired a tryptophan-oxidizing enzyme. In addition, various inhibitors influenced tyrosinase activity. In Foster's words: "From the results so far obtained with mouse skin homogenates a relatively simple picture of hair-pigmentation emerges: 1) Both the yellow and dark pigments are continually produced in pigmented skins. 2) The dark pigment masks the presence of the yellow pigment. 3) When the tyrosinase system is blocked yellow pigment becomes evident. 4) When both systems are blocked the albino type results. 5 ) The yellow band of the agouti hair might then represent a period of abrupt suspension and resumption of tyrosinase activity. 6) Spotting rcprcscnts rcgiond failurc of either the tyrosinase system or of both systems. 7) Red pigment represents the products of a preponderant
w).
58
W. C. Quevedo et al.
tryptophan-oxidizingsystem and a weak tyrosinase system."
an inhibitor resulting in the unexpectedly great increase in activity. Since the potentially high levels of tyrosinase activity in albino tissues were held in check by an inhibitor, Hearing concluded that "the c (albino) locus might more correctly be regarded as a regulator rather than a structural locus for tyrosinase". Ih 1974, Pomerantz and Li(12) confirmed that tyrosinase was present in the skin of albino mice. The rate of tyrosine hydroxylation was about 2-3% of wild type tyrosinase. However, in albino skin homogenates, 'Triton' X-100 failed to increase tyrosinase activity to lhal of wild type mice.
In 1959, Fitzpatrick, Brunet and Kukita(6) used autoradiography to measure genetic influences on tyrosinase activity within mouse hair bulb melanocytes. The incorporation of tyrosine-2-Cl4 into melanin varied, with brown (hm) = dilute brown (dld;bm) > yellow @Y/a;B/BJ = black & / a ; m> pink-eyed dilute black &/jxD-;d~) > albino WE). No uptake of labelled tyrosine was found in the albino. Their model for melanogenesis was similar to Foster's(5). They tentatively proposed that eumelanin and pheomelanin were formed by two distinct but coupled In 1981, Townsend et al.(13) questioned Coleman's metabolic pathways with tyrosine the precursor for the former (7) evidence in favor of designating the c-locus as the and tryptophan for the latter. structural locus for tyrosinase. They found that c-locus alleles influenced total tyrosinase activity and the subcellular In 1962, Coleman(7) reported that tyrosine-2-C1 distribution of tyrosinase. Since the kinetic parameters of was incorporated into pheomelanin but tryptophan-Cl4 was tyrosinase were not influenced by the plocus alleles, they not. Measuring the amounts of radioactivity in skin concluded that the & c u s is probably not the structural locus homogenates, Coleman confirmed Fitzpamck et al's.(6) for tyrosinase. Their data could not distinguish between possible alternative explanations such as a "decrease in earlier autoradiographic finding that tyr0sine-2-C~~ was tyrosinase translation [or] an increase in tyrosinase turnover incorporatedinto eumelanin and concluded that tyrosine was due to a possible deficiency in post-translationalprocessing." the major precursor both of pheomelanin and eumelanin. In In 1985, supporting this interpretation of c-locus function, addition, Coleman tested a larger number of coat color genes Townsend et al.( 14) provided evidence indicating that than Fitzpamck et a1.(6) for their influence on tyrosinase activity. To a large extent, Coleman confirmed Fiupatrick et himalayan (ch)tyrosinase had the same temperature al. (6) on genotypes overlapping with his study. His detailed sensitivity as the wild-type (Q tyrosinase characteristic of analysis of the c-locus alleles revealed differing levels of C57BW6J mice. tyrosinase activity > E h > ch > ~e > d. Focusing on the Following the shift to the molecular genetics of tyrosinase associated with the action of the himalayan (&) pigment cell biology during the latter half of the 198Os, the allele, h e provided biochemical evidence that it was uncertainty surrounding c-locus function has been significantly more thermolabile than wild-type Q tyrosinase. significantlyreduced. However, it is important to remember Coleman states: "This thermolability of the enzyme produced that direct knowledge of the gene for tyrosinase and its allelic under the influence of the Himalayan allele suggests that this variants leads to expectationsregarding the performance of its allele and thus, by inference, all of the alleles at the C-locus, products. Therefore it is important to match studies on control the structure and not the quantity of the enzyme tyrosinase performed prior to the major impact of molecular produced." In 1963, Foster(8) concluded that the h-locus genetics on pigment cell biology with those performed after to regulated the availability of endogenous substrate for determine whether studies subsequent to the "molecular melanogenesis and the number or effectiveness of melanin genetic revolution" differ because of interpretation or because binding sites on the proteinaceous melanosomal matrix. of real differencesin the biological material examined. If this is not done, older work will be considered to be superceded In the late 1960s, Prota and Nicolaus(9) brought by the new with the result that valuable insights into pigment order to conflicting reports about pheomelanogenesis when cell function will be ignored. they demonstrated that pheomelanin synthesis involved a deviation from the eumelanin pathway. Dopa quinone, In 1986, Shibahara et a1.(15) reported cloning and derived from the tyrosinase catalyzed oxidation of DOPA, sequencingof the cDNA for mouse tyrosinase. However the combined with cysteine to produce cysteinyldopas, sequence of the cDNA did not closely correspond with the intermediates in pheomelanogenesis, rather than forming primary structure of tyrosinase and was later mapped to the bleucodopachrome, an intermediate in eumelanogenesis(9). locus by Jackson(l6). In 1987, Yamamoto et a1.(17) More recent studies [ 19861 by Lamoreux et a1.(10) indicate published a cDNA sequence which differed from that of that not only is overall tyrosinase activity significantly Shibahara et a1.(15) but mapped at the c-locus and matched reduced in yellow mice but that, compared to wild type the primary structure of tyrosinase. In 1989, Kwon et a1.(18) (black) tyrosinase, the DOPA-oxidase activity of AYIperformed a nucleotide sequence analysis of the albino (c) tyrosinase is substantially reduced relative to its tyrosine allele of Balb/c mice detecting a G to C substitution which hydroxylase activity. This finding seemingly contradicts L. results in the replacementof cysteine by serine at a critical site B. Russell and W. L. Russell's(4) observation that the in tyrosinase(18). The himalayan allele ($) has also been intensity of the DOPA-reaction was the same in eumelanic sequenced and a point mutation replacement of A by G W& and pheomelanic &Y/& mice otherwise identical in identified which results in histidine being replaced by arginine major pigmentation genes. at a key position in tyrosinase(l9). The demonstration that cDNA capable of coding for tyrosinase maps to the plocus In 1973, Hearing(l1) demonstrated tyrosinase activity and that G-locus mutant alleles show point mutations in albino tissue homogenates that could be enhanced by potentially capable of altering the functional properties of treatment with 'Triton' X-100. In a fraction containing tyrosinase appears to establish that the s-locus is the structural premelanosomes from the eyes of albino mice, tyrosinase locus for tyrosinase. Although the h-locus protein expresses activity almost equaled that of wild type (black) following considerable point and sequence homology with tyrosinase, treatment with 'Triton' X-100. He suggested that the Halaban and Moellmann(20) have concluded that it is a detergent released albino tyrosinase from its association with
W. C. Quevedo et al. catalase [catalase B]. Jiminez et al.(21) and Hearing(22) have reported that it is a tyrosinase with markedly reduced activity compared to G-locus (wild type) tyro&=. In 1988, Halaban et al.(23) determined that albino cd) tyrosinase did not display enzymatic activity and was a b d y sensitive to proteolytic cleavage. ~n conmt, himalayan ($1 tyrosinase expressed defects in N-linked glycosylation which rendered it unstable and abnomdy heat sensitive. Therefore, to this point,rhree major issues relating to tyrosinase which have been addressed by molecular geneticists but with their roots in physiological genetics remain to be adjudicated. First, is there an inhibited tyrosinase in albino mice at least potentially capable of approximating wild-type tyrosinase activity( 1l)? Second, is the tyrosinase of himalayan mice abnormally sensitive to heat inactivation(7,13,14)? Third, is the h-locus protein a determinant of melanin binding sites on the melanosomal maaix(8). a catalase(20),or a weak tyrosinase-likeenzyme which may supplement the ~-1ocusin melanogenesis(21,22)? Fourth, based on the discussion to follow, is there a structural difference between yellow and wild type tyrosinase which would m u n t in part for its reduced overall activity in BY mice? Answers to these questions are important for they determine the credibility of models that m a y be generated to visualize how genes regulate melanogenesis. In 1989, Tamate et al.(24) demonstrated that the levels of immunoprecipitable tyrosinase were reduced in the skin of agouti @/A)mice compared to wild type and tyrosinase messenger RNA (mRNA) levels were about the same in the two genotypes. They concluded that the agouti locus exerts its influence on tyrosinase by controlling its translation from mRNA and/or its post-translational modification. The suggestion that alleles at the a-locus may influencepost-translationalmodifcation of tyrosinase lends credence to L a m o ~ u xet al.ls(l0) proposal that there may be structuraldifferencesbetween yellow (BY/-) and black (wild type)tyrosinase.
In albino WE) mice, immunologically-precipitable tyrosinase was absent although normal levels of tyrosinase mRNA were present. Albino tyrosinase exhibited little tyrosine hydroxylase and no DOPA-oxidase activity. Although, they performed their assays of dopa oxidase activities on preparations treated with 'Triton' X-100. the preparations which were tested for tyrosine hydroxylase activity, unfommately, were not so trcated. Tamate et al.(24) concluded that, as in the case of the 8-locus, the s-locus in some way regulated translation of tyrosinase mRNA andor the post-translational modificaton of the enzyme rendering it unreactive to the tyrosinase-specific antibody. The failure of Tamate et al.(24) to demonstrate immunoprecipitable tyrosinase in albino mice conflicts with Takeuchi's(25) and Halaban et al.'s(23) publications to the contrary. Halaban et al.(23) found that tyrosinase, following proteolytic cleavage, retained rtactivity to the tyrosinase antibody. In contrast with their observations on the a- and Eloci, Tamate et al.(24) found that in brown (hm) mice and dilute black (dld;BIB)mice the levels of tyrosinase mRNA and immunopxxipitable tyrosinascwere elevated compared to wild type black (B/B) mice. They believe that these loci regulate transcription of the tyrosinase gene leading to increased mRNA and tyrosinase. It is not clear how this action of the b-gene would produce the well documented brown color of melanosomes and their ultrastructure which
59
also differs from wild type. In view of the far-reaching implications of Tamate et A's wak for the gemtic regulation of tymhasc activity, it would sctm ofthe urmost imporcanct to determine why their findings on the cross-reactivity of albino tyrosinase with tyrosinase antibody differfrom those of Takeuchi(25) and Halaban et al.(23).
In 1990, Tanaka et al.(26) accomplished the difficult feat of microinjecting a mouse synthetic minigene mg-Tyrs-J for mouse tyrosinase into fertilized eggs of albino (ddmice producing transgenic mice which synthesizd melanin only at sites w h m pigment is normally found in wild type mice. The ability to transplant the tyrosinase gene at will, opens up new possibilities for determiningthe mechanisms by which genes regulate pigmentation, for the balance of the genome can be altmd and its consequence evaluated by examination of the altered pigmentationof transgenic mice. A historical approach to the current understanding of tyrosinase genetics meals steady progress paralleling the development of more sophisticated techndogy. The important message arising from this approach is that current achievements, no matter how complicated the technology upon which they are based, do not automaticaUy replace past ones. The testing and assimilation, where appropriate, of "older" ideas about tyrosinase promises to reveal a complexity of melanogenesis that has not been generally appreciated. This paper has attempted to identify sevetal key areas where this effort might begin.
RefereRussell ES. A quantitative histological study of the pigment found in the coat-color mutants of the house mow. I. Variable attributes of the pigment granules. Genetics 1946331:327-46. 2. Russell ES.A quantitative study of genic cf€cctson coat color in the house mouse. Genetics 194&31:227. 3. Russell ES. A quantitative histological study of the pigment found in the coat-color mutants of the house mouse. II. Estimates of the total volume of pigment. Genetics 1948:33:228-36 4. Russell LB, Russell WL.A study of the physiological genetics of coat color in the mouse by M B ~ S of the dopa reaction in frozen sections of skin. Genetics 1948:33:237-62. 5 . Foster M. Enzymatic studies of pigment-forming abilities in mom skin. J Ex@ Zoo1 1951:117:211-46. 6. Fitzpaaick TB, Brunet P, Kukita. A. The n&turcof hair pigment. In: Montagna W.Ellis RA. eds. The Biology of Hair Growth. New York: Academic Press, 1958~255-303. 7. Coleman DL. Effect of genic substitution on the incorporation of tyrosine into the melanin of mouse skin.Archiv Biochem Biophys 1%2:69562-8. 8. Foster M. Some physiological genetic aspects of mammalian melanogenesis. In: Riley V, Former JG, eds. The Pigment Cell. Annals New York Acad Sci, 1963:100:743-8. 9. Prota G. Strucnue and biogenesis of phemmelanins. In: Riley V, ed. Pigmentation: Its Genesis and Biological Control. New York: Appleton-Centurycrofts, 1972:615-30. 10. Lamorcux ML, Wooll~yC, Pcndcrgaat P. Genetic controls over activities of tyl~sinaseand dopachrome conversion factor in murine melanocytes. Genetics 1986:113:967-84. 1.
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1 1. Hearing VJ. Tyrosinase activity in subcellularfractions of black and albino mice. Nature New Biology 1973:245:81-3. 12. Pomerantz SH, Li JP-C. Tyrosinase in the skin of albino hamsters and mice. Natrrre 1974252941-3. 13. Townsend D, Witkop CJ,Jr., Mattson J. Tyrosinase subcellular distribution and kinetic parameters in wild type and ~-1ocusmutant c57BU6J mice. J Exptl Zoo1 1981:216113-9. 14. Townsend D, Guillery P. King RA. Himalayan tyrosinase does not demonstrate temperature sensitivity. In: Bagnara J, Klaus SN, Paul E, Schartl M, 4 s . Pigment Cell 1985. Tokyo: U. Tokyo Press, 1985121C
15. ihibahara S, Tomita T, Sakakura C, Nager C, Chaudhuri BB, Muller R. Cloning and expression of cDNA encoding mouse tyrosinase.Nucleic Acids Res 1986 142413-27. 16. Jackson IA. A cDNA encoding tyrosinase-related protein maps to the brown locus in mouse. Pnx: Natl A d Sci USA 1988~854392-6. 17. Yamamom H, Takeuchi S, Kudo T,et al. Cloning and sequencing of mouse tyrosinase cDNA. Jpn J Genet 1987:62271-4. 18. Kwon BS, Haq AK, Wakulchik M, et al. Isolation, chromosomal mapping, and expression of the mouse tyrosinase gene. J Invest Dermatol1989:93:589-94. 19. Kwon BS, Halaban R, Chintamaneni C. Molecular basis of mouse himalayanmutuation. Biochem Biophys Comm~n1989161:252-60. 20. Halaban R, Moellmann G. Murine and human h locus pigmentation genes encode a glycoprotein (gp75) with catalase activity. Proc Natl Acad Sci USA 1990:87:4809-13. 21. Jim6nez M, Maloy WL, Hearing VJ. Specific identification of an authentic clone for mammalian tyrosinase. J Biol Chem 1989:264:3397-403. 22. Hearing, V. The nature of tyrosinase isozymes. Mishima Y, ed. Abstracts of the XIVth International Pigment Cell Conference. Pigment Cell Res 1991: (in press). 23. Halaban R, Moellmann G, Tamura A, et al. Tyrosinases of murine melanocytes with mutations at the albino locus. Proc Natl Acad Sci USA 1988:85:7241-5. 24. Tamate HB, Hirobe T, Wakamatsu K, It0 S, Shibahara S,I s h h w a K. Levels of tyrosinase and its mRNA in coat-colamutants of C57BU6T congenic mice: E B m of genic substitution at the agouti, brown, albino, dilute. and pint-eyed dilution loci. J Exptl Zoo1 1989:25O:3O4-11. 25. Takeuchi T. Yamamoto H. Sato S, Ishikawa K. Demonstration of the tyrosinase CRh4 in albino skin by use monoclonal antibodies. In: Jimbow K, ed. Structure and Function of Melanin, vol 1. Sapporo: Fuji-shoin Co. 1984.56-8. 26. Tanaka S, Yamamoto H, Takeuchi S, Takeuchi T. MelaniZation in albino mice transformed by introducing cloned mouse tyrosinase gene. Development 1990:108~223-7.
The Shift from Physiological Genetics to Molecular Genetics in the Study of Mouse Tyrosinase WALTER C. QUEVEDO, Jr AND THOMAS J. HOLSTEIN Division of Biology and Medicine, Brown University, Providence RI 02912 and Department of Biology, Roger Williams College, Bristol, RI 02809
In general, molecular genetics has focused on the central dogma pathway of DNA --> mRNA --> Protein. The much older field of physiological genetics has examined the path from protein (the initial phenotype) to the derived phenotype (a trait expressed at some higher level of developmental complexity). In the case of mouse tyrosinase, for more than 40 years, physiological geneticists have examined the enzymatic properties of intracellular and extracted tyrosinase emphasizing genetic influences on its contribution to the generation of ocular and integumentary melanin pigmentation. They could not penetrate more closely to the tyrosinase gene for the required methodology was not available. In recent years, this deficiency has been corrected largely due to brilliant analytical and technological advances in biochemistry, molecular and cell biology. Molecular geneticists have centend their attention on the isolation of the tyrosinase gene, determination of its structure, transcription of its information into mRNA, translation of the mRNAencoded message into tyrosinase protein, post-translational modification of nascent tyrosinase, and the structure, kinetics and stability of the enzyme.
This paper provides a very brief, selective account of how physiological genetic studies on the pigmentationof the mouse helped to establish the agenda for molecular genetic studies of tyrosinase, some of the accomplishments thus far, and important questions that remain unanswered. A major task confronting pigment cell biologists is that of assembling the many fragmentary insights provided by molecular and physiological genetics into an uninterrupted epigenetic pathway stretching from its origin in the tyrosinase gene to its termination in ocular and integumentarypigmentation. In the mouse, this entails linking the numerous c-locus alleles to the heritable coat color variations which some time ago revealed their existence. Among the first questions asked about the physiological genetics of mice were "What influence do coat color genes have on tyrosinase activity?" and "Does tyrosinase catalyze early steps in the synthesis of eumelanin and pheomelanin?" In the late 1940s, E. S. Russell(1-3), examined the quantitative and qualitative properties of melanin granules (melanosomes) in the hair of a great variety of mouse coat
color mutants. The properties evaluated included melanosome color, size, shape, volume and distribution along the hair shaft. E. S. Russell's study established the foundation upon which all subsequent studies of mouse coat color genetics have been based. In 1948, L. B. Russell and W. L. Russell(4) started building on this foundation by examining histochemically the DOPA-oxidasehyrosinase activity (tyrosinase had not as yet gained universal recognition as the catalyst in the initial stages of melanogenesis) in hair bulb melanocytes. They found that only the c-series of alleles significantly modified the intensity of the DOPA-reaction within melanocytes. No DOPAmelanin was deposited in albino WE) melanocytes. The DOPA-reaction was approximately equal in otherwise corresponding da (eumelanic) and AYla (pheomelanic) genotypes. Surprisingly, the intensity of the DOPA-reaction correlated better with gene action on the amount of pheomelanin in the yellow hair coats of the AY/a (pheomelanic) gene series than it did with eumelanin in coats of the da (eumelanic) series. Foster[ 19511 used manometric techniquesto measure tyrosinase activity as a function of oxygen consumption when tyrosine was the principal melanogenic substrate(5). He demonstratedthat % consumption (tyrosinase activity) varied with coat color (e.g., black IB] > brown [hl > yellow BY] > albino Very little tyrosinase activity was found in yellow or albino skin specimens. Based on a variety of experiments, Foster concluded that eumelanin synthesis was catalyzed by tyrosinase whereas phmmelanogenesisrequired a tryptophan-oxidizing enzyme. In addition, various inhibitors influenced tyrosinase activity. In Foster's words: "From the results so far obtained with mouse skin homogenates a relatively simple picture of hair-pigmentation emerges: 1) Both the yellow and dark pigments are continually produced in pigmented skins. 2) The dark pigment masks the presence of the yellow pigment. 3) When the tyrosinase system is blocked yellow pigment becomes evident. 4) When both systems are blocked the albino type results. 5 ) The yellow band of the agouti hair might then represent a period of abrupt suspension and resumption of tyrosinase activity. 6) Spotting rcprcscnts rcgiond failurc of either the tyrosinase system or of both systems. 7) Red pigment represents the products of a preponderant
w).
58
W. C. Quevedo et al.
tryptophan-oxidizingsystem and a weak tyrosinase system."
an inhibitor resulting in the unexpectedly great increase in activity. Since the potentially high levels of tyrosinase activity in albino tissues were held in check by an inhibitor, Hearing concluded that "the c (albino) locus might more correctly be regarded as a regulator rather than a structural locus for tyrosinase". Ih 1974, Pomerantz and Li(12) confirmed that tyrosinase was present in the skin of albino mice. The rate of tyrosine hydroxylation was about 2-3% of wild type tyrosinase. However, in albino skin homogenates, 'Triton' X-100 failed to increase tyrosinase activity to lhal of wild type mice.
In 1959, Fitzpatrick, Brunet and Kukita(6) used autoradiography to measure genetic influences on tyrosinase activity within mouse hair bulb melanocytes. The incorporation of tyrosine-2-Cl4 into melanin varied, with brown (hm) = dilute brown (dld;bm) > yellow @Y/a;B/BJ = black & / a ; m> pink-eyed dilute black &/jxD-;d~) > albino WE). No uptake of labelled tyrosine was found in the albino. Their model for melanogenesis was similar to Foster's(5). They tentatively proposed that eumelanin and pheomelanin were formed by two distinct but coupled In 1981, Townsend et al.(13) questioned Coleman's metabolic pathways with tyrosine the precursor for the former (7) evidence in favor of designating the c-locus as the and tryptophan for the latter. structural locus for tyrosinase. They found that c-locus alleles influenced total tyrosinase activity and the subcellular In 1962, Coleman(7) reported that tyrosine-2-C1 distribution of tyrosinase. Since the kinetic parameters of was incorporated into pheomelanin but tryptophan-Cl4 was tyrosinase were not influenced by the plocus alleles, they not. Measuring the amounts of radioactivity in skin concluded that the & c u s is probably not the structural locus homogenates, Coleman confirmed Fitzpamck et al's.(6) for tyrosinase. Their data could not distinguish between possible alternative explanations such as a "decrease in earlier autoradiographic finding that tyr0sine-2-C~~ was tyrosinase translation [or] an increase in tyrosinase turnover incorporatedinto eumelanin and concluded that tyrosine was due to a possible deficiency in post-translationalprocessing." the major precursor both of pheomelanin and eumelanin. In In 1985, supporting this interpretation of c-locus function, addition, Coleman tested a larger number of coat color genes Townsend et al.( 14) provided evidence indicating that than Fitzpamck et a1.(6) for their influence on tyrosinase activity. To a large extent, Coleman confirmed Fiupatrick et himalayan (ch)tyrosinase had the same temperature al. (6) on genotypes overlapping with his study. His detailed sensitivity as the wild-type (Q tyrosinase characteristic of analysis of the c-locus alleles revealed differing levels of C57BW6J mice. tyrosinase activity > E h > ch > ~e > d. Focusing on the Following the shift to the molecular genetics of tyrosinase associated with the action of the himalayan (&) pigment cell biology during the latter half of the 198Os, the allele, h e provided biochemical evidence that it was uncertainty surrounding c-locus function has been significantly more thermolabile than wild-type Q tyrosinase. significantlyreduced. However, it is important to remember Coleman states: "This thermolability of the enzyme produced that direct knowledge of the gene for tyrosinase and its allelic under the influence of the Himalayan allele suggests that this variants leads to expectationsregarding the performance of its allele and thus, by inference, all of the alleles at the C-locus, products. Therefore it is important to match studies on control the structure and not the quantity of the enzyme tyrosinase performed prior to the major impact of molecular produced." In 1963, Foster(8) concluded that the h-locus genetics on pigment cell biology with those performed after to regulated the availability of endogenous substrate for determine whether studies subsequent to the "molecular melanogenesis and the number or effectiveness of melanin genetic revolution" differ because of interpretation or because binding sites on the proteinaceous melanosomal matrix. of real differencesin the biological material examined. If this is not done, older work will be considered to be superceded In the late 1960s, Prota and Nicolaus(9) brought by the new with the result that valuable insights into pigment order to conflicting reports about pheomelanogenesis when cell function will be ignored. they demonstrated that pheomelanin synthesis involved a deviation from the eumelanin pathway. Dopa quinone, In 1986, Shibahara et a1.(15) reported cloning and derived from the tyrosinase catalyzed oxidation of DOPA, sequencingof the cDNA for mouse tyrosinase. However the combined with cysteine to produce cysteinyldopas, sequence of the cDNA did not closely correspond with the intermediates in pheomelanogenesis, rather than forming primary structure of tyrosinase and was later mapped to the bleucodopachrome, an intermediate in eumelanogenesis(9). locus by Jackson(l6). In 1987, Yamamoto et a1.(17) More recent studies [ 19861 by Lamoreux et a1.(10) indicate published a cDNA sequence which differed from that of that not only is overall tyrosinase activity significantly Shibahara et a1.(15) but mapped at the c-locus and matched reduced in yellow mice but that, compared to wild type the primary structure of tyrosinase. In 1989, Kwon et a1.(18) (black) tyrosinase, the DOPA-oxidase activity of AYIperformed a nucleotide sequence analysis of the albino (c) tyrosinase is substantially reduced relative to its tyrosine allele of Balb/c mice detecting a G to C substitution which hydroxylase activity. This finding seemingly contradicts L. results in the replacementof cysteine by serine at a critical site B. Russell and W. L. Russell's(4) observation that the in tyrosinase(18). The himalayan allele ($) has also been intensity of the DOPA-reaction was the same in eumelanic sequenced and a point mutation replacement of A by G W& and pheomelanic &Y/& mice otherwise identical in identified which results in histidine being replaced by arginine major pigmentation genes. at a key position in tyrosinase(l9). The demonstration that cDNA capable of coding for tyrosinase maps to the plocus In 1973, Hearing(l1) demonstrated tyrosinase activity and that G-locus mutant alleles show point mutations in albino tissue homogenates that could be enhanced by potentially capable of altering the functional properties of treatment with 'Triton' X-100. In a fraction containing tyrosinase appears to establish that the s-locus is the structural premelanosomes from the eyes of albino mice, tyrosinase locus for tyrosinase. Although the h-locus protein expresses activity almost equaled that of wild type (black) following considerable point and sequence homology with tyrosinase, treatment with 'Triton' X-100. He suggested that the Halaban and Moellmann(20) have concluded that it is a detergent released albino tyrosinase from its association with
W. C. Quevedo et al. catalase [catalase B]. Jiminez et al.(21) and Hearing(22) have reported that it is a tyrosinase with markedly reduced activity compared to G-locus (wild type) tyro&=. In 1988, Halaban et al.(23) determined that albino cd) tyrosinase did not display enzymatic activity and was a b d y sensitive to proteolytic cleavage. ~n conmt, himalayan ($1 tyrosinase expressed defects in N-linked glycosylation which rendered it unstable and abnomdy heat sensitive. Therefore, to this point,rhree major issues relating to tyrosinase which have been addressed by molecular geneticists but with their roots in physiological genetics remain to be adjudicated. First, is there an inhibited tyrosinase in albino mice at least potentially capable of approximating wild-type tyrosinase activity( 1l)? Second, is the tyrosinase of himalayan mice abnormally sensitive to heat inactivation(7,13,14)? Third, is the h-locus protein a determinant of melanin binding sites on the melanosomal maaix(8). a catalase(20),or a weak tyrosinase-likeenzyme which may supplement the ~-1ocusin melanogenesis(21,22)? Fourth, based on the discussion to follow, is there a structural difference between yellow and wild type tyrosinase which would m u n t in part for its reduced overall activity in BY mice? Answers to these questions are important for they determine the credibility of models that m a y be generated to visualize how genes regulate melanogenesis. In 1989, Tamate et al.(24) demonstrated that the levels of immunoprecipitable tyrosinase were reduced in the skin of agouti @/A)mice compared to wild type and tyrosinase messenger RNA (mRNA) levels were about the same in the two genotypes. They concluded that the agouti locus exerts its influence on tyrosinase by controlling its translation from mRNA and/or its post-translational modification. The suggestion that alleles at the a-locus may influencepost-translationalmodifcation of tyrosinase lends credence to L a m o ~ u xet al.ls(l0) proposal that there may be structuraldifferencesbetween yellow (BY/-) and black (wild type)tyrosinase.
In albino WE) mice, immunologically-precipitable tyrosinase was absent although normal levels of tyrosinase mRNA were present. Albino tyrosinase exhibited little tyrosine hydroxylase and no DOPA-oxidase activity. Although, they performed their assays of dopa oxidase activities on preparations treated with 'Triton' X-100. the preparations which were tested for tyrosine hydroxylase activity, unfommately, were not so trcated. Tamate et al.(24) concluded that, as in the case of the 8-locus, the s-locus in some way regulated translation of tyrosinase mRNA andor the post-translational modificaton of the enzyme rendering it unreactive to the tyrosinase-specific antibody. The failure of Tamate et al.(24) to demonstrate immunoprecipitable tyrosinase in albino mice conflicts with Takeuchi's(25) and Halaban et al.'s(23) publications to the contrary. Halaban et al.(23) found that tyrosinase, following proteolytic cleavage, retained rtactivity to the tyrosinase antibody. In contrast with their observations on the a- and Eloci, Tamate et al.(24) found that in brown (hm) mice and dilute black (dld;BIB)mice the levels of tyrosinase mRNA and immunopxxipitable tyrosinascwere elevated compared to wild type black (B/B) mice. They believe that these loci regulate transcription of the tyrosinase gene leading to increased mRNA and tyrosinase. It is not clear how this action of the b-gene would produce the well documented brown color of melanosomes and their ultrastructure which
59
also differs from wild type. In view of the far-reaching implications of Tamate et A's wak for the gemtic regulation of tymhasc activity, it would sctm ofthe urmost imporcanct to determine why their findings on the cross-reactivity of albino tyrosinase with tyrosinase antibody differfrom those of Takeuchi(25) and Halaban et al.(23).
In 1990, Tanaka et al.(26) accomplished the difficult feat of microinjecting a mouse synthetic minigene mg-Tyrs-J for mouse tyrosinase into fertilized eggs of albino (ddmice producing transgenic mice which synthesizd melanin only at sites w h m pigment is normally found in wild type mice. The ability to transplant the tyrosinase gene at will, opens up new possibilities for determiningthe mechanisms by which genes regulate pigmentation, for the balance of the genome can be altmd and its consequence evaluated by examination of the altered pigmentationof transgenic mice. A historical approach to the current understanding of tyrosinase genetics meals steady progress paralleling the development of more sophisticated techndogy. The important message arising from this approach is that current achievements, no matter how complicated the technology upon which they are based, do not automaticaUy replace past ones. The testing and assimilation, where appropriate, of "older" ideas about tyrosinase promises to reveal a complexity of melanogenesis that has not been generally appreciated. This paper has attempted to identify sevetal key areas where this effort might begin.
RefereRussell ES. A quantitative histological study of the pigment found in the coat-color mutants of the house mow. I. Variable attributes of the pigment granules. Genetics 1946331:327-46. 2. Russell ES.A quantitative study of genic cf€cctson coat color in the house mouse. Genetics 194&31:227. 3. Russell ES. A quantitative histological study of the pigment found in the coat-color mutants of the house mouse. II. Estimates of the total volume of pigment. Genetics 1948:33:228-36 4. Russell LB, Russell WL.A study of the physiological genetics of coat color in the mouse by M B ~ S of the dopa reaction in frozen sections of skin. Genetics 1948:33:237-62. 5 . Foster M. Enzymatic studies of pigment-forming abilities in mom skin. J Ex@ Zoo1 1951:117:211-46. 6. Fitzpaaick TB, Brunet P, Kukita. A. The n&turcof hair pigment. In: Montagna W.Ellis RA. eds. The Biology of Hair Growth. New York: Academic Press, 1958~255-303. 7. Coleman DL. Effect of genic substitution on the incorporation of tyrosine into the melanin of mouse skin.Archiv Biochem Biophys 1%2:69562-8. 8. Foster M. Some physiological genetic aspects of mammalian melanogenesis. In: Riley V, Former JG, eds. The Pigment Cell. Annals New York Acad Sci, 1963:100:743-8. 9. Prota G. Strucnue and biogenesis of phemmelanins. In: Riley V, ed. Pigmentation: Its Genesis and Biological Control. New York: Appleton-Centurycrofts, 1972:615-30. 10. Lamorcux ML, Wooll~yC, Pcndcrgaat P. Genetic controls over activities of tyl~sinaseand dopachrome conversion factor in murine melanocytes. Genetics 1986:113:967-84. 1.
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W. C. Quevedo et al.
1 1. Hearing VJ. Tyrosinase activity in subcellularfractions of black and albino mice. Nature New Biology 1973:245:81-3. 12. Pomerantz SH, Li JP-C. Tyrosinase in the skin of albino hamsters and mice. Natrrre 1974252941-3. 13. Townsend D, Witkop CJ,Jr., Mattson J. Tyrosinase subcellular distribution and kinetic parameters in wild type and ~-1ocusmutant c57BU6J mice. J Exptl Zoo1 1981:216113-9. 14. Townsend D, Guillery P. King RA. Himalayan tyrosinase does not demonstrate temperature sensitivity. In: Bagnara J, Klaus SN, Paul E, Schartl M, 4 s . Pigment Cell 1985. Tokyo: U. Tokyo Press, 1985121C
15. ihibahara S, Tomita T, Sakakura C, Nager C, Chaudhuri BB, Muller R. Cloning and expression of cDNA encoding mouse tyrosinase.Nucleic Acids Res 1986 142413-27. 16. Jackson IA. A cDNA encoding tyrosinase-related protein maps to the brown locus in mouse. Pnx: Natl A d Sci USA 1988~854392-6. 17. Yamamom H, Takeuchi S, Kudo T,et al. Cloning and sequencing of mouse tyrosinase cDNA. Jpn J Genet 1987:62271-4. 18. Kwon BS, Haq AK, Wakulchik M, et al. Isolation, chromosomal mapping, and expression of the mouse tyrosinase gene. J Invest Dermatol1989:93:589-94. 19. Kwon BS, Halaban R, Chintamaneni C. Molecular basis of mouse himalayanmutuation. Biochem Biophys Comm~n1989161:252-60. 20. Halaban R, Moellmann G. Murine and human h locus pigmentation genes encode a glycoprotein (gp75) with catalase activity. Proc Natl Acad Sci USA 1990:87:4809-13. 21. Jim6nez M, Maloy WL, Hearing VJ. Specific identification of an authentic clone for mammalian tyrosinase. J Biol Chem 1989:264:3397-403. 22. Hearing, V. The nature of tyrosinase isozymes. Mishima Y, ed. Abstracts of the XIVth International Pigment Cell Conference. Pigment Cell Res 1991: (in press). 23. Halaban R, Moellmann G, Tamura A, et al. Tyrosinases of murine melanocytes with mutations at the albino locus. Proc Natl Acad Sci USA 1988:85:7241-5. 24. Tamate HB, Hirobe T, Wakamatsu K, It0 S, Shibahara S,I s h h w a K. Levels of tyrosinase and its mRNA in coat-colamutants of C57BU6T congenic mice: E B m of genic substitution at the agouti, brown, albino, dilute. and pint-eyed dilution loci. J Exptl Zoo1 1989:25O:3O4-11. 25. Takeuchi T. Yamamoto H. Sato S, Ishikawa K. Demonstration of the tyrosinase CRh4 in albino skin by use monoclonal antibodies. In: Jimbow K, ed. Structure and Function of Melanin, vol 1. Sapporo: Fuji-shoin Co. 1984.56-8. 26. Tanaka S, Yamamoto H, Takeuchi S, Takeuchi T. MelaniZation in albino mice transformed by introducing cloned mouse tyrosinase gene. Development 1990:108~223-7.
