336 BIOCHEMICAL GENETICS 



no doubt that a systematic analysis of mammalian pigmentation still provides one of the 

 best methods in elucidating the many diverse ways that genie action can influence the 

 behavior of a single cell. 



DISCUSSION 



Dr. Burdette : Dr. Morris Foster will discuss Dr. Silvers' paper. 



Dr. Foster: I find myself in the quandary of finding something to say when Dr. 

 Silvers' work is so clear that it drastically reduces the number of possible questions any 

 fertile imagination could produce. Therefore I should like to defer any questions 

 directed to Dr. Silvers in favor of anyone in the audience who has perhaps less favorable 

 bias toward his work and to provide instead a very brief supplement describing some 

 recent advances in methodology relating to the genetically controlled biochemistry of 

 mammalian melanin pigmentation. 



At the subcellular level, we are confronted with processes which occur in at least 

 three distinct echelons of organization in the sequence from tyrosine to melanin. At 

 the first, small molecular level within differentiated and functioning melanocytes, we 

 deal with substrates such as tyrosine. The precursor undergoes a series of trans- 

 formations, and we then proceed to a second organizational level, that of the macro- 

 molecule, when the late intermediates being to polymerize. We finally arrive at a 

 third level of organization, that of a fully formed cytoplasmic organelle, when a poly- 

 merizing product is conjugated with an already formed protein matrix to produce a 

 mature melanin granule. 



It should be emphasized at the outset that it is quite naive to equate tyrosinase 

 activity with the whole process of melanin formation ; it is asking too much of mam- 

 malian material to expect that assayed tyrosinase activity and the amount of melanin 

 would be modified either to the same degree or, indeed, in the same direction by given 

 allelic substitutions. Thus, before trying to relate a given genotype to a given amino- 

 acid sequence of the tyrosinase molecule, it might be useful first to find out which 

 genie substitution affects which part or parts of the pigment-building system, and at 

 least to obtain, for biochemical purposes, a tyrosinase-rich tissue. At least one such 

 genotype was unexpectedly found, and it seems to offer now the greatest hope for 

 extracting and solubilizing the enzyme and, ultimately, for determining its amino-acid 

 sequence (perhaps also permitting analysis of secondary and tertiary structural features). 



Our own recent approach to objective and quantitative assays for genetically 

 controlled, melanogenic attributes involves the use of lyophilized individual skins, 

 from each of which we obtain four, equal, dry-weight subsamples. These subsamples 

 are assayed respirometrically for tyrosinase and dopa-oxidase activity, and subsequently 

 they are also assayed for melanin content by means of turbidimetric measurements of 

 alkaline thioglycolate suspensions of unincubated tissue or of tissue which had been 

 incubated in the Warburg vessels and removed after a standard period of incubation. 

 The genetic materials we used involved separate and combined substitutions at the 



