230 PHYSIOLOGIC GENETICS 



can be used. In the study of the dilute gene, we were very fortunate in that mutation 

 to the intense color has occurred several years ago in the DBA stocks. Thus we had 

 genetic homogeneity with an intense animal, DD, a heterozygote, Dd, and the normal 

 dilute animal, dd, all available within the same strain. We were fortunate also here in 

 having a multiple allelic series to work with since there is another allele of this locus, the 

 dilute lethal. This allele, although not available on the DBA background, was in the 

 process of being inbred on another stock. 



Also, we made use of pleiotropism. Both dilute lethal and dilute mice have diluted 

 pigment, but the dilute lethal dies at about 3 weeks and is subject to spontaneous epilepti- 

 form seizures. The DBA strains are also subject to seizures under audiogenic stimuli 

 and it seemed that there should be some relationship between this type of seizure and 

 the pigmentation defect if dilute were just a lesser dose of dilute lethal. The diluted 

 pigmentation further suggested a possible abnormality in aromatic amino-acid meta- 

 bolism. Thus, one of the first things tried was incorporation studies of radioactive 

 tyrosine (the normal pigment precursor) into the pigment granules of both dilute and 

 intense mice. It was found that the rate of pigment formation was the same in both 

 genotypes when tyrosine is used as the pigment precursor. Next the enzymes involved 

 in the formation of tyrosine were examined. The enzyme, phenylalanine hydroxylase, 

 which forms tyrosine from phenylalanine was found to be deficient in dilute strains of 

 mice, having an activity of about 50 per cent in dilute mice, dd, and about 14 per cent 

 that of the normal in dilute lethal. This, at first glance, provided a ready explanation 

 for the diluted pigmentation. However, it was pointed out by Dr. Russell that there 

 is not less pigment in the dilute animal but rather that the granules are in a clumped 

 formation. Also, calculations showed that a leaky enzyme which allowed a 50 per 

 cent production of tyrosine would allow normal pigment formation under most of 

 the conditions that we know, especially when one considers that some tyrosine is 

 derived from the food. Thus, it appeared that we were working with a secondary 

 effect of the original genie action. Further studies on the actual amount of the 

 enzymes present indicated that there was no actual difference between the amount 

 found in dilute and nondilute animals; one function of the dilute gene seemed to be the 

 production of an inhibitor of the enzyme, phenylalanine hydroxylase. 



In any event, the situation is somewhat analogous to that found in phenylketonuria, 

 diluted pigmentation and inhibition of this enzyme with a subsequent accumulation of 

 phenylacetic acid and other phenylalanine metabolites. Phenylacetic acid is a com- 

 pound toxic to the central nervous system which suggests an explanation for the 

 seizure in dilute mice. There are several areas of research which we now are attempting. 

 Could phenylalanine or a metabolite in abnormal concentrations cause an abnormal 

 development of the brain or neural crest which then leads to these seizures? Could 

 such abnormal concentrations cause an irreversible change in the mode of pigmentation, 

 thus preventing the animal from ever forming normal pigment ? Or, on the other 

 hand, could the decreased levels of tyrosine be critical at an early period of develop- 

 ment, thus causing these changes ? The final answers to these problems will only be 



