foy April 30, 1959 Dr, Katherine Wilaon, Executive Secretary Genetica Study Section Division of Research Grants National Institutes of Health Bethesda 14, Maryland In ret Cm2440 (C0451) Dear Dr, filgons In reaponse to your request, the following is a summary report of progress achieved by the above project durins the past two years, Only major ;oints sre incluied, The work of the project has cantered around she problezs of the genetic control of ;rotein synthesis, From a senetic point-of-view, the most ig :ortant preplems of --ene-to-):rotein inforreation transfer are those of the determination of amino acid se,uence ani the control of folding of the polypeptide chain, From a biochemical point-of-view, the problems consist of demonetrating the involvement of the biochemical processes involvei in protein synthesis, i,e., activation sf amino acids by ATP, transfer of amino acida to and transport by “soluble” RNA, condensation of amino acids into a polypeptide chain presumably on a glerosonal RNA template, removal of the polypeptide chain from the RNA surface, and formation of foldinzs and cross protyrosinase ————______» tyrosinase Sta sel 3tase 2 Dr. #ilson< It was proposed thit each of these staves is under szenetic control, Staze 1 couid involve establishment of amino acid secuence and preliminary folilnz, while stsaise 2 could involve establishment of final tertiary coafisuration, The major features of this proposal have now been validated (Fox and Burnett, 1959). Frotyrosinase, a large, enzymatically inactive polypeptide, electrophoretically sep- arable from tyrosinase, has been demonstrated and isolated, In mycelial extracts it is activited to form tyrosinase of the game speciticityas isa formed in vivo, The kinetics of activation are first order, susgsgestins a mon>molecular or pseudomono- molecular process, Activation is meiiated by one or more enzymes, electrophoretically separable from both pypotyrosinase and tyrosinase, Oultures which ere senetically incapable of foruing tyrosinase either fall to fore protyrosinage or fail to form activating enayme, These results are in marked contrist to those reported by Horowits ani Fling, ,. These, ¥orkers report a pair .f contrasting slleles, T” and T°, producing respectively therno- atabile ani Gaernolabile tyrosinase, Since only one of the two enzyses could be found in a hosecaryon (jepending on which allele wis present), and since both ensymes were produced in unaltered fora in heteroc:ryons, it wes concluded that each of the slleles produces its respective enazywe without interaction, de@e BY Means of a teu. late mecLaniem,. No differences in specificity (Michaelis conatant, turnover number) could be found between the thermostable ani thermolabile ensymes, and strip paper electrophoresis yielded no evidence of hetcro- geneity in either case, Until recently, the connection between the analysis of Horowits and Fling ani our own was not apparent, We had also been unable to ieconstrate heterogeneity in strip paper electro- phoresis, but it appeared that only the ther:ostabile tyro~ Sinuse was present in our material, We aow find, however, th-t continuous paper electrophoresis serves to separate the tyro- Sinase extracted from homocaryona into three cum: onents of indistinguishsole specificity. One of these (2, ) exhibits an enersy of thernal inactivation of 95,300 calv/mole, virtually identical with the value reportei by Horowits and Fiing for their thermolabile tyrosinase, The other tro (T, and tT) exbibit energies of inactivation e.usl to 60,900" and 57,300 cal, /fmole respectively, closely simiiar to the values reported by Horowita and Flinz for their tuermostabile engyne, Together, T. and fy constitute 96% of tne trrosinsse extracted. Phe presenc® of P,“cannot be Jetected withioub’ prior fractisn- ation by continuotts paper electrophoresia. MoreoveR, during incubetion of eruwie extruc.s at low temperatures (5%=25"), fT, and T., diaappear, Quantitetive measurements disclose thst the teerBese in ft, and tT, is exactly comsensatel by an increuse Dr, #ilson- in Tye asp if both af the formar are cunverte! inte the Llatters (2; 1) 91, oP Ty Bs Ta Overall, the terminal stsges of tyrogiunse ayntnesis seen to be as follows: p—*_9(?,—>t,) >, or E , E Fy Erotyrosi nase EB, activotin; enayme or enzymes taersolabile® tyrocinises TB. and T3e *thernostabile” ny inage,) - it is entirely possible that the converstoe. of zy and Tt to Tt, ia also ensynatically cavalyaed, » Phe identical specificities of ty » and T, would seem to render the first of these achertes Ieretatie ani would indicate thut gpeeificity is jetermined in earlier stazes of synthesis, de> at the tine of detersinstion of anino acid sequence or “uring preliminary folding, The oft ee in tuernoatability and electrophoretic wobility of T ’ and sugcest dif erences in te:tiory structure rath t A an thes active centers, The conversion of the two former into the latter suggests that the T locus of Horowitz, and Fling produces ita effects by controlin.: the rate of these terminal stages in tyrosinase synthesis, thu: lesiins te the predone {nance of one or the other of the enzynes, rather than by means of a tesclsate mechanism, (These results will be re:orted at the fortheoning AM IT. B. 5. neetinys,) The second of the two oases whiea we have investi sated has dealt with antigenic effects of the sex chromosomes in Poss Tassy. elinozaster (fox and Yoon, 1958; Pox, 19583 ox anres in Xechromogsome gosa56 result in gualis. oLve siifte in : anticen pattern, as is summarized in ie, tileon= the following table, Antizens Sex Chromocomes G~]} Gea2 G=l Gad xX/x ~ a - + vhs ~ - + + er'z.r - . + + X/fK-+T ~ ~- + + KA + + - - rrp + + - - xxi > * - - XT /O + + - - Bach of the four antigens is a protein and possesses distinct: entizenia specificity - there is no cross-reaction of one with antibody to any of the others, As may be sean, iniividusls with one X-chromogome, resarcless of the presence or absence of all or part of the Y, possess antisens Om] and c@2, Individuals with two Xechromososea, regordless of Y conatisution, posseas Qe] and 9-2, The Y chro: esore is thus not involved, anc the effect ia attributeble to the difference in doaase of one or nore euchromatic loci on the Z, possibly the sex~ determining loci thenmgelvea, ne effect ia obviously one on the configuration of the antisgenie sgrouplose themselves, fhe sisnificance of she existence of two specific anutisenga in each case is not yet understood, but they may be true alternutives, This ta another instance of an effeot of euchrosatic loci on the antizenic apecifieity of proteins, but since it is a cualitative effect of dosaze it is. bable thut a ginple te:plate mechanian is involved, It seema Likely that the differences involve either differences in amino acid sequence or in prelininary folding (or perhaps in both), The hecerovhromatic Yechronosome hus ao detectable effect on the antigenic specificity of proteina, However, the immunological ani physical properties of a particular protein are subject to a maternal influence of the Tecnromosgome, Dr, #vilson- When a female lacke a Y-chromosome (X/X) her progeny exhibit an antigenic apecificity (¥-1) associated with a protein capable of inducing antibody formation in rabbits, and capable of uniting with and precipitating the specific antibody, The term “somplete” ia used to describe the properties of the antisen under these concitions, When a female possesses a Y-chromogome (XZ/¥) her progeny exhibit the same antisenic specificity (Y¥<-1), but it is associated with a protein which is incapable of precipitating the specific antibody, althou sh it 4e capable of iniucing formation of the antibody and of uniting (inhibiting) with it in v . The tera:*ineanplete” ‘$@ applied to this situation, fhe effect seems to be localized in the proximal portion of the short arm of the Y-chromosome, i.e. to that portion carrying the nucleolar organizer. fhe difference between complete and incomplete Y-1 probably reflects a physical difference between the raspective molecules of a sort not involvins a (difference of their antizenic group= ings but rather in their number, Both forma of Yel are non- dialyzable, heat labile, and precipitated by protein pre- cipitante, Complete x-1 is precipitated from crude extracts by simple dialysis ageinst water, A physic:i difference between complete and incomplete Yel is sugsested by a greater resistance of the former to imactivation during lyophilization. The two forns could differ either in the sise of their respective molecules or in the way in which the polypeptiie chain is folded, It ia interestins to note thst it is the presence of the Y-chromosome in the oocyte of a female that 1s reaponsible for this effect, but that the effect is observed in her progeny as adults, The lose of the ¥ during meiosia does not alter the result. Thus, a self~perpetuating mechanism must be est~ ablished in the oocyte, capable of maintaining itself in the goma during all of development. These observations are probsbly associated with those of Sehulte and coworkers that the Yechromosome influences the base constitution of RNA synthesized in the oocyte. Since euchromatic chanzses heve effects on specificity, while heterc- chromatic chanzes seem to affect only tertiary structure, it 4s possible to suggest that each produces an RNA of different function, Euchromatin may produce RNA concerned with the determination of amino acid sequence and preliminary folding, while heterochromatin mitht produce RNA concerned with the determination of final tertiary structure, Both of these kinds of RNA could bgp the constiteents of the cytoplasmic (microsomal) ribonucleoprotein particles, and some kind of mechanism of somatic self=-perpetuation is conceivable, These observations and specul:tions serve to indicate the iireczcion of future work and provicse a connection with De. Wilson= the recent biochemical developments referred te above, fhe following publicationa have resuited from the work of the project during the past two years! Fox, Ay 5S, 1957. Genes, The Enoylopedia of Chemistry, é, L, Clark and G, G. Hawley, eds., Reinhold, New York, PPe 442-444, Fox, A. S.y and 3, B, Yoon, 1957, Application of azar- diffusion techniques to the analysis of Drosophila anticens, Anat, Rec, 1281552 (Abstract). Fox, A. Sy and &. B. Yoon, 1957, Genetic mechanisns responsible for antizenic differences between males and females in Drosovhila melanogsster. Genetics 42: 370 (abstract). Fox, A. 85, and J, B, Burnett, 1957, The components of the protyrosinase activation system in Neuroapg: iy atrain 15500, an: their production by genetically aifferent cultures, Genetics 42:370 (Abstract), Pascaldo, K, & 1957, A technique for the collection of bacteriologically sterile flies, Drosophila Information Bervice 31:174—175, Bead, C. G, 1957, The «ffect of Bar, Enhancer of Bar and chanses in thelr positions on the free amino acids and peptides of 3. melanogaster. Drosophila Information Service 31:1133<154, Fox, Ae See and 8. 3B. Yoon. 1958, Antisenie a4fferences between males and females in Drosophila not attributable to the ¥ chromosome. Transplant. Bull, 525255, Fox, Ae 3. 19585, The genetics of tis ue specificity, hranspiant. Bull. 5:77 (Abstract), Fox, A. S,, and J, B. Burnett. 1958, Kinetics of tyrosine oxidation by erudie tyrosinase preparations from Neurospora gregsa., Proc, Soo, Exp, Biol. Med. 981110-114, — Fox, Ae 3. 1958, Immunogenetic studies in relation to problems of protein synthesis, fFroe, X Int, Conz, Gen, 21846885 (Abstract). Pox, A. S. 1954. Genetica of tissue specificity, Annals Pox, As Bes and Je 3B. Burnett, 1959. The genetics and biocheristry of tyrosinase in Neurospora crassa. In Pigment Cell dology, kK. Gordon ed,, Acadente BS, PPe 2hI~277 « br, Wilson- Fox, As Sas Mead, C, Gey ami I. L. Kunyon,. 1959. The sex peptide of Drosophila melanogaster. Sealence, in press, Fox, Ae 3. 1959, Genetic determination of sex-specific antigens, Jour, Nat. Cancer Inet., in press. Sincerely yours, Allen 3B, Pox