DROSOPHILA MELANOGASTER. V. AMINO ACID METABOLISM and TUMOR FORMATION in the Tu Bw ; St Su-Tu STRAIN

Home , Anthranilic acid, Phenylalanine

PHYSIOLOGICAL GENETICS OF MELANOTIC TUMORS IN DROSOPHILA MELANOGASTER. V. AMINO ACID METABOLISM AND TUMOR FORMATION IN THE tu bw ; st su-tu STRAIN

BARRIE BURNET AND JAMES H. SANG

Department of Genetics, University of Sheffield,' and School of Biological Sciences, Uniuersity of Sussex, England Received October 23, 1967

HE tu buJ; st su-tu strain carries a tumor gene located on the second chromo- Tsome at locus 83.9 ( GLASS1954), and a recessive suppressor on chromosome three, so that tumors appear in only a small proportion of individuals. When the strain is propagated on live yeast medium the level of tumor penetrance is variable, and depends upon a number of factors in the external environment. BURNET and SANG(1964a and b) and SANGand BURNET(1967) have shown that when the strain is cultured under germ-free conditions, on synthetic culture media, tumor incidence is quantitatively controlled by the availability of certain essential nutrients. Deficiencies of protein (casein), cholesterol. phospholipid, niacin, pyridoxine, thiamine, and magnesium, or high dietary concentrations of tryptophan, all increase tumor incidence. Further investigations are required to establish whether (1) the tumorigenic effect of dietary amino acid excess is restricted to a limited range of amino acids or their dissimilation products, and (2) how the action of these compounds may be related to the effects of other nutritional treatments known to control tumor incidence in this strain. Evidence bearing on the former question is described by PLAINEand GLASS(1955) who obtained a marked iixrease in tumor incidence by feeding excess dietary tryptophan, phenylalanine, and tyrosine, and a number of compounds related to txyptophan, including kynurenine, anthranilic acid, and indole. A difficulty in the )way of interpreting these results, however, is that compounds were added to undefined media in the presence of yeasts and other microorganisms, and consequently it it uncertain whether any of the observed effects are actually due directly to the compounds being tested, or to altered by-products of yeast metabolism. Chromosome substitution analysis showed that the tumorigenic effect of nutr- tional treatments depends on the presence of the tu gene on the second chromosome, rathci than an indirect effect caused by interfering with the action of the suppressor (BURNETand SANG1964a; SANGand BURNET1967). Although sug- gestive, the evidence that the tu gene itself is implicated in the response is by no means conclusive, since it was not possible to separate effects attributable to the presence of the tumor gene from those attributable to the second chromosome as

' Address of the first author

Genetics 59: 211-235 June 1968 212 BARRIE BURNET AND JAMES H. SANG a whole. Comparative studies showed that both tryptophan excess and cholesterol deficiency increase tumor incidence in the tuKstrain, which is known to be free of any major tu gene mutation expressed under normal environmental conditions. However, the F, between the tuKand tu bw ;st su-tu strains also responds to these treatments, indicating that allelic mutant genes control treatment sensitivity in both parental strains. Tumorigenic effects of dietary tryptophan in strains normally free of melanotic tumors are reported by SIMMONSand GARDNER (1958), and by HINTON,NOYES and ELLIS(1951). It is known that a number of loci in the second chromosome are potentially capable o€ initiating tumor formation, and some of these, like tuTc,are isoalleles with phenotypic effects detectable only under conditions of physiological stress. These considerations point to the possibility that the range of nutritional treatments affecting tumor incidence in the tu bw ; st su-tu strain may represent a mixed array of gene- environment interactions involving several loci in the second chromosome, some of which are independent of the presence of the major tu gene mutation. The aim of the present study is to examine the relationship between tumor incidence and genetic and environmental factors affecting amino acid metabolism, and to achieve a more precise characterization of the metabolic basis of tumor gene action.

MATERIALS AND METHODS

Culture media Experiments were carried out at 25°C using germ-free larvae cultured on chemically defined medium, of which technical details are given by SANG (1956). Cultures in 15 x 2.5 cm boiling tubes containing 5 ml of medium were each inoculated with 50 larvae, and control and treatment groups each contained 8-10 replicate cultures. As shown in Table 1 the medium contains 5.5% vitamin-free bovine casein. The figures given for the amino acid content of the medium are based upon analyses of the amino acid content of hydrolysates of this casein, allowing for moisture and ash content of the sample used. The protein-free amino acid medium used to determine the essential requirements of the larvae for arometic amino acids was slmilar to that described by GEER (1966). Thermolabile compounds were sterilized by Seitz filtration and added to the autoclaved basic medium. The usual checks for sterility were made, and infected cultures were discarded. Strains: A description of the tu bw ;st su-tu strain is given in preceding publications of this series. Incorporation of the genes vermilion (U) and yellow (y) into this strain involved complete substitution of chromosome 1 by the marker-bearing first chromosome, and consequently is more than a single locus substitution. Chromosomes 2 and 3 were kept isogenic during this process by balancing over the complex multiple inversion containing chromosomes SM5 and TM3 which effectively suppress crossing over throughout each of these major autosomes. Complete replacement of the second chromosome background containing tu was achieved by crossing the tumor strain tu bw ;TM3/Sb to a strain homozygous for al dp b pr c px sp to synthesize the strain al dp b pr tu px sp ;TM3/Sb. This strain was then crossed to the inbred Edinburgh wild strain and one double crossover chromosome lacking all visible markers to the left and right of tu was extracted from the backcross. The resulting lu strain was serially backcrosssd to Edinburgh wild for 3 generations, and the tu gene in the genetic background of the Edinburgh wild second chromosome used to synthesize the tu ; su-tu strain using suitable combinations of balancer chromosomes at each stage. Amino acid analyses: Extracts for amino acid analysis were prepared following the procedure of CHEN and HANIMANN(1965) using approximately 2-3 g freshweight of larvae from aseptic media, and 5-log of larvae from live yeast medium. These were synchronized larvae in all AMINO ACIDS AND TUMORS IN DROSOPHILA 21 3

TABLE 1

Composition of the aseptic medium

Medium Amino acids in casein

g agar 3.00 alanine 0.014 casein 5.50 arginine 0.178 sucrose 0.75 aspartic acid 0.357 cholesterol 0.03 cystine 0.014 lecithin 0.40 glutamic acid 1 .om ribonucleic acid 0.40 glycine 0.086 thiamine, HC1 O.ooo2 histidine 0.153 riboflavine 0.001 isoleucine 0.244 niacin 0.0012 1e u c i n e 0.396 Ca pantothenate 0.0016 lysine 0.441 pyridDxine, HCl 0.00025 methionine 0.128 biotin O.ooOo16 phenylalanine 0.238 folic acid 0.0003 proline 0.396 NaHCO, 0.336 sxine 0.270 KH,PO, 0.063 threonine 0.175 Na,HPO, 0.266 tryptophan 0.094. MgSO, 0.012 tyrosine 0.344 Water to 100ml valine 0.267

The amino acids present in the casein are given as g of amino acid per 100 ml of medium. cases. Extractions in methanol were made at -3O"C, and dried at 28°C. Dried extract was taken up in 1 ml of double distilled water per 1 g of initial freshweight, washed with chloroform, and after centri,fugation (60,000 x g for 30 min) 0.9 ml of sodium citrate buffer (pH 2.2) per 1 g of initial freshweight was added with final adjustment to pH 2.2 with ~/10HCl. The final extract volume was standardized at 2 ml per g initial freshweight. Charcoal extraction was omitted, since preliminary tests showed that this leads to differential loss of aromatic amino acids, and that yellow pigment present in larval extracts does not interfere with other fractions on the column. Amino acid analyses were performed by DR.W. FERDINANDon an EEL amino acid analyzer, programmed 'according to the system of SPACKMAN,STEIN and MOORE(1958). 1-2 ml of prepared extract was applied to the columns in 0.2~sodium citrate buffer, pH 2.2. Neutral and acidic amino acids were separated on a 150 cm column at pH 3.25 and 30"C, followed by buffer at pH 4.25 and 50°C; the buffer and temperature changes were made after 11% hours. Basic amino acids were separated on a 50 cm column at pH 4.26 with a temperature change from 30°C to 50°C after 10 hours. Known standards were run in this system to ideatify peaks for which data are given in Tables 6 and 7. In addition to these identified fractions, there were three major unidentifiesd peaks of which one (running between methionine sulfoxide and aspartic acid) is probably identical with the "peptid" peak of CHEN and HANIMANN(1965). Under our conditions tyrosine phosphate (Tyr. P) is not distinguishable from the major peak of glycerophosphoethanolamine (GPEA). In both the standard and in the wild-type extracts the major peak of GPEA is associated with a faster running minor peak. The areas under these two GPEA peaks appear to be directly prqmrtionsl, ar.d this fact has been used to estimate the amount of GPEA in the combined major GPEA + Tyr. P peak. This procedure clearly involves certain assumptions, and consequently the combined estimate for GPEA and Tyr. P is subject to some reservation. The combined valua for phosphoserine .+ cysteic acid, and glutamine + asparagine, was derived using an average color constant for the two compounds. There was same variation 214 BARRIE BURNET AND JAMES H. SANG in total yield of ninhydrin positive substan in p mole/g initial freshweight between samples from different treatment media, so to faci te comparisons the value for each compound is expressed as percentage of total yield. Paper chromatograms were run using a descending system with two solvents. (i) Isopropanol : ammonia : water (v/v. 20:1:2), and (ii) Butanol : pyridine : water (v/v. 1:l:l). Kynurenme was identified by Rf value, fluorescence in ultraviolet light (UV) at 254 P. and reaction with ninhydrin and EHRLICH’Sreagent. Known standards were run in each case. Quantization of the phenotype: Melanotic tumors are typical of a group of dichotomous threshold characters of the “all-or-none” type, in the sense that the phenotype of any given individual is normal or tumorous. Observations are limited to a measurement of incidence (p), which is the proportion of individuals which show the mutant character. For comparison of tumor incidences in the control and any treatment group the usual x* test has been applied, and the result expressed as the difference in percentage of tumorous individuals in the two groups. This method is satisfactory for some purpos-s, but more discriminating methods are required for quantizing gene-environment interactions. The proportion of a population showing the mutant character, is related to the magnitude of an environmental component z, as shown in Figure 1. When we are free to choose a series of values for I, the data will consist of sets of observations each one representing a proportion of the underlying distribution falling below a given threshold dsse of x. If the distribution of tolerances is normal cn the z dosage scale (or on some simple transformation of dosage), when we plot ca:h percentag- of mutant indivlduals against dose we obtain a normal sigmoid, which transforms to a straight line when the percentages are converted to probits or normal equivalent deviates. The probit of the percentage of mutant individuals is now related to the concentration, or dose of z, by the linear equation: 1 probit p = 5 f - [F(z) - m] S where F(z) is a normalizing transformati-n of z, and m and s are the mean and standard deviation of the distribution on the transform-d scale. A successful normalizing transformation of z is indicated by the linearity of probit p on F(z). In all data presented here it has been necessary to transform the dosage scale from x to log z. The constants m and s are estimated by fitting a straight line to the plotted pwints. The procedure is described by FINNEY(1962), and consists of maximum likelihood estimates of the regression coefficient b and median effective dose m.

...... CONCENTRATION of X X’ FIGURE1.-Individual tolerances to a treatment are assumed to be normally distributed in a population, such that individuals with tolerances below z‘ will show the mutant phenotype. The proportion (p) of the population responding to treatment is related to the area of the curve falling below 2’;whilst (l--p) do not respond. AMINO ACIDS AND TUMORS IN DROSOPHILA 215

The median effective dose is a measure of the mean of the tolerance distribution whilst the variance is related to the regression- coefficient as: variance sz =

Table 11 shows that in several strains a substantial proportion of individuals were phenotypically tumorous iri the absence of any treatment. Under these circumstances the proportion of tumorous individuals attributable to dietary supplements is not equal to the observed incidence in the treatment groups. If the proportion of individuals with tumors in the control is c, and the proportion with tumors at any treatment level is p’, then the proportion actually responding to treatment is: (P’ - c) PI- (1 --c) The adjustment is important because our uhject is to determine the mean and variance of tolerance to the tumorigenic activity of a specific environmental treatment, and consequently it is the responze of the phenotypically ngrmal population at risk which is used in computing the probit regression.

RESULTS Indiuidrial amino acids in excess: The effects of adding single amino acids to the medium are shown in Table 2. Each amino acid was supplied at a concentration of 40 mM irrespective of the concentration already present in whole casein. Of the 18 amino acids tested, 10 significantly retarded the rate of larval development, and only one, L-a-alanine, significantly improved it compared with the optimum casein level. Tryptophan and phenylalanine cause an increase in tumor incidence, as previously found by PLAINEand GLASS(1955), whereas lysine. aspartic acid and serine cause a smaller but significant rise. Lysine severely reduces larval viability at this concentration which appears to be the maximum tolerated by the larvae. An interesting feature here is the apparent lack of effect of tyrosine. Tyrosine itself is insoluble in the medium at normal pH and consequently may not be available to the larvae at an effective concentration. If phenylalanine is readily converted to tyrosine in uiuo, the bulk of the larval growth requirement might be derived primarily from this source. This point was investigated by replacing the protein (casein) normally provided in the larval culture medium by a balanced amino acid mixture containing phenylalanine and tyrosine, or double the concentration of either singly. The results are summarized in Table 3. Phenyl- alanine alone satisfies the growth requirement for phenylalanine and tyrosine, and therefore Drosophila larvae must be readily capable of carrying out the orthohydroxylation of phenylalanine. Tyrosine alone does not support growth, either because the rate of uptake is too low or because it cannot be converted to phenylalanine, or both. Thus, lack of effect of excess dietary tyrosine on tumor incidence is not in itself decisive since increased levels of dietary phenylalanine may raise the level of free tyrosine in the haemolymph amino acid pool. Only the L-isomers of tryptophan and phenylalanine have a significant effect. As shown in Table 4 the respective D-isomers are ineffective at a concentration of 40 mM and equimolar racemic mixtures consisting of 20 miv of each isomer 21 6 BARRIE BURNET AND JAMES H. SANG TABLE 2 The effectof dietary supplements of 40 mM of individual L-amino acids on the tu bw ; st su-tu strain

Tumor incidence Larval period percent log days Control 37.2 0.81% Aniino acid supplement: a-alanine + 5.06 -0.014.5 * * glycine - 4.05 f0.0079 isoleucine - 3.60 +0.0138 leucine - 5.79 +0.0144* * valine - 6.25 $0.0127 serine + 8.97* -0.0068 threonine + 0.45 +O.O"* aspartic acid +13.1** +0.0513** glutamic acid + 2.92 +0.0416** arginine + 5.35 +0.0022 lysine $18.69" +0.0596* * cystine - 7.29 $0.0185 methionine - 0.76 +O.ooeS phenylalanine +38.6** +0.0284** tyrosine + 5.60 +O.OlW histidine + 6.84 +0.0596** proline - 9.12 +0.0088 tryptophan +48.1** +0.0841**

In this and in subsequent tables * and ** indicate significance at the 5% and 1 % levels respec- tively.

do not differ significantly in their effect from 20 mM of the L-isomer alone. The effects of siinultaneous administration of 20 mM of both L-isomers together is by no means equivalent to the effect of 40 miv of either L-isomer alone, indeed tumor incidence is not significantly higher than for a single dose of 20 mM of phenylalanine. Consequently, tryptophan and phenylalanine act in a non-addi-

TABLE 3 Survival to adult on a protein-free synthetic diet containing arginine, cystine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, threonine, and valine

Amino acid mixture plus tu bw , st su tu Pacific wild tryptophan, phenylalanine, tyrosine + + phenylalanine tyrosine - - phenylalanine, tyrosine, anthranilic acid - - tryptophan, tyrosine - - tryptophan, phenylalanine + + AMINO ACIDS AND TUMORS IN DROSOPHILA 21 7

TABLE 4

The effects of D and L-isomers of tryptophan and phenylalanine on tumor incidence in the tu bw; st su-tu strain

20 mM 4.0 mM Control 29.3

L- tryp tophan + 5.43 +53.7** D-tryptophan - 9.87 L-phenylalanine +12.4** +38.6** D-phenylalanine + 8.86 L-tryptophan + D-tryptophan + 0.229 L-phenylalanine + D-phenylalanine +16.6** L-tryptophan f >phenylalanine +14.5**

Amino acids were supplied at concentrations of 20 miv and 40 mM and as racemic mixtures consisting of 20 mM of each isomer. Tumor incidence (percent) is expressed as the deviation from control. tive fashion, and although their effects on the phenotype may be connected by a common mechanism of action, the amino acids must operate through one or more dissimilar intermediate reactions. The difference between the effects of D- and L-phenylalanine on tumor incidence is interesting in view of the report that both isomers are utilized by Dro- sophila larvae (GEER1966). That the tumor strain does not lack the ability to utilize D-phenylalanine for growth is shown by the data in Table 5. The rate of larval development and survival of the tumor strain is poor, compared with wild type, on the protein-free balanced amino acid diet. D- and L-phenylalanine are distinguishable by their effect on larval growth rate and survival which is lower when the D-isomer is supplied. These results could be explained on the assumption that the larvae are able to convert D- to L-phenylalanine, but that the rate of racemization is slow, such that most of the resulting L-isomer is utilized for growth. Uader these circumstances the concentration of free L-phenylalanine in the larval amino acid pool may remain below the threshold for tumorigenesis. The free amino acid pool in tumorous laruae: The action of tryptophan and phenylalanine raises the question of what effect high dietary concentrations of these amino acids may have on the composition of the free amino acid pool of the

TABLE 5 Larval development rat? and survival of Edinburgh wild and the tu bw; st su-tu strains on a protein-free amino acid medium containing L- or D-phenylalanine

larval period log days survival percent.

Edinburgh tu bw ; st su-tu Edinburgh tu bw ; st su-tu L-phenylalanine 1.0807 t 0.0021 1.1513 -C 0.0035 91.17 38.00 D-phenylalanine 1.0703 _t 0.0022 1.1660 & 0.0039 86.50** 30.55'; 21 8 BARRIE BURNET AND JAMES H. SANG treated larvae. Before examining this, it is worth while to determine to what extent there is variation in composition of the pool between genotypes, since differences in incidence between tumor strains may already be a reflection of gene controlled differences in composition of their respective amino acid pools. It is desirable that this comparison be made as close to the phenocritical period as possible. Accordingly synchronized second instar larvae, cultured on live yeast medium, and with median age of 45 hours after hatching from the egg were used for analysis. The strains used were: Edinburgh wild, the unsuppressed tumor strain tu bw ; and the suppressed tumor strain tu bw ; st su-tu, these having tumor frequencies on live yeast medium of 0.1 %, 97% and 17%, respec- tively. The results are summarized in Table 6. The two tumor strains which are isogenic for the same second chromosome,

TABLE 6 Free ninhydrin-positive substances in late second instar larvae cultured on live yeast medium. Each value is expressed as a percentage of total recovery which varied from 57.7 to 66.3 .umole/g freshwight

Edinburgh tu bw ; +su-tu tu bw ;st SU-~U phosphoserine + cysteic acid 0.15 0.08 0.41 glycerophosphoethanolamine + tyrosine phosphate 3.25 3.36 3.79 phosphoethanolamine 3.70 3.74 5.208 taurine 0.46 1.45 1.74 urea 2.70 3.28 2.4.6 methionine sulfoxide 0.69 0.78 0.73 aspartic acid 1.42 1.64, 2.92 threonine 3.568 3.60 2.88 serine 3.50 3.14 9.16 asparagine + glutamine 19.2 11.'7 16.1 proline 5.80 5.84 6.82 glutamic acid 4.93 6.30 6.76 glycine 4.44. 5.29 4.45 a-alanine 21.7 22.9 13.0 valine 2.67 2.41 2.47 cystathionine 0.15 0.15 0.13 methionine 0.52 0.53 0.38 isoleucine 1.32 1.41 0.87 leucine 1.83 2.01 1.20 tyrosine 2.97 3.34 3.40 phenylalanine 0.80 0.99 0.36 P-alanine 1.14 0.89 1.11 y-aminobutyric acid 0.79 0.47 0.32 ornithine 0.56 0.52 0.53 ethanolamine 0.45 0.81 0.22 ammonia 1.21 1.47 2.18 lysine 5.52 6.14 3.95 histidine 3.24 3.36 4.65 tryptophan trace tracc 0.04 arginine 1.33 2.39 1.85 AMINO ACIDS AND TUMORS IN DROSOPHILA 219 differ from wild type in having lower levels of 7-aminobutyric acid, and higher levels of arginine, glutamic acid and taurine. The tumor strains differ from one another in that the suppressed strain shows noticeably lower levels of a-alanine, ethanolamine, isoleucine, lysine, methionine, phenylalanine, and threonine; and increased levels of aspartic acid, histidine, phosphoethanolamine, phosphoserine 4- cysteic arid, and serine. In addition, the suppressed strain is distinguished by a characteristic ratio of tyrosine/phenylalanine of approximately 9-10 compared with approximately 34in wild type and the unsuppressed strain. This difference in the balance of aromatic amino acids in favor of tyrosine is maintained in early third instar larvae (Table 7). The date clearly indicate that there are real differences in the composition of the amin6 acid pools of the different genotypes at the same physiological age. The extent, if any, to which these differences are related to the level of tumor incidence can be revealed only by a more searching analysis of induced vari- ations in pod composition. Analysis of larvae of the tu bw;st su-tu strain summarized in Table 7 shows that the composition of the amino acid pool is markedly influenced by the larval diet. The series includes data for larvae fed on three treatment media, supplemented with tryptophan, phenylalanine, or arginine, to a concentration of 40 mM, the latter being included as an additional control to assess the effect of a L'n~n-t~morigeni~'7amino acid. Larvae from tryptophan supplemented media show a high level of free haemolymph tryptophan. a-alanine, a possible breakdown product of tryptophan, is also increased. Most of the amino acids potentially capable of yielding acetyl-coA (serine, aspartic acid, leucine, isoleucine, phenylalanine, tyrosine, and glycine) are similarly increased in amount, possibly because the requirement is met primarily by a-alanine. As might be expected, there is a rise in the level of free ammonia. Although ammonia levels tend to be rather variable between groups, there is little doubt that the observed increase is biologically significant. Of special importance is the decrease in methionine and methionine sulfoxide. The high level of taurine indicates a considerable breakdown of methionine, and/or cystine, but this seems insufficient to account for the notable deficiency of sulfur containing amino acids. Phenylalanine supplementation of the diet raises the level of free haemolymph phenylalanine, but not of tyrosine, and this implies a restriction on tyrosine biosynthesis at this stage, or else that any excess tyrosine which is formed is rapidly dissimilated. The level of ammonia is also significantly increased, but urea is correspondingly below the control level. This implies an inhibition of urea synthesis which might be a consequence of higher levels of fumaric acid derived from the breakdown of phenylalanine or tyrosine. The increased levels of aspartic acid and arginine would be in line with this interpretation, although the existence of a urea cycle in Drosophila is conjectural. The levels of other amino acids are similar to those of the control except for threonine, serine. valine and leucine which are somewhat lower, and there is again a deficiency 220 BARRIE BURNET AND JAMES H. SANG

TABLE 7 Free ninhydrin-positive substances in early third instar larvae of the tu bw ; st su-tu strain cultured on meptic synthetic medium supplemented with tryptophan, phenylalanine, or arginine to a concentration of 40 mM. Each value is expressed as a percentage of the total recovery

~~~ ~- Control tryptophan phenylalanine arginine phosphoserine f cysteic acid 0.55 0.41 0.25 0.38 glycerophosphoethanolamine + tyrosine phosphate 13.89 5.66 10.47 11.12 phosphoethanolamine 3.58 3.42 2.99 3.78 taurine 0.87 1.16 0.74 0.88 urea 1.74 1.45 0.98 7.58 methionine sulfoxide 1.80 0.30 1.32 1.70 aspartic acid 1.11 2.34 2.56 1.87 threonine 3.64 3.32 2.34 2.92 serine 4.10 5.02 3.58 4.20 asparagine + glutamine 14.2 18.1 10.6 11.6 proline 26.5 14.9 21.7 25.6 glutamic acid 5.60 6.53 5.51 4.45 glycine 2.88 4.54 2.86 2.68 a-alanine 4.10 7.03 4.37 3.41 valine 1.34 1.77 0.75 0.94 cystathionine 0.20 0.09 0.16 0.14 methionine 0.46 0.22 0.25 0.41 isoleucine 0.28 0.63 0.22 0.28 leucine 0.67 1.27 0.46 0.69 tyrosine 3.34 4.59 2.58 2.54 phenylalanine 0.34 0.82 7.34 0.28 p-alanine 0.58 0.78 0.67 0.57 y-aminobutyric acid 0.13 trace trace ornithine trace 0.19 0.05 0.59 ethanolamine 0.75 trace trace ammonia 0.84 2.20 7.40 0.72 lysine 2.22 2.75 3.03 1.96 histidine 3.33 3.73 3.96 4.53 tryptophan 3.30 ... arginine 1.93 2.62 2.80 4.44

of methionine and methionine sulfoxide but in this case not associated with an increase in taurine. It is interesting that excess dietary arginine leads to an increase in the levels of ornithine and urea thus confirming the presence of an active arginase in the larvae. There is some reduction of threonine, glutamic acid, a-alanine, and valine, which may all be accounted for by an increase in the utilization of KREBScycle precursors in the metabolism of excess arginine. There is no alteration in the levels of methionine and methionine sulfoxide in the arginine supplemented series. These data indicate that excess dietary concentrations of tryptophan, phenylalanine, or arginine cause changes in the relative composition of the pool of free AMINO ACIDS AND TUMORS IN DROSOPHILA 22 1 amino acids in the larvae, but these changes tend to be particular to each dietary treatment. All three dietary excesses appear to alter the pattern of amino acid supplies to the KREBScycle, but in different ways. Tryptophan and phenylalanine treatments both cause a reduction in the level of methionine and its sulfoxide, but the effect is not obviously related to the difference in methionine levels in untreated tumor strains (Table 6). Dietary amino acid interactions: The decrease in methionine, associated with abnormally high levels of tryptophan and phenylalanine in the free amino acid pool, would be significant if it could be shown that increased availability of methionine 'was attended by a change in phenotypic expression of the tumor gene. Whilst examining this possibility it would be unwise to lose sight of the complex array of gene-environment interactions shown by this strain (SANGand BURNET1967). The availability of dietary amino acids is related to the growth requirement for certain B-vitamins as SANG(1962) has shown, and it is already known that both pyridoxine and thiamine deficiencies lead to an appreciable rise in tumor incidence. Also relevant in this connection is the report of HINTON, NOYES,and ELLIS( 1951 ) that excess dietary tryptophan raises the RNA requirement of Drosophila larvae on aseptic defined media. The amino acid treatments may, therefore, create an abnormally high requirement for some metabolite which, being in fixed supply from the defined diet, may then become a limiting factor on the expression of the normal developmental pathway, and the relevant metabolite need not be an amino acid. To test these possibilities twelve amino acids, including methionine, have been added as individual supplements at a concentration of 10 mM on media containing 40 mM of tryptophan or phenylalanine. In addition a fivefold increase in all vitamin levels and a twofold increase of RNA concentration, were included as shown in Table 8. Arginine, threonine, and valine supplements counteract the effect of tryptophan to some extent, and serine slightly reduces the effect of phenylalanine. Methionine unequivocally reduces tumor incidence in both cases, but neither increased RNA, nor increased vitamin levels, are effective. Supplementary methionine has been found to reduce tumor incidence only in the presence of excess tryptophan or phenylalanine, indicating a close similarity in their mode of action on the tumor system. Larvae raised on aseptic medium have a higher relative concentration of free methionine than larvae raised on live yeast, but tumor penetrance is also some 20% higher in the germ-free larvae. Increasing the methionine content of the medium over an extended dose range has no effect in reducing this difference in tumor incidence between the TWO environments, which could be attributed to factors with a different mechanism of action from tryptophan and phenylalanine. The dissimilation products of tryptophan: The tumorigenic effects of kynurenine and anthranilic acid reported by PLAINEand GLASS(1955) and KANEHISA (1956) suggest that tryptophan may operate via anthranilic acid with kynurenine as an intermediate as shown in Figure 2. The kynurenine pathway is important in view of reports that 3-hydroxykynurenine may be tumorigenic in certain other systems (BRYAN,BROWN and PRICE,1964). An alternative possibility. that 222 BARRIE BURNET AND JAMES H. SANG TABLE 8

Effectof increasing the levels of individual nutrients on media containing tryptophan or phenylalanine at a concentration of 40 mM

~- ~ ~ ~~

tryptophan 41) mM phenylalanine 40 m~

Control 86.67 77.044 Supplement: RNA x2 + 4.70 +12.2** Vitamins x5 + 2.64 + 0.09 arginine -1 1.6* + 3.84 cystine - 7.98 + 1.43 glycine - 7.24 - 0.18 histidine + 0.39 + 4.39 isoleucine - 4.95 + 1.30 methionine -28.4* * -21.9* * phenylalanine - 3.08 proline - 6.48 - 5.83 serine - 0.52 + 7.l* threonine - 9.45f + 4#.5 tryptophan - 3.14 tyrosine + 3.53 + 1.46 valine - 9.64' - 3.42

The supplementary amino acids are each supplied at a concentration of 10 miw. Tumor incidence (percent) is expressed as the deviation from control. anthranilic acid may be used as a precursor for tryptophan synthesis, is excluded by the failure of anthranilic acid to support larval development on an amino acid diet which lacks tryptophan as shown in Table 3. Tryptophan pyrrolase, the first enzyme of the oxidative pathway to kynurenine, is under the control of the vermilion locus in Drosophila melamgaster. The enzyme has been isolated and shown to be adaptive, such that the level of enzyme activity is related to the availability of the substrate (KAUFMAN1962). Since the next enzyme of the pathway, kynurenine formamidase, is always present in amounts which are unlikely to be limiting ( GLASSMAN1956), increased levels of dietary tryptophan might be expected to accelerate the rate of kynurenine synthesis. Kynurenine itself has been shown to accumulate in the larval fat body in the late third instar, prior to hydroxylation to 3-hydroxykynurenine and utilization in ommochrome synthesis (RIZKI and RIZKI 1963), and both kynurenic acid (DANNEELand ZIMMERMANN1954) and xanthurenic acid (UME- BACHI and TSUCHITANI1955) have been identified in Drosophila. Table 9 shows the effect of a number of tryptophan derivatives. Anthranilic acid and hydroxyanthranilic acid were the only compounds causing a significant increase in tumor incidence and these are approximately mice as effective as tryptophan on an equimolar basis. The negative results obtained with m- and p-aminobenzoic acid show that the ortho-arrangement of amino and carboxyl AMINO ACIDS AND TUMORS IN DROSOPHILA 223

HOTr!hOH, ~

decarboxylase H H 5- HYDROXYTRYPTOPHAN 5-HY DROXYTRY PTAMINE (SEROTONI N) dphanhydroxylase -TRYPTOPHAN FORMY-L-KYNURENINE NH 12 CH2C HCOO H (vermilion)

tryptophan pyrrolase H kynurenine Jformamidase

ANTHRANILIC ACID L-KYNURENINE KYNURENIC ACID

FIGURE2.-Pathways of tryptophan dissimilation.

groups is necessary for activity. Tests with these compounds are complicated by their toxicity to Drosophila larvae and the concentrations used were the maximum permitting a reliable estimate of tumor incidence to be made. Niacin has no effect on tumor incidence. The tumor strain cannot survive in the absence of dietary niacin, and must consequently lack the ability to synthesize the vitamin. Kynurenic, xanthurenic, and indoleacetic acid were ineffective at the concentrations tested. The result for indoleacetic acid is in line with that of HARTUNG (1955) who found no influence of this compound on the mt4 tumor strain, although he did observe some increase with certain wild type strains. Tryptamine, and isatin cause a significant reduction in tumor incidence. Indole, which proved to be toxic at levels above 0.5 mM, had no significant tumorigenic effect. At a concentration of 40 mM kynurenine actually brings about a reduction in tumor incidence of some 21 percent. This result is surprising in view of the reports by PLAINEand GLASS(1955) and KANEHISA(1 956) of the converse effect. The situation was examined more closely by testing kynurenine on a killed yeast medium containing 80 mM of DL-kynurenine, but in this case non-sterile first instar larvae were inoculated on to the medium so that the usual microflora developed. Under these conditions a significant increase in incidence was observed, suggesting that the increase observed by these authors is due to some by-product of microbial metabolism of kynurenine. Under our conditions kynu- 224 BARRIE BURNET AND JAMES H. SANG TABLE 9

Effectof possible derivatives of tryptophan on tumor incidence in thz tu bw ;st su-tu strain

20 mM 40 mM .- Yeast Medium Control 6.85 m-kynurenine . .. + 9.75** Synthetic Medium Control 34.8 L-kynurenine - 5.23 -21.1 * * kynurenic acid - 2.10 xanthurenic acid + 0.80 tryptamine -17.5** toxic indoleacetic acid + 8.00 toxic isatin + 7.81 -15.4** anthranilic acid +28.8* * toxic hydroxyanthranilic acid +25.5** toxic m-aminobenzoic acid + 1.87 toxic p-aminobenzoic acid -10.01 toxic niacin + 6.21

Compounds were supplied at a concentration of 20 mM or 40 mM in the diet. Tumor incidence (percent) is expressed as the deviation from control.

renine msat the same position as lysine and consequently would not be detected in the amino acid analyses of larval homogenates discussed above. However, chromatographic tests have established the presence of kynurenine, and a second fluorescent compound which may be kynurenic acid, in tryptophan treated larvae, but it has not been possible to detect anthranilic acid under the same conditions. Ommochrome synthesis is blocked in this strain, which is homozygous for scarlet, and the presence of kynurenic acid would indicate that kynurenine is diverted into the quinoline pathway by kynurenine transaminase. Although failure to detect the presence of anthranilic acid is not in itself decisive, the feeding tests with kynurenine are against the hypothesis that tryptophan is converted to anthranilic acid via kynurenine, and the effects of the vermilion block discussed below seem to settle the issue. This does not, of course, affect the possibility that anthranilic acid may be derived from tryptophan by some alternative route. If the kynurenine pathway provides an important dissimilation route for tryptophan, a genetically controlled block caused by mutation at the U locus could lead to accumulation of this amino acid, or to its diversion into an alternative pathway. The substitution of U into the tumor strain is attended by a pro- nounced increase in tryptophan sensitivity (Figure 3) and a significant reduction in the median effective dose for this compound from 41.4 to 27.4 mM (Table 10). Taken together with the dietary evidence, the effect of the U block shows that AMINO ACIDS AND TUMORS IN DROSOPHILA 225

TABLE 10

The median effectiue dose (m) in log. mM, and regression coefficient (b), with standard errors, from dose responses for tumorigenic treatments

m b

Tryptophan tu bw ;st su-tu 1.617 t 0.014 5.604 t 0.896 U ;iu bw ;st su-tu 1.438 t 0.009 6.327 i- 0.582 y ;tu bw ;st su-tu 1.6228 & 0.010 7.391 t 1.040 tu ;st su-tu 1.699 & 0.015 6.973 2 1.040

Phenylalanine tu bw ; st SU-~U 1.500 t 0.023 5.104 k 0.746 U ;tu bw ;st su-tu 1.643 & 0.01 1 7.649 t 1.610 y ; tu bw ;st su-tu 1.606 & 0.073 3.455 f 0.500 tu ;st su-tu no response

Anthranilic acid tu bw ;st su-tu 1.126 & 0.028 3.098 f 0.654 U ;tu bw ;st su-tu 1.545 t 0.262 2.474 t 1.450 y ; tu bw ;st su-tu 1.301 t 0.032 3.308 t 0.639 tu ;st su-tu no response

the tumorigenic effects of tryptophan and anthranilic acid cannot be connected through the kynurenine pathway. This pathway, which evidently provides a metabolic overspill for dissimilation of excess dietary tryptophan in strains, must be available quite early in larval life since the tryptophan sensitive period is between late second and early third instar (Figure 4). The presence of the U mutation increases the median effective dose for phenylalanine and anthranilic acid (Table 10) but, as the y marked first chromosome substitution has a closely similar effect, this is unlikely to be due to the U mutant itself. That sensitivity to these two compounds should be independent of tryptophan sensitivity is additional evidence that the tumorigenic effects of the three compounds are indirectly related. The sensitive periods: In an earlier publication of this series (BURNETand SANG196413) we have defined the phenocritical period during which the penetrance of tu can be modified by external agencies. Along with other dietary environmental manipulations the sensitive period for tryptophan treatment is restricted to the close of the second and beginning of the third larval instars, just prior to the changes in haemocyte morphology associated with tumor formation. If tryptophan and phenylalanine affect tumor incidence by influencing the same developmental reaction, this would imply an identity of the timing of their sensitive periods within the limits of resolution for the material. Accordingly larvae were inoculated on the media containing 40 mM of tryptophan or phenyl- 226 BARRIE BURNET AND JAMES H. SANG

6.0 / !z m 2 5.0 n

4.0

3.0 I I I !5 30 35 40 45 50 CONC. TRYPTOPHAN m M

FIGURE3.-Relation between tumor incidence (in probits) and the concentration of dietary tryptophan on a log scale. U ; tu bw ; st su-tu strain (squares), y ; tu bw ; st su-tu strain (diamonds), tu bw ; st su-tu strain (circles), and tu ;st su-tu strain (triangles). alanine, or 20 mM of anthranilic acid, in parallel experiments. The larvae were transferred after successively longer intervals to a killed yeast culture medium to complete development. The results for the three treatments summarized in Figure 4 are in close agree- ment, but it should be noted that the changes in incidence include the difference between yeasted media and the control aseptic synthetic medium as well as that due to the amino acid treatments. The dietary treatments have no effect when larvae are removed from the treatment media before 36 hours, whereas the effect of treatment is irreversible after 55 hours of larval life. The sensitive periods for the amino acid treatments appear to lie in the interval between 36-55 hours after hatching from the egg as measured on the standard 96 hour time scale. It is not possible to delimit the sensitive periods with any greater accuracy by this method because of the spread in physiological age of larvae within replicate cultures. The evidence is therefore compatible with the view that tryptophan, phenylalanine, and anthranilic acid influence the same developmental reaction, or different reactions which are closely similar in their timing, and which precede the first appearance of atypical cytological changes leading to tumor formation. The relationship between tumor incidence and cuticular melanogenesis: The AMINO ACIDS AND TUMORS IN DROSOPHILA 22 7

10 20 30 40 50 60 70 AGE IN HOURS

FIGURE4.-Sensitive periods for tumorigenic treatments. Larvae plated initially on media containing 44l mM tryptophan (circles), 40 mM phenylalanine (triangles), or 20 mM anthranilic acid (squares), were transferred to yeast medium after successively longer intervals. The time scale on the abcissa is standardized to a 96 hour larval period.

effects of the body color mutant yellow on the responses of the tumor strain is shown in Table 10. The y; tu bw ; st su-tu strain shows a small decrease in sensitivity to phenylalanine and anthranilic acid treatments. Since the U marked first chromosome has a similar effect, this is unlikely to be due to the y mutant itself. The induced tumors are in all respects identical in intensity of black pig- mentation to those of the non-y strains, whilst neither of the amino acid treatments affected the expression of yellow body color. ENCKE(1958) suggests that phenotypic differences between yellow and wild type are due to an inhibitor of melanogenesis. If this is the case, the presence of melanized tumors in y flies might be accounted for in a variety of ways. (i) The inhibitor may be tissue specific in occurrence. (ii) There may be differences in the structural organization of melanin in the two tissues. (iii) There may be two different melanins, each synthesized by a different biosynthetic pathway, only one of which is sensitive to the inhibitor. The genetic basis of the treatment responses: The chromosomal location of the 228 BARRIE BURNET AND JAMES H. SANG major gene or genes implicated in the response to amino acid treatments can be inferred from the data summarized in Table 11. Substitution of chromosomes 2 of the tu bw ;st su-tu strain by a wild-type second chromosome yields a +tu ;st su-tu strain which shows no response to supplementary amino acids over a comprehensive dose range. In contrast the tu bw ;+su-tu strain, which carries a wild type third chromosome, exhibits the same treatment sensitivity as the original suppressed strain, confirming that the response to treatment is controlled by a gene, or genes, located on the tu bw second chromosome. The tu-B3 strain carries a mutant allele of independent origin at locus tu (2-83.9), which also interacts with the suppressor (BURNET 1966). The tu-B3 ; st su-tu strain responds to tryptophan and phenylalanine, and anthranilic acid, suggesting that the treatment responses may depend on a gene-environment interaction involving the tu gene alone. Additional evidence does not favor this possibility. Substitution of part of the left arm of chromosome 2 to incorporate the mutant black into the tu bw second chromosome (b tu bw ; strain) is attended by a loss of sensitivity to anthranilic acid. The response to this compound is evidently controlled independently of tu, or depends upon an interaction between the major locus and a linked modifier. More compelling evidence pointing to a multifactorial basis to the complex of treatment responses comes from the Pacific wild strain which shows a high tumor frequency in the presence of tryptophan, but is un- affected by phenylalanine. The Edinburgh wild strain is not affected by any of the treatments. Whilst suggesting that the sensitivity to each treatment may be under inde-

TABLE 11 Tumor incidence (percent) on aseptic synthetic medium in the presence of tryptophan, phenylalanine, or anthranilic acid. Tumor incidence on the supplemented media is expressed as the deviation from control

Tryptophan Phenylalanine Anthranilic Control 40 miv 40 miv acid 20 mM

~~ Unsuppressed strains tu bw; fsu-tu 65.1 +25.0* * f32.1; * +16.4** b tu bw; fsu-tu 75.2 f14.5** +17.4** - 2.41

Suppressor strains tu bw; st su-tu 37.2 +48.1** +38.6** +28.8* * tu-B3; st su-tu 26.4 +20.7** +25.5** toxic +tu ;st su-tu 2.06 f 0.51 - 0.25 + 0.47

Wild strains Pacific 1.15 +56.2** + 0.73 toxic Edinburgh 2.80 - 2.68 - 2.20 - 1.79 tu bw; st su-tu x Pacific 0.45 f97.2'; - 0.02 + 0.18

~~~~~~

* * denotes significance at the 1% level of probability. AMINO ACIDS AND TUMORS IN DROSOPHILA 229 pendent genetic control, these observations immediately raise the question of whether mutation at tu is essential to any part of the array of gene-environment interactions observed in the original tu bw ; st su-tu strain. In order to answer this question it is necessary to determine whether the response to different environmental treatments is controlled by different independently acting loci, or by a series of modifying genes which interact with the major tu locus. This can be done by transposing tu into different genetic backgrounds, and in particular by examining the effect of residual variation in the second chromosome on phenotypic expression. Substitution of tu into the second chromosome background of the Edinburgh wild strain (MATERIALS AND METHODS) was followed by reconstruction of the original genome outside chromosome 2 to give the tu ;st su-tu strain. This strain is distinguished by the absence of any significant change in tumor incidence over a comprehensive dose range of phenylalanine, or anthranilic acid, whilst the median effective dose for tryptophan is increased to 50 miu compared with 41.4 miu in the original strain (Table 10 and Figure 3). These facts conclusively establish the multifactorial genetic basis of the response to amino acid treatments shown by the tu bw ; st su-tu strain, and establish that the major tu gene is at the locus controlling tryptophan sensitivity, whereas the responses to phenylal- nine and anthranilic acid are controlled by other mutant genes present in the background of the tu bw and tu-B3 second chromosomes. If the response to tryptophan depends only upon the presence of the major gene as seems to be indicated by these results, does the response to tryptophan by the Pacific wild strain have the same genetic basis? The F, bemeen two inbred strains of Pacific wild and tu bw ; st su-tu responded strongly to increasing concentrations of dietary tryptophan as shown in Table 11, but phenylalanine and anthranilic acid had no effect on tumor frequency over a wide dose range. A simple explanation of this result would be that the genes responsible for sensitivity in the two strains are at the same locus. In other words the Pacific wild strain is homozygous for an isoallelic mutant at locus tu which has a range of phenotypic expression which overlaps with wild type under the normal range of environmental conditions. The failure of the F, to respond either to phenylalanine or anthranilic acid could be explained on the assumption that mutants controlling the response to these treatments are recessive, and only the dominant normal alleles are present in Pacific wild.

DISCUSSION Three amino acids have a significant effect on tumorigenesis. L-tryptophan and L-phenylalanine increase tumor incidence whilst L-methionine counteracts their tumorigenic effect. None of the more likely metabolic derivatives of tryp iophan so far tested, with the exception of anthranilic acid, have been found to have tumorigenic activity when added to the diet, but nutritional and genetic evidence in the present analysis shows that anthranilic acid is unlikely to be derived from tryptophan in larvae of the tumor strain. Consequently, the tumori- 230 BARRIE BURNET AND JAMES H. SANG genic activity of tryptophan is attributable either to the amino acid itself or to some as yet unidentified dissimilation product. The hypothesis that the effective amino acids act directly, in the sense that they complement the action of the tu gene, is not supported by the data, but a more likely hypothesis is that tryptophan and phenylalanine modify larval metabolism in a similar manner and affect tumorigenesis indirectly. The similarity of the sensitive periods, together with the effects of these amino acids on the level of free methionine in the treated larvae, goes some way towards supporting this idea. The data are, of course, by no means conclusive and limited by some of the difficulties inherent in the “whole organism approach.” Thus, many of the dissimilation products particularly of phenylalanine are unsuitable for dietary administration, whilst homogenates of whole larvae are unlikely to disclose localized and tissue specific changes in the levels of free ninhydrin-positive substances. The composition of the free amino acid pool in larvae of comparable physiological age reared on normal medium shows a quite unexpected range of variation (Tables 6 and 7). Although the data do not permit any conclusions to be drawn about the genetic basis of this variation, they do emphasize the importance of environmental standardization for future comparative studies. The quantitative differences of amino acid pool composition in this, compared with the study of CHENand HANIMANN(1965), are probably both nutritional and genetic in origin. It is also worth noting that the results using synthetic medium show that larvae will tolerate considerable changes in haemolymph composition before departing from their normal pattern of development. A direct connection between the metabolic pathways of tryptophan and phenylalanine is disclosed by the study of phenylketonuria. In phenylketonuria a deficiency of phenylalanine hydroxylase leads to the accumulation of phenylalanine, phenylpyruvic acid, and certain other dissimilation products which alter the pattern of synthesis of pharmacodynamic amines including serotonin (see KNOX1966 for a discussion of mechanisms). Such amines are known to occur in insects (COLHOUN1963), but their lability makes them unsuitable for dietary administration. It is interesting, however, that the monoamine oxidase inhibitor tranylcypromine ( phenylcyclopropylamine) which is specific in its action ( PLETSCHER1966) , and would be expected to cause an increase of endogenous monoamines, causes a marked increase in the proportion of normal individuals (Figure 5). In this experiment larvae were cultured in media containing excess tryptophan, and they respond to tranylcypromine in proportion to dose. This implies that tumorigenesis on tryptophan supplemented media is attributable to a deficiency of endogenous monoamine. That the relevant amine may well be serotonin is suggested by the results summarized in Table 12 which show that lysergic acid diethylamide (LSD) reduces tumor incidence on tryptophan and phenylalanine supplemented media. Direct evidence is now required about endogenous serotonin under the two treatment conditions, since the precise mode of action of LSD is still rather problematical. If the effect of tranylcypromine on tumorigenesis is attributable to accumulation of serotonin, then LSD would appear to potentiate rather than inhibit the physiological action of serotonin. AMINO ACIDS AND TUMORS IN DROSOPHILA 23 1

5.5 -

, / 5.0 -

-a I 4.5 , Ye -!- sm 4.0 -,

3.5 , I’0 3oj30 3 I 0,004 0.008 0.02 0.04 0.08 CONC. TRANYLCYPROMINE FIGUREj.-The relation between the incidence (in probits) of non-tumorous individuals and the concentration (percent) of dietary tranylcypromine, on media containing 25 mM tryptophan.

Special interest attaches to the role of methionine in reducing the tumorigenic activity of tryptophan and phenylalanine, and the reduced titer of free methionine in the amino acid pools of larvae treated with the tumorigenic amino acids. A possibility here is that methionine is involved in some reaction for the dissimilation of excess tryptophan and phenylalanine, possibly by a process requir- ing methyl group transfer. The importance of labile methyl groups is also suggested by the tumorigenic effect of choline deficiency described by SANGand BURNET(1967). This poses the question of {whetherit is a relative deficiency of methionine which is tumorigenic, in which case the protective effect of additional dietary methionine would not be surprising; or whether supplementary methio-

TABLE 12 The effectof lysergic acid diethylamide on tumor incidence (percent) in larvae of the tu bw ; st su-tu strain cultured on media containing tryptophan or phenylalanine

Lysergic acid diethylamide nil 20 ma/ml difference

Tryptophan 45 mM 97.0 32.4 54.6** Phenylalanine 4.0 miw 77.0 53.1 23.9*’

** Difference significant at the 1 percent level of probability. 232 BARRIE BURNET AND JAMES H. SANG nine facilitates the detoxication or excretion of excess tryptophan and phenylalanine, or their dissimilation products. The evidence tends to support the latter possibility, in that tumor incidence is not consistently correlated with the pool titer of methionine in all environments, and supplementary methionine is only effective in the presence of excess tryptophan or phenylalanine. More detailed observations are required to establish whether the high level of taurine observed in larvae homozygous for the tu bw second chromosome is due to the tu gene or to the residual background. If the former possibility is confirmed the significance of the findings in relation to methionine may have to be re-examined. The tumorigenic effect of tryptophan is independent of the presence of the suppressor su-tu as we have previously shown, and transposition of the mutant allele tu into the second chromosome background of a non-responsive strain positively identifies the tumor gene as the locus controlling tryptophan sensitivity. The presence of an isoallelic mutant at the same locus in the Pacific wild strain, which does not respond either to phenylalanine or anthranilic acid, is independent confirmation that sensitivity to these compounds depends upon the presence of other mutant genes in the second chromosome. Mutant genes of this kind may well act upon the mechanisms regulating transport of phenylalanine or anthranilic acid to their site of action, or alter the balance between pathways by which these compounds are dissimilated. We may visualize that both the major tu gene and isoallelic gene are responsible for different degrees of mutational damage to the same enzyme protein so that the activity of the altered protein is sensitive to inhibition by tryptophan or its metabolic derivatives, and tolerance, measured in terms of the threshold concentration at which the mutant phenotype is expressed, is modulated by the balance of a number of pathways regulating the utilization of the amino acid. Thus tryptophan sensitivity is controlled by mutation at one gene locus whilst tolerance, measured by the characteristics of the dose response, should generally be under multifactorial control. Different strains have characteristic tolerance variances with respect to the same amino acid treatment, as shown in Table 10, which would be expected if substitution of part of the genome of the original strain has led to a change in the array of tolerance modifiers. The extent to which variances are similar for different treatments within the same strain is an additional indication of how far the action of the different treatments is mediated through similar metabolic routes. These considerations permit a reassessment of the mutant tuK (SANGand BURNET 1963) since the F, between the tuK and tu bw ; st su-tu strains also responds to tryptophan (BURNETand SANG1964). By the same criteria that were applied to the results for the Pacific wild strain, tuK may also be regarded as an isoallele at the sametlocus as the major tu gene. The two strains (tuKand tu bw ;st su-tu) are, however, by no means concordant in their respective arrays of gene-environment interactions (SANGand BURNET1967). Such differences may be attributable to residual variation between the two strains, and the importance of interactions of the major tu gene mutation and residual genetic variation in the second chromosome, in determining the pattern of amino acid AMINO ACIDS AND TUMORS IN DROSOPHILA 233 sensitivities, affords reasonable grounds for this expectation. We are dealing here with a major gene mutation and an isoallele at the same locus, and these could be heteroalleles which cause greater and lesser degrees of “mutational damage” to the same enzyme protein. To the extent that the enzyme may not be equally defective in the two types of mutant homozygote we would not expect their patterns of response to physiological stress to be identical. More comprehensive data bearing on these possibilities are discussed by BURNETand WILLOTT (in preparation). It is probable that the tumorigenic effect of excess tryptophan reported by SIMMONSand GARDNER(1958) and by HINTON,NOYES and ELLIS(1951), in strains normally free of melanotic tumors, is attributable to the presence in these strains of mutants of the same isoallelic series. The phenomenon is not a pheno- copy, since induction of melanotic tumors by tryptophan has a definite genetic basis, and not all strains are capable of response as is well illustrated by the data summarized in Table 11. This, and other evidence for the existence of isoallelic genes responsible for destabilizing individual developmental pathways under specific conditions of physiological stress, is greatly illuminated by the rapidly expanding body of direct evidence for the widespread occurrence of enzyme protein polymorphism revealed by gel electrophoretic techniques (HUBBYand LEWONTIN1966, MACINTYREand WRIGHT1966).

This work was supported by grants from the Medical Research Council, and the Agricultural Research Council, of Great Britain. Thanks arc due to DR. W. FFXLXNAND,Department of Bio- chemistry, University of Sheffield, who carried out the amino acid analyses of larval extracts, and to MRS.D. J. DRURYfor technical assistance.

SUMMARY High concentrations of tryptophan, or phenylalanine, in the diet of second instar larvae of the tu bw ;st su-tu strain cause an increase in tumor incidence. Only the L-isomers of these amino acids have tumorigenic activity, although D-phenylaianine is readily utilized for larval growth. Accumulation of tryptophan, or phenylalanine, in the free amino acid pool of the larvae is associated with changes in the balance of a number of free ninhydrin positive substances, notably by reduced levels of methionine and its sulfoxide, whilst increased dietary intake of methionine, on tryptophan or phenylalanine supplemented media, counteracts the tumorigenic effects of those amino acids. Anthranilic acid and hydroxyanthranilic acid also increase tumor incidence, Larvae are, however, unable to synthesize tryptophan from anthranilic acid, and it seems unlikely that anthranilic acid is a dissimilation product of the amino acid, since mutational inactivation of tryptophan pyrrolase enhances the tumorigenic effect of tryptophan. Consequently, tryptophan, phenylalanine, and anthranilic acid, probably alter the incidence of the mutant phenotype independently rather than by giving rise to a common dissimilation product. Preliminary results using tranylcypromine, and lysergic acid diethylamide, suggest that the action of the tumorigenic amino acids may be mediated through a change in the metabolism 234 BARRIE BURNET AND JAMES H. SANG of serotonin.-Changes in tumor incidence induced by dietary amino acids have a complex genetic basis. Sensitivity to tryptophan depends upon a mutant gene indistinguishable from the tumor gene at locus 83.9 in the tu bw second chromosome, whereas sensitivity to phenylalanine, and anthranilic acid, depends upon the presence in the second chromosome of additional mutant genes which interact with the tu gene. At least two major gene mutations and two isoallele mutations are known at this locus.

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