Genetic and Biochemical Studies of Genes Controlling the Synthesis of Threonine and Methionine in Saccharomyes'

GENETIC AND BIOCHEMICAL STUDIES OF GENES CONTROLLING THE SYNTHESIS OF THREONINE AND METHIONINE IN SACCHAROMYES'

H. DE ROBICHON-SZULMAJSTER, Y. SURDIN AND R. K. MORTIMER Laboratoire d'Enzymologie du C.N.R.S.Gif-sur-Yvette (S. & 0.)France, and Division of Medical Physics, Donner Laboratory, University of California, Berkeley Received November 24, 1965

ranched pathway for the biosynthesis of threonine and methionine in T:fai, as described by BLACKand WRIGHT(1955 a,b,c), includes three steps leading to the common precursor homoserine: I. Aspartokinase ATP : L-aspartate 4phosphotransferase-2.7.2.4: ATP + L-aspartate e ADP + 4 phospho-L-aspartate (Asp-PO,) 11. Aspartate semialdehyde (ASA) dehydrogenase L-aspartate p-semialdehyde : NADP oxidoreductase (phosphorylating) 1.2.1.11 : L-aspartate p-semialdehyde (ASA) + phosphate + NADP e L-P-aspartylphosphate (Asp- PO,) + NADP, 111. Homoserine (HS) dehydrogenase L-homoserine : NAD oxidoreductase-1.1.1.3: L-homoserine (HS) + NAD or NADP + L-aspartate p-semialdehyde (ASA) + NADH, or NADPH, Mutants at four apparently unlinked loci, thr,, thrs, thr, and thr6, require liomoserine or threonine and methionine for growth. Strains with each of the four mutations have been studied for the presence of the enzymes associated with the above three reactions. This is a report of these studies.

MATERIALS AND METHODS

a. Genetic analysis. All mutants were induced, with ultraviolet light, in haploid strains of Saccharomyces cerevisiae. The techniques for isolating hybrids, dissecting asci, and determining gene-gene and gene-centromere linkage by tetrad analysis have been described previously (HAWTHORNEand MORTIMER1960; PERKINS1953). b. Media and growth conditions. YPGA : Difco yeast extract, 5 g; Bacto Peptone, 5 g; glucose, 30 g; adenine, 20 mg; per liter. GO : minimal medium defined by GALZYand SLONIMSKI (1957). G21 : GO plus uracil and adenine, 20 mg/l. The following amino acids have been added to GO or G21 : m-arginine, L-histidine, DL-leucine, L-lysine, L-tryptophan, m-methionine, m-threonine, DL-homoserine, L-aspartate or L-glutamate at concentrations given in the text. Growth was at 28°C with good aeration (obtained by strong shaking). The amount of growth was estimated turbidimetrically at 650 mp using special flasks equipped with a diverticule. For the preparation of cell-free extracts, cells were collected at the end of exponential growth, centrifuged, washed twice with Tris buffer (0.1 M, pH 7.4) and suspended in the same buffer.

The present work has been supported by the DBlegation GBnerale B la Recherche Scientifique et Technique (Grant 61-FR-063), The Commissariat B 1'Eneree Atomique, and by the United States Atomic Energy Commmlon

Genetics 59: 609419 March 1966 610 H. DE ROBICHON-SZULMAJSTER et al.

Extracts were made using glass beads in a Nossal shaker, as dascribed by DE ROBICHON- SZULMAJSTER(1961). c. Estimation of enzymic actiuities. Aspartokinase : The P-aspartyl-phosphate formed is trapped by hydroxylamine in the form of hydroxamate as described by STADTMANet al. (1961). The amounts of aspartyl-phosphate formed are deduced from the standard curve established with synthetic aspartyl-hydroxamate. ASA-dehydrogenase : The reaction was followed in the reverse direction by appearance of NADPH, at 341) mp, pH 8.6 (0.1 M diethanolamine and 28°C). Aspartic semialdehyde was pre- pared according to BLACKand WRIGHT(1955b), and its concentration titrated by using purified homoserine dehydrogenase from yeast as described by KARASSEVITCHand DE ROBICHON-SZUL- MJASTER (1963). Homoserine dehydrogenase : The reaction was followed by NADPH, appearance or dis- appearance, at 28"C, according to KARMSEVITCHand DE ROBICHON-SZULMAJSTER(1963). Glutamic dehydrogenases : L-glutamate : NAD oxidoreductase (deaminating) 1.4.1.2. and L-glutamate NADP oxidoreductase (deaminating) 1.4.1.4., were estimated at 28°C by NADH, or NADPH, appearance, as described by COLOWICKand KAPLAN(1962). Aspartate aminotransferase : L-aspartate : 2-oxoglutarate aminotransferase 2.6.1 .l. was estimated according to KARMEN (1955), from aspartate to glutamate by coupling with malic dehydrogenase. AZanine aminotransferase : L-alanine : eoxoglutarate amino-transferase 2.6.1.2. was estimated according to WROBLEWSKIand LADU(1956). Amino acid permease was measured as described by SURDINet al. (1964), at 30°C.

RESULTS AND DISCUSSION Genetic analyses: All the mutants or their meiotic segregants that confer a requirement for threonine or threonine plus methionine were intercrossed, and the resultant diploids tested for growth in the absence of threonine. The mutants fell into six complementation groups. Two of these corresponded to a requirement for only threonine, while four corresponded to a requirement for both threonine and methionine. The threonine mutants, thr, and thr,, have been mapped by tetrad analysis on chromosomes I11 and VI1 respectively (HAWTHORNEand MORTIMER1960). The threonine-plus-methionine mutants corresponding to the other four complementation groups were examined for linkage, to determine if

TABLE 1

Genetic analysis of the threonine-methionine genes

Number of asci Fmt-division Second-division Percent Gene segregation segregation second-divisionsegregation ihr, 78 172 68.8 thr, 83 164 66.4 thr, 129 48 27.1 thr, 26 4.9 65.3

Gene-pair Parental &type Nonparental ditype Tetratype thr,-thr, 5 6 37 ihr,-thr, 3 2 17 ihr,-thr, 3 5 27 THREONINE-METHIONINE GENES IN SACCHAROMYCES 61 1 some of the complementation observed was not inter- but intralocus. thr, was centromere-linked (second-division segregation frequency less than e/3) while the other three genes were unlinked to their centromeres, and thus are not closely linked to thr,. When thr2, thr, and thr, were intercrossed, they segregated independently of each other (Table I), establishing that four separate genes are involved in the steps common to the synthesis of threonine and methionine. Three of these genes have already been located (HAWTHORNEand MORTIMER1960; MORTIMERand HAWTHORNE1966) : thr, is linked to hi, (chromosome V), thr, is linked to the centromere of XII, and thr, is linked to tyPon a section of chromosome not yet identified with a centromere. Selection of strains used for biochemical studies: For reasons not well under- stood, some of the strains bearing recessive alleles of the four genes studied were not able to grow at a “reasonable” rate on the minimal medium supplemented with homoserine. For example, two of the 13 thr, strains tested needed a small supplement of methionine or threonine in order to grow in the presence of homoserine. This additional requirement appears. however, not to be a property of particular alleles at this locus, since only one of the four strains bearing the thr7-, allele shows this pecular behavior. (The authors are grateful to DR.M. LUZZATI for kindly supplying representatives of different heteroalleles at locus thr,.) Also, on homoserine alone, two among the five thr, strains tested grew slowly, only one among the seven thr, strains tested was able to grow, and the two thr, did not grow or grew very poorly. These differences in growth response do not seem to be relatable either to the presence or absence of the known additional markers which were present in the

TABLE 2 Growth requirements of selected homoserineless strains

Additions to the basal medium (C)’ (pg/ml) m-methionine (20) plus DL-thre- DL-me- DL-homoserine nL-threonine onine thionine Locus Mutant strainsf (0) (75) (20) (10) (25) (75) (250) (10) (25) (75) (250) thr, JE2 tr + ++ ++ JE42 tr + ++ ++ 19-11 tr + ++ ++ thr, JE16 tr + ++ ++ Ill-6A tr + ++ ++ thr, JA41-1 + + ++ ++ JE61 tr tr 2 + thr, JD20-6 tr + ++ ++ JD24-1 tr + ++ ++ JD43-1 tr + ++ ++ JD144-1 tr + ++ ++ JE67 tr + ++ ++

+=good growth. -t+ =very good growth; *=weak growth’ trztrace of growth. -=no growth. + Mutants with different numbers in each group were obtain& independently bu;map at the same locus. * Medium (C) : G,, supplemented with DL-leucine 10 pg/ml, L-tryptophan 20 pg/ml, L-arginine 10 pg/ml, L-histidine 10 pg/ml, L-lysine 40 pg/ml. 612 H. DE ROBICHON-SZULMAJSTERet al. different strains tested. Nevertheless, it seems likely that undetected genetic differences, present in the parent strains, have influenced the growth of certain strains. Different levels of amino acid permease (AAP) might be such a factor. More recent studies (SURDINd al., 1964, 1965) on the phenotypic effects of low AAP content on different strains, render this hypothesis quite plausible, particu- larly because, as can be seen in Table 2, the concentration of homoserine required for optimal growth is very high (250 pg/ml). In order to avoid alterations in the phenotype from possible undetected modi- fier genes, the strains used for biochemical studies were chosen from a group of those that responded to homoserine alone. Growth behavior of such strains is given in Table 2. Gene-enzyme relationships for thrz, thr, and thrs: Extracts of strains bearing recessive alleles of thr,, thr,, thr, and thr6 were analyzed for their content of the first three enzymes of the pathway of methionine and threonine biosynthesis. Results of a typical experiment are shown in Table 3. It can be seen that for each of thrs, thr, and thr6,a specific enzyme, corresponding to one of the three known biochemical steps, is deficient. The values reported in these cases represent the lower limit of detection for each enzyme and the actual levels may be somewhat lower. The deficient enzymes are aspartokinase for thr,, ASA dehydrogenase for thr,, and homoserine dehydrogenase for thr,. The standard deviations of enzyme levels calculated for eight different extracts of the same strain (4094-B) are as follows: aspartokinase i. lo%, ASA dehydrogenase * 24%,homoserine dehydrogenase 13 %. The levels of aspartokinase and ASA dehydrogenase are lower in the thr, and thr, strains than expected from these standard deviations. This might be due to the fact that these strains have different origins. On the other hand, it is known (DE ROBICHON-SZUL- MAJSTER et al. 1963) that threonine is a very effective repressor of aspartokinase and probably also controls the synthesis of ASA dehydrogenase in 4094-B. The mutant strains used here require threonine for growth and might be more sus- ceptible to repression than the control strain 4094-B. For example, the enzyme levels obtained for the cells grown on complex medium (YPGA) are lower than those reported in Table 3 for cells grown on synthetic medium. Our experience

TABLE 3 Activities of the enzymes from the aspartate homoserine pathway in different homoserine requiring strains

Homoserineless mutant strains

Enzymes Control ihrK ihr, thr, ihr, Aspartokinase 56 15 <.5 39 5 ASA dehydrogenase 47 12 37 <.2 10 HS dehydrogenase 457 234 486 555 <.5

Extracts and assays used are described under METHODS. Specific actiyities are expressed as mpmoles min-' mg protein-'. Control: Strain 4094-B was &wn on G, medium. ihr, strain ill-GA, ihr, strain 19-11, and thr, strain JD144 were grown on G, medium complemented with DL-threonine 75 pg/ml and DL-methionine 20 pg/ml. fhr, strain JA41-1 was grown on the medium (C) defmed in the legend of Table 2. THREONINE-METHIONINE GENES IN SACCHAROMYCES 613 with the strain 4094-B has shown that such a rich medium leads to repressed values of aspartokinase and ASA dehydrogenase. However, other explanations are still possible, In any case, even the low values reported in Table 3 for aspartokinase and ASA dehydrogenase activity indicate that significant amounts of enzyme are present. Gene-enzyme relationships for thr,: For the thr, strain each of the first three enzymes of the pathway are present (Table 3). It seemed likely then that the missing step would be one preceding aspartate synthesis, and that, correspond- ingly, the requirement would be satisfied by aspartate. (a) Nutritional requirements: As already mentioned, thr, mutants grew poorly on the synthetic medium supplemented only with threonine and methionine, but much better on the complete medium containing many other amino acids. It seemed, then, that to study the enzymic deficiency of thr,, it was preferable to define first a convenient minimal medium for thr, strains. The addition of aspartate to certain combinations of amino acids improved the growth of strain JA 41-1 (th~--~).However, aspartate alone was not better than threonine + methionine. Also, growth rates equivalent to those on YPGA medium were always observed when glutamate in addition to aspartate was present. Glutamate alone had no effect in promoting growth. (b) Terminal steps in aspartate and glutamate biosynthesis: The double requirement defined above is difficult to explain as the result of a single mutation. A defect in glutamate dehydrogenase or at a step inside the Krebs cycle, which could explain a glutamate requirement, should be satisfied by addition of glutamate only, as in the glt, mutants already described in S. cerevisiae (OGUR, COKER,and OGUR 1964). On the other hand, a defect in aspartate aminotransferase should lead only to an aspartate requirement. Levels of these enzymes were then determined in two thr, mutants. It can be seen in Table 4 that aspartate aminotransferase activity is missing in both mutants. The values are the results obtained from two duplicate experiments. Again, the lower values for alanine aminotransferase found in the thr, mutants relative to the control might be due to differences in the origin of the

TABLE 4

Specific activities of enzymes for glutamate synthesis or degradation

Strains ihr, mutants Enzymes Control 4094-8 JA 41-1 JE-61 Glutamate dehydrogenase (NADP-dependent) 7.2' 6.9 6.4 6.0 7.3 Alanine aminotransferase 42.5 15.5 59.5 7.5 17.1 Aspartate aminotransferase 252 <3 <3 268

Specific activitm are expressed as mymoles min-' mg protein-'. Cells were grown on YPGA medium. 614 H. DE ROBICHON-SZULMAJSTER et al. strains. However, it might be related to the aspartate aminotransferase deficiency. That the defect is not a general transaminase deficiency which could be due to failure in pyridoxal-phosphate synthesis, is shown by the fact that alanine-aminotransferase is present in these strains. (Incidentally, these results indicate that these two transaminations are probably catalyzed by two different enzymes in yeast.) The above results do not explain the glutamate requirement. As shown in Table 5, the NADP-dependent glutamate dehydrogenase was always present. This enzyme appeared in cells grown with glutamate as the only nitrogen source (HOLZERand HIERHOLZER1963) at a level comparable to the control strain. Glutamate biosynthesis or degradation does not seem, then, to be impaired in thr, mutants. In addition (Table 5), growth on glutamate as the only nitrogen source does not seem to modify the enzymatic pattern of those strains (except for the expected change in NAD-dependent glutamic dehydrogenase). The absence of aspartate aminotransferase is maintained, when, instead of exogenous threonine and methionine, aspartate is used as a source for these end-products. It seems unlikely then that the absence of this enzyme is due to repression by either end-product. (c) Dissociation of glutamate and aspartate requirements: At this stage, it be- came necessary to determine if the aspartate requirement and glutamate effect were genetically dissociable. The following cross was made: JA 41-1 (athr, ad, ‘petite’) x D24O-1B (aZy, ‘grande’) . Segregation of the genes involved was found to be 2:2 in most of the tetrads. However, some showed a 3: 1 segregation which

TABLE 5

Influence of differentgrowth conditions on the specific activity of diflerent enzymes in the wild type and a thr, strain

Modification of G21 basal medium, and strains Ammonium salts No ammonium salts Aspartate+ Aspartate+ Glutamate Glutamate Glutamate 4094-B JA 41-1 40%-B JA41-1 Enzymes (Control) (thr,) (Control) (thr,) Glutamate dehydrogenase (NADP-dependent) 24+ 10 13 16 Glutamate dehydrogenase (NAD-dependent) 0 0 9 10 Aspartate aminotransferase 250 <3 460 <3 Aspartokinase 14 15 12 12 Aspartic semialdehyde dehydrogenase 11 12 9 12 Homoserine dehydrogenase 376 380 360 4.00 Amino-acid permease 1W%t 96%t

Specific activities are expressed as mfimoles min-1 mg protein-’. In the medium with ammonium salts, aspartate and glutamate are each added at final concentration of 100 pg/ml; in the medium without ammonium salts, they are each added at a fmal concentration of 10 fig/ml. In percent of reference strain. THREONINE-METHIONINE GENES IN SACCHAROMYCES 615 TABLE 6

Segregation ratios in tetrads from the cross JA41-I (athr, ad,) x D240-IB (a ly,)

Segregation ratio AD:ad LY:ly ASP:asp Number of asci 2:2 2:2 2:2 13 2:2 3:1 3: 1 6 3: 1 3: 1 2:2 I 3: 1 2: 2 3:1 1 always concerned two different segregating traits simultaneously (Table 6). This peculiar segregation might be due to the presence of a super-suppressor gene (HAWTHORNEand MORTIMER1963) in one of the parent strains, although more 4: 0 and 3: 1 asci would be expected if this were the case. Alternatively, these irregular segregations could be the result of polysomy or polyploidy. Addition of glutamate slightly improved the growth of some of the thr, segre-

JA 41-1 CH 104-15 NCH 104-22 - aCH 104-98 0.3 L OCH 104-3 3 0

0 ~ ASP ASP T T M, + M + LYS + GLU +M +M GLU, + ASP t ASP LYS + GLU + LYS

FIGURE1.-Comparative growth of different thr, strains in various conditions T = DL- threonine 75 pg/ml, M = DL-methionine 20 pg/ml, ASP = L-aspartate 100 pg/ml, GLU = t-glutamate 100 pg/ml, LYS = L-lysine 4 pg/ml. thr, strains : JA41-1 “petite” ad-, CH104-15 ‘“grande”, CH104-22 “petite” ad-, CH104-9B “petite”, CH104-3 “grande”. 61 6 H. DE ROBICHON-SZULMAJSTERet al. gants, but less than expected from the response of the parent strain JA-41-1. The only difference between this and previous experiments was the presence of lysine, suggesting that the glutamate effect could somehow be modified by lysine. Since lysine appears to be synthesized in S. cerevisiae from keto-glutarate exclusively (MATTOON, MOSNIER,and KREISER 1961; MATTOONand HAIGHT1962; STRASS- MAN and CECI 1964; STRASSMAN,CECI, and SILVERMAN1964), a defect in keto- glutarate synthesis might produce a double requirement for glutamate and lysine. If the block is not complete, one might expect a sparing effect of one of these compounds on the other. Lysine-independent segregants were plated in the presence or absence of glutamate, lysine, or both. It was found that for eight strains (out of 14 lysine- dependent) the growth is greatly improved by glutamate and slightly by lysine. However, for the six remaining strains growth with aspartate was equivalent to growth with glutamate and/or lysine in addition to aspartate. These results show that the aspartate requirement can be separated from the glutamate and the lysine effects. This conclusion was verified by following growth rates in liquid cultures of some of the strains. Results, in divisions per hour, are shown in Figure 1. For the strain CH 104-3, aspartate alone is sufficient to provide a growth rate equal to that obtained with addition of lysine or glutamate. On the contrary, growth of CH 104-9B and the parent JA 41-1, are at least doubled by the addition of glutamate. As mentioned before, the lysine effect is smaller than the glutamate effect, and is observed for segregants but not for JA 41-1. In addition, this experiment shows that there is no relation between the glutamate and lysine effects and the adenine-requirement or respiratory-deficiency associated with markers also present in the parent strain. It is then tempting to assign the glutamate effect

0.01 YI 0 10 20 30 40 50 60 70 Hours FIGURE2.-Growth response to aspartate of a thr, segregant, expressed as optical density. Strain CH104-3 (thr,). Mean generation time: aspartate 10 pg/ml-21 hours; 50pg/ml-9 hr; 100 ,ug/ml-5 hr; 1 mg/ml-5 hr; 10 mg/ml-4.5 hr. THREONINE-METHION INE GENES IN SACCHAROMYCES 61 7

TABLE 7 Specific activities of aspartate aminotransferase and the aspartate to homoserine enzymes in a wild-type and in a thr, strain

Enzyme Aspartate Aspartic semialdehyde Homoserine Source of extract aminotransferase Aspartokinase dehydrogenase dehydrogenase - ~___ wild type 4094-B 360' 56 47 457 thr, segregant CH104-3 3 31 11 427 4093.-B + CH104-3 360

Specific activities are in mp moles min-1 mg protein-' Strain CH 104-3 was grown m minimal medium GO supplemented with 3M) pg/ml aspartate. to a gene present in JA 41-1, but distinct from thr,. We have not yet been able to determine its segregation with certainty, because spore viability was very poor. (d) Phenotypic expression of thr, gene: The results of the growth experiments described above allow us to relegate the glutamate effect to the phenotypic expression of some unknown gene and to consider the aspartate requirement as the only phenotypic effect of the thr, allele. Figure 2 shows the growth response in relation to aspartate concentration for the strain CH 104-3 which is able to grow on minimal medium (GO) plus aspartate. From this it is clear that the aspartate requirement can be explained by the deficiency in aspartate aminotransferase found in the original mutant strains (see Table 5). This conclusion has been verified by enzymatic analysis of segregant CH 104-3. As shown in Table 7, the first three enzymes of the aspartate to homoserine part of the pathway are present and the only observed deficiency is aspartate aminotransferase. Mixed extracts from the mutant and the wild-type strain 4094-B gives the same activity as the extract from 4094-B. Thus, the absence of activity in thr, mutants cannot be due to the presence of an inhibitor. (e) Partial requirements for uracil and arginine: It was surprising that a deficiency in such a metabolically important enzyme would lead only to the threo-

TABLE 8 Effects of arginine and uracil on growth of a thr, mutant

Complete medium Minimal medium Mean generation Divisions Mean generation Divisions Omissions time, hrs per hour Additions time, per hour -~ hrs .,.. 3.5 0.286 . .. 6.5 0.154 Threonine and methionine 11.0 0.091 Asp, Glut 4.2 0.238 Ur 2.5 0.400 Ur 6.0 0.167 Arg 4.0 0.250 Arg 5.0 0.200 Ur. Arg 5.6 0.179 Ur, Arg 4.7 0.212

Strain JA 41-1. Complete medium : see legend of Table 2. Mmimal medium : GO + adenine + threonine + methionine, at same concentrations as in complete medium. Uracil and arginine are also added at the same concentrations as in the complete medium; L-aspartate and L-glutamate are added at 100 gg/ml. 618 H. DE ROBICHON-SZULMAJSTER et al. nine and methionine requirements. Since aspartate is known as a precursor of uracil synthesis and as the source of the amino group of arginine, om of the mutant strains was checked for response to uracil and arginine. As shown in Table 8, omission of uracil or arginine from complete medium has practically no effect, but the omission of both increases the generation time by 60%. In parallel, addition of uracil or arginine to a minimal medium shows no effect, but addition of both reduces the generation time by 28%. It seems then, that uracil and arginine are not strictly required by a thr, mutant. It is possible that the small amount of aspartate aminotransferase found in this mutant (1 to 2% of the control strain) is still sufficient to provide a preferential biosynthesis of uracil and arginine. However, it might still be possible that different pathways can assume the biosynthesis of these two end-products, when aspartate aminotransferase is absent. Two other genes, thr, and thr,, which give a requirement for only threonine and are probably concerned with the two steps between homoserine and threonine, are now under investigation. In Figure 3 are summarized our findings on the interrelations between genes and enzymes for the threonine and methionine biosynthesis pathway in S. cerevisiae.

SUMMARY Mutants at six loci have been identified, two of which require threonine alone, while four require both threonine and methionine. Five of these have been mapped: thr, on chromosome VII, thr, on a chromosome section identified as fragment 11, thr, on chromosome V, thr, on chromosome 111, and thr, on chromosome XII. thr, is unmapped and is not linked with the other five. Biochemical defects have been identified for four loci: thr,-aspartate aminotransferase; thr, -aspartokinase; thr,-aspartate semialdehyde dehydrogenase; and thr,- homoserine dehydrogenase. An effect of glutamate or lysine on growth of thr, strains is probably due to another mutation which manifests itself only under the growth conditions required for thr, mutants.

thr,

ARGININE Aspartate'r' I 'i" aminotransferase t Aspartokinase ASA-dehydrogenase GLUTAMATE ASPARTATE '/3-ASPARTYL - PHOSPHATE L ASPARTIC - - - SEMI-ALDEHYDE (ASA) 1 ATP- ADP NADPH, NADP URACIL

thr, thr, and thr, h 1 HOMOSERINE-PHOSPHATE -' THREONINE -ISOLEUCINE HS-dehydrogenore n- HOMOSERINE (HS) NADPH, NADP or NADH, or NAD CYSTATHIONINE -HOMOCYSTEINE -METHIONINE CYSTEINE6 FIGURE3.-Biosynthetic pathway for threonine and methionine showing role of different genes. THREONINE-METHIONINE GENES IN SACCHAROMYCES 619

LITERATURE CITED

BLACK,S., and N. G. WRIGHT,1955a p-aspartokinase and p-aspartyl-phosphate. J. Biol. Chem. 213 : 27-38. - 1955b Aspartic p-semialdehyde dehydrogenase and aspartic p-semialdehyde. J. Biol. Chem. 213: 39-50. - 1955c Homoserine dehydrogenase. J. Biol. Chem. 213: 51-60. COLOWICK,S. P., and N. 0. KAPLAN,(eds.) 1962 Methods in Enzymology, Volume 2, p. 220. Academic Press, New York. GALZY,P., and P. SLONIMSKI,1957 Evolution de la constitution enzymatique de la levure cultivke sur acide lactique ou sur glucose comme seule source de carbone. Compt. Rend. 245: 25562558. HAWTHORNE,D. C., and R. K. MORTIMER,1960 Chromosome mapping in Saccharomyces : centromere-linked genes. Genetics 45: 1085-1 1IO. - 1963 Super-suppressors in yeast. Genetics 48: 61 7-620. HOLZER,H., and G. HIERHOLZER,1963 Hemmung der Synthese von DNP-abhangiger Gluta- matdehydrogenase in Hefe durch Actinomycin C. Biochim. Biophys. Acta 77: 329-331. KARASSEVITCH,Y., and H. DE ROBICHON-SZULMAJSTER,1963 RBgulations mktaboliques de la biosynthhse de la mkthionine et de la threonine chez Saccharomyces cereuisiae. Biochim. Biophys. Acta 73: 414-426. KARMEN,A., 1955 A note on the spectrophotometric assay of glutamic-oxalacetic transaminase in human blood serum. J. Clin. Invest. 34: 131-133. MATTOON,J. R., T. A. MOSNIER,and T. H. KREISER,1961 Separation and partial characteriza- tion of a lysine-precursor produced by a yeast mutant. Biochim. Biophys. Acta 51: 615-617. MATTOON,J. R., and R. D. HAIGHT,1962 Glutaric acid accumulation by a lysine-requiring yeast mutant. J. Bid. Chem. 237: 34863490. MORTIMER,R. K., and D. C. HAWTHORNE.1966 Genetic mapping in Saccharomyces. Genetics 53: 165-173. OGUR,M. L., L. COKER,and S. OGUR,1964 Glutamate auxotrophs in Saccharomyces. I. The biochemical lesion in the glt, mutants. Biochem. Biophys. Res. Comm. 14: 193-197. PERKINS,D. D., 1953 Detection of linkage in tetrad analysis. Genetics 38: 187-197. DE ROBICHON-SZULMAJSTER,H., 1961 Contribution h 1'Ctude gBnCtique et physiologique du mCtabolisme du galactose chez la levure. Ann. Technol. Agr. 10: 81-142; 185-256. DE ROBICHON-SZULMAJSTER,H., Y. SURDIN,Y. KARASSEVITCH,and D. CORRIVAUX,1963 RBgula- tions metaboliques chez Saccharomyces cereuisiae. Biosynthhse de la methionine et de la thkronine. Coll. Intern. C.N.R.S. N 124: 255-269. STADTMAN,E. R., G. N. COHEN,G. LE BRAS,and H. DE ROBICHON-SZULMAJSTER,1961 Feed-back inhibition and repression of aspartokinase activity in Escherichia coli and Saccharomyces cereuisiae. J. Biol. Chem. 236: 2033-2038. STRASSMAN,M., and L. CECI, 1964. Enzymatic formation of homocitric acid, an intermediate in lysine biosynthesis. Biochem. Biophys. Res. Comm. 14: 26%267. STRASSMAN.M., L. CFCI, and B. E. SILVERMAN,1964 Enzymatic conversion of homoisocitric acid into a-ketoadipic acid. Biochem. Biophys. Res. CO". 14: 268-271. SURDIN,Y., W. SLY, J. SIRE, A. M. BORDES,and H. DE ROBICHON-SZULMAJSTER,1964 Systhme d'accumulation des acides amines chez Saccharomyces cereuisiae: proprietks et contr6le genetique. Compt. Rend. 258: 50%-504.9. __ 1965 Proprietks et contr6le genetique du systhme daccumulation des acides amines chez Saccharomyces cereuisiae. Biochim. Bio- phys. Acta. 107: 546-566. WROBLEWSKI.F., and J. S. LADu, 1956 Serum glutamic-pyruvic transaminase in cardiac and hepatic diseases. Proc. Soc. Exptl. Biol Med. 91: 569-571.