ISOLATION AND PROPERTIES OF SELENOMETHIONINE-RESISTANT MUTANTS OF NEUROSPORA CRASSA

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1 ISOLATION AND PROPERTIES OF SELENOMETHIONINE-RESISTANT MUTANTS OF NEUROSPORA CRASSA GREGORY S. CHENl AND ROBERT L. METZENBERG The Department of Physiological Chemistry, University of Wisconsin Medical School, Madison, Wisconsin Manuscript received January 15, 1974 Revised copy received April 23, 1974 ABSTRACT Mutants resistant to selenomethionine were isolated, and their properties studied. Mapping studies indicate that the mutation sites are located near the eth-27 locus in linkage group I, about ten map units away from the mating type locus. The sites of new mutation are either allelic to or very close to eth-it. They are resistant not only to selenomethionine but also to ethioinine, while the ethionine-resistant mutant, eth-2'; is sensitive to selenomethionine. The selenomethionine-resistant mutants are also temperature-sensitive mutants. However, they can grow at higher temperatures in medium containing 1 M glycerol.-it is very unlikely that the resistance is due to a change in the permeability of the membrane. Aryl sulfatase of se-met' mutants is not repressed by a high concentration of methionine (5 mm), although inorganic sulfate (2 mm) still can cause total repression. The y-cystathionase levels of the mutants are normal, but the S-adenosylmethionine synthetase levels are only one-tenth of that observed in the wild-type strain. The heat-stability of this enzyme in the mutant is also different from that o the wild-type enzyme suggesting that the mutation might affect the structural gene of S-adenosylmethionine synthetase. ELENOMETHIONINE, an analog of methionine, has been found in Escherichia coli (COWIE and COHEN 1957; HUBER, SEGEL and CRIDDLE 1967; TUVE and WILLIAMS 1961), yeast (BLAU 1961), and plants (PETERSON and BUTLER 1962) when they were grown in the presence of inorganic selenium compounds. The physical and chemical properties of selenomethionine are quite similar to those of methionine and it is not surprising that this analog can be incorporated into protein in place of methionine by strains of E. coli which require methionine for growth ( COWIE and COHEN 1957) or by a strain which is resistant to selenite (HUBER, SEGEL and CRIDDLE 1967; TUVE and WILLIAMS 1961). In contrast, the properties of selenocysteine and selenocystine are substantially different from those of their sulfur analogs with respect to pk, values, redox potential, reactivity and stability to acid (HUBER and CRIDDLE 1967a). Substitution of selenohydryl groups for the sulfhydryl groups in proteins could result in an altered biological activity, eventually killing the cell. Neither selenocystcinn nor selenocystine was found in protein hydrolysates of the same E. coli strains (COWIE and COHEN 1957; HUBER, SEGEL and CRIDDLE 1967), even when enzymatic hydrolysis was Present address Department of Biochemistry, Roche Instltute of Molecular Biology, Nutley, New Jersey 071 IO. Genetics 77: August, 1974.

2 628 G. S. CHEN AND R. L. METZENBERG employed (HUBEP: and GRIDDLE ; TUVE and WILLIAMS 1961). Selenomethionine is highly toxic to mammals as well as to Neurospora. Probably this is because selenomethionine is converted to selenocysteine much more rapidly in these organisms than it is in E. coli. This is consistent with the fact that Neurospora readily uses methionine as the sole sulfur source, whereas E. coli uses it only very slowly. Unlike E. coli, Neurospora contains two enzymes similar to those mediating reverse trsns-sulfuration in animal liver: P-cystathionine synthetase (EC ), which converts serine and homocysteine to cystathionine; and y-cystathionase (EC ), which forms cysteine and hoinoserine from cystathionine (FLAVIN and SLAUGHTER 1967). Therefore, the conversion of methionine to cysteine occurs by a different pathway, and involves different enzymes from the conversion of cysteine to methionine (see Scheme I). HOMOSERINE (r-cy stathionase) CYSTATHIONINE A (p-cystathionine j synthetase SERINE HOMOCYSTEINE T S-ADENOSY LHOMOCYSTEINE X-CH3 S-ADENOSY LMETHlONlNE 4- CYSTATHIONINE ACETYL CoA O-ACETYL- HOMOSERINE -cystathionine synthetase 1 (g -cystathionasej SERINE 1 N METHYLPOLY - '-GL"TAMYL THFA HOMOCYSTEINE ATP ET HlON IN E Scheme I. Transsulfuration in Neurospora It would be desirable to isolate mutants blocked in the pathway from methionine to cysteine. Such mutants would be helpful in deciding whether cysteine or methionine or both are corepressors for the regulation of the synthesis of aryl sclfatase, sulfite reductase, sulfate permease and choline sulfatase, as postulated earlier by one of us ( METZENBERG and PARSON 1966). If the toxicity of selenomethionine is due to its conversion to selenocysteine, then mutants resistant to

3 SELENOMETHIONINE-RESISTANT MUTANTS 629 selenomethionine might bc expected to be blocked at some step in the conversion of methionine to cysteine. One might also expect to find permease mutants or membrane-defective mutants which would not allow selenomethionine to get into rhe cell (LESTER 1966; STADLER 1966). In this paper we will report on the isolation, mapping, and properties of two of these mutants. As will be evident, both are able to transport selenomethionine into the cell, and have lower than normal levels of S-Edenosylmethionine synthetase. MATERIALS AND METHODS Materials: Seleno-DL-methionine, ATP, ethionine, p-fluorophenylalanine, cycloheximide, L-cysteine, L-methionine, cysteic acid, and N-acetyl-DL-methionine were obtained from Sigma Chemical Co. Sorbose was obtained from Pfanstiehl Chemical Co. 1%-labelled ATP (specific activity 17.5 mci/mmole), methionine (specific activity 12.4 mci/mmole), and ethionine (specific activity 2.4 mci/mmole) were purchased from New England Nuclear Corp. Membrane filters (25 mm diameter, 0.22 pm pore size) were purchased from the Millipore CO. Neurospora strains: The wild-type strains of the two mating types, 74-ORB-lA and 74- OR8-la, were of the Oak Ridge genetic background, and were essentially isogenic. The temperature-conditional lethal mtuant, un-3 (55701t), the methionineless mutant, me-2, and fluffy, a highly fertile, non-conidiating strain were furnished by the Fungal Genetics Stock Center, presently at Humboldt State College, Arcata, Calif. The eth-lr mutant was originally isolated in our laboratory (METZENBERG, KAPPY and PARSON 1964). Media Fries medium (described by BEADLE and TATUM 1945) was used for vegetative growth, with sucrose (1.5%) as the carbon source. Medium was solidified, when appropriate, with 1.5% Difco agar. The carbon source for all metabolic experiments was autoclaved separately from the salts and the amino acids. Fries medium sans sulfate is a medium in which the usual sulfur source, MgSO,.7H,O (2 mm), was replaced by an equimolar amount of MgC1,. The particular sulfur source desired, e.g., cysteic acid, methionine, or N-acetyl-DL-methionine, was then added. Plate counts of either conidial suspensions or heat-shocked ascospores were made by spreading aliquots onto medium P and incubating the plates for 24 to 48 hours at 34 as described before (METZENBERG 1968). Medium P contains ordinary Fries salts with 0.05% each of glucose and fructose as carbon sources, as well as 1% sorbose to induce colonial growth, and in addition, 1 mm L-methionine and 1% Difco yeast extract. The latter addition gives faster growing, more opaque colonies even with prototrophs. The medium of WESTERGAARD and MITCHELL (1947) was used for all crosses. The medium for the selection of resistant mutants (selenomethionine medium) was Fries salts sans sulfur supplemented with 0.2 mm cysteic acid, 0.2 mm N-acetyl-DL-methionine and 0.1 mm seleno-dl-methionine (the highest concentration at which wild-type strain and me-2 mutant could grow was 4 x 10-5 M) and either 1% sorbose with 0.05% glucose and 0.05% fructose or 1.5% sucrose as carbon source, as specified in the text. Growth of Neurospora and preparation of crude extracts: Erlenmeyer flasks (500 ml) containing 100 ml Fries medium supplemented with the desired sulfur source were inoculated with a conidial suspension so as to give a final absorbancy of 0.1 at 420 nm, when measured in a Coleman spectrophotometer with a 1.7 cm light path. The cultures were shaken (200 cycles per minute) at 25 for approximately 12 hours on a New Brunswick Gyrorotatory Shaker (New Brunswick Scientific Co., New Brunswick, N. J.). The cells were harvested at A.42O = 1.0. The mycelia were ground with Tris-HC1 buffer (ph 7.5, 0.05 M) as has been described previously (METZENBERG 1968). The homogenates were then centrifuged for 20 minutes at 14,500 x g at 0 to 3. The supernatant solutions were used for enzyme assays. The linear growth rate of Neurospora was measured by the method of RYAN, BEADLE and TATUM (1943). The growth tubes were charged with the minimal medium of Fries with 1.5% sucrose as the carbon source and various supplements as specified in the text. Isolation of mutants: It was hoped that strains which were resistant to selenomethionine might be isolated by virtue of the fact that they lacked the enzyme y-cystathionase and could not convert selenomethionine to selenocysteine. Since the biosynthetic enzyme, P-cystathionase,

4 630 G. S. CHEN AND R. L. METZENBERG might also catalyze this reaction to some degree, we reasoned that it would be advantageous to use a mutant that was already missing the P-cystathionase as the parental strain. The appropriate mutant, me-2, was grown on plates of Fries' medium supplemented with 5 mm methionine for 5 to 7 days. Conidia were harvested, washed with water, and diluted to a concentration of roughly 106/ml. The suspension was irradiated with an ultraviolet germicidal lamp to approximately 60% killing. Then sorbose, glucose, fructose, cysteic acid, N-acetyl-DL-methionine, and selenomethionine were added to give the desired final concentration, and aliquots of the suspension (IO ml) were pipetted into Petri dishes and incubated for 3 days at 25". The conidia which grew in this liquid medium were transferred to small tubes charged with selenomethioninesucrose medium. Those cultures that grew vigorously were crossed to wild-type Neurospora of the opposite mating type. Selenomethionine-resistant sporelings were picked, and again outcrossed to wild type to insure that the strains were homdkaryotic, and to remove the me-2 allele, which was no longer desired in the genetic background. The new mutants were again outcrossed to wild type to render them reasonably free of secondary mutations that might have been induced by the irradiation. To assure independent origin of the mutants, only one strain from each Petri dish was saved. All of the final strains could grow on either methionine, cysteic acid, or inorganic sulfate as sole sulfur source. Ascospores of the last cross were heat-shocked at 60" for 30 minutes and plated on medium P. A second aliquot was plated on selenomethionine-sorbose plates to determine the fraction that was resistant to the analog. All the plates were incubated at 25" for 24 hours. The medium P plates were used for scoring total percent germination under non-selective conditions. The selenomethionine-sorbose plates showed two types of germinated ascospores: those that were resistant and gave rise to colonies, and those that germinated but stopped growing at the germling stage. When the latter were transferred to ordinary Fries' medium, they gave cultures that were confirmed to be sensitive to selenomethionine, and, unlike the resistant progeny, could grow at 40". The ratio of resistant to sensitive progeny was about 1:l (see Table 1). This suggested that resistance to selenomethionine in these strains was attributable to a single gene change. Assay for enzymes: Aryl sulfatase (EC ) extraction and assay was as described previously (METZENBERG 1968) except that 0.5 M Tris-acetic acid buffer at ph 7.5 was used. y-cystathionase (EC ) activity was measured on crude extracts as described by FLAVIN (1962). S-Adenosylmethionine synthetase (EC ) activity was determined on crude extracts OI the strains grown on 5.0 mm methionine and 1.5% sucrose as carbon source, as described in the growth of mycelia and preparation of crude extracts. Supernatant solution containing 0.12 to 2.0 mg of protein was incubated for 30 minutes at 25" with 2 pmoles of methionine, 1.25 pmoles of ATP with 50 nci of 14C-ATP, 10 &moles of MgCl,, 200 pmoles of KC1 and 25 pmoles of HEPES (N-Z-hydroxy-ethylpiperazine-N'-2-ethanesulf~nic acid) buffer ph 7.4, and 1 mole of glycerol in a final volume of 1.0 ml. The reaction was stopped by adding 1 ml of absolute ethanol. The reaction mixture was cooled at 0" for 10 minutes and the denatured protein was removed by centrifugation in a clinical centrifuge at full speed for 5 minutes. One ml of the supernatant solution was put onto a small column (6 mm in diameter) containing about 1 cm of Dowex 50 equilibrated with 0.1 M NaC1, followed by 10 ml of 0.1 N HC1. S-Adenosylmethionine was then quantitatively eluted with two 5 ml portions of 6 N HC1 over a period of abut 10 to 15 minutes. The HC1 eluates were evaporated to dryness on a hot plate at 70" to 90". The residue was dissolved in 1 ml water and subjected to liquid scintillation counting. A detailed discussion of this method will be published elsewhere. TABLE 3 Classification of progeny from the cross, se-metr-a X 74-OR23-IA Cross Progeny on selenomethionine-sorbose plates Resis:ant Sensitive Gemination se-metr-'-a x 74-Om-IA % se-met'-s-a x 74-OR2-1A %

5 SELENOMETHIONINE-RESISTANT MUTANTS 63 1 Other methods: Protein was assayed by the method of LOWRY et al. (1951), using bovine serum albumin as a standard. Uptake experiments were performed as described by KAPPY and METZENBERG (1965); the incubation temperature was IO" for reasons which have been previously discussed (KAPPY and METZENBERG 1967). RESULTS Mapping of mutants: During the outcrossing of selenomethionine-resistant mutants se-met'--l and se-metr-2, resistant ascospores were isolated from each cross and their mating types were determined. From each cross, resistant progeny of both mating types were found bnt the parental combination was much more frequent than the recombinant type, the proportion of parental types being 136/151 and 120/130, respectively. This indicates that the mutation occurred in linkage group I, roughly 10 centimorgans from the mating type locus. eth-i' is also linked to mating-type locus. To determine whether these mutation sites were allelic, se-met' mutants wcre crossed with eth-i '. In scoring for wild-type recombinants, we took advantage of the fact that neither selenomethionine-resistant mutants nor the ethionine-resistant mutants could grow on the Fries' sorbose medium at 40". One-half of the usual salts concentration was used to assure that the inability of the parental types to grow at 40" would not be repaired by high osmolarity. Thus, only wild-type recombinants should grow at 40, and they would be sensitive to both drugs. Only a few wild-type recombinants were seen among several thousand germinated ascospores. Therefore, the two mutation sites are either allelic, or located very close together in separate cistrons (Table 2). In order to test whether the two mutations resistant to selenomethionine were allelic, a cross between these mutants was carried out. From thz results shown in Table 2, it was concluded that they must be very closely linked, and could be at the same site. Resistance to methionine analogs: Growth in the prescnce of various inhibitors was followed by spotting conidial suspensions on solid media and measuring growth after constant growth rate was reached at 25" (usually after 48 hours incubation). Results given in Table 3 showed that only the new mutants are resistant to selenomethionine. eth-i ', a mutant resistant to ethionine, is sensitive to selenomethionine although it is probably allelic to se-met'. un-3 (5570It), a mutant which shows altered uptake of several nutrients and is resistant to TABLE 2 Results of pair-wise crosses between two selenomethionine-resistant mutants and eth-1 l' Cross se-metr-'-a x se-metr-*-a se-met'-'-a x eth-1'-a se-metr-2-a x eth-1'-a Wild-type Ascospores' recombinant+ Germination 5, % 6, % 4, % * Ascospores germinated and grown at 25" on medium P plates. j- Wild-type progeny were defined as those that could grow at 40" on plates containing half the usual concentratition of salts plus 1% sorbose, 0.05% glucose, and 0.05% fructose. Separate tests showed that these were sensitive to both ethionine and selenomethionine.

6 632 G. S. CHEN AND R. L. METZENBERG TABLE 3 Growth of colonies on solid Fries' medium containing inhibitors Growth (mm/hr) Inhibitor Wild tvde eth-1' un lt) se-met'-' se-mep2 None mm seleno-dl-methionine mm ethionine * mm p-fluorophenylalanine 0 0 * 0 0 * Not tested in this experiment. Previous reports indicated un-3(55701t) was also resistant to these drugs (JACOBSON and METZENBERC 1968; KAFFY and METZENBERG 1965, 1967). ethionine and p-fluorophenylalanine (JACOBSON and METZENBERG 1968; KAPPY and METZCNBERG 1965, 1967), is also sensitive to selenomethionine. Permeability properties: Resistance to selenomethionine and ethionine could be due to an altered permeability of the mutants to these analogs (LESTER 1966; STADLER 1966). Uptake experiments were performed to test for this possibility. Because radioactive selenomethioninc was not available, we used 14C-ethionine to test the permeability of these mutants. We assume that the mechanism of resistance to both analogs is the same. Uptake of 14C-methionine was measured to test whether these mutants also had normal ability to transport this amino acid. As shown in Figure 1, se-mep and se-metr+ mutants both transport methionine, albeit at a rate somewhat slower than that of the wild-type strain. r wild-type se-met'; I I / se-met'-' TIME(min1 FIGURE 1.-Uptake of 14C-methionine by germinated conidia of the indicated strains at 10". Cycloheximide (0.1 mm) was added to inhibit protein synthesis.

7 SELENOMETHIONINE-RESISTANT MUTANTS 633 Comparable results were obtained when the uptake of ethionine was tested, as shown in Figure 2. We consider that the decreased uptake of methionine analog by the two mutants is too slight to explain their resistance t~ these drugs, especially since un-3 (55701t) transports ethionine much more slowly than do the two new mutants (KAPPY and METZENBERG 1967), yet it is sensitive to selenomethionine. Temperature-conditional lethality of selenomethionine-resistcznt mutants: During the isolation process, we noticed that the resistant mutants grew very well either in liquid Fries' medium or on solid agar plates at 25". They did not grow in the same medium at 40". Howcver, 1 M glycerol in the medium restored the ability to grow at this temperature. Aryl sulfatase activity of selenomethionine-resistant mutants grown on diflerent sulfur sources: Recently we have obtained evidence that cysteine or a related compound is the only corepressor for the repression of aryl sulfatase synthesis (CHEN, JACOBSON and METZENBERG, in preparation) instead of there being an "early" corepressor related to sulfide and a "late" corepressor related to methionine as suggested earlier (METZENBERG and PARSON 1966). If a partial block occurred in the pathway from methionine to cysteine, theii the mutant might not be able to synthesize cysteine from methionine rapidly enough to repress the synthesis of aryl sulfatase. However, it should still be subject to repression by inorganic sulfate, since it could convert the latter to cysteine as fast as the wildtype strain. As shown in Table 4, aryl sulfatase synthesis by the wild-type strain was repressed at a high concentration of methionine (5 mm), and of inorganic F TIME (min) FIGURE 2.-Uptake of 14C-ethionine by germinated conidia of the indicated strains at IO". Cycloheximide (0.1 mm) was added to inhibit protein synthesis.

8 634 G. S. CHEN AND R. L. METZENBERG TABLE 4 Aryl sulfatase activity of selenomethionine-resistant mutants grown on different sulfur sources Sulfur soupce 0.25 mm methionine 5.0 mm methionine 1.O mm cysteic acid 2.0 mm sulfate Aryl sulfatase activity (nmoles/mh/mg protein) Wild tme se-met"-' se-mety-* sulfate (2 mm), while it was derepressed at low concentration' of methionine (0.25 mm) and on cysteic acid at any concentration. As predicted, aryl sulfatase activity of both selenomethionine-resistant mutants was high when grown on any concentration of methionine and low in 2 mm inorganic sulfate. This result suggested that both mutants had a partial failure in the conversion of methionine to cysteine. y-cystathionase activity of selenomethionine-resistant mutants: If the toxicity of selenomethionine in wild type is due to its conversion to selenocysteine in the cell, the mutants resistant to selenomethionine should be partly or completely blocked in the pathway from methionine to cysteine. The high level of aryl sulfatase of selenomethionine-resistant mutants grown on 5 mm methionine encouraged us to examine the levels of some of the enzymes on the pathway from methionine to cysteine. One crucial enzyme in this conversion would be y-cystathionase, and one hypothesis might be that the activity of this enzyme is low in the mutants. However, as seen in Table 5, se-metr-'-' and se-metr-* mutants had enzyme levels as high or higher than that of the wild-type strain. Thus, the mutation might block a step between methionine and cystathionine. S-Adenosylmethionine synthetase activity of selenomethionine-resistant mutants: S-Adenosylmethionine synthetase is a particularly important enzyme on the pathway from methionine to cysteine because it not only catalyzes the first step of the conversion but also is essential as the methyl donor of the cell. Alteration of this enzyme by mutation could reduce selenocysteine formation and cause resistance to selenomethionine. As seen in Table 5, the S-adenosylmethionine synthetase of both selenomethionine-resistant mutants showed only about onetenth of the activity of that of the wild-type strain under the same growth conditions. The result indicated that this step might be parially blocked in the TABLE 5 y-cystathionase and S-adenosylmethionine synthetase actiuity of se-metr mutants grown on 5 mm methionine ycystathionase activity SAM synthetase activity Strain (nmoles/min/mg protein) (nmoles/mg protein/30 min) Wild type eth-1' se-met'-' se-metr-$

9 SELENOMETHIONINE-RESISTANT MUTANTS 635 FIGURE 3.-Heat HEAT TREATMENT tmin) inactivation of S-adenosylmethionine synthetase of wild-type and se-metr-2 crude extract at 37" with and without sulfhydryl group protecting reagent. Cleland's reagent (1 mm) was used. 0: wild type + SH; 0: wild type - SH; A: se-met+-* +SH; A: se-met?-'-" -- SH. mutants. Heat stability of S-adenosylmethionine synthetase in the crude extract of a selenomethionine-resistant mutant (se-metr+) was also different from that of the enzyme from the wild-type strain (Figure 3). This suggests that the structure of the enzyme is altered in this mutant. DISCUSSION SHRIFT and SPROUL (1963) reported that Chlorella vulgaris B, a green unicellular alga, could adapt to a medium containing 3.1 X 10-5 M selenomethionine by 2 to 3 serial transfers, and that the resistance persisted or 220 generations in the absence of selenomethionine. Despite the relative stability of the adapted form, it could be deadapted simply by sulfur starvation. The adapted cells had lower ability to transport methionine, and a regimen of sulfur starvation restored permeability to methionjne. The authors concluded that the adaptation and resistance to selenometllionine was due to a reduction in the permeability of the cells to this analog. Similarly the resistance of some other selenomethionineresistant mutants may be due to the alteration of a permease (unpublished results). The uptake experiment with the present mutants indicated, however, that the resistance of se-met+ mutants to ethionine was not the result of an inability to transport this analog. After examining the properties of selenomethionine-resistant mutants, we found that they were similar to the ethionine-resistant mutant, eth-z+, in many respects. Both eth-1' and se-met+ mutants are temperature-sensitive, but can be repaired by 1 M glycerol (METZENBERG 1968). Aryl sulfatase is not repressed

10 636 G. S. CHEN AND R. L. METZENBERG by a high concentration of methionine (5 mm) but is still subject to repression by inorganic sulfate (2 mm). Both eth-1' and se-met" mutants are located in linkage group I rather near to the mating-type locus (METZENBERG, KAPPY and PARSON 1964), and are quite possibly allelic. However, there are some significant differences between se-met' and eth-1 mutants. First, the ethionine-resistant mutant cannot grow on a medium containing selenomethionine. Second, both se-met' and eth-1' mutants have low S-adenosylmethionine synthetase activity when grown on 5 mm methionine, but the enzyme from mutant eth-1" is much more unstable than the enzyme from se-met" mutant. Heat-inactivation patterns of S-adenosylmethionine synthetase of the crude extract of mutants eth-1' and se-met' at 37" in the presence and absence of a mercaptan were very different from one another ( JACOESON, CHEN and METZENBERG, in preparation). KERR and FLAVIN (1970) reported that one of the ethionine-resistant mutants, eth-1', had a smaller pool of S-adenosylmethionine than did the wild-type strain under the same growth condition, and the level was restored to normal when the mutant was grown on the same medium with glycerol added to a concentration of 1 M. They suggested that eth-1' might be a structural gene mutant for S-adenosylmethionine synthetase. In harmony with this, we found that growth on 1 M glycerol increased the level of this enzyme in the eth-1" mutant two- to three-fold, to a value about one-third to one-fifth of that of the wild-type strain (JACOBSON, CHEN and METZENBERG, in preparation). In addition, we note that the thermostability of S-adenosylmethionine synthetase is altered in selenomethionineresistant mutants, and that se-met" is also very likely a structural gene mutant of this enzyme. It is puzzling that eth-1' is not resistant to selenomethionine as well as to ethionine, since the levels of S-adenosylmethionine synthetase in this strain, measured under our assay conditions, are not higher than those in se-met" strains. Possibly under conditions that obtain inside the cell, the activity of the enzyme is lower in se-met' mutants than in eth-1'. Alternatively, the changes in the enzyme in se-met' may reduce its activity in the adenosylation of selenomethionine by an even greater factor than the reduction in the adenosylation of methionine, which might not be the case in eth-1'. If it is true that selenomethionine exerts its toxicity by being converted to selenocysteine, cysteine should reverse the toxicity of selenomethionine in wild type Neurospora. Unfortunately, this cannot be tested, because at moderately high concentrations cysteine itself is quite toxic to Neurospora. In addition, cysteine is probably transported into the cell by some, and perhaps all of the permeases that could transport selenomethionine ( STADLER 1966; WILEY and MATCHETT 1966; PALL 1969, 1971). Hence, failure to grow might be due to cysteine toxicity, and ability to grow might be explained by competition between cysteine and selenomethionine for transport, rather than by internal competition between cysteine and selenocysteine. This investigation was supported by a Public Health Service Grant, GM One of the authors (R.L.M.) was supported by a Public Health Service Career Development Award, K3- GM

11 SELENOMETHIONINE-RESISTANT MUTANTS 637 LITERATURE CITED BEADLE, G. W. and E. L. TATUM, 1945 Neurospora. 11. Methods of producing and detecting mutations concerned with nutritional requirements. Am. J. Botany 23 : BLAU, M., 1961 Biosynthesis of (Se75) selenomethionine and (Se75) selenocystine. Biochim. Biophys. Acta 49 : COWIE, D. B. and G. N. &HEN, 1957 Biosynthesis off Escherichia coli o'f active altered protek containing selenium instead of sulfur. Biochim. Biophys. Acta 26: FLAVIN, M., 1962 Microbial transsulfuration: the mechanism of an enzymatic disulfide elimination reaction. J. Biol. Chem. 237: FLAVIN, M. and C. SLAUGHTER, 1967 The derepression and function of enzymes of reverse trans-sulfuration in Neurospora. Biochim. Biophys. Acta 132 : HUBER, R. E. and R. S. CRIDDLE, 1967a Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch. Biochem. Biophys. 122: , 1967b The isolation and properties of P-galactosidase from Escherichia coli grown on SOdium selenate. Biochim. Biophys. Acta 141 : HUBER, R. E., I. H. SEGEL and R. H. CRIDDLE, 1967 Biochim. Biophys. Acta 141 : Growth of Escherichia coli on selenite. JACOBSON, E. S. and R. L. METZENBERG, 1968 A new gene which affects uptake of neutral and acidic amino acids in Neurospora crassa. Biochim. Biophys. Acta 156: KAPPY, M. S. and R. L. METZENBERG, 1965 Studies on the basis of ethionine resistance in Neurospora. Biochim. Biophys. Acta 107: , 1967 Multiple alterations in metabolite uptake in a mutant of Neurospora crassa. J. Bacterid. 94: li37. KERR, D. S. and M. FLAVIN, 1970 The regulation of methionine synthesis and the nature of cystathionine y-synthetase in Neurospora. J. Biol. Chem. 245: LESTER, G., : Genetic control of amino acid permeability in Neurospora crassa. J. Bacteriol. LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, 1951 with the Folin phenol reagent. J. Biol. Chem. 193: Protein measurement METZENBERG, R. L., 1968 Repair of multiple defects of a regulatory mutant of Neurospora by high osmotic pressure and by reversion. Arch. Biochem. Biophys. 125 : METZENBERG, R. L. and J. W. PARSON, 1966 Altered repression of some enzymes of sulfur utilization in temperature conditional lethal mutant. Pmc. Natl. Acad. Sci. US. 55: METZENBERG, R. L., M. S. KAPPY and J. W. PARSON, 1964 Irreparable mutations and ethionine resistance in Neurospora. Science 145: PALL, M. L., 1969 Amino acid transport in Neurospora crassa. I. Properties of two amino acid transport systems. Biochim. Biophys. Acta 173: , 1971 Amino acid transport in Neurospora crassa. IV. Properties and regulation of a methionine transport system. Biochim. Biophys. Acta 233: PETERSON, P. J. and G. W. BUTLER, 1962 The uptake and assimilation of selenite by higher plants. Australian J. Biol. Sci. 15: RYAN, R. J., G. W. BEADLE and E. L. TATUM, 1943 The tube method of measuring the growth rate of Neurospora. Am. J. Botany 30: SHRIFT, A. and M. SPROUL, 1963 Nature of the stable adaptation induced by selenomethionine in Chlorella vulgaris. Biochim. Biophys. Acta 71 : STADLER, D. R., 1966 Genetic control of the uptake of amino acids in Neurospora. Genetics 54:

12 638 G. S. CHEN AND R. L. METZENBERG TUVE, T. and H. H. WILLIAMS, 1961 Metabolism of selenium by Escherichia coli: biosynthesis of selenomethionine. J. Biol. Chem. 236: WESTERGAARD, M. and H. K. MITCHELL, 1947 Neurospora. V. A synthetic medium favouring sexual reproduction. Am. J. Botany 34: WILEY, W. R. and W. H. MATCHETT, 1966 Tryptophan transport in Neurospora crmsa. I. Specificity and kinetics. J. Bacteriol. 92: Corresponding editor: R. H. DAVIS