Chapter 16
Isolation and Characteriiation of Mzctants of Saccharomyes cerevisiae Able to Grow after Inhibition of dTMP Synthesis M. BRENDEL,' W. W. FATH,'
I. Introduction
.
.
.
.
.
.
AND
. . .
W. LASKOWSKIZ
.
11. Mutants Able to Grow after Inhibition of dTMP Synthesis 111.
IV. V. VI.
A. Reversible Inhibition . . . . B. Isolation Procedure . . . . Characterization of typ or tup Mutants . . . A. Genetic Characterization . . . . B. Biochemical Characterization . , . . Isolation of dTMP Low Requirers (zyp tlr Mutants). Mutants Auxotrophic for dTMP (typ rmp Mutants) . A. Theory . . . . . . . B. Procedure of Isolation . . . . . dTMP Auxotrophs with Low Requirement for dTMP References , . , . . . .
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. . . . .
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. . . . . . . . . .
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. .
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. .
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287 288 288 289 291 291 291 292 292 292 293 293 293
I. Introduction When bacteria are inhibited or genetically blocked in thymidylate biosynthesis, this condition can be overcome by exogenously supplied thymine (Thy) or deoxythymidine (dThd) since these organisms can take up Thy and dThd and utilize them for thymidylate synthesis via the enzymes thymidine phosphorylase (tpp) and thymidine b a s e (tk), or via tk alone (O'Donovan and Neuhard, 1970).
i Fachbereich Biologie der J. W.GoethaUniversittlt, Frankfurt am Main, Germany. 'Zentralinstitut 5 der Freien Universitilt, Berlin, Germany.
287
288
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
The yeast Saccharomyes cerevisiae obviouslylacks tk(Grivel1and Jackson, 1968) and also seems to take up poorly both Thy and dThd (Grenson, 1969; Jannsen et al., 1968; Lochmann, 1965). It is therefore not possible in this organism to overcome a blockage in thymidylate biosynthesis by offering either molecule. Hence, if blockage of dTMP synthesis is to be overcome in yeast, one must offer deoxythymidine phosphoric acids, at least in the form of deoxythymidine 5'-monophosphate (dTMP). In 1968, Jannsen et al. reported DNA-specific labeling by exogenous dTMP in strain 211 of S. cerevisiae. Further experiments verified the DNA-specific nature of this labeling (Jannsen et al., 1970;Brendel and Haynes, 1972),but showed rather poor efficiency of utilization of the offered dTMP molecules (Filth and Brendel, 1974). DNA-specific labeling is achieved with 'H- or '42-labeled dTMP and also with dTMP-32P when derepression of acidic phosphatase is inhibited (Brendel and Haynes, 1973). In order to obtain more economical utilization of dTMP in S . cerevisiae, it was necessary to develop a screening procedure for the isolation of mutants able to grow with exogenous dTMP after inhibition of dTMP biosynthesis. Such mutants would allow further screening for low requirers of exogenous dTMP.
11. Mutants Able to Grow after Inhibition of dTMP Synthesis A.
Reversible Inhibition
In bacteria thymidylate synthesis may be inhibited by folic acid antagonists such as aminopterin (APT), which strongly interferes with dihydrofolic acid (DHFA) reductase, thus preventing DHFA from being reduced to tetrahydrofolic acid (THFA) (O'Donovan and Neuhard, 1970; Brown, 1970; Kit, 1970). Thymidylate biosynthesis by thymidylate synthetase (ts) is known to be the only C ,transfer reaction in which THFA is oxidized (Brown, 1970). All the other C, transfer reactions leave THFA in its reduced state, i.e., they are THFA-conservative. Thus the continued action of ts in the presence of APT rapidly depletes the supply of THFA within the cell, thereby stopping growth. In yeasts total inhibition of growth could not be achieved by APT alone (Laskowski and Lehmann-Brauns, 1973; Filth and Brendel, 1974). However, this is possible by the simultaneous use of APT and asulfonamide (e.g., sulfanilamide, SAA), which interfere synergistically with THFA production. In the presence of APT -+ SAA, the cell obviously is not able to perform any step of THFA-dependent metabolism once ts has oxidized the remaining THFA to DHFA. Therefore one would expect restoration of growth in yeasts inhibited by APT t SAA after addition of dTMP plus those products requiring THFA-conservative metabolism for synthesis (Brown, 1970).
16.
C1-metabolism
INHIBITION OF dTMP SYNTHESIS
289
purine biosynthcsis amino acid metabdim initiaion ot protein synthesis in mitochondria
FIG. 1. Putative folic acid metabolism and its relation to thymidylate biosynthesis in yeast. The scheme was designed according to the data given by O'Donovan and Neuhard (1970), Kit (1970), Hartman (1970). Brown (1970), and Baglioni and Colombo (1970). The numbers given represent the following enzymes: 1, ribonucleoside diphosphate reductase (BI subunit); 2, nucleoside diphosphate kinase; 2a. ribonucleoside triphosphate reductase; 2b, dCTP deaminase; 3, dUTP pyrophosphatase: 3a. dCMP deaminase; 4, thymidylate synthetase; 5, DHFA reductase; 6, thymidine phosphorylase; 7, thymidine kinase. Reactions 1 to 4 constitute the putative main pathway, and reactions 2a, 2b, and 3a possibleshunts for thymidylate biosynthesis. Reactions 6 and 7 (broken lines) have not been found in yeasts but were shown to exist in prokaryotes. CDP, dCMP, dCDP, dCTP, Ribo- and deoxyribonucleosidephosphoric acids of cytosine; UTP, UDP, dUMP, dUTP, rib* and deoxyribonucleosidephosphoric acids of uracil; dTMP, deoxythymidine-5' monophosphate; Thy, thymine; dThd, deoxythymidine; DHFA/THFA, dihydro-/tetrahydrofolic acid.
Among these are adenine (to compensate for the inhibition of purine biosynthesis) and the amino acids glutamic acid, glycine, and methionine (Fig. 1). In the yeast S. cerevisiue, however, an offer of the above-mentioned substances does not lead to restoration of growth in an APT + SAA-inhibited culture (Fig, 2a). But mutants can be found that are able to grow under these conditions (Fig. 2b).
B. Isolation Procedure Between lo5 and lo8 stationary cells, depending on strain and ploidy, are plated on medium C, the composition of which is given in Table I. A few large colonies will arise after a 3-6 days' incubation at 30°C. Among these
290
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
0
14 22 26 t (hours of incubation)
8
32
c
.-0
c
0
.c-c 0.1
0.01 t ( h o u r s of incubation 1
FIO. 2. Growth curves of strain 211-laM (upper part) and a typ mutant (lower part) in media A, B, and C (from Laskowski and Lehmann-Brauns, 1973).
one will find mutants able to synthesize DNA from external dTMP only (Ftith et al., 1974). Such mutants are called TMP-per by Jannsen et al. (1973), typ by Laskowski and Lehmann-Brauns (1973), and tup by Wickner (1974). TMP-per mutants have not yet been genetically characterized. Growth curves of a typ mutant and the corresponding parental strain in liquid media A, B, and C are shown in Fig. 2.
16. INHIBITION OF dTMP SYNTHESIS
29 1
TABLE I
MEDIUMFOR Component
I
I1 111
THE ISOLATION OF
typ MUTANTS'
Compound
Medium
Yeast nitrogen base without amino acids (Difco), 6.7 gm Casamino acids without vitamins (Difco), 2.0 gm D( +)-Glucose, 20 gm Adenine, 50 mg Bacto agar (Difco), 20 gm APT, 20 mg SAA, 4-6 gm dTMP, 10 mg
C
'Dissolve the compounds of component I in 400 ml of distilled water and autoclave. Allow mixture to cool to 60°C and add: for medium B, a filtersterilized solution of SAA in 600 ml of distilled water and a solution of 20 mg APT in 1 ml of dimethyl sulfoxide; for medium C, add to medium B an adequate amount of filter-sterilized dTMP. Prepare plates immediately. Liquid media A, B, and C are prepared in a similar fashion, the agar being omitted.
111. Characterization of typ or tup Mutants
A. Genetic Characterization So far, all typ or tup mutants have been found to be recessive, and nearly all are respiration-deficient (petite). The latter may be a result of the procedure of isolation, since both folic acid analogs and dimethyl sulfoxide are known to be inducers of cytoplasmic petite mutations (Wintersberger and Hirsch, 1973a,b; Yee et al., 1972). The mutants isolated by Wickner (1974) fall into four, and those isolated by Laskowski and Lehmann-Brauns into three, complementation groups. Whether these complementation groups are identical or similar has not yet been established. Their localization in the yeast genome seems to vary widely, ryp mutants apparently being linked (Laskowski and Lehmann-Brauns, 1973), while ?up mutants seem to be located on several chromosomes (Wickner, 1974).
B.
Biochemical Characterization
typ or tup mutants take up and incorporate labeled dTMP specifically into DNA in the presence as well as in the absence of APT and SAA, provided the cells are supplied with a medium sufficiently rich in inorganic phosphate, e.g., medium A or C.
292
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
Strain 21 1-laMT2, a well-characterized typl mutant, requires approximately 30 pg dTMP/ml to grow optimally in the presence of APT + SAA (Fgth et al., 1974). This suffices to obtain cellular DNA whose Thy content is totally derived from exogenous dTMP. When labeling is performed in the same medium without APT + SAA and adenine, a three times the amount of exogenous dTMP is needed to obtain the same result.
IV. Isolation of dTMP Low Requirers (typ tlr mutants) dTMP as a crucial parameter for growth versus no growth in the presence of APT + SAA makes it possible to select for mutants with a low requirement for dTMP. This can simply be achieved by the use of gradient plates containing the inhibitory medium described above and a linear gradient of dTMP. Approximately 106 typ mutant cells are plated onto freshly prepared gradient plates, and the plates are then incubated for 4-6 days at 300C. Single colonies growing beyond the borderline of dense growth are putative typ tlr mutant colonies (Fgth et al., 1974). The typ tlr mutants so far obtained require approximately 10 pg dTMP/ml for optimal growth in the presence of APT + SAA, i.e., they utilize exogenous dTMP three times as well as the originally isolated haploid typ mutants. This means that, to obtain cellular DNA maximally labeled with exogenous dTMP, one has to offer only five times the amount of macromolecular cellular Thy content under APT + SAA conditions (Filth et al., 1974).
V. Mutants Auxotrophic for dTMP (typ tmp Mutants) A. Theory The rationale for the isolation of such mutants may be derived from Fig. 1. If adenine is omitted from the inhibitory medium described above and only dTMP and the essential amino acids are offered, cells in which thymidylate biosynthesis via ts is possible would not be expected to grow. Cells in which thymidylate biosynthesis is partially or totally blocked by a malfunction of ts itself or by a poor supply of its substrate (Fig. l), however, would be expected to grow. In such mutant cells the amount of THFA oxidized by ts is more or less drastically reduced. Thus they are more able to perform the THFA-conservative biosynthesis of purine nucleotides and have a growth advantage over parents prototrophic for dTMP.
16.
INHIBITION OF dTMP SYNTHESIS
29 3
B. Isolation Procedure A stationary culture of any typ or tup mutant is prepared in medium A (without adenine). The cells are collected, washed three times in phosphate buffer (0.067 M,pH 7), and then treated as follows. They are suspended in a 10 -7 dilution of ethyl methanesulfonate (EMS) in phosphate buffer to give a titer of 10’ celldm1 and incubated for 2 hours at 30oC with continuous shaking. Then an equal volume of 10% sodium thiosulfate is added, and neutralization of EMS takes place for 15 minutes. The mutagenized cells are pelleted, washed three times in phosphate buffer, and resuspended in liquid medium A (without adenine) supplemented with 20-30 pg dTMP/ml to a final titer of approximately 107 cells/ml. Incubation takes place for 4 hours at 300C. Then lo5to 106 cells are plated onto solid medium C described in Table I (but without adenine) and incubated for 4-7 days at 300C. One will find many small colonies and a few large ones on the plates, the large ones being potential dTMP auxotrophs. These are removed and streaked onto solid medium A which additionally contains 20-3Opg dTMP/ ml. Incubation takes place for 1-2 days at 30°C. This master plate is replica-plated onto solid medium A. Clones not growing on this medium are dTMP auxotrophs.
VI. dTMP Auxotrophs with a Low Requirement for dTMP The isolation procedure of typ tmp tlr mutants is analogous to that described in Section IV,with the exception that medium-A plates with a linear gradient of dTMP are used.
RERERENCES Baglioni, C., and Colombo, B. (1970). In “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed., Vol. 4, pp. 278-349. Academic Press, New York. Brendel, M., and Haynes, R. H. (1972). Mol. Gen. Genet. 117,3944. Brendel, M., and Haynes, R. H. (1973). Mol. Gen. Genet. 126, 337-348. Brown, G . M. (1970). I n “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed., Vol. 4, pp. 369410. Academic Press, New York. Fiith, W. W., and Brendel, M. (1974). Mol. Gen. Genet. 131, 57-67. Fath, W. W., Brendel, M., Laskowski, W., and Lehmann-Brauns, E. (1974). Mol. Gen. Genet. 132,335-345. Grenson, M. (1969). Eur. J. Biochem. 11,249-260. Grivell, A. R., and Jackson, J. F. (1968). J. Gen. Microbiol. 54, 307-317. Hartman, S. C. (1970). I n “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed.,Vol. 4, pp. 1-58. Academic Press,New York. Jannsen, S., Lochmann, E.-R., and Laskowski, W. (1968). Z.Nuturforsch. B 23, 1500-1507. Jannsen, S., Lochmann, E.-R., and Megnet, R. (1970). FEBS (Fed. Eur. Biochem. Soc.) Lett. 8,113-115.
294
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
Jannsen, S., Witte, I., and Megnet, R. (1973). Biochim. Biophys. Ada 299,681-685. Kit, S. (1970). In “Metabolic Pathways” (D. M. Greenberg, ed.),3rd ed.,Vol. 4, pp. 70-252. Academic Press, New York. Laskowski, W., and Lehmann-Brauns, E. (1973). Mol, Gen. Genet. 125, 275-277. Lochmann, E.-R.(1965). Naturwissenschajien 52,498. O’Donovan. G . A., and Neuhard, J. (1970). Baderiol. Rev. 34,278-343. Wickner, R. B. (1974). J. Baderiol. 117, 252-260. Wintenberger, U., and Hinch, J. (1973a). Mol. Gen. Genet. 126.61-70. Wintcnberger, U., and Hirsch, J. (1973b). Mol. Gen. Genet. 126, 71-74. Yee, B., Tsuyumu, S., and Adams, B. G. (1972). Biochem. Biophys. Res. Commun. 49, 1336-1 342.
Isolation and Characteriiation of Mzctants of Saccharomyes cerevisiae Able to Grow after Inhibition of dTMP Synthesis M. BRENDEL,' W. W. FATH,'
I. Introduction
.
.
.
.
.
.
AND
. . .
W. LASKOWSKIZ
.
11. Mutants Able to Grow after Inhibition of dTMP Synthesis 111.
IV. V. VI.
A. Reversible Inhibition . . . . B. Isolation Procedure . . . . Characterization of typ or tup Mutants . . . A. Genetic Characterization . . . . B. Biochemical Characterization . , . . Isolation of dTMP Low Requirers (zyp tlr Mutants). Mutants Auxotrophic for dTMP (typ rmp Mutants) . A. Theory . . . . . . . B. Procedure of Isolation . . . . . dTMP Auxotrophs with Low Requirement for dTMP References , . , . . . .
.
. . . . .
.
. . . .
. . . . . . . . . .
.
. .
.
. .
. .
. .
.
. . . .
.
. .
. . . . . .
. . . . .
. . . . . . . . . . .
. .
287 288 288 289 291 291 291 292 292 292 293 293 293
I. Introduction When bacteria are inhibited or genetically blocked in thymidylate biosynthesis, this condition can be overcome by exogenously supplied thymine (Thy) or deoxythymidine (dThd) since these organisms can take up Thy and dThd and utilize them for thymidylate synthesis via the enzymes thymidine phosphorylase (tpp) and thymidine b a s e (tk), or via tk alone (O'Donovan and Neuhard, 1970).
i Fachbereich Biologie der J. W.GoethaUniversittlt, Frankfurt am Main, Germany. 'Zentralinstitut 5 der Freien Universitilt, Berlin, Germany.
287
288
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
The yeast Saccharomyes cerevisiae obviouslylacks tk(Grivel1and Jackson, 1968) and also seems to take up poorly both Thy and dThd (Grenson, 1969; Jannsen et al., 1968; Lochmann, 1965). It is therefore not possible in this organism to overcome a blockage in thymidylate biosynthesis by offering either molecule. Hence, if blockage of dTMP synthesis is to be overcome in yeast, one must offer deoxythymidine phosphoric acids, at least in the form of deoxythymidine 5'-monophosphate (dTMP). In 1968, Jannsen et al. reported DNA-specific labeling by exogenous dTMP in strain 211 of S. cerevisiae. Further experiments verified the DNA-specific nature of this labeling (Jannsen et al., 1970;Brendel and Haynes, 1972),but showed rather poor efficiency of utilization of the offered dTMP molecules (Filth and Brendel, 1974). DNA-specific labeling is achieved with 'H- or '42-labeled dTMP and also with dTMP-32P when derepression of acidic phosphatase is inhibited (Brendel and Haynes, 1973). In order to obtain more economical utilization of dTMP in S . cerevisiae, it was necessary to develop a screening procedure for the isolation of mutants able to grow with exogenous dTMP after inhibition of dTMP biosynthesis. Such mutants would allow further screening for low requirers of exogenous dTMP.
11. Mutants Able to Grow after Inhibition of dTMP Synthesis A.
Reversible Inhibition
In bacteria thymidylate synthesis may be inhibited by folic acid antagonists such as aminopterin (APT), which strongly interferes with dihydrofolic acid (DHFA) reductase, thus preventing DHFA from being reduced to tetrahydrofolic acid (THFA) (O'Donovan and Neuhard, 1970; Brown, 1970; Kit, 1970). Thymidylate biosynthesis by thymidylate synthetase (ts) is known to be the only C ,transfer reaction in which THFA is oxidized (Brown, 1970). All the other C, transfer reactions leave THFA in its reduced state, i.e., they are THFA-conservative. Thus the continued action of ts in the presence of APT rapidly depletes the supply of THFA within the cell, thereby stopping growth. In yeasts total inhibition of growth could not be achieved by APT alone (Laskowski and Lehmann-Brauns, 1973; Filth and Brendel, 1974). However, this is possible by the simultaneous use of APT and asulfonamide (e.g., sulfanilamide, SAA), which interfere synergistically with THFA production. In the presence of APT -+ SAA, the cell obviously is not able to perform any step of THFA-dependent metabolism once ts has oxidized the remaining THFA to DHFA. Therefore one would expect restoration of growth in yeasts inhibited by APT t SAA after addition of dTMP plus those products requiring THFA-conservative metabolism for synthesis (Brown, 1970).
16.
C1-metabolism
INHIBITION OF dTMP SYNTHESIS
289
purine biosynthcsis amino acid metabdim initiaion ot protein synthesis in mitochondria
FIG. 1. Putative folic acid metabolism and its relation to thymidylate biosynthesis in yeast. The scheme was designed according to the data given by O'Donovan and Neuhard (1970), Kit (1970), Hartman (1970). Brown (1970), and Baglioni and Colombo (1970). The numbers given represent the following enzymes: 1, ribonucleoside diphosphate reductase (BI subunit); 2, nucleoside diphosphate kinase; 2a. ribonucleoside triphosphate reductase; 2b, dCTP deaminase; 3, dUTP pyrophosphatase: 3a. dCMP deaminase; 4, thymidylate synthetase; 5, DHFA reductase; 6, thymidine phosphorylase; 7, thymidine kinase. Reactions 1 to 4 constitute the putative main pathway, and reactions 2a, 2b, and 3a possibleshunts for thymidylate biosynthesis. Reactions 6 and 7 (broken lines) have not been found in yeasts but were shown to exist in prokaryotes. CDP, dCMP, dCDP, dCTP, Ribo- and deoxyribonucleosidephosphoric acids of cytosine; UTP, UDP, dUMP, dUTP, rib* and deoxyribonucleosidephosphoric acids of uracil; dTMP, deoxythymidine-5' monophosphate; Thy, thymine; dThd, deoxythymidine; DHFA/THFA, dihydro-/tetrahydrofolic acid.
Among these are adenine (to compensate for the inhibition of purine biosynthesis) and the amino acids glutamic acid, glycine, and methionine (Fig. 1). In the yeast S. cerevisiue, however, an offer of the above-mentioned substances does not lead to restoration of growth in an APT + SAA-inhibited culture (Fig, 2a). But mutants can be found that are able to grow under these conditions (Fig. 2b).
B. Isolation Procedure Between lo5 and lo8 stationary cells, depending on strain and ploidy, are plated on medium C, the composition of which is given in Table I. A few large colonies will arise after a 3-6 days' incubation at 30°C. Among these
290
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
0
14 22 26 t (hours of incubation)
8
32
c
.-0
c
0
.c-c 0.1
0.01 t ( h o u r s of incubation 1
FIO. 2. Growth curves of strain 211-laM (upper part) and a typ mutant (lower part) in media A, B, and C (from Laskowski and Lehmann-Brauns, 1973).
one will find mutants able to synthesize DNA from external dTMP only (Ftith et al., 1974). Such mutants are called TMP-per by Jannsen et al. (1973), typ by Laskowski and Lehmann-Brauns (1973), and tup by Wickner (1974). TMP-per mutants have not yet been genetically characterized. Growth curves of a typ mutant and the corresponding parental strain in liquid media A, B, and C are shown in Fig. 2.
16. INHIBITION OF dTMP SYNTHESIS
29 1
TABLE I
MEDIUMFOR Component
I
I1 111
THE ISOLATION OF
typ MUTANTS'
Compound
Medium
Yeast nitrogen base without amino acids (Difco), 6.7 gm Casamino acids without vitamins (Difco), 2.0 gm D( +)-Glucose, 20 gm Adenine, 50 mg Bacto agar (Difco), 20 gm APT, 20 mg SAA, 4-6 gm dTMP, 10 mg
C
'Dissolve the compounds of component I in 400 ml of distilled water and autoclave. Allow mixture to cool to 60°C and add: for medium B, a filtersterilized solution of SAA in 600 ml of distilled water and a solution of 20 mg APT in 1 ml of dimethyl sulfoxide; for medium C, add to medium B an adequate amount of filter-sterilized dTMP. Prepare plates immediately. Liquid media A, B, and C are prepared in a similar fashion, the agar being omitted.
111. Characterization of typ or tup Mutants
A. Genetic Characterization So far, all typ or tup mutants have been found to be recessive, and nearly all are respiration-deficient (petite). The latter may be a result of the procedure of isolation, since both folic acid analogs and dimethyl sulfoxide are known to be inducers of cytoplasmic petite mutations (Wintersberger and Hirsch, 1973a,b; Yee et al., 1972). The mutants isolated by Wickner (1974) fall into four, and those isolated by Laskowski and Lehmann-Brauns into three, complementation groups. Whether these complementation groups are identical or similar has not yet been established. Their localization in the yeast genome seems to vary widely, ryp mutants apparently being linked (Laskowski and Lehmann-Brauns, 1973), while ?up mutants seem to be located on several chromosomes (Wickner, 1974).
B.
Biochemical Characterization
typ or tup mutants take up and incorporate labeled dTMP specifically into DNA in the presence as well as in the absence of APT and SAA, provided the cells are supplied with a medium sufficiently rich in inorganic phosphate, e.g., medium A or C.
292
M. BRENDEL, W. W. FATH, AND W. LASKOWSKI
Strain 21 1-laMT2, a well-characterized typl mutant, requires approximately 30 pg dTMP/ml to grow optimally in the presence of APT + SAA (Fgth et al., 1974). This suffices to obtain cellular DNA whose Thy content is totally derived from exogenous dTMP. When labeling is performed in the same medium without APT + SAA and adenine, a three times the amount of exogenous dTMP is needed to obtain the same result.
IV. Isolation of dTMP Low Requirers (typ tlr mutants) dTMP as a crucial parameter for growth versus no growth in the presence of APT + SAA makes it possible to select for mutants with a low requirement for dTMP. This can simply be achieved by the use of gradient plates containing the inhibitory medium described above and a linear gradient of dTMP. Approximately 106 typ mutant cells are plated onto freshly prepared gradient plates, and the plates are then incubated for 4-6 days at 300C. Single colonies growing beyond the borderline of dense growth are putative typ tlr mutant colonies (Fgth et al., 1974). The typ tlr mutants so far obtained require approximately 10 pg dTMP/ml for optimal growth in the presence of APT + SAA, i.e., they utilize exogenous dTMP three times as well as the originally isolated haploid typ mutants. This means that, to obtain cellular DNA maximally labeled with exogenous dTMP, one has to offer only five times the amount of macromolecular cellular Thy content under APT + SAA conditions (Filth et al., 1974).
V. Mutants Auxotrophic for dTMP (typ tmp Mutants) A. Theory The rationale for the isolation of such mutants may be derived from Fig. 1. If adenine is omitted from the inhibitory medium described above and only dTMP and the essential amino acids are offered, cells in which thymidylate biosynthesis via ts is possible would not be expected to grow. Cells in which thymidylate biosynthesis is partially or totally blocked by a malfunction of ts itself or by a poor supply of its substrate (Fig. l), however, would be expected to grow. In such mutant cells the amount of THFA oxidized by ts is more or less drastically reduced. Thus they are more able to perform the THFA-conservative biosynthesis of purine nucleotides and have a growth advantage over parents prototrophic for dTMP.
16.
INHIBITION OF dTMP SYNTHESIS
29 3
B. Isolation Procedure A stationary culture of any typ or tup mutant is prepared in medium A (without adenine). The cells are collected, washed three times in phosphate buffer (0.067 M,pH 7), and then treated as follows. They are suspended in a 10 -7 dilution of ethyl methanesulfonate (EMS) in phosphate buffer to give a titer of 10’ celldm1 and incubated for 2 hours at 30oC with continuous shaking. Then an equal volume of 10% sodium thiosulfate is added, and neutralization of EMS takes place for 15 minutes. The mutagenized cells are pelleted, washed three times in phosphate buffer, and resuspended in liquid medium A (without adenine) supplemented with 20-30 pg dTMP/ml to a final titer of approximately 107 cells/ml. Incubation takes place for 4 hours at 300C. Then lo5to 106 cells are plated onto solid medium C described in Table I (but without adenine) and incubated for 4-7 days at 300C. One will find many small colonies and a few large ones on the plates, the large ones being potential dTMP auxotrophs. These are removed and streaked onto solid medium A which additionally contains 20-3Opg dTMP/ ml. Incubation takes place for 1-2 days at 30°C. This master plate is replica-plated onto solid medium A. Clones not growing on this medium are dTMP auxotrophs.
VI. dTMP Auxotrophs with a Low Requirement for dTMP The isolation procedure of typ tmp tlr mutants is analogous to that described in Section IV,with the exception that medium-A plates with a linear gradient of dTMP are used.
RERERENCES Baglioni, C., and Colombo, B. (1970). In “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed., Vol. 4, pp. 278-349. Academic Press, New York. Brendel, M., and Haynes, R. H. (1972). Mol. Gen. Genet. 117,3944. Brendel, M., and Haynes, R. H. (1973). Mol. Gen. Genet. 126, 337-348. Brown, G . M. (1970). I n “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed., Vol. 4, pp. 369410. Academic Press, New York. Fiith, W. W., and Brendel, M. (1974). Mol. Gen. Genet. 131, 57-67. Fath, W. W., Brendel, M., Laskowski, W., and Lehmann-Brauns, E. (1974). Mol. Gen. Genet. 132,335-345. Grenson, M. (1969). Eur. J. Biochem. 11,249-260. Grivell, A. R., and Jackson, J. F. (1968). J. Gen. Microbiol. 54, 307-317. Hartman, S. C. (1970). I n “Metabolic Pathways” (D. M. Greenberg, ed.), 3rd ed.,Vol. 4, pp. 1-58. Academic Press,New York. Jannsen, S., Lochmann, E.-R., and Laskowski, W. (1968). Z.Nuturforsch. B 23, 1500-1507. Jannsen, S., Lochmann, E.-R., and Megnet, R. (1970). FEBS (Fed. Eur. Biochem. Soc.) Lett. 8,113-115.
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