A simple method for the isolation and characterization of thymidylate uptaking mutants in Saccharomyces cerevisiae.

Molec. gen. Genet. 147, 209-215 (1976) © by Springer-Verlag 1976

A Simple Method for the Isolation and Characterization of Thymidylate Uptaking Mutants in Saccharomyces cerevisiae Martin Brendel Arbeitsgruppe Mikrobengenetik im Fachbereich Biologie, J.W. Goethe-Universit/it, D-6000 Frankfurt/Main, Federal Republic of Germany

Summary. The mutant tmpl lO tS which confers thermosensitive auxotrophy for thymidylate is employed for the selection of 5'-dTMP uptaking mutants. At the nonpermissive temperature yeast cells phenotypically wild type for thymidylate uptake can grow for only 3 to 4 generations in the presence of 10-ZM 5'-dTMP. Thymidylate utilizing mutants (turn mutants) were isolated which can grow in the presence of 12 to 24 lag 5'-dTMP/ml. Genetical analysis revealed one of these mutant strains to be a double mutant, tuml turn2. For normal growth haploid thymidylate auxotrophic strains require approximately 360 gg 5'-dTMP/ml when tuml and 24 lag 5'-dTMP when turn2 is present, respectively. Cells prototrophic for thymidylate ( T M P ) harbouring tuml turn2 will also take up 5'-dTMP and incorporate it specifically into their DNA. Thymidylate utilization in such strains is independent of functional mitochondria, as similar incorporation of labelled 5'-dTMP is found in isogenic strains with rho +, rho- and rho ° status. Optimal stimulation of the 5'-dTMP uptaking principle in haploid T M P strains is found at 4 gg 5'-dTMP/ ml when tuml and turn2 are present.

Introduction

Since the first demonstration of DNA-specific labelling in yeast by exogenously supplied 5'-dTMP (Jannsen, Lochmann and Laskowski, 1968) and its dependence on a nuclear gene (Brendel and Haynes, 1972) several yeast strains have been isolated which take up 5'-dTMP and specifically incorporate it into their DNA (Jannsen, Witte, and Megnet, 1973 ; Laskowski and Lehmann-Brauns, 1973; Wickner, 1974). Some of these 5'-dTMP uptaking strains have been genetically analyzed and it appears likely that four to seven nuclear genes control the uptake or utilization of 5'-

dTMP (Brendel, Frith, and Laskowski, 1975). Selection of mutants with 5'-dTMP low-requirement has led to economic DNA-specific labelling in yeast (Frith, Brendel, Laskowski, and Lehmann-Brauns, 1974; Frith and Brendel, 1975). The fact that several discrete stages of 5'-dTMP low-requirement could be observed (Frith and Brendel, 1976) also suggests that a multitude of genes may be involved in the uptake and utilization of 5'-dTMP in yeast. A genetical study of the phenomenon of thymidylate utilization requires the isolation of a large number of 5'-dTMP uptaking mutants. We wish to report here a simple method for the isolation of such mutants and to describe briefly some characteristics of two of them.

Materials and Methods Strains. The yeast strains employed are given in Table 1. They were all derived by cross-breeding strains of Saccharomyces eerevisiae from the Berkeley culture collection (KC series of Resnick (1969)). Standard Growth Conditions. Strains were grown in medium N with appropriate supplement of 5'-dTMP (Frith and Brendel, 1974). When necessary adenine (Ade) and uracil (Ura) at concentrations of 50 gg/ml were added. Media were supplemented with 51-dTMP (Merck) from a filter sterilized 10-2M stock solution in medium N. All cultures were agitated in a New Brunswick waterbath

Table 1. Description of yeast strains

Name

Genotype

MB1076-2B MB1076-2D MB I076-20D MB 1076-24C MB1001-1D MB 1092-6B MB1002-1C

a t m p l - l O ts ade2 a t m p l - l O ts ade2 c~ t m p l - l O ~ ade2 t m p l - l O t` rho ° ade2 arg4 his5 a ade2 arg4 his5 c~ ade2 arg4 his5

adex arg4 his5 leul rho ° arg4 l y s l rho ° rho ° lysl ural rad2 rho + lysl leul radl tad2 p h r l rho + lysl leul radl rho +

210

M. Brendel: Thymidylate Uptaking Mutants in Yeast

shaker (model G76). After 24 h growth was determined by microscopic cell count.

Test for 5'-dTMP Requirement. Cells of 24 h cultures grown to a titer of approximately l0 s cells/ml at 23 ° in medium N (at which temperature strains harbouring the tmpl lO ts allele do not require 5'-dTMP) were microscopically counted and 106 cells/ml were inoculated in the test flasks (2 ml medium in 25 ml Pyrex flasks). Thymidylate requirement (or uptake) was determined by incubating at 36 ° (at which temperature the tmpl-lO ~sallele confers thymidylate auxotrophy). Genetic Analysis. Methods were essentially those described by Johnston and Mortimer (1959) and Mortimer and Hawthorne (1966). Sporulation, micromanipulation and spore growth were carried out at 23 ° .

Assay of Radioactivity. This was essentially done as described by Brendel and Haynes (1973) and F/ith and Brendel (1974). (MethyI3H)-5'-dTMP was purchased from Amersham (specific activity 20 Ci/mmole).

Results and Discussion

The isolation of 5'-dTMP uptaking mutants requires a system in which endogenous thymidylate biosynthesis is inhibited. The method used so far has been that of stopping 5'-dTMP biosynthesis by blocking the enzyme dihydrofolic acid reductase with the help of aminopterin (APT) and sulfanilamide (SAA) (Laskowski and Lehmann-Brauns, 1973; Jannsen et al., 1973; Wickner, 1974; Frith et al., 1974). For the isolation of 5'-dTMP uptaking mutants ( = r u m strains) we prefer a genetical block of the eiazyme thymidylate synthetase as the more direct and more ecconomical method. Cells auxotrophic for 5'-dTMP, however, cannot serve for the isolation of 5'-dTMP uptaking mutants as they simultaneously must be 5'-dTMP uptaking (at least to some extent) in order to be able to grow at all (Brendel and FMh, 1974). The isolation of conditionally 5 ' - d T M P auxotrophic mutants (tmp ts mutants; Brendel and Frith, 1974) has enabled us to

construct strains that are viable at the permissive temperature (23 °) and that undergo "Thymineless Death ( T L D ) " at the nonpermissive temperature (36 °) regardless of absence or presence of 5'-dTMP. At 36 ° even a 5'-dTMP concentration as high as 1 0 - 2 M will allow growth for 3-4 generations only, i.e. from an inoculum of 1 x 106 to maximally 1 x 10 v cells/ml. At the permissive temperature such strains will reach a cell titer in medium N of 1-2 x 10S/ml independent of 5'-dTMP presence. Strains harbouring the t m p l - 1 0 *s allele can therefore serve for the selection or 5'-dTMP uptaking mutants without application of APT and SAA: The haploid tmp1-10 ts strains are pre-grown at 23 ° (permissive temperature) and the cells are then plated on solid medium containing a certain amount of 5'-dTMP and incubated at the nonpermissive temperature (36°). With this procedure it was possible to isolate a large number of putative 5'-dTMP uptaking strains (turn mutants) on a few petri dishes containing medium N supplemented with 36 gg 5'-dTMP/ml (Table 2). The frequency of putative turn mutants lies between 5 x 10 .4 and 10-6 per cell plated. Mutation frequency cannot be determined accurately as the non 5'-dTMP uptaking ( T U M ) strains at the nonpermissive temperature are still able to undergo one to two mitotic cycles before T L D occurs (cf. Fig. 3). Most of the putative tum mutants are able to take up 5'-dTMP and have retained the t m p l - l O ~s allele (no growth at 36 ° in the absence of thymidylate) while a minority has reverted to TMP prototrophy and therefore is able to grow without 5'-dTMP at 36 °. turn mutants isolated were of two qualities: Most were extremely flaky while only a few had retained the creamy character of the T U M parental strain. Quantitative analysis of the flaky strains proved to be impossible as cell titers in response to varying 5'-dTMP supplement could not be counted correctly. Therefore, only a few creamy t m p l - l O ts tum mutants

Table 2. Isolation of turn mutants from 4 haploid strains Strain

MB1076-2B MB1076-2D MB1076-20D MB1076-24C

Number of cells plated

Number of colonies at 36 °

turn mutants (designation)

2.1 x l0 T

182

96 (tumA 1 12 (tumB 1 78 (tumC1 20 b (tumD 1 -

3.5 × l06 1.1 x 108 2.7 x 108

14 ~ 120 a 660"

tmpl-lO~TMP revertants

86 96) 2 12) 42 78) 0b 20)

" Mutants of tumB, tumC and tumD were not further characterized as most subcultures were flaky b Only 20 tumD mutants were tested

M. Brendel: Thymidylate Uptaking M u t a n t s in Yeast

were further analyzed in more detail (all derived from strain MB1076-2B). Two principal ways of further analysis are possible: (i) One can isolate large numbers of turn mutants at one concentration of 5'-dTMP and search for complementation amongst the isolated strains. This procedure of a fixed 5'-dTMP concentration for screening purposes has been chosen by Laskowski and Lehmann-Brauns (1973) and by Wickner (1974). (ii) One can subject the first set of turn mutants to another screening procedure with a lowered offer of 5'-dTMP. According to results obtained with other strains is our laboratory one would expect to find turn mutants of lower 5'-dTMP requirement (F~ith etal., 1974; Frith and Brendel, 1976). This procedure could have the advantage to isolate turn mutant genes that might otherwise not be able to confer the ability to grow at 36 gg 5'-dTMP/ml. We chose the second approach in order to obtain clarity whether 5'-dTMP low-requirement always leads to sterility of the strains (all previously isolated tlr strains have lost their mating type (FS_th and Brendel, 1976)). Sixteen isolates (tumA1 to tumA16) were pregrown in medium N (50 gg Ade/ml) at 23 ° and 106 cells were plated on one petri dish each containing medium N (50 gg Ade/ml). The plates were dried for one hour and then a center piece of the agar removed (diameter 1 cm). 720 gg 5'-dTMP dissolved in 0.2 ml distilled water were added into the center well and the plates incubated at 36 ° for 4 to 5 days. These

211

is rich growth around the center well with colonies becoming smaller towards the outer perimeter of the petri dish (where there is less 5'-dTMP due to the diffusion gradient). However, a few large colonies can be detected in this outer rim and these were isolated as putative 5'-dTMP low-requirers. Each of these isolates was grown in medium N (50 gg Ade/ml) at 23 ° and rechecked on its 5'-dTMP uptake quality using the same procedure described above. This time, however, only 72 ~tg 5'-dTMP were added into the center well. Only a few isolates exhibited growth covering 2/3 to 3/4 of the surface of the petri dish. Of these four substrains each of tumA1, tumA3 and tumA6 were further tested in liquid culture for optimal growth after supplement of various concentrations of 5'-dTMP. Table 3 shows the results obtained with three of these tumA substrains in comparison to the growth response obtained with T U M and tumA1. Clearly there are two characteristic requirements of 5'-dTMP for optimal growth. Mutant tumA1 exhibits growth dependence at 36 ° on 5'-dTMP supplement and reaches the optimal titer of approx. 108 cells/ml at the concentration of 72 I-tg 5'-dTMP/ml. Substrains tumA1 7, tumA1-8 and tumA3-7 show optimal growth at 5'-dTMP concentrations between 12 and 36 lag/ml. At higher concentrations these strains exhibit reduced growth, a phenomenon observed for some 5'-dTMP low-requiring strains (Langjahr, Hartmann, and Brendel, 1975; Frith and Brendel, 1976). Parental strain 1076-2B tmpl-lO t~ T U M cannot grow

Table 3. Growth dependence on supplement of 5'-dTMP of five yeast strains at 36 ° 5'-dTMP (gg/ml)

Cell titer x ml 1 after 24 h at 36 ° (morphology of cells) TUM 1076-2B

tumA 1

t u m A 1 -- 7

tumA 1- 8

tumA3 - 7

0

2.2 x 106 (all double cells ; TLD)



1.4x 106 (all double cells; TLD)

1.7 x 106 (cells totally deformed)

3

1.8 x 106 (all double cells; TLD)


5.9 x 10 v (some cells show TLD)

4.7 x 10 v (many double cells; enlarged)

3.8 x 107 (many double cells ; enlarged)

6

3.1 x i06 (all double cells; TLD)


8.9 x 107 (few cells show TLD)

7.0 × 107 (some cells show TLD)

6.9 x 10 v (cells normal; some TLD)

12


2 . 2 x 10 v (normal plus swollen cells)

1.2 x 108 (cells normal)

8.5 x 10 v (few cells show TLD)

7.9 x 107 (some cells show TLD)

36

2.1 x 106 (alI double cells ; TLD)

6.2x 107 (cells normal)

8.9 x 10 v (cells normal)


1.4x l0 s (cells normal)

72


9 . 0 x 10 v (cells normal)




212

M. Brendel: Thymidylate Uptaking M u t a n t s in Yeast

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108 o •

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XX

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Fig. 1 a-d. Growth in the presence of exogenous 5'-dTMP of yeast strains phenotypically T U M or turn and auxotrophic or prototrophic for thymidylate. Cells were grown in medium N (5'-dTMP; 50 gg Ade/ml; 50 gg Ura/ml) at 36 ° for 24 h, the initial titers being 106/ml

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Fig. 3. Influence of rho status on utilization of exogenous 5'-dTMP. • o, growth at 36 ° after A P T + S A A mediated inhibition of thymidylate biosynthesis in the presence of exogenous 5'-dTMP. x - x , 5'-dTMP utilization with thymidylate biosynthesis permitted as monitored by the incorporation of (3H)-5'-dTMP into alkali stable TCA precipitable material. Cells were grown in medium N (0-60 gg 5'-dTMP/ml; 2.5 gCi (3H)-5'-dTMP/ml; 50 p,g Ade/ml) at 36 ° for 24 h, the initial titer being 106/ml. o - - o , cell titer after 24 h

I

30

Fig. 2. Isopycnic banding in CsC1 of D N A from strain MB1093-8A with different rho status. • - e , rho + ; x - - x , r h o - (spontaneous petite) ; o - - - ©, rho ° (ethidium bromide induced petite)

able to grow at 36 ° in the presence of 36 gg 5'-dTMP/ ml. In the past all 5'-dTMP low-requiring strains were derived from either thymidylate auxotrophic strains (already able to grow at high 5'-dTMP concentation) or after A P T + S A A treatment of prototrophic strains. Both procedures have always led to the loss

of mitochondrial D N A and have resulted in petite strains (Brendel and Frith, 1974; Fath et al., 1974). Strains harbouring the tmpl-lO ts allele and grown at 23 ° are also mostly petite (Brendel and Frith, 1974) and 5'-dTMP low-requiring grande strains have not been found. Transfer of genes conferring 5'-dTMP low-erquirement was also impossible as all strains isolated for excellent uptake of 5'-dTMP had lost their mating type (F~ith et al., 1974; Frith and Brendel, 1975). It was thus interesting to obtain grande strain MB1093-8A that carries tuml turn2 by genetic con-

M. Brendel: Thymidylate Uptaking Mutants in Yeast

struction. Now it was possible to test whether there is an influence on 5'-dTMP uptake by the genetic information residing in mitochondrial DNA (mtDNA), i.e. whether the petite mutation (mostly rho °) is a prerequisite for the isolation of 5'-dTMP low-requiring mutants. A spontaneous petite mutant and ethidium bromide induced (A1-Aidros, Somers and Bussey, 1973) neutral petite mutants were isolated from grande strain MB1093-8A. While the spontaneously selected petite strain still contains m t D N A the ethidium bromide induced petite mutant shows no significant presence of m t D N A (Fig. 2). All three strains grow equally well in medium N (50 gg Ade/ml) at 23 ° or 36 ° in the presence as well as in the absence of 5'-dTMP. When the uptake of 5'-dTMP by these isogenic strains, differing only in informational contents of mtDNA, is compared one finds no or little change. The concentration of 5'-dTMP required for optimal utilization of the nucleotide is the same, the optimal offer being 4 lag of 5'-dTMP/ml. Obviously isotope dilution is more than compensated by the stimulation of the 5'-dTMP uptaking principle (Frith and Brendel, 1976) so that we can witness, at least in the concentration range from 0 to 4 gg 5'-dTMP/ml, an increase in the radioactivity incorporated into the yeast DNA. Only at concentrations higher than 8 gg/ml one finds the expected effect of isotope dilution, indicative of the saturation of the 5'-dTMP uptaking principle (Fig. 3). Under APT + SAA conditions, where endogenous thymidylate synthesis is blocked, all three strains show the same growth dependence on exogenous 5'dTMP, regardless of their rho status (Fig. 3). It thus seems probable that the elimination of the m t D N A is not a prerequisite for the induction or selection of 5'-dTMP low-requirement. It may be noted that substrains of MB1093-8A, regardless of rho status, do not show impaired growth at high concentrations of 5'-dTMP (Fig. 3) as can be witnessed in the originally isolated strain t u m A l - 7 (Fig. 1 a). At present we cannot exclude the possibility that the genetic background of the strains (or possibly a third turn gene not detectable with our screening procedures) plays an important role in this phenomenon. It is hoped that this problem can be solved with the isolation of further turn genes and the then possible construction of triple ot quadruple turn mutant strains. The good thymidylate utilization of grande strains carrying turnl and turn2 mutant genes as well as the fact that these genes can be readily transferred into other genetic backgrounds will greatly facilitate studies on yeast DNA, especially when labelling with high specific activity is mandatory.

215 Acknowledgements. I wish to thank Mrs. Gabriele Polzer-Jung for excellent technical assistance. I enjoyed many discussions with Mr. Wolfgang W. Frith and appreciate his critical reading of this manuscript. This work was supported by a grant of the Deutsche Forschungsgemeinschaft.

References A1-Aidros, K., Somers, J.M., Bussey, H. : Retention of cytoplasmic killer determinants in yeast cells after removal of mltochondrial DNA by ethidium bromide. Molec. gen. Genet. 122, 323-330 (1973) Brendel, M., Frith, W.W.: Isolation and characterization of mutants of Saccharomyces cerevisiae auxotrophic and conditionally auxotrophic for 5'-dTMP. Z. Naturforsch. 29c, 733-738 (1974) Brendel, M., F~ith, W.W., Laskowski, W. : Isolation and characterization of mutants of Saccharomyces cerevisiae able to grow after inhibition of dTMP synthesis. In: Methods in cell biology (D.M. Prescott, ed.), Vol. 11, p. 287 294. New York and London: Academic Press 1975 Brendel, M., Haynes, R.H.: Kinetics and genetic control of the incorporation of thymidine monophosphate in yeast DNA. Molec. gen. Genet. 117, 39-44 (1972) Brendel, M., Haynes, R.H.: Exogenous thymidine 5'-monophosphate as a precursor for DNA synthesis in yeast. Molec. gen. Genet. 126, 337 348 (1973) Brendel, M., Langjahr, U.G.: "Thymineless death" in a strain of Saccharomyces cerevisiae auxotrophic for deoxythymidine-5'monophosphate. Molec. gen. Genet. 131, 351-358 (1974) Frith, W.W., Brendel, M. : An improved assay Of UV-induced thymine-containing dimers in Saccharomyces cerevisiae. Z. Naturforsch. 30e, 804-810 (1975) Frith, W.W., Brendel, M. : Isolation and properties of yeast mutants with highly efficient thymidylate utilization. Z. Naturforsch. 31e, in the press (1976) F~ith, W.W., Brendel, M., Laskowski, W., Lehmann-Brauns, E.: Economizing DNA-specific labelling by deoxythymidine-5'monophosphate in Saccharomyces cerevisiae. Molec gen. Genet. 132, 335-345 (1974) Janssen, S., Lochmann, E.-R., Laskowski, W. : DNS-Synthese nach R6ntgenbestrahlung bei homozygoten Hefestrimmen verschiedenen Ploidiegrades. Z. Naturforsch. 23 b, 1500-1507 (1968) Janssen, S., Witte, I., Megnet, R. : Mutants for the specific labelling of DNA in Saccharomyces cerevisiae. Blochim. biophys. Acta (Amst.) 299, 681-685 (1973) Johnston, J.R., Mortimer, R . K : Use of snail digestive juice in isolation of yeast spore tetrads. J. Bact. 78, 292 (1959) Langjahr, U.G., Hartmann, E.-M., Brendel, M.: Nucleic Acid Metabolism in Yeast. I. Inhibition of RNA and DNA synthesis by high concentrations of exogenous deoxythymidine 5'-monophosphate in 5'-dTMP low-requiring strains. Molec. gen. Genet. 143, 113-118 (1975) Laskowski, W., Lehmann-Brauns, E.: Mutants of Saccharomyces able to grow after inhibition of thymidine phosphate synthesis Molec. gen. Genet. 125, 275~77 (1973) Mortimer, R.K., Hawthorne, D.C. : Genetic mapping in Saccharomyces. Genetics 53, 165 173 (1966) Resnick, M.A. : Genetic control of radiation sensitivity in Saccharomyces cerevisiae. Genetics 62, 519-53I (1969) Wickner, R.B. : Mutants of Saccharomyces eerevisiae that incorporate deoxythymidine-5'-monophosphate into deoxyribonucleic acid m vivo. J. Bact. 117, 252-260 (1974)

Communicated by F. Kaudewitz Received March 29, 1976

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