Thymidine kinase-deficient mutants of Physarum polycephalum

Copyright 0 lY80 by Academx Proa. Inc. 411 right\ of reproduction in any form reserved 0014-48?7/80/0403~I-07c0?.00/0

Experimental

THYMIDINE

Cell Research 126 (1980) 351-3.57

KINASE-DEFICIENT PHYSARUM Biochemical

J. MOHBERG,’ Institute

qf Biochemistry

MUTANTS

OF

POL YCEPHALUM Characterization

E. DWORZAK’ and W. SACHSENMAIER”

and Experimentnl

Cancer

Research,

University

of Innsbruck,

Austria

SUMMARY Thymidine kinase (TK) and deoxycytidine kinase (dCK) activity levels, [“Hlthymidine (TdR) and 5-bromo-2’-deoxyuridine (BUdR) incorporation and 5-fluoro-2’-deoxyuridine (FUdR) sensitivity have been compared in TK-deficient (TU63 and TU84) and normal (TU291 and M,b) strains of the myxomycete, Physarum polycephalum. The mutants had about 2% of the TK and 100% of the dCK activity of wild-type (wt) strains. They incorporated some TdR into both nuclear (nDNA) and mitochondrial DNA (mtDNA) but incorporated too little BUdR to give a buoyant density shift in nuclear DNA. They grew in the presence of levels of FUdR which completely blocked DNA synthesis in TU291. The FUdR sensitivity of strain M,b could be increased by supplementing growth medium with folic acid.

Thymidine kinase activity in the plasmodial slime mold, Physarum polycephalum, shows a characteristic pattern of change during the mitotic cycle, rising from a minimum in mid-G2 phase to a peak in early S phase and dropping again during late S and early G2 phase [14, 19, 331. The increase in enzyme activity represents de novo synthesis, since it is blocked by inhibitors of RNA and protein synthesis [35] and is accompanied by incorporation of deuterated amino acids into enzyme [31]. Physarum TK is made up of several electrophoretically separable variants, and at the time of telophase, which in Physarum is immediately before the beginning of S phase, there is a dramatic shift in the relative amounts of the individual variants [ 141. These results suggest that TK, although not in the direct pathway of DNA synthesis, is nonetheless involved in DNA synthesis in

some way, possibly in a regulatory capacity [331. Much of the information now available on function and localization of TK variants in mammalian cells has been obtained by the use of TK-deficient mutants because most, if not all, still have normal levels of TK in their mitochondria but lack cytoplasmic TK, which comprises the bulk of the total TK in proliferating wild-type cells [l, 2, 8, 231. In this paper we report the results of a study of TK and dCK levels, of C3H]TdR and BUdR incorporation and of FUdR sensitivity in some mutant and wild-type (wt) strains of Physarum. It was found that the ’ Present address: Colleee of CAS. Governors State University, Park Forest South, IL 66466, USA. * Present address: Institute of Medical Chemistry, University of Innsbruck, Austria. 3 To whom reprint requests should be addressed.

352

Mohberg,

Dworzak and Sachsenmaier

mutants incorporated no detectable amount of BUdR and little r3H]TdR and grew in the presence of a level of FUdR which completely blocked the wt strains. In spite of the FUdR resistance of the mutants, in both mutant and wt strains FUdR enhanced incorporation of [3H]TdR into mtDNA to a greater extent than into nuclear DNA. MATERIALS

AND METHODS

Culture strains and methods Strains used were the Tromso University mutants, TU63 and TU84 [16, 171 and wt TU291 and M,b. MBb is from the same McArdle isolate as the subline used in most previous work from this laboratory, but it was grown fresh from spherules made in 1962. It is the ‘LMRhlr”used by Tyson et al. 1371. All strains were maintained in submersed culture in semi-defined medium. “N+C” rlll was used for all but the last two experiments with‘FUdR, which were done in a different laboratorv where toxicitv nroblems with “N+-C” forced use of the simpler medium of Brewer & Prior [5]. Both media were supplemented with hemoglobin (0.013%) instead of with hematin. In exoeriments involvina folic acid suodementation. a freshly prepared, filter sterilized 4x’iOm3 M (100x concentrated) solution of the vitamin in 1 N NaHCO, was added to all media, including the control medium used for the initial feeding to ensure equilibration of intracellular pools before FUdR was given. Plasmodia were grown in Petri dishes on filter paper supported either by stainless steel grids [28] or by glass beads (when inhibitors were given). Heterokaryons (l+ 1) of TU291 and TU84 were made by mixing equal volumes of microplasmodial suspension [ 181, then inoculating Petri dishes as usual.

Labeling

techniques

BUdR labeling was done by incubating plasmodia on media containing BUdR (50 fig/ml), FUdR (5 &ml) and UdR (100 &ml) through MI S phase, beginning about 1 h before MI and ending 2 h before MII. Nuclei were isolated [29] and DNA was extracted according to McCormick et al. [27]. DNA was analysed in CsCl gradients (initial refractive index, 1.4000; 1J-3 pg DNA; about 0.4 ml/cell), centrifuged (44800 rpm, 25”C, 18 h) in the Centriscan 75 ultracentrifuge (MSE Ltd, Crawley, England). Buoyant densities were calculated by means of a Streptococcus griseus internal standard. Plasmodia were labeled with r3H]TdR by incubating for 1 h on medium containing either r3H]TdR (methyl label; 5 &i/ml) or r3H]TdR (5 &i/ml), FUdR (5 pg/ml) and UdR (100 pg/ml). Plasmodia were harvested by dipping in ice-water and scraping onto dry ice. DNA was isolated as from nuclei, except that a-amylase (0.5 mglml) was included in the RNAase Ex/, Cdl Res 126 ( 1980)

incubation mixture. DNA was fractionated in preparative CsCl gradients with about 10 pg of [3H]DNA and 0.1 pg ‘Vlabeled main band DNA (1000 cpm) in a 6 ml step gradient [6]. [r4C]marker was prepared from nuclei of cultures grown on [‘“C]TdR (50 &i/ml) for 2 h in MI1 S phase. Tubes were centrifuged at 40000 rpm for 21 h at 25°C in a 10~ 10 ml angle rotor in a Centriscan 75 ultracentrifuge. Tubes were pierced and dripped and carrier (100 pg of bovine serum albumin) was added to each fraction. DNA was precipitated and washed with TCA, then dissolved in 0.1 N KOH, added to cocktail and counted in a liquid scintillation spectrometer. The correction for 14C spillover into the YH channel was determined by counting a gradient of the [14C]DNA alone. Recovery of 14Cfrom gradients was 1OOrfr 15%. Specific activities of nDNA and mtDNA were calculated by means of data from Keck DNA analyses and analytical ultracentrifugation profiles. G2 phase preparations contained about 10% mtDNA, 4% nucleolar DNA and 86% main band or chromatin DNA, similar to preparations of Hoh & Gurney [20]. Mass (in pg) of DNA in a peak from a preparative gradient was estimated by multiplying pg DNA (from Keck assay) applied to gradient by 0.1 for mtDNA and by 0.9 for nDNA (=nucleolar+main band). Specific activity (in cpm/pg) was then equal to peak area (in cpm)tpg DNA.

Analytical

techniques

Extracts to be analysed for TK were prepared according to Griibner & Sachsenmaier [14] except that samples were disrupted by sonication (MSE, low power; amplitude 1; 4~ for 3 set each time; 0°C) instead of by Potter-Elvehjem homogenization. Protein in the extracts was precipitated and washed with TCA to remove mercaptoethanol (Dworzak & Sachsenmaier, in preparation), then was dissolved in 0.4 N NaOH and analysed by the Lowry method [25]. DNA in solution in saline-citrate was estimated by the Keck procedure [22], done with two amyl acetate and one chloroform extraction. Whole plasmodial DNA and protein were determined by the Burton [7] and Lowry [25] methods, with modifications described previously [28].

RESULTS TK levels in TU63 and TU84 were 2 % or less of those of the wt cultures [32]. This activity in mutants was not owing to a TK inhibitor or a TMP nucleotidase [2], judging from results of mixing extracts of wt and mutant strains (table 1). TU291 extracts showed the same activity, whether analysed alone or in mixture with extracts of TU84 or 84B [30]. When analysed by polyacrylamide iso-

Biochemistry of TK-less Physarum

353

electric focusing (PAGIF) (fig. 1), extracts of mutants seemed comprised of the same major enzyme variants-A, B, C-that are found in M3b [14], although the bands were much broader because resolution of the gels was reduced by the heavy protein load which had to be applied to give measurable enzymatic activity in the gel slices. The figure suggests that peak A, is unique to TU84, but this band is usually also seen in M,b extracts [ 141. CsCl gradient profiles of [3H]TdR-labeled cultures (fig. 2) showed that TU84, like McArdle wild-type, M,c [4], incorporated TdR primarily into nDNA during S phase and into both nDNA and mtDNA during G2 Fig. 1. Isoelectric focusing patterns of TK in TU84 phase [ 121. Since all FUdR-treated samples and MBb plasmodia. Extracts were made at telophase were fractionated by isoelectric focusing in polyshowed a more prominent mtDNA peak and acrylamide [ 141. than did the untreated member of each pair, rough approximations of specific activity were made (see Materials and Methods). in FUdR-treated than in untreated plasmodia. The fraction of thymidine label in Although the specific activities of mtDNA and nDNA in M,b were several times higher mtDNA in the 84 S phase control was too than in the mutant (table 2), relative specific small to measure so we could not comactivities of mtDNA : nDNA in both mutant pare specific activities of the two DNAs and wild-type were about 1.5 times greater in this sample. In the FUdR-treated culture, however, specific activities of the two peaks were about equal. Table 1. Effect on TK activity of mixing When TU84 and a TU84 polyploid, 84B, extracts of wild type and mutant strains were incubated on medium containing Extracts of TU291, TU84 and 84B [30], all containing 3 mg of protein/ml, were assayed for TK alone BUdR, FUdR and UdR during mitosis I or in combination. Each assay vessel received 20 ~1 S phase (see Materials and Methods), inof substrate and the indicated volumes of extracts, corporation of density label (data not with Tris-glycerol-mercaptoethanol buffer [ 141 being added to make 50 ~1 of sample shown) was negligible, the main band DNAs having a density of about 1.703, as TK activity; punits/ml compared with 1.700 in unlabeled DNA extract Volume [ 131. Under the same labeling conditions extract Found Expected Strain (/-a most of the main band of TU291 was shifted to a density of 1.75 g/cm3, although more 68.1 TU291 alone 25 than 20% remained in the LL band and 1.2 TU84 alone 25 1.5 84B alone 25 presumably failed to replicate. Al+ 1 hetTU291+ TU84 25+25 66.3 69.3 erokaryon of 291 and 84 behaved similarly. 73.8 69.6 TU291+84B 25-1-25 12.5+ 12.5 32.0 34.6 TU291+TU84 TK-deficient mammalian cells are re12.5+ 12.5 35.2 34.8 TU291+84B sistant to FUdR because they cannot phos-

354

Mohberg,

Dworzak arid Sachsenmaier

Fig. 2. Incorporation of [3H]-

*

:

:s1/

:, ;,I :I ~:’

L

10

20

Jo

phorylate FUdR to FUdMP, the entity actually responsible for blocking thymidine synthetase and DNA synthesis [9]. TU84 and 63 showed a similar resistance to FUdR (fig. 3A, C) and continued to synthesize DNA at nearly the control rate when exposed to concentrations of FUdR (5 pg/ml with 100 pg/ml UdR to prevent incorporation of FUdR into RNA [34]) which gave a complete block of DNA synthesis in TU291 (fig. 3B) and a 60% or greater inhibition of M,b (fig. 3E). Since sensitivity of L1210 leukemia cells to FUdR can be increased by supplementing the growth medium with folinic or folic acid [38], folic acid supplementation was tried with M,b. In the presence of 4x lop5 M folic acid, 5 pg/ml FUdR gave complete inhibition of DNA synthesis in M,b (fig. 3F) and had no noticeable effect on TU63 E.r/’

Cell

Rr.\

I26 f 1980)

D

m

30

thymidine into DNA in TU84 and M,b. TU84 plasmodia were incubated on [3H]TdR medium in the presence (top) and absence @orof FUdR (see Materials and -400 tom) Methods) for l-h periods in S phase (M II+45 to 105min) or in G2 3oo phase (M II+6-7 h). M,b was labeled from M 11+7-8 h (early prophase). DNA was isolated from whole plasmodia, centrifuged in preparative CsCl gradients and assayed for “H and I%. -, 3H corrected for 14Cspillover; - - -, “‘C marker.

(fig. 3 C, expt 2). The FUdR-folic acid treatment drastically delayed MI11 in both wt strains, giving a delay of 12 h in Msb and 21 h in TU291. Such prolonged exposure to FUdR caused visible damage, beyond causing the nucleolar vacuolation already noted by Sachsenmaier & Rusch [34].

DISCUSSION Selection of the Physarum mutants which we used was done by a procedure based on Kao & Pucks method [21] for isolating nutritionally deficient mammalian cell lines. Amoebae were mutagenized with N-methyl-N’-nitro-N-nitrosoguanidine (NMG), grown on normal L xherichia co/i for a short time to fix the mutation, and then were fed on BUdR-labeled bacteria and ex-

Biochemistry oj’ TK-less Physarum

355

Fig. 3. Growth of mutant and wt strains in the presence of FUdR. Replicate cultures were started and at the times indicated by arrows were transferred either to fresh control medium or to medium containing 5 pg/ml FUdR and 100 &ml UdR. “N+C” medium [II] was used with TU84 and TU291 (L?) and Brewer-Prior’s [5] for the remainder. Experiments involving folic acid are marked “FOL”. Whole plasmodial DNA was determined by the Burton method [7] and data plotted with circles (0) for controls, triangles (A) for drug-treated. (B) Gives data for duplicate cultures, while all other panels give means of two experiments, the first in open symbols and the second in filled symbols.

posed to long-wavelength ultraviolet light. Cells which survived, those unable to utilize the labeled bacteria and thus insensitive to the irradiation, were isolated and mated

Table 2. Specific activities of mitochondrial and nuclear DNA in [3H]TdR-labeled mutant and wild-type cultures Areas (in cpm) of mtDNA and NDNA peaks in fig. 2 were converted to specific activities by the equation: Spec. act.=areax% of total DNA in fractionspg DNA in fraction. “Relative” spec. act.=spec. act. mtDNA+spec. act. nDNA Spec. act. (cpmlwg) Sample

NUclear

Mitochondrial

TU84; S phase + FUdR -FUdR

I10 235

130 Cannot be measured

TU84; G2 phase + FUdR - FUdR M,b; G2 phase + FUdR -FUdR

“Rel.” spec. act. mtDNA/ nDNA 1.3 -

29 31

1 160 760

40 24

91 235

8 100 14 000

89 59

to produce plasmodia [17]. Plasmodia thus had never been exposed to BUdR before it was used in these experiments. The mutation appeared to be very stable, for despite almost 10 years’ growth in control medium the mutants still had only about 2% of the wt TK activity at the time these experiments were done and they did not incorporate enough BUdR into main band nuclear DNA to give a buoyant density shift. Lunn et al. [26] found that whole plasmodia in S phase incorporated only between 2 and 10% as much [“H]TdR as did normal strains, and we observed a similar difference in the rate at which G2 phase mutant and wt strains incorporated TdR into mtDNA and nDNA. In TK-deficient mammalian cells the small amount of TK activity which remains is in the mitochondria, where it is present at roughly normal levels [ 1, 2, 8, 231. Whether this is also true of Physurum mutants is still uncertain. We have analysed isolated Myb mitochondria [lo], both with and without Triton X-100 treatment [2], and have found 3-5 punits of TK per mg of

3.56

Mohberg,

Dworzak and Sachsenmaier

protein, whereas TU84 mitochondria prepared similarly had O-O.5 punits of TK per mg (unpublished data of the authors). We cannot, however, rule out that some, if not all, of the activity in the MBb preparations came from cytoplasmic contamination, because polyacrylamide isoelectric focusing (PAGIF) showed that the same major bands were present in mitochondria as in whole cultures. The results of the r3H]TdR incorporation experiment suggest that the Physarum mutants are low in mitochondrial TK because mitochondria labeled to roughly onetenth the specific activity of the wild-type. However, the same difference between TK+ and TK- mouse cells has been observed by Clayton and co-workers [2, 31 and they have suggested that in TK+ cells both cytoplasmic and mitochondrial TK contributed TMP for incorporation into mtDNA. They proposed further that there was an extramitochondrial thymidine synthetase which supplied thymidylate nucleotides to both mitochondria and nuclei, since in TK+ cells nDNA and mtDNA labeled to roughly the same specific activity. The much greater FUdR resistance of the TKcells resulted from the fact that before the drug could act on thymidine synthetase it first had to be transported into the mitochondria to be phosphorylated to FUdMP by mtTK, then be exported back into the cytoplasm. Since we were unable to calculate the specific activity of mtDNA in most S phase samples because it was not sufficiently well separated from nDNA by our CsCl gradients, we have made some rough estimates of S phase specific activities from autoradiographic data of Guttes et al. [15], which were obtained with a McArdle wildtype, labeled with [“H]TdR in the absence of FUdR. If it is assumed that (a) mtDNA

constitutes 10% of the total DNA throughout the mitotic cycle; (b) nDNA content is 0.5 pg in S phase and 1.0 in G2 phase [29]; and (c) there are five times as many mitochondria as nuclei in S phase and ten times as many in G2 phase [ 15, 241, the ratio of specific activity of mtDNA : nDNA (both expressed in grainslpg), in S phase is 270: 300 or about 1: 1 and in G2 phase, 400 : 12 or about 33 : 1. (If these calculations are done using Kuroiwa’s value for mtDNA content, 2~ lop3 pg [24], ratios are five times higher.) Rough as these calculations are, they would still seem to bear out the conclusions that (a) the rate of mtDNA synthesis is approximately equal to the rate of nDNA synthesis during S phase and is much greater in G2 phase; and (6) FUdR enhances [“H]TdR incorporation into mtDNA to a greater extent than into nDNA. These results would fit more simply with the idea that there are two distinct thymidine synthetases, one synthesizing mtDNA and the other nDNA, rather than that there is a single extramitochondrial synthetase [2] for both DNAs. Data on thymidine synthetase levels are clearly needed to clarify this point but attempts at analysing the enzyme have thus far been unsuccessful, owing to the very low concentration in Physarum (H. Wolf, unpublished data). The sensitivity of the wild-type TU291 to FUdR was unexpected, in view of early results with McArdle strain which showed that FUdR, regardless of concentration, could never completely block DNA synthesis [34]. We now believe that this difference in sensitivities among wt strains may be due to differences in intracellular levels of 5,10-methylene tetrahydrolate, which is required for complexing of FUdMP and thymidine synthetase [36], since supplementation of growth medium with folic

Biochemistry of TK-less Physarum acid makes M,b as sensitive as TU291 to FUdR. We wish to thank Mrs Janice Forde for technical assistance during this project and to acknowledge financial support from the “Fund of Austrian Cancer Research Institutes”.

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17. Haugli, F B & Dove, W F, Mol gen genet 118 (1972) 109. Haugli, F B, Dove, W F & Jimenez, A, Mol gen genet 118(1972) 97. 19. Hildebrandt, A & Sauer, H, Biochim biophys acta 294 (1973) 8. 20. Holt, C E & Gurney, E G, J cell biol40 (1969) 485. 21. Kao, F T & Puck, T T, Proc natl acad sci IJS 58 (1967) 1227. 22. Keck, K, Arch biochem biophys 63 (1956) 446. 23. Kit, S, Leung, W-C &Kaplan, LA, Eurj biochem 39 (1973) 43. 24. Kuroiwa, T, Hizume, M & Kawano, S, Cytologia 43 (1978) 119. 25. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall. R J. J biol them 193 (1951) 265. 26. Lunn, Ai Cooke, D & Haugli, F, Genet res Cambr 30 (1977) 1. 27. McCormick, J J, Marks, C & Rusch, H P, Biochim biophys acta 287 (1972) 246. 28. Mohberg, J & Rusch, H P, J bact 97 (1969) 1411. 29. Mohberg, J & Rusch, H P, Exp cell res 66 (1971) 305. 30. Mohberg, J, Dworzak, E, Sachsenmaier, W & Haugli, F B, Cell biol intematl reports. In press. 31. Oleinick, N L, Radiat res 51 (1972) 638. 32. Sachsenmaier, W & Dworzak, E, Radiation and cellular control processes (ed J Kiefer) p. 229. Springer-Verlag, Berlin (1976). 33. Sachs&maier,W & Ives, D H, Biochem Z 343 (1965) 399. 34. Sachsenmaier, W & Rusch, H P, Exp cell res 36 (1964) 124. 35. Sachsenmaier, W, v Fournier, D & Giirtler, K F, Biochem biophys res commun 27 (1967) 655. 36. Santi, D V & McHenry, C S, Proc natl acad sci US 69 (1972) 1855. 37. Tyson, J, Garcia-Herdugo, G & Sachsenmaier, W, Exp cell res 119 (1979) 87. 38. Ullman, B, Lee, M, Martin, D W, Jr & Santi, D V, Proc natl acad sci US 75 (1978) 980.

Received July 26, 1979 Revised version received November 7, 1979 Accepted November 19, 1979