Molec. gen. Genet. 173, 307-313 (1979)

MGG

© by Springer-Verlag 1979

Mitochondrial Activity of 2,6-Diaminopurine in Saccharomyces cerevisiae Cheryl Wallis and David Wilkie Department of Botany and Microbiology, University College London, Gower Street, London WC1E 6BT, England

Summary. 2,6-diaminpurine (DAP) selectively inhibited mitochondrial protein synthesis in yeast cells with concomitant failure of cells to grow in non-fermentable (yeast extract, glycerol) medium. The selectivity was pronounced in all strains tested (15) nearly all of which were able to grow in yeast extract, glucose medium containing 5 mg/ml DAP (maximum solubility) whereas growth was arrested in all strains at 250 500 gg/ml DAP in the glycerol medium. The inhibition was reversed by further addition of adenine to the culture medium. RNA synthesis in rat liver mitochondria was depressed by DAP suggesting that the analogue affected RNA polymerase activity. There was no evidence of nuclear mutagenicity by DAP but resistance to the antibiotics chloramphenicol and oligomycin was induced by the drug. Genetic evidence, although limited, inclicated that the resistance mutations were cytoplasmic. The mitochondrial petite mutation was also induced by DAP but only at comparatively high concentrations. The mutagenic effects were seen only in the glycerol medium.

Introduction 2,6-diaminopurine (DAP) is of historical importance as the first purine analogue to exhibit inhibition of neoplastic growth (Burchenal et al., 1949, 1951 ; Bisele et al., 1951) but because of a low therapeutic index its use clinically has not been pursued. The compound has also been shown to inhibit growth or multiplication in a wide variety of other biological systems including mammals, birds, plants and numerous bacteria (see Hitchings and Elion, 1%3). The analogue inhibited the intracellular replication of DNA and RNA viruses in mouse fibroblast L cells suggesting For offprints contact: C. Wallis

that DAP interferes with both types of nucleic acid metabolism (Balduzzi and Morgan, 1964). DAP was identified as an adenine antagonist by inhibition and reversal studies using Lactobacillus casei (Elion et al., 1953). The ribonucleotide of DAP is probably the key factor in growth inhibition by DAP following the study of DAP-resistant mutants in Salmonella typhimurium (Kalle and Gots, 1963) and cultured human fibroblasts (Rappaport and DeMars, 1973) where resistance was correlated with the loss of the enzyme, AMP pyrophosphorylase. Studies with 14C-labelled diaminopourine have shown labelling of nucleic acid guanine and adenine in yeast (Kerr and Chernigoy, 1953) and E. coli (Kaplan et al., 1961) and most of the DAP on entering the cell will be converted to guanine whence its inhibitory action will be blocked (Hartenstein and Fridovich, 1967). There is little evidence for the incorporation of unchanged DAP into nucleic acids except to a small extent in E. coli DNA (Kaplan et al., 1961) and T4 bacteriophage DNA (Freese, 1959). Thus, DAP has a dual metabolic fate as it may serve as an inhlbitor or, when its inhibitory action is blocked, as a precursor for nucleic acid purines. DAP may exert its inhibitory properties by being converted into inhibitory analogues of adenine containing cofactors such as adenine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD +) and flavin adenine dinucleotide (FAD +). The competition between adenine and DAP may occur either in the formation of the nucleotides or in their function. Wheeler and Bowden (1966) have demonstrated the formation of an analogue of NAD + in which the adenine moiety was replaced by DAP when cultured mammalian cells were grown in the presence of DAP. Diaminopurine ribonucleoside-5'-triphosphate has been isolated and identified from stored human erythrocytes incubated with the analogue (Tatibana and Yoshikawa, 1962; Blair and Dommasch, 1969). Korn-

0026-8925/79/0173/0307/$01.40

308

c. Wallis and D. Wilkie : Mitochondrial Activity of 2,6-Diaminopurine in S. cerevisiae

b e r g a n d P r i c e r (1951) d e m o n s t r a t e d t h a t D A P r i b o side m a y b e e n z y m a t i c a l l y p h o s p h o r y l a t e d t o a D A P analogue of ATP but this analogue was able to replace ATP in the hexokinase reaction and also in the phosphorylation of DAP ribonucleoside. Diaminopurine a n d its r i b o n u c l e o s i d e , i n t h e p r e s e n c e o f t h e m e t h y l donor, S-adenosyl methionine, was found to inhibit E. coli e n z y m e s w h i c h m e t h y l a t e g u a n i n e i n t r a n s f e r R N A ( t R N A ) W a i n f a n a n d L a n d s b e r g , 1973). A deficiency of methylated bases in tRNA results in ambig u i t y in r e s p o n s e t o m e s s e n g e r R N A b u t it is n o t known whether this mechanism has a significant role in vivo. D A P h a s b e e n s h o w n t o b e m u t a g e n i c f o r b a c t e r i o p h a g e T4, g r o w i n g o n E. coli, a n d t h e ' m u t a bility spectrum' o f T~ r I I - t y p e p h a g e m u t a n t s , induced by DAP, has been studied in some detail b y F r e e s e ( 1 9 5 9 ) a n d B e n z e r (1961). H o w e v e r , s p e c i f i c mechanisms for the inhibitory activity of DAP have not yet been defined. T h e y e a s t , Saccharomyces cerevisiae, h a s p r o v e d a very useful tool in screening drugs for primary antim i t o c h o n d r i a l a c t i v i t y since, as a f a c u l t a t i v e a n a e r o b e , it c a n g r o w a n d d i v i d e in t h e a b s e n c e o f m i t o chondrial repiration provided fermentable substrate is a v a i l a b l e . H o w e v e r , u n d e r c o n d i t i o n s o f m i t o c h o n drial deficiency growth cannot proceed if the carbon s o u r c e is n o n - f e r m e n t a b l e (e.g. g l y c e r o l , p y r u v a t e ) . Preliminary work indicated anti-mitochondrial activity o f 2 , 6 - d i a m i n o p u r i n e i n t h e y e a s t cell ( L e e a n d W i l k i e , 1974). D e t a i l s a n d e x t e n s i o n o f t h e s e s t u d i e s a r e d e s c r i b e d in t h e p r e s e n t p a p e r .

Materials and Methods Organisms and Cultural Conditions. Haploid strains of Saccharomyces cerevisiae of this laboratory were used. All growth media, buffered with 50 mM Tris maleate, pH 6.5, contained Difco yeast extract (0.5%) supplemented with either 2% (w/v) glucose (YED) or 4% (v/v) glycerol (YEG) as carbon sources, solidified where required with 2% (w/v) Difco agar. Petite determining medium (PDM) contained 0.2% (w/v) glucose and 4% (v/v) glycerol. Petite colonies were distinguished from respiratory sufficient coionies on this medium by their smali size and white colour. Wickerham's minimal medium (MM) (Wickerham, 1946) was used with ammonium sulphate as nitrogen source and glucose (2% w/v) as carbon source. 2,6-diaminopurine (Sigma) was added directiy, up to maximum solubility (5 mg/ml), to the medium before antoclaving. Antibiotic media consisted of YEG with 2 mg/ml erythromycin (Abbot Laboratories Ltd.), 2 mg/ml chloramphenicol (Sigma) and 1 gg/ml oligomycin (Sigma). Tests of growth inhibition using a multiple inoculation device are described elsewhere (Wilkie, 1972). Standard methods of crossing, sporulation and tetrad analysis were used in genetic studies (Mortimer and Hawthorne, 1969).

Cytoehrome Absorption Spectra. Cells grown in liquid glucose medium were harvested at stationary phase, washed twice with distilled water, resuspended in distilled water (approximately 109

cells/ml) and the absorbance measured at room temperature in the Unicam SP 1800 recording spectrophotometer.

Measurement of Respiratory Activity. Oxygen uptake by both whole yeast cells and of isolated rat liver mitochondria was measured polarographically in a Clark-type oxygen electrode (Rank) coupled to a pen recorder. Yeast cells were suspended in the electrode chamber in 50 mM Hepes buffer, pH 6.8, and isolated mitochondria were treated as described by Chappell and Hansford (1972).

Mutagenesis by DAP. Cells, grown overnight on YED-agar plates, were first grown for 6 h in liquid YED-peptone (2% w/v) medium (initial concentration 1 × 1 0 6 cells/ml) and then for 18 h in liquid YEG medium containing 0,50 and 100 gg/ml DAP (initiai concentration 5 x 105 cells/ml) at 30 ° C on a shaker. Undiluted aliquots (0.1 ml per plate) of these cultures were then plated onto antibiotic containing y E G medium to select the resistant cells. Appropriately, diluted samples were also plated onto PDM for viability counts and petite estimation. Erythromycin, chloramphenicol and oligomycin resistant mutants were scored after 18 days' incubation at 30 °.

Rat Liver Mitochondria. [5,6-3H] uridine triphosphate as the ammonium salt of specific ativity 50 Ci/mMole was obtained from the Radiochemical Centre, Amersham; cold nucleotides as their trisodium salts were from Boehringer and all reagents were Analar grade. Mitochondria were prepared from the livers of young, 70 g Wistar rats (female) according to the method of Chappell and Hansford (1972). Throughout the preparation, which lasted about 90 rain, the liver homogenate and mitochondria were maintained at ice temperature and sterile solutions used in order to avoid bacterial contamination (Kroon et al., 1968) In the RNA polymerase assay, incubations were carried out at 30°C in conical ended 10 ml centrifuge tubes. The procedure was performed in a water bath with vigorous shaking and each incubation contained approximately 1.5 mg mitochondrial protein in a total vloume of 100 gl. Other components in the system were as follows: 53 mM Tris-hydrochloride pH 7.6; 0.3 mM each of ATP, CTP, GTP, UTP and 5 ~tCi [3H]UTP; 64 mM potassium chloride, 10 mM potassium succinale, 3 mM magnesium chloride. Incubation tubes were prepared in the cold and the assay was started by addition of the label and transfer to the shaker bath. Drugs were added as 10 Ixl aliquots from a stock solution at the start of the incubation. After incubation for 20 min at 30 ° C the reaction was stopped by the addition of 0.5 ml 0.i M sodium pyrophosphate and precipitated with 5 ml of ice-cold 5% trichloracetic acid (TCA). In estimating incorporated radioactivity, the acid-insoluble material was collected on Millipore filters (1.2 g pore size, 2.4 cm diameter) by suction and washed five times with 10 ml aliquots 5% TCA. The filters were dried at 60 ° C for 2 days, placed in a vial, immersed in 10 ml of scintillant (butyl-PBD (CIBA-Geigy), 6 g; toluene, 900 ml; methanol, 100 ml) and radioactivity counted with a Corumatic 2000 liquid scintillation spectrophotometer for 20 rain in the tritium channel at about 55% efficiency. Checks for bacterial contamination of the mitochondrial suspension were made by withdrawing samples from the assays at the beginning and end of the incubation period and plating suitably diluted suspensions on nutrient broth (Oxoid) agar. The plates were incubated for 48 h at 37 ° C before scoring. Protein estimates were made using the method of Lowry et al. (1951) using Fraction V bovine serum albumin (Sigma) as standard.

C. Wallis and D. Wilkie: Mitochondrial Activity of 2,6-Diaminopurine in S. cerevisiae

309 2,6-DAP (Nlml)

Results 5-

Growth Studies. Agar medium with glucose (YED) and glycerol (YEG) as respective carbon sources and containing DAP in concentrations ranging from 50 gg/ml to 5 mg/ml were prepared in 2 petri dish series. These were inoculated with 15 strains of S. cerevisiae and incubated. All strains tested were remarkably sensitive to DAP but oa~Jy in glycerol medium. Thirteen strains were inhibited and gave no indications of being able to grow at a drug concentration of 250 pg/ml in Y E G while the two remaining strains were inhibited at 500 gg/ml in YEG. In contrast, nearly all strains were able to maintain growth in YED at the maximum concentration of 5 mg/ml. Only two strains were inhibited in YED at concentrations of 3 and 2 mg/ml DAP respectively. The results obtained with the petri dish series were extended by measuring growth of some strains in liquid medium in the presence and absence of DAP. Typical growth curves of Y E G cultures of strain D6 are shown in Fig. 1 in which serious inhibition by 100 and 250 pg/ml DAP can be seen. In YED on the other hand, it was found tlqat at the highest concentration used (500 gg/ml), there was little effect on growth but that the final yield of cells was reduced by about 25% compared to control cultures as determined by dry weight measurements, haemocytometer counts and optical density readings. These results provided the first indication that the mitochondria were being selectively inhibited by DAP in the yeast cell. Effect of Adenine. The view that DAP was acting as an analogue of adenine was confirmed by showing that adenine, but not guanine, was able to reverse the inhibition of growth by DAP on Y E G medium. All strains inhibited by 250 pg/ml DAP showed some growth when 50 pg/ml adenine was added and showed full growth on addition of 100 pg/ml adenine to the Y E G medium containing 250 pg/ml DAP. In glucose medium containing 250 gg/ml DAP, similar additions of adenine had no effect on growth (which was already proceeding). It may be emphasised that yeast extract contains adenine and the amount in the medium used here has been estimated as 50 pg/ml approximately based on information supplied by the manufacturer. It is noteworthy that Johnson and Harris (1968) found as little as 0.1% yeast extract in the culture medium caused 95% reversal of growth inhibition of leptospires by DAP.

Cytochrome Spectra. Cytochrome aa3 and b, but not cytochrome c, failed to develop in cells growing in glucose medium in the presence of the analogue clearly indicating that DAP was acting to arrest

0 4-

c

3--

1-

50

0

-

I

10

0

I

I

z I

20

30

40

-"

100

25O I

50

Time ( h o u r s )

Fig. 1. Growth curves of strain D6 in non-fermentable medium containing 2,6-diaminopurine (DAP)

550 562

"l

% [AT

605

C

I

i

500 520 540

[ I 560 580 600 I

I 620

Wavelength ( nm}

Fig. 2. Absorption spectra of strain B41. a)YED culture; b) Y E D + 5 0 gg/ml D A P ; c) Y E D + 1 0 0 pg/ml DAP. Peaks at 605, 562 and 550 n m are c~peaks of cytochromes a+a3, b and e respectively while the /~ peaks of cytochromes b and c occur at 530 and 520 n m respectively; absorption at 585 n m represents a precursor of cytochrome a+a3

protein synthesis in mitochondria while other cellular biosynthetic processes were apparently unaffected. Cytochrome spectra of treated and untreated cells are shown in Fig. 2.

Respiratory Activity. The addition of DAP up to a concentration of 250 pg/ml to respiring cells of strain

310

C. Wallis and D. Wilkie: Mitochondrial Activity of 2,6-Diaminopurine in S. eerevisiae

Table 1. Effect of DAP on the frequencies of mutants resistant to chloramphenicol (cR); erythromycin (E a) and oligomycin (O R) in strains B41 and B/B in glycerol medium Treatment a

Number of resistant mutants per 107 cells B41

Control 50 p,g/ml DAP 100 gg/mi DAP

a

B/B

CR

Ea

O~

CR

ER

OR

21 (78/3.8 x 1 0 7 )

171 (563/3.3 x 1 0 7 )

21 (306/1.5 x 108)

77 (123/1.6 x 1 0 7 )

594 (95/1.6 × 1 0 6 )

6 (120/2.0 x l0 s)

144 (i 15/8.0 × 1 0 6 )

130 (259/2.0 × 1 0 7 )

129 (553/4.3 x 107)

330 (231/7.0 × 1 0 6 )

540 (232/4.3 × 1 0 6 )

34 (102/3.0 × 107)

25 (12/4.8 x 1 0 6 )

49 (166/3.4 x 107)

683 (198/2.9 x 1 0 6 )

467 (84/1.8 x 1 0 6 )

53 (148/2.8 x 107)

46 (24/5.2 x 106)

See Materials and Methods

B/B in the oxygen electrode had no effect on the uptake of oxygen by these cells. Oxygen uptake of isolated rat liver mitochondria was also found to be unaffected by added D A P up to 250 lag/ml. These results taken together indicate that the inhibitory effect of D A P in Y E G medium does not result from a direct effect on the function of mitochondria but rather from interference with their biogenesis, possibly at the level of transcription.

Induction of Antibiotic Resistant Mutants. The results obtained with T4 bacteriophage by Freese (1959) and Benzer (1961) lead one to expect that D A P might be mutagenic in the yeast cell. Mitochondrial mutation to antibiotic resistance is well documented in the literature (Wilkie, 1975) and in these present studies specific mitochondrial inhibitors, the antibiotics erythromycin, chloramphenicol and oligomycin, were used to test possible induction of resistance by DAP. Table 1 shows the results of induction tests in which cells of strains B41 and B/B were grown in Y E G medium containing sub-inhibitory concentrations of D A P (50 and 100 gg/ml). The data indicate that D A P is mutagenic in the induction of oligomycin and chloramphenicol resistant mutants, but not for erythromycin resistance mutations, in both strains. Indeed, D A P appears to depress the numbers of erythromycin resistant mutants in strain B41. These tests were repeated more than once (with the exclusion of screening for the induction of oligomycin resistance in B/B) and, although there was variation in the overall numbers of resistant mutants, the relative proportions a m o n g treated and untreated cultures were essentially similar. Finally, tests for induction of resistance to chloramphenicol and erythromycin by D A P in strains D6 and B/A were carried out and once again there was evidence for induction of chloramphe-

nicol resistance but not erythromycin resistance. Preliminary investigations showed that antibiotic resistance mutations were only induced when cells were grown in glycerol medium in the presence of the analogue. There was little, if any, induction of chloramphenicol resistant mutants by D A P with glucose as carbon source in strain B4a. At these concentrations of D A P (50 and 100 gg/ ml) there was no significant effect on nuclear mutations (reversion to prototrophy of his in strain B41 and arg in strain D6) nor on cell viability. A modification of the fluctuation test as described by Luria and Delbruck (1943) was carried out to test that D A P was actually inducing resistance mutations and not selecting pre-existing antibiotic resistant cells. Cells of strain B41 were grown under identical conditions as for the mutagenesis experiments. Rand o m samples from a ' bulk culture' treated with DAP, as expected, showed similar numbers of oligomycin resistant mutants (ratio of variance to mean: 1.06) whereas numbers of oligomycin resistant colonies arising from samples taken from individually DAPtreated cultures showed a wide fluctuation about the mean (ratio of variance to mean: 5.61). The evidence so far suggests that D A P is inducing antibiotic resistance in the mitochondrial genome and genetic analysis of a few DAP-induced chloramphenicol and oligomycin resistant mutants of strain B41 was carried out to test for cytoplasmic inheritance of drug resistance. Five chloramphenicol and four oligomycin resistant mutants when crossed to a sensitive strain showed segregation of resistant and sensitive diploid colonies which is a first indication of cytoplasmic control. Attempts to follow up this analysis were discontinued because of very low spore viability in tetrads and poor growth of viable spores on glycerol medium.

C. Wallis and D. Wilkie: Mitochondrial Activity of 2,6-Diaminopurine in S. cerevisiae Table 2. Effect of DAP treatment a on petite frequency in strains of D6, B41, B/A and B/B in glycerol medium Treatment

311

6× c ~

5

% petite D6

B41

B/A

B/B

0.27 (2/748)

2.03 3.54 (26/1280) (24/678)

50 gg/ml DAP

0.17 (2/i156)

4.55 14.17 0.24 (83/1822) (147/1038) (3/1258)

100 lag/ml DAP

0.22 (2/929)

3.87 4.21 (112/2893) (40/950)

£ 4 x=

Control

"

0.15 (2/1322)

0.76 (7/922)

See Materials and Methods

E

3

F 2

8 ~n_ 8 =

0

I

I

I

5

10

15

I

I

I

20 25 30

I

I

35 40

Incubation time ( minutes}

Induction of the Petite Mutation. Induction of the petite (p-) mutation by D A P in glycerol medium was investigated. Table 2 shows the petite frequencies in 4 strains grown under the same conditions as those known to induce antibiotic resistant mutants by D A P (50 and 100 lag/ml). These data indicate weak induction of petite in three of the strains tested. More extensive treatment with D A P was carried out with strain B ~ in YEG. Cells were exposed for 48 h to concentrations of 250 and 500 lag/ml. Cell viability of the treated cells was about 35% at each concentration used, while petite frequency was 33.1 and 42.3% respectively against a spontaneous frequency of 2.0% in controls. Thus D A P was petite inducing at comparatively high concentrations in Y E G medium. Although these concentrations of D A P effectively inhibited growth in Y E G medium, under the microscope micromanipulated cells were seen to form a few buds before growth was arrested. Petite mutants were relatively more frequent under conditions which had a pronounced lethal effect. In contrast, there was little induction of petite, and little or no loss in viability, when cells were grown in glucose medium containing 500 gg/ml DAP. The possibility that the p-cells originally present in the culture had a selective advantage over the p + cells with regard to the killing effect of 250 and 500 gg/ml D A P in glycerol medium was not substantiated since both spontaneous and 'DAP-induced petite strains derived from B¢1 were found to be more sensitive to the lethal effects of D A P than the parent strain. RNA Synthesis in Rat Liver Mitochondria. The evidence that D A P competes with adenine inhibiting the synthesis of cytochromes a+a3 and b in glucose medium but does not significantly induce the petite mutation suggests a primary inhibitory effect 9 n mitochondrial RNA, rather than D N A , synthesis. Thus,

Fig. 3. Time course of uptake of [ZH]UTP by isolated rat liver mitochondria. The proteins were incubated in the standard reaction mixture and the reaction stopped at the time indicated. Incorporation was measured as described in Materials and Methods in preliminary experiments, the effect of D A P on the incorporation of labelled R N A precursor by isolated rat liver mitochondria was studied. The time course of incorpoation of [3H]UTP into a rat liver mitochondrial preparation is given in Fig. 3. The reaction proceeded almost linearly for up to 20 min incubation but declined quite rapidly thereafter. The incorporation ofnucleotides was not dependent on the addition of an A T P generating system (4 m M phosphoenalpyruvate plus 40 lag/ml pyruvate kinase) if actively phosphorylating mitochondria were used and a respiratory substrate (10 m M potassium succinate) supplied. A possible source of R N A synthesis with isolated miochondria is contaminating bacteria. The isolated mitochondrial preparations contained a m a x i m u m of 900 colony-forming organisms per mg mitochondrial protein. This does not represent gross contamination and should not contribute significantly to R N A synthesis. It has been shown that mitochondrial preparations contaminated with up to 105 bacteria per mg mitochondrial protein were indistinguishable from sterile preparations in incorporation rates (Kroon et al., 1968). The incubation medium used here is not suitable for bacterial growth and there was little increase in bacterial number after 20 min incubation in the assay medium. The effect of acriflavine, a well-known inhibitor of R N A synthesis in mitochondrial systems (Fukuhara and Kujawa, 1970), on the incorporation after 20 min incubation is given in Table 3. Although l o w . (25 gg/ml acriflavine inhibited 23% of the incorporation) the inhibition of incorporation increased with increasing concentrations of the dye. Table 3 also shows the effect of DAP, at various concentrations, on the incorporation of radioactive

C. Wallis and D. Wilkie: Mitochondrial Activity of 2,6-Diaminopurine in S. cerevisiae

312

Table 3. Effect of acriflavine and 2,6-diaminopurine (DAP) on incorporation of [3H]UTP into rat liver mitochondria over a 20 rain incubation period Acriflavine (l,tg/ml)

c.p.m./mg mitochondria protein % inhibition

DAP (gg/ml)

0

10

15

20

25

0

100

150

200

250

524

436

440

415

402

442

382

350

272

192

0

12

I6

21

23

0

14

21

38

57

precursor. Clearly, DAP was inhibiting RNA synthesis in isolated rat liver mitochondria. Inhibition was about 50% at a concentration of 250 gg/ml DAP but the effects of higher concentrations could not be tested due to the relative insolubility of the analogue in the system. It was considered useful to study the effect of DAP on isolated animal mitochondria firstly because of the relative ease of isolation, secondly so that conclusions could be drawn as to the general nature of these effects of the analogue. It has been reported that in vitro synthesis of RNA and proteins by isolated organelles mimics with fidelity the processes occurring in vivo (Groot and Van Harten-Loosbroek, 1976).

Discussion Given the choice between DAP and adenine, the mitochondrial system is clearly much less able to discriminate against the analogue than is the nuclear system. Unlike amino acid analogues (Wilkie, 1970), mitochondrial sensitivity is general and not strain dependent. The findings suggest that DAP depresses the activity of R N A polymerases of the mitochondrion while having little or no effect on transcription in the nucleus, assuming that the differential effect is not due to selective restriction of entry of the analogue into the nucleus. If this is true, these findings emphasise the differences, already cited (Scragg, 1974), between RNA polymerases of organelle and nucleus and point to DAP as a useful tool in further investigations of the transcription process in mitochondria both in vitro and in vivo. In the latter context, Kobiler and Allweiss (1974) have shown that macromolecular RNA synthesis was inhibited in rats treated with DAP. DAP is also mutagenic in the mitochondrial system presumably as a consequence of its incorporation into DNA. Lesions tend to be point mutations to antibiotic resistance rather than the deletions that characterise the petite mutation. Point mutations would be expected if DAP is substituting for adenine in replicating DNA. Faye et al. (1974) concluded from

a study of the segment of mitochondrial D N A coding for chloraphenicol resistance that this segment had more A-T pairs than that coding for erythromycin resistance. The fact that resistance to chloramphenicol was more readily induced by DAP than erythromycin resistance would seem to fit these observations. The apparent selectivity of DAP indicates that this mutagen could be a useful adjunct to manganese which has been used with some success as a mitochondrial mutagen (Putrament et al., 1973), but with the advantages of little or no nuclear mutagenicity and low petite inducibility at effective concentrations. Why these mutagenic effects are seen only under conditions where mitochondria are fully functional, i.e. in glycerol medium, is not understood, particularly since the effects on mitochondrial protein synthesis are seen in glucose medium. A factor may be the lowering of up to 3-fold in the number of mitochondrial genomes in glucose-repressed cells (Goldthwaite et al., 1974) so that there would be fewer targets for induction of resistance compared with glycerol culture. These findings in themselves underline the mitochondrial activity of DAP since it would not be expected that the activity of a general mutagen (such as nitrosoguanidine) would be much influenced by the state of the mitochondria. Acknowledgements. These studies were supported by a Research Studentship of the University of London awarded to C.W. and by a grant to D.W. from the Cancer Research Campaign. The valuable technical assistance of Mrs. D. Collier is gratefully acknowledged.

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Communicated by F. Kaudewitz

Received February 12, 1979