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Biogenesis of mitochondria
J. Mol. Biol. (1973) 78, 335-350
Biogenesis of Mitochondria XXV.? Studies on the Mitochondrial Genomes of Petite Mutants of Yeast using Ethidium Bromide as a Probe PHILLIP
NAOLEY, ELLIOT B. GINGOLDS, H. B. LUKINS AND ANTHONY W. LINNANE
Department of Biochemistry, Mona.sh University Clayton, Victoria, 3168, Australia (Received 21 August 1972, and in revised form 24 April 1973) This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of fkccharomyees cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (p” cells). The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondriat DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones. It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (p-ER’) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two p-ERr clones, and in both the gene elimination rate is slower than in the parent p+ER’ strain, It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is t’he possible purification of erythromycin resistance genes.
1. Introduction A number of features of the mitochondrial genomes of cytoplasmically inherited respiratory deficient petite mutants of Saccharomyces cerevisiae have been recognized t Paper XXIV in this series is Dixon et al. (1972). $ Present address: Departrtment of Biochemistry, University 335
of Leicester,
Leicester,
England.
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ET AL.
and it may be said, in general, that petite mutations are essentially deletions of genetic information from the mtDNAt of the particular parent respiratory competent grande cells (for reviews see Linnane et al. (1972) and Nagley & Linnane (1973)). The concept of information loss in petite cells was initially recognized by identification of petite cells derived from a p+ERr strain as being p-ER’ (retaining the ERY resistance gene) or p-ER” (lacking this gene) (Gingold et al., 1969; Saunders et aZ., 1971; cf. also Rank, 1970; Bolotin et aZ., 1971). In different petite cells the possible changes in genetic information retained in the mtDNA are reflected by the wide range in physical properties which has been described for the mtDNA of p- cells (for reviews see Borst (1972) and Nagley & Linnane (1973)). These physical changes may be manifested as a reduction in the kinetic complexity of mtDNA judged by the rapid renaturation rates of mtDNA from some p- cells (Mehrotra & Mahler, 1968; Bernardi et al., 1970; Hollenberg et al., 1972a). In other cases a reduction in size of p- mtDNA compared with p+mtDNA has been reported (Billheimer & Avers, 1969 ; Goldring et al., 1971; Hollenberg et al., 19726). Additionally, the mtDNA of many p- cells shows an average base composition different from p+ mtDNA, the change being in the direction of increased adenine and thymine content (Mounolou et al., 1966; Mehrotra & Mahler, 1968 ; Bernardi et al., 1968 ; Carnevali el al., 1969 ; Bernardi et al., 1970). Two quite distinct classes of petite cells have recently been defined (Nagley & Linnane, 1972). One class, the suppressive petites (termed p-), retain mtDNA in cellular amounts approximately equal to that of the grande parent strain (Nagley & Linnane, 1972), but the mtDNA may be considerably different from that of the p+ parent. The second class of petite cells, termed p”, contain no mtDNA (Nagley & Linnane, 1970; Goldring et al., 1970), and all other indicators of mitochondrial genetic information are undetectable (Nagley $ Linnane, 1970). These cells are of zero suppressiveness (Nagley $ Linnane, 1970,1972; Michaelis et al., 1971), and represent the complete deletion of mitochondrial genetic information. Calculations made recently of the number of molecules of mtDNA per cell (Williamson, 1970; Nagley & Linnane, 1972) lead to the idea that the mitochondrial genome of a p+ haploid cell consists of about 50 individual molecules, assuming each is of molecular weight 50 x 10” (Hollenberg et al., 1970). In p- cells containing mtDNA of reduced size, but maintaining a total quantity of mtDNA similar to that of the grande parent, it is clear that there are many more than 50 molecules of mtDNA in such petite cells (Nagley & Linnane, 1972). The molecular population is thus of the order of hundreds of molecules, each molecule representing a fragment of the mtDNA of the p+ parent strain. The molecular population in different p- clones may constitute a heterogeneous array of a large spectrum of fragments, or in other cases, a relatively homogeneous population of a few specific base sequences. Thus, one might expect that in some stabilized p-ERr clones the fragments of mtDNA carrying the base sequence specifying resistance to erythromycin could be considerably amplified within the cells of a particular clone. One method of probing the structure of the mitochondrial genome in yeast is to use the specific mitochondrial mutagen EthBr. This agent has been used previously on p+ cells to induce petite mutants efficiently (Slonimski et al., 1968) and to cause elimination of ERY resistance genes from p+ER’ cells (Saunders et al., 1971; Nagley t Abbreviations used: mtDNA, mitochondrial DNA; ERY, erythromycin; bromide. Uses of the symbols p and ER are explained in the text.
EthBr,
ethidium
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& Linnane, 1972). EthBr inhibits the replication of p+ mtDNA and causes it to break into smaller molecules (Goldring et al., 1970,1971; Perlman & Mahler, 1971). In addition, EthBr treatment converts some of the p+ cells into p” cells (Nagley & Linnane, 1970,1972; Goldring et al., 1970; Michaelis et al., 1971; Hollenberg et al., 1972b). In this paper we report studies on the effect of EthBr on the mitochondrial genomes of a number of different p- clones, all of which are susceptible to mutagenesis by EthBr. EthBr action has the same effect on p- clones as in the p + parent : namely, inhibition of replication of mtDNA, and the production of p” cells. Examination of the genetic effects of EthBr action show firstly that when mtDNA is eliminated from p- cells, the over-all suppressiveness of the culture decreases, thus confirming the previously suggested relation between suppressiveness and mtDNA (Saunders et al., 1971; Nagley & Linnane, 1972; see also Discussion herein). Second, the quantitative effects of the elimination of ERY resistance genes from p-ER’ clones are consistent with an increase in the absolute number of copies of ERY resistance genes in such clones as compared with the p +ER’ parent.
2. Materials and Methods (a)Strainsand petite clones All clones are derived from S. cerev&ze strain L411 (a ura his ; p + ERP) (Linnane et al., 1968). K4.5 (p-ERo; 99o/o supp ressive) is a subclone of a spontaneously arising petite clone (K4) from L411; El7 (p-ER”; 20% suppressive) was derived by EthBr treatment of L411 (see Nagley & Linnane, 1972, for details). El82 (P-ERO; 70% suppressive) and El810 (P-ERO; 90% suppressive) are two subclones of E18, an EthBr-induced petite clone (Nagley & Linnane, 1972). K55 and K5272 were derived by subcloning a spontaneously arising petite clone (K5; p-ERr) from L411; this involved one subcloning step in the case of K66, and three steps in the case of K6272 (see Saunders et al., 1971, for details). K56 (p-ER’) showed 45% suppressiveness, and K5272 (p-ER’) showed 8% suppressiveness . (b) Growth media The standard liquid growth medium is a yeast extract/salts medium (Wallace et aE., 1968) supplemented with histidine (60 pg/ml) and uracil (25 pg/ml), and containing glucose (6%) as carbon source. Other media are as follows: medium I, Difco yeast extract (lx), Bactopeptone (2x), glucose (2%); medium II, Difco yeast extract (lo%), BactoDifco yeast nitrogen base (0*67%), peptone (2%), glycerol (2%): minimal-glucose, glucose (2%) ; minimal-petite, Difco yeast nitrogen base (O-67 %), glucose (O.lo/o), glycerol (4%). Erythromycin was included where required in minimal-petite medium or in medium II at 4 mg/ml (added after autoclaving). Solid media contained agar (2%). (c) Genetic tests (i) Determination
of auppressiveness
Suppressiveness determinations were carried out on cultures of petite cells by mating with strain L2200 (a ode lys trp; p+ERs) in liquid medium and analyzing the zygotes for the frequency of respiratory deficient zygotic clones (Ephrussi et al., 1955). The details of the procedure used are as follows. From overnight cultures (early stationary phase) of the petite and of strain L2200, samples each containing about l-5 mg dry weight of cells were mixed into 10 ml of liquid medium I and shaken at 28 to 3O*C for 4.5 h. A O-OS-ml portion of a 1 in 100 dilution (in water) of this mixture was spread on duplicate minimal-petite plates. This technique of prototrophic selection only allows diploid colonies to form. The colonies (about 200 to 300 per plate under these conditions) were
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scored after 4 to 5 days growth as microcolonies (< 0.5 mm diameter), or large colonies (> 2 mm in diameter). Experiments using the tetrazolium overlay technique (Ogur et al., 1957) confirmed that microcolonies were respiratory deficient and that large colonies were composed of respiratory competent cells. Size was taken as the sole criterion of colo~ly type in the experiments described herein. Grande strains continuously and spontaneously give rise to some petite clones, and it is necessary to correct estimations of suppressiveness for this background of spontaneously arising petites. This correction was made by measuring the frequency of petite zygotic clones when a reference petite strain of zero suppressiveness (E5) derived from strain L411 (Nagley & Linnane, 1970) was crossed with strain L2200 each time a suppressiveness measurement was made on some other clones. This correction throughout the series of experiments fell in the range 1 to 5%, on different occasions. Details of the calculations are as follows. If the minimal-petite plates from the test cross yield M microcolonies and L large colonies, and the reference E5 cross yields m microcolonics and 1 large colonies, the corrected suppressiveness of the test cross (expressed as a percentage) is given by
100 X
M --L + M 1-z
m lfm 1 + T?b
(ii) Measurement of the level of erythromycin resistance genes in petite cultures It is not possible to detect directly ERY resistance genes in petite cells since they are unable to respire under any conditions, hence a genetic test for the presence of such genes in petite cells must be carried out. The petite culture was mated with strain I,2200 (p+ERs) and the frequency of p+ER’ zygotic clones was measured after plating the mixture onto both minimal-petite and minimal-petite-ERY medium. Plates were scored for the number of large colonies and the number of microcolonies. From the minimalpetite plates, the frequency of p + zygotes is obtained, and from the minimal-petite-ERY plates the frequency of p+ERP zygotes is determined. The level of ERY resistance genes in the original haploid p- culture is then calculated by dividing the frequency of p+ER’ zygotes by the frequency of p+ zygotes, and this parameter is usually expressed as a percentage. (d) Extraction
and analysis of cellular
DXA
The cellular DNA from lo-ml cultures grown to a density of 5 mg dry weight/ml at early stationary phase was extracted and analysed in analytical CsCl gradients for the relative proportions of nuclear and mtDNA exactly as described by Nagley & Linnane (1972).
3. Results (a) Studies on changes in suppressiveness
induced by ethidium
bromide
(i) Studies on clone K45 A maximally suppressive petite clone (K45, 990/ suppressive) was chosen for initial study. After growth of this clone in the presence of EthBr (10 pg/ml) for 20 generations the suppressiveness of the cultures did not change. Furthermore, no effect of EthBr on the cellular level of mtDNA in K45 was observed under these conditions. The growth of K45 for eight generations in the presence of EthBr at concentrations considerably greater than 10 pg/ml leads to a reduction in the suppressiveness of the culture (Fig. l(a)). When EthBr is present at 50 pg/ml a value of only 10 to 15% in suppressiveness is exhibited by the culture. These results can be contrasted with the behaviour of the parent strain L411 growing in the presence of EthBr (10 pg/ml),
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fb
FIG. 1. Effect of EthBr on the suppressiveness and mtDNA replication of clone K46. Cult,ures of K46 were grown through 8 generations (18 h) in the presence of various concentrations of EthBr, and washed portions of the cultures were either (a) crossed with strain L2200 for determination of the suppressiveness of (b) used for extraction of total cellular DNA. On the ordinate of (b) the level of mtDNA is expressed as percentage of the oellular love1 of &DNA present in untreated cells of K46 (namely, 17% of total DNA). The greater the degree of inhibition or mtDNA replication by EthBr, the smaller is the cellular level of mtDNA after treatment; that is, mtDNA becomes diluted from the culture relative to nuclear DNA. Nuclear DNA can be considered as un&ected by EthBr under these experimental conditions, since all cells underwent the same number of divisions. Hence the same number of rounds of nuclear DNA replication had occurred in every culture.
where after one to two generations extensive disruption of the mitochondrial genet’ic system occurs together with severe inhibition of the replication of mtDNA (Nagley & Linnane, 1972; cf. also Goldring et al., 1970; Perlman & Mahler, 1971). It was also observed that at concentrations of EthBr above 10 pg/ml the cellular level of mtDNA in the culture of K45 declined (Fig. l(b)), indicating an inhibition of mtDNA replication. At 50 pg EthBr/ml the cellular level of mtDNA in the culture is close to the limits of detection. The similarity between the sets of data obtained for elimination by EthBr from K45 of suppressiveness and of mtDNA (Fig. 1) strongly suggests that there is a close relationship between these two parameters. More information on the effect of EthBr on clone K45 is obtained by examining t’he properties of individual subclones induced by EthBr, both immediately and aft,er further growth in the absence of EthBr. Cells of K45 were grown for eight generations in the presence of EthBr (35 pg/ml), and then were washed twice in water (culture A). A portion of this culture was allowed to grow for eight generations in growth medium minus EthBr to give culture B, a portion of which was inoculated into further growth medium (no EthBr) and grown for another eight generations to give culture C. The suppressiveness of cultures A, B and C was 21, 18 and 20%, respectively. The EthBr treatment thus reduced t,he suppressiveness of K45 from 99% to about 20% and this remained essentially unchanged during subsequent growth in the absence of EthBr for at least 16 generations. The composition of these cultures was obtained by measuring the suppressiveness of individual clones isolated from cultures A, B and C. Data for 20 clones selected at random from each of the three cultures are shown in Figure 2. It is evident that two main classes of cells ultimately result from EthBr treatment of K45, namely petites of very high suppressiveness similar in this property to K45, and petites of very low
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IL)
s 6 4 2 0L 0
20
.---
i ;o+*o
Suppresslveness (%)
100
FIG. 2. Analysis of the population of cells resulting from EthBr treatment of clone K46. Portions of cultures A, B and C (see text) were plated onto medium I, to allow isolation of single colonies. 20 oolonies from each culture were selected at random and tested for suppressiveness by crossing individually with strain L2200. (a) Clones from culture A; that is, clones taken immediately after EthBr treatment of K46 (36 pg EthBr/ml for 8 generations). (b) Clones from culture B; that is, clones isolated after 8 generations of cellular growth subsequent to EthBr treatment. (c) Clones from oulture C; that is, clones isolated after 16 generations of cellular growth subsequent to EthBr treatment.
and zero suppressiveness. The petite cells of low suppressiveness (10 to 20%) induced under these conditions, and apparent in culture A (Fig. 4(a)) are unstable and are no longer found after further growth of the EthBr-treated culture in EthBr-free growth medium. We have previously reported that a number of zero suppressive petite clones lack mtDNA (Nagley & Linnane, 1970,1972; cf. Michaelis et al., 1971) and that p- clones purified to eliminate cells of zero suppressiveness contain a relatively constant cellular level of mtDNA approximating that of the parent p+ strain (Nagley 6 Linnane, 1972). In p- clones from strain L411 considered here, this level is about 16% of total cellular DNA. Herein we observe a large reduction in the over-all cellular level of mtDNA in the culture of K45 treated with EthBr at 35 pg/ml (Fig. l(b)), namely to about 3% of total yeast DNA (that is, one-fifth of the initial level of mtDNA). Moreover, large numbers of petite cells of zero suppressiveness are induced under these conditions. The mtDNA level of 3% of total DNA is the average of the mtDNA levels of the cells of the culture, which is thus composed of zero suppressive petite cells lacking mtDNA (p”) and the suppressive cells which retain mtDNA. (ii) Studies on petite clones of a range of initial suppressiveness The results described above have shown that both suppressiveness and mtDNA in one petite clone are susceptible to EthBr. We have also carried out studies on
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petite clones of other suppressiveness values to test the generality of this response to EthBr. Cells of clones E17, K55, E18.2, E18.10 (and K45 for comparison) were grown for eight generations in the presence of various concentrations of EthBr. The suppressive-
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20
30
40
50
[EthBr](pg/ml)
FIQ. 3. Reduction of suppressiveness induoed by EthBr in clones of a range of initial suppressiveness values. Cultures of each clone were grown for 8 generations (18 h) in the presence of various oonoentrations of EthBr and washed samples were crossed with strain L2200 for measurement of suppressiveness. -E-W--, El7 (20%); --n--A--, KS6 (46%); -O-o---, El32 (70%); -A-A-, El8.10 (90%); -.-a---, K46 (99%). The numbers in parentheses are the suppressiveness values exhibited by the untreated clones.
ness of the treated cultures was determined and the data obtained (Fig. 3) show that the suppressiveness in all clones tested is susceptible to reduction by EthBrt. This process generally takes place at lower EthBr concentrations than required for a comparable degree of elimination from K45 (Fig. 3), except in the 90% suppressive clone El&O, which is similar to K45. t The data shown in Fig. 3, and in experiments of similar design where the extents of elimination of various parameters as a function of EthBr concentration are determined (Figs 1, 4 and 6) are difficult to reproduce precisely in quantitative terms. Qualitatively the results are highly reproduoible; the relative response of alones tested is always maintained. The results presented are those of single typical experiments in which the data shown were obtained from all the olones in any one Figure within a single experiment. The uncertainties in measurements of individual parameters are as follows: suppressiveness, &2% (p- zygotic olones); oellular level of mtDNA, + 1% (of total yeast DNA); level of ERY resistance genes, &2% (p+ER’ zygotia clones/total p + zygotic clones).
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The dichotomy between petites of very high suppressiveness and pebites of lower suppressiveness as regards reduction of suppressiveness of EthBr is reflected in the extent of inhibition of mtDNA synthesis by EthBr at different concentrations in clones of different initial suppressiveness (Fig. 4). Inhibition of mtDNA synthesis in clones K5272 (8% suppressive) and K55 (45% suppressive) takes place at lower (but’ similar) concentrations of EthBr t,han does inhibition of synthesis of mtDNA in K45. These DNA inhibition studies were carried out under the same growth conditions as
100
00
20
0
5
IO [Ether]
15
20
Cpg/ml)
FIG. 4. Inhibition by EthBr of replication of mtDNA in different clones. Cultures of each clone were grown for 8 generations in the presence of various concentrations of EthBr, and analysed for the percentage of total cellular DNA present as mtDNA. Other details of this type of measurement are given in the legend to Fig. 1. The initial cellular levels of mtDNA in the clones were: L411, 18% of total DNA; K6272, 16%; KSS, 12%; K46, 17%. -O-O--, L411; --A--A-, K.5272; -A-A-, KS; -.-a-, K4B.
the suppressiveness reduction experiments, and it can be seen comparing Figures 3 and 4 that the data for the two classes of suppressive petites are displaced along the abscissa (EthBr concentration) to roughly similar extents in both cases. These results establish that suppressiveness in general is closely associated with mtDNA in petite clones. It will be shown in a later part of this paper that EthBr acts on clone K5272 (8% suppressive) to yield large numbers of p” cells ; data on the reduction of suppressiveness in clone K5272 at different EthBr concentrations are not shown in Figure 3 because the low initial suppressiveness precludes precise measurements of changes in this parameter. It is to be observed in Figure 4 that mtDNA replication in strain L411 is even more susceptible to EthBr inhibition than its derived p- clones. The reasons for these differences between the p+ and p- cells, as well as between various p- cells, are not yet understood.
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(b) Studies cm clones carrying erythromyein resistance genes The preceding experiments have centred around the reduction in suppressiveness by EthBr in p- cells, together with the inhibition of mtDNA synthesis by EthBr. An alternative approach to obtaining information about the mitochondrial genomes of p- yeast cells is to follow the fate of specific base sequences in mtDNA as marked by known genes, such as the ERY resistance genes in p-ER’ cells. (i)
Elimination
of erythromycin resistance genes by ethidium bromide
The elimination by EthBr of ERY resistance genes from the p-ER’ clones K5272 and K55, as well as from the parent p+ERP strain L411, is shown in Figure 5. For
[EthErI
(pg/ml)
FIG. 6. Elimination of ERY resistanoe genes in clones K6272, K66 and Strain L411. Cultures of eech clone were grown for 8 generations (18 h) in the presenceof vsrious concentretiona of EthBr, then washed twioe in sterile water and crossed with L2200 to measure the level of ERY resistance genes. Initial levels of ERY resistance for K6272, KS6 and L411 were measured at SSY’, S&J%, 86%, respectively. -A--A-, K6272; -A--A---, K66; --O-O--, L411.
these experiments cells were grown through eight generations in the presence of various concentrations of EthBr. It is seen from Figure 5 that in all casea the ERY resistance genes can be almost completely eliminated by EthBr, but the sensitivity of these genes at a given EthBr concentration varies considerably amongst the three cell types tested. The p- clones are less sensitive than the p+ strain, but a variation in the sensitivities of p- clones also exists; K5272 is far less sensitive than K56 as regards elimination of ERY resistance genes by EthBr. In order to interpret these differenoes in sensitivities of ERY resistance genes towards EthBr elimination, a study was carried out on the process of elimination of these genes from p-ERr cells, using clone K5272. 3
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K5272 was grown for ten generations in the presence of EthBr (20 pg/ml), washed in water, and grown for a further 20 generations in EthBr-free medium. The level of ERY resistance genes retained in the culture was monitored during the experiment, and the results are presented in Figure 6. It is evident that during the EthBr treatment substantial elimination of ERY resistance genes occurs, to a residual level of about 10% of the initial value. Subsequent growth in the absence of EthBr leads to a very small change in the level of ERY
resistance
genes in the culture.
The same be-
haviour is manifested by L411 treated with Et,hBr under the same conditions (Fig. 6)
+ EthBr -10
0 Number
I IO
I 20
-I
of generations
Fro. 6. Pattern of ohanges in the level of ERY resistance genes in clone K6272 and strain L411 during recovery from EthBr treatment. Cells of each clone were grown for 10 generations (24 h) in the presence of EthBr (20 pg/ml), washed twioe in sterile water, and reinoouleted into fresh growth medium without EthBr, at low cell density. One further sub-culture W&B made after 10 generations growth to yield oells grown 20 generations subsequent to EthBr treatment. The level of ERY resistance genes W&B determined at different stages of the experiment by withdrawing samples and orossing with L2200. Note the logarithmic scale on t,he ordinate. (A) K6272; (0) L411.
although the L411 ERY resistance genes are apparently more than two orders of magnitude more sensitive than those of K5272. Individual clones resulting from EthBr treatment of K5272 were analysed in order first to investigate whether the residual resistance genes were distributed equally among the population, or alternatively, if they were concentrated in a small proportion of stable p-ERr cells. A culture of Kb272 was grown 10 generations in the presence of EthBr (20 pg/ml), and after a further 20 generations growth in the absence of EthBr, it was found to retain a level of 5% ERY resistance genes, and was of 5% suppressiveness. Samples of this tial culture were diluted and plated on culture medium I to allow growth of single colonies, and three plates containing a total of 197 colonies were used. A replica plating crossing technique was used to identify colonies containing p-ER’ cells (see legend to Table 1). The resulta obtained aa shown in Table 1 were that only 15 of the 197 colonies contain P-ER’ cells (that is, 7.5% of the population). As summarized in Table 1, 13 of these 15 clones are almost pure for p-ERr cells, whilst only two are clearly mixed for p-ERr and ER” cells. These results show that extensive (but not quite complete) stabilization of cells with respect to ERY resis-
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TABLE 1 Analysis of the pop&&on
Colony type
P-J=’ (PuM
p-ER’, p -ER”, pa (mixed)? p-ER”, p0(mixed)$ P0 (pure)S
of cells derived from clone K527.2 treated with ethidium bromide Number
of colonies 13
($2, (24/32)
yo of total colonies 6.6 1.0 23.1 69.3
K6272 was grown for 10 generations (24 h) in the presence of EthBr (20 pgjml), then allowetl to grow for a further 20 generations in the absenoe of EthBr. Samples of this final culture were plated onto medium I, and 197 colonies from 3 seperate plates were analysed by a replica plating crossing technique BFI follows. Each plate was replioated by the velveteen pad technique onto minimal-glucose plates upon whioh a lawn of strain L2200 (10s cells) had been spread. The diploitl colonies which grew up were in turn replica plated onto a medium II-erythromycin plate. Colonies growing up on this latter plate represented oolonies containing p-ER’ cells from the initial medium I master plete. Control medium II replicas of the minimal gluaose plates served to show that each of the initial petite colonies gave one corresponding diploid colony containing sufficient p+ cells to grow on medium II. t To classify further colonies containing p-ERr cells, each of these was mated individually with L2200 to measure the level of ERY resistanoe genes present. Colonies yielding >SO% p+ zygotes of the type p + ERr are classified aa pure p - ER’. Colonies yielding < 80% of p + zygotes of the type p+ERr are classified mixed for p-ER’ and ERO oells. (The two mixed clones identified in this way both yielded about 36 y. p + ER’ zygotes.) $ To determine the frequency of p” colonies amongst the colonies classified 89 ER”, 32 colonies selected at random were tested individually for their suppressiveness by crossing with L2200 (see Fig. 7(a)). Colonies exhibiting <2% suppressiveness were classified as p”.
t’ance genes did occur during the 20 generations growth after EthBr treatment. This finding permits the properties of p-ERr cells and ER” cells to be examined separately. The suppressiveness of the individual clones in the population of EthBr-treated cells of K5272 waa determined, since this data would yield information concerning the nature of mtDNA in the clone. In particular, a change in suppressiveness from a mother clone to a daughter clone would indicate that a change in mtDNA has occurred (cf. Saunders et al., 1971) and second, zero suppressiveness is highly indicative of a loss of mtDNA (Nagley & Linnane, 1972; Michaelis et aZ., 1971). Thirty-two clones classified as ER” by the replica plating technique (Table 1) were selected at random from the medium I plates and tested individually for their suppressiveness. The results are shown in Figure 7(a) and it can be seen that 24 of the ER” clones are zero suppressive petites (p”), the remainder showing very low suppressiveness values. Thus, since ER” cells account for about 90% of the population (Table l), then it may be concluded that about 70% (24 out of 32 tested) of this population is of the type pot probably lacking mtDNA. This correlates with the loss of mtDNA from cultures of K5272 treated with EthBr (Fig. 4). By contrast the suppressiveness distribution of the 13 pure p-ER’ clones, as is shown in Figure 7(b), reveals that such clones fall into the low to medium range of suppressiveness (from 5 to 66%) and there are no clones of zero suppressiveness. Most clones show the same level of suppressiveness as the parent clone K5272 (namely 5 to 8% suppressive), but a number of large changes have also occurred.
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Ill I I 20 30 40 Suppressiveness ( % I
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FIQ. 7. Analysis of the suppressiveness of the population of cella derived from clone K6272 treated with EthBr. K6272 was grown for 10 generations (24 h) in the presenoe of EthBr (20 &ml) and then grown for a further 20 generations in EthBr-free medium, and single colonies obtained from this oulture were olaeeified aa p-ER’, or ER” aa described in Table 1. Selected colonies were tested for suppressiveneas by crossing with L2200 individually. (a) Data for 32 clones selected at random from the 182 classified as ER”; (b) data for the 13 clones classified as containing only p-ER’ cells.
These results indicate that the action of EthBr on the mitochondrid genome of clone K5272 leads to the fragmentation of its mtDNA. This conclusion can be drawn because Srstly, many p-ER” cells have been induced and are present in the culture; these are suppressive cells which retain mtDNA but from which have been deleted resistance. Second, a number of p-ER’ sequences determining erythromycin clones are found which have large alterations in their suppressiveness, indicating changes in mtDNA other than in the ERY resistance gene region. These interpretations taken together with the direct observations of inhibition of mtDNA synthesis by EthBr in p-ER’ cells (Fig. 4) lead us to conclude that the mechanism of elimination of mitochondrial genes ad mtDNA from p- cells occurs by the same mechanism as from p+ cells. (ii) Quantitative aqects of elimination of eythromycin resistance genesand mitodwdial DNA by ethidium bromide This work has demonstrated differences in the sensitivities of ERY resistance genes towards EthBr in p+ER’ ad p-ERr clones (Figs 5 and 6). It was considered that if there were a greater absolute number of copies of ERY resistance genes in cells of clone K5272 as compared with K55, and with I.&l, then elimination of these genes by EthBr would be less efficient in the case of K5272, as has been observed. To clarify this argument by means of a specific example consider a particular grade clone (say
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L411) containing 60 copies of the ERY resistance gene per cell, assuming each of the 50 molecules of mtRNA (see Williamson, 1970; Nagley $ Linnane, 1972) to carry this gene. Let us consider further a stabilized p-ER’ clone derived from L411 containing mtDNA of one tenth the complexity of p+ mtDNA, with each sequence including an ERY resistance gene. Since the stable petite cells contain the same total cellular amount of mtDNA (Nagley t Linnane, 1972) it would then follow that 500 copies of the ERY resistance gene are present in each such petite cell. On treatment of the grande and such a petite with EthBr, the probability of elimination of ERY resistance genes is clearly much greater for the grande than for the petite. This interpretation depends on the differences in the susceptibility of mtDNA towards damage caused by EthBr. The extent of inhibition of mtDNA synthesis (Fig. 4) may be used as an indicator of the susceptibility of mtDNA to EthBr action. To relate the elimination of ERY resistance genes to the susceptibility of mtDNA in clones K5272, K55 and L411, we have taken the data in Figures 4 and 5 and calculated, for each clone at each EthBr concentration, the expression residual ERY resistance genes (“/ initial level) residual mtDNA (% initial level)
5
IO
15
20
[ElhBr] (pg/ml)
FIG. 8. Normalization of the date for elimination of ERY resistance genes with respect to the susceptibility of mtDNA to EthBr in clones K6272, K66 end strain L411. Data from Figures 4 and 5 were taken and for each concentration of EthBr the function residual level of ERY resistence genes ( y0 initial level)/residual was calculated, and plotted aa the ordinate in this Figure. ---n---n---, K6272; --A--A--, K66; -O-O-, L411.
level of mtDNA
( y0 initial
level)
348
P. NAGLEY
This expression is plotted as results show clearly that even the sensitivities to elimination order K5272 < K55 < L4lI. K5272 contains more copies of than L411.
ET
AL.
a function of EthBr concentration in Figure 8. The allowing for the different mtDNA responses to EthBr, by EthBr of ERY resistance genes is clearly in the We therefore interpret these data to indicate that ERY resistance genes than K55, and K55 in turn more
4. Discussion (a) Relationship between supprwsiveness and mtDNA The results obtained in this work concerning the simultaneous reduction in suppressiveness and loss of mtDNA from p- cells treated with EthBr reinforces previous proposals that suppressiveness is determined by the mtDNA of p- cells. Sherman & Ephrussi (1962) showed that suppressiveness was associated only with cytoplasmically inherited petite strains, and not with nuclear determined respiratory deficient mutants in which mtDNA is not changed. More recently it was shown that only petites containing mtDNA exhibit suppressiveness, whereas cells that lack mtDNA do not show suppressiveness (Nagley & Linnane, 1970,1972; Michaelis et al., 1971). Third, Saunders et al. (1971) demonstrated that when a p-E& cell spontaneously lost the base sequence specifying ERY resistance to become p-ERO, a change in suppressiveness to a higher or lower value almost always resulted. These latter findings demonstrated that the particular base sequences retained in the mtDNA of a p- cell play a role in determining the suppressiveness of that cell. So far no evidence has been obtained which links a particular trend in the physical properties of mtDNA (for example base composition) in p- clones to the suppressiveness of such clones (compare data of Mehrotra & Mahler (1968), Bernardi et aZ. (1970), Michaelis et al. (1971), Nagley & Linnane (1972) and data collated by Nagley & Linnane (1973)). The foregoing discussion has emphasized the cytoplasmic nature of suppressiveness and the possible relationships with p- mtDNA. However, many aspects of the replication and maintenance of mtDNA depend on nuclear function. It is well established that the rate of spontaneous mutation of p + to p - depends upon certain nuclear genes (see Ephrussi, 1953; Sherman, 1963). It follows that when a cross is made between a p- and a p+ strain the frequency of petite zygotic clones will depend not only upon “suppressive” interactions stemming from the p- mtDNA, but also upon interactions of nuclear genes in the zygotes which have been generated by combination of the two sets of nuclear genes from the haploids. In crosses using petite cells of very low or zero suppressiveness the nuclear regulation of the frequency of petite zygotes assumes significant importance. For this reason it is very important when measuring the suppressiveness of a petite clone derived from a given p+ strain, say strain A, by crossing with a standard p+ strain B, to determine simultaneously the frequency of petite zygotic clones obtained when a p” clone derived from strain A is crossed with strain B (p’). In order to obtain a reliable measurement of the suppressiveness of the tested petite clone, the frequency of petite zygotic clones must be corrected for that frequency obtained in the cross using the p” cells, as is described in Materials and Methods. This correction is preferable to that solely utilising measurements of the petite frequency in the p+ partner of the cross.
MITOCHONDRIAL
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MUTANTS
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(b) Concept of increased gene redundancy in p- cede The available evidence supports a general description of petite mitochondrial genomes consisting of some fragments of the grande mtDNA. Since the total amount of mtDNA in stable petite clones closely approximates that of the grande parent cells (Nagley t Linnane, 1972), then at least some sequences retained in the mitochondrial genomes of such petite cells must be present in higher multiplicity than in the grande cells. The validity of this generalization relies on the p- mtDNA not containing sequences which are not found in p + mtDNA. Evidence that this condition does hold for some p- cells has been obtained by Michaelis et al. (1972) who tested the ability of RNA transcribed in vitro from mtDNA of a grande strain and one particular p- clone to hybridize to both p+ and p- mtDNA. The results indicated that whilst the petite lacked 50 to 60% of the wild-type mitochondrial genome, no additional sequences differing from those in the p+ mtDNA could be detected in the p- mtDNA. The quantitative effects of EthBr action on p-ER’ cells are consistent with the notion that the cells studied contain a higher multiplicity of ERY resistance genes than in the parent p+ER’ strain. Our proposal of gene reiteration is in accord with the results of Cohen et al. (1972) who tested the mtDNA of a number of p- clones for the presence of genes coding for the mitochondrial leucyl-tRNA. It was found that in some clones this gene accounted for a substantially higher proportion of mtDNA than in the parent p+ clone. These findings have raised the possibility of biologically concentrating, and thus purifying, specific base sequences in mtDNA. REFERENCES Bernardi, G., Carnevali, F., Nicolaieff, A., Piperno, G. & Tecce, G. (1968). J. Mol. Biol. 37, 493. Bernardi, G., Fames, M., Piperno, G. & Slonimski, P. P. (1970). J. Mol. Biol. 48, 23. Billheimer, I?. E. & Avers, C. J. (1969). Proc. Nat. Acad. Scsci.,U.S.A. 64, 739. Bolotin, N., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E. & Slonimski, P. P. (1971). Bull. Inet. Pasteur, 69, 215. Borst, P. (1972). Annu. Rev. Biochem. 41, 333. Carnevali, F., Morpurgo, G. & Tecce, G. (1969). Science, 163, 1331. Cohen, M., Casey, J., Rabinowitz, M. & Getz, G. S. (1972). J. Mol. BioZ. 63, 441. Dixon, H., Kellerman, G. M. & Linnane, A. W. (1972). Arch. Biochem. Biophye. 152, 869. Ephrussi, B. (1953). Nu~Zeocy~~l~rn~c ReZations in Micro-organisms, Clarendon Press, Oxford. Ephrussi, B., Margerie-Hottinguer, H. & Roman, H. (1955). Proc. Nut. Acad. Sci., U.S.A. 41, 1065. Gingold, E. B., Saunders, G. W., Lukins, H. B. & Linnane, A. W. (1969). Genetics, 62,735. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R. & Marmur, J. (1970). J. Mol. BioZ. 52, 323.
Goldring, E. S., Grossman, L. I. & Marmur, J. (1971). J. Bacterial, 107, 377. Hollenberg, E. P., Borst, P. & Van Bruggen, E. F. J. (1970). Biochim. Biophys. Acta, 209, 1. Hollenberg, C. P., Borst, P., Flavell, R. A., Van Kreijl, C. F., Van Bruggen, E. F. J, & Arnberg, A. C. (1972a). Biochim. Biophys. Acta, 277, 44. Hollenberg, C. P., Borst, P. & Van Bruggen, E. F. J. (19723). Biochim. Biophye. Acta, 277, 35. Linnane, A. W., Saunders, G. W., Gingold, E. B. & Lukins, H. B. ( 1968). Proc. Nut. Acd Sci.,
U.S.A.
59, 903.
Linnane, A. W., Haslam, J. M., Lukins, H. B. & Nagley, P. (1972). Annu. 26, 163.
Rev. Microbial.
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ET AL.
Mehrotra, B. D. & Mahler, H. R. (1968). Arch. B&hem. Biophya. 128, 685. Michaelis, G., Douglass, S., Tsai, M. & Criddle, R. S. (1971). Biochem. Genet. 5, 487. Michaelis, G., Douglass, S., Tsai, M., Burchiel, K. & Criddle, R. S. (1972). Biochemistry, 11, 2026. Mounolou, J. C., Jakob, H. & Slonimski, P. P. (1966). B&hem. Biophya. Res. Commun. 24, 218. Nagley, P. & Lirmane, A. W. (1970). Biochem. Biophya. Res. Commun. 39, 989. Nagley, P. & Linnane, A. W. (1972). J. Mol. Biol. 66, 181. Nagley, P. & Linnane, A. W. (1973). In Advancee in Molec~Z~r Genetica (Falconer, I. R. & Cove, D., eds), Paul Elek Ltd, London, in the press. Ogur, M., St. John, R. & Nagai, S. (1967). Science, 125, 928. Perlman, P. S. t Mahler, H. R. (1971). Nuture New Biol. 231, 12. Rank, G. H. (1970). Can&. J. Genet. Cytol. 12, 129. Saunders, G. W., Gingold, E. B., Trembath, M. K., Lukins, H. B. & Linuane, A. W. (1971). In Autonomy and Bbgeneais of Mitochondriu and ChZoroplaeta (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds), p. 185, North Holland Publishing Co, Amsterdam. Sherman, F. (1963). Genetics, 48, 375. Sherman, F. & Ephrussi, B. (1962). Genetics, 47, 675. Slonimski, P. P., Perrodin, G. & Croft, J. H. (1968). B&hem. Biophys. Res. Commun. 30, 232. Wallace, P. G., Huang, M. & Linnane, A. W. (1968). J. Cell BioZ. 37, 207. Williamson, D. H. (1970). In Control of OrguneZZeDevelopment (Miller, P. L., ed.), p. 247, University Press, Cambridge. This work was supported (D65115930).
by a grant from the Australian
Research Grants Committee
Biogenesis of Mitochondria XXV.? Studies on the Mitochondrial Genomes of Petite Mutants of Yeast using Ethidium Bromide as a Probe PHILLIP
NAOLEY, ELLIOT B. GINGOLDS, H. B. LUKINS AND ANTHONY W. LINNANE
Department of Biochemistry, Mona.sh University Clayton, Victoria, 3168, Australia (Received 21 August 1972, and in revised form 24 April 1973) This paper describes investigations into the effects of ethidium bromide on the mitochondrial genomes of a number of different petite mutants derived from one respiratory competent strain of fkccharomyees cerevisiae. It is shown that the mutagenic effects of ethidium bromide on petite mutants occur by a similar mechanism to that previously reported for the action of this dye on grande cells. The consequences of ethidium bromide action in both cases are inhibition of the replication of mitochondrial DNA, fragmentation of pre-existing mitochondrial DNA, and the induction, often in high frequency, of cells devoid of mitochondrial genetic information (p” cells). The susceptibility of the mitochondrial genomes to these effects of ethidium bromide varies in the different clones studied. The inhibition of mitochondriat DNA replication requires higher concentrations of ethidium bromide in petite cells than in the parent grande strain. Furthermore, the susceptibility of mitochondrial DNA replication to inhibition by ethidium bromide varies in different petite clones. It is found that during ethidium bromide treatment of the suppressive petite clones, the over-all suppressiveness of the cultures is reduced in parallel with the reduction in the over-all cellular levels of mitochondrial DNA. Furthermore, ethidium bromide treatment of petite clones carrying mitochondrial erythromycin resistance genes (p-ER’) leads to the elimination of these genes from the cultures. The rates of elimination of these genes are different in two p-ERr clones, and in both the gene elimination rate is slower than in the parent p+ER’ strain, It is proposed that the rate of elimination of erythromycin resistance genes by ethidium bromide is related to the absolute number of copies of these genes in different cell types. In general, the more copies of the gene in the starting cells, the slower is the rate of elimination by ethidium bromide. These concepts lead us to suggest that petite mutants provide a system for the biological purification of particular regions of yeast mitochondrial DNA and of particular relevance is t’he possible purification of erythromycin resistance genes.
1. Introduction A number of features of the mitochondrial genomes of cytoplasmically inherited respiratory deficient petite mutants of Saccharomyces cerevisiae have been recognized t Paper XXIV in this series is Dixon et al. (1972). $ Present address: Departrtment of Biochemistry, University 335
of Leicester,
Leicester,
England.
336
P. NAGLEY
ET AL.
and it may be said, in general, that petite mutations are essentially deletions of genetic information from the mtDNAt of the particular parent respiratory competent grande cells (for reviews see Linnane et al. (1972) and Nagley & Linnane (1973)). The concept of information loss in petite cells was initially recognized by identification of petite cells derived from a p+ERr strain as being p-ER’ (retaining the ERY resistance gene) or p-ER” (lacking this gene) (Gingold et al., 1969; Saunders et aZ., 1971; cf. also Rank, 1970; Bolotin et aZ., 1971). In different petite cells the possible changes in genetic information retained in the mtDNA are reflected by the wide range in physical properties which has been described for the mtDNA of p- cells (for reviews see Borst (1972) and Nagley & Linnane (1973)). These physical changes may be manifested as a reduction in the kinetic complexity of mtDNA judged by the rapid renaturation rates of mtDNA from some p- cells (Mehrotra & Mahler, 1968; Bernardi et al., 1970; Hollenberg et al., 1972a). In other cases a reduction in size of p- mtDNA compared with p+mtDNA has been reported (Billheimer & Avers, 1969 ; Goldring et al., 1971; Hollenberg et al., 19726). Additionally, the mtDNA of many p- cells shows an average base composition different from p+ mtDNA, the change being in the direction of increased adenine and thymine content (Mounolou et al., 1966; Mehrotra & Mahler, 1968 ; Bernardi et al., 1968 ; Carnevali el al., 1969 ; Bernardi et al., 1970). Two quite distinct classes of petite cells have recently been defined (Nagley & Linnane, 1972). One class, the suppressive petites (termed p-), retain mtDNA in cellular amounts approximately equal to that of the grande parent strain (Nagley & Linnane, 1972), but the mtDNA may be considerably different from that of the p+ parent. The second class of petite cells, termed p”, contain no mtDNA (Nagley & Linnane, 1970; Goldring et al., 1970), and all other indicators of mitochondrial genetic information are undetectable (Nagley $ Linnane, 1970). These cells are of zero suppressiveness (Nagley $ Linnane, 1970,1972; Michaelis et al., 1971), and represent the complete deletion of mitochondrial genetic information. Calculations made recently of the number of molecules of mtDNA per cell (Williamson, 1970; Nagley & Linnane, 1972) lead to the idea that the mitochondrial genome of a p+ haploid cell consists of about 50 individual molecules, assuming each is of molecular weight 50 x 10” (Hollenberg et al., 1970). In p- cells containing mtDNA of reduced size, but maintaining a total quantity of mtDNA similar to that of the grande parent, it is clear that there are many more than 50 molecules of mtDNA in such petite cells (Nagley & Linnane, 1972). The molecular population is thus of the order of hundreds of molecules, each molecule representing a fragment of the mtDNA of the p+ parent strain. The molecular population in different p- clones may constitute a heterogeneous array of a large spectrum of fragments, or in other cases, a relatively homogeneous population of a few specific base sequences. Thus, one might expect that in some stabilized p-ERr clones the fragments of mtDNA carrying the base sequence specifying resistance to erythromycin could be considerably amplified within the cells of a particular clone. One method of probing the structure of the mitochondrial genome in yeast is to use the specific mitochondrial mutagen EthBr. This agent has been used previously on p+ cells to induce petite mutants efficiently (Slonimski et al., 1968) and to cause elimination of ERY resistance genes from p+ER’ cells (Saunders et al., 1971; Nagley t Abbreviations used: mtDNA, mitochondrial DNA; ERY, erythromycin; bromide. Uses of the symbols p and ER are explained in the text.
EthBr,
ethidium
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& Linnane, 1972). EthBr inhibits the replication of p+ mtDNA and causes it to break into smaller molecules (Goldring et al., 1970,1971; Perlman & Mahler, 1971). In addition, EthBr treatment converts some of the p+ cells into p” cells (Nagley & Linnane, 1970,1972; Goldring et al., 1970; Michaelis et al., 1971; Hollenberg et al., 1972b). In this paper we report studies on the effect of EthBr on the mitochondrial genomes of a number of different p- clones, all of which are susceptible to mutagenesis by EthBr. EthBr action has the same effect on p- clones as in the p + parent : namely, inhibition of replication of mtDNA, and the production of p” cells. Examination of the genetic effects of EthBr action show firstly that when mtDNA is eliminated from p- cells, the over-all suppressiveness of the culture decreases, thus confirming the previously suggested relation between suppressiveness and mtDNA (Saunders et al., 1971; Nagley & Linnane, 1972; see also Discussion herein). Second, the quantitative effects of the elimination of ERY resistance genes from p-ER’ clones are consistent with an increase in the absolute number of copies of ERY resistance genes in such clones as compared with the p +ER’ parent.
2. Materials and Methods (a)Strainsand petite clones All clones are derived from S. cerev&ze strain L411 (a ura his ; p + ERP) (Linnane et al., 1968). K4.5 (p-ERo; 99o/o supp ressive) is a subclone of a spontaneously arising petite clone (K4) from L411; El7 (p-ER”; 20% suppressive) was derived by EthBr treatment of L411 (see Nagley & Linnane, 1972, for details). El82 (P-ERO; 70% suppressive) and El810 (P-ERO; 90% suppressive) are two subclones of E18, an EthBr-induced petite clone (Nagley & Linnane, 1972). K55 and K5272 were derived by subcloning a spontaneously arising petite clone (K5; p-ERr) from L411; this involved one subcloning step in the case of K66, and three steps in the case of K6272 (see Saunders et al., 1971, for details). K56 (p-ER’) showed 45% suppressiveness, and K5272 (p-ER’) showed 8% suppressiveness . (b) Growth media The standard liquid growth medium is a yeast extract/salts medium (Wallace et aE., 1968) supplemented with histidine (60 pg/ml) and uracil (25 pg/ml), and containing glucose (6%) as carbon source. Other media are as follows: medium I, Difco yeast extract (lx), Bactopeptone (2x), glucose (2%); medium II, Difco yeast extract (lo%), BactoDifco yeast nitrogen base (0*67%), peptone (2%), glycerol (2%): minimal-glucose, glucose (2%) ; minimal-petite, Difco yeast nitrogen base (O-67 %), glucose (O.lo/o), glycerol (4%). Erythromycin was included where required in minimal-petite medium or in medium II at 4 mg/ml (added after autoclaving). Solid media contained agar (2%). (c) Genetic tests (i) Determination
of auppressiveness
Suppressiveness determinations were carried out on cultures of petite cells by mating with strain L2200 (a ode lys trp; p+ERs) in liquid medium and analyzing the zygotes for the frequency of respiratory deficient zygotic clones (Ephrussi et al., 1955). The details of the procedure used are as follows. From overnight cultures (early stationary phase) of the petite and of strain L2200, samples each containing about l-5 mg dry weight of cells were mixed into 10 ml of liquid medium I and shaken at 28 to 3O*C for 4.5 h. A O-OS-ml portion of a 1 in 100 dilution (in water) of this mixture was spread on duplicate minimal-petite plates. This technique of prototrophic selection only allows diploid colonies to form. The colonies (about 200 to 300 per plate under these conditions) were
338
P. NAGLEY
E2’ AZ/.
scored after 4 to 5 days growth as microcolonies (< 0.5 mm diameter), or large colonies (> 2 mm in diameter). Experiments using the tetrazolium overlay technique (Ogur et al., 1957) confirmed that microcolonies were respiratory deficient and that large colonies were composed of respiratory competent cells. Size was taken as the sole criterion of colo~ly type in the experiments described herein. Grande strains continuously and spontaneously give rise to some petite clones, and it is necessary to correct estimations of suppressiveness for this background of spontaneously arising petites. This correction was made by measuring the frequency of petite zygotic clones when a reference petite strain of zero suppressiveness (E5) derived from strain L411 (Nagley & Linnane, 1970) was crossed with strain L2200 each time a suppressiveness measurement was made on some other clones. This correction throughout the series of experiments fell in the range 1 to 5%, on different occasions. Details of the calculations are as follows. If the minimal-petite plates from the test cross yield M microcolonies and L large colonies, and the reference E5 cross yields m microcolonics and 1 large colonies, the corrected suppressiveness of the test cross (expressed as a percentage) is given by
100 X
M --L + M 1-z
m lfm 1 + T?b
(ii) Measurement of the level of erythromycin resistance genes in petite cultures It is not possible to detect directly ERY resistance genes in petite cells since they are unable to respire under any conditions, hence a genetic test for the presence of such genes in petite cells must be carried out. The petite culture was mated with strain I,2200 (p+ERs) and the frequency of p+ER’ zygotic clones was measured after plating the mixture onto both minimal-petite and minimal-petite-ERY medium. Plates were scored for the number of large colonies and the number of microcolonies. From the minimalpetite plates, the frequency of p + zygotes is obtained, and from the minimal-petite-ERY plates the frequency of p+ERP zygotes is determined. The level of ERY resistance genes in the original haploid p- culture is then calculated by dividing the frequency of p+ER’ zygotes by the frequency of p+ zygotes, and this parameter is usually expressed as a percentage. (d) Extraction
and analysis of cellular
DXA
The cellular DNA from lo-ml cultures grown to a density of 5 mg dry weight/ml at early stationary phase was extracted and analysed in analytical CsCl gradients for the relative proportions of nuclear and mtDNA exactly as described by Nagley & Linnane (1972).
3. Results (a) Studies on changes in suppressiveness
induced by ethidium
bromide
(i) Studies on clone K45 A maximally suppressive petite clone (K45, 990/ suppressive) was chosen for initial study. After growth of this clone in the presence of EthBr (10 pg/ml) for 20 generations the suppressiveness of the cultures did not change. Furthermore, no effect of EthBr on the cellular level of mtDNA in K45 was observed under these conditions. The growth of K45 for eight generations in the presence of EthBr at concentrations considerably greater than 10 pg/ml leads to a reduction in the suppressiveness of the culture (Fig. l(a)). When EthBr is present at 50 pg/ml a value of only 10 to 15% in suppressiveness is exhibited by the culture. These results can be contrasted with the behaviour of the parent strain L411 growing in the presence of EthBr (10 pg/ml),
MITOCHONDRIAL
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MUTANTS
33’3
fb
FIG. 1. Effect of EthBr on the suppressiveness and mtDNA replication of clone K46. Cult,ures of K46 were grown through 8 generations (18 h) in the presence of various concentrations of EthBr, and washed portions of the cultures were either (a) crossed with strain L2200 for determination of the suppressiveness of (b) used for extraction of total cellular DNA. On the ordinate of (b) the level of mtDNA is expressed as percentage of the oellular love1 of &DNA present in untreated cells of K46 (namely, 17% of total DNA). The greater the degree of inhibition or mtDNA replication by EthBr, the smaller is the cellular level of mtDNA after treatment; that is, mtDNA becomes diluted from the culture relative to nuclear DNA. Nuclear DNA can be considered as un&ected by EthBr under these experimental conditions, since all cells underwent the same number of divisions. Hence the same number of rounds of nuclear DNA replication had occurred in every culture.
where after one to two generations extensive disruption of the mitochondrial genet’ic system occurs together with severe inhibition of the replication of mtDNA (Nagley & Linnane, 1972; cf. also Goldring et al., 1970; Perlman & Mahler, 1971). It was also observed that at concentrations of EthBr above 10 pg/ml the cellular level of mtDNA in the culture of K45 declined (Fig. l(b)), indicating an inhibition of mtDNA replication. At 50 pg EthBr/ml the cellular level of mtDNA in the culture is close to the limits of detection. The similarity between the sets of data obtained for elimination by EthBr from K45 of suppressiveness and of mtDNA (Fig. 1) strongly suggests that there is a close relationship between these two parameters. More information on the effect of EthBr on clone K45 is obtained by examining t’he properties of individual subclones induced by EthBr, both immediately and aft,er further growth in the absence of EthBr. Cells of K45 were grown for eight generations in the presence of EthBr (35 pg/ml), and then were washed twice in water (culture A). A portion of this culture was allowed to grow for eight generations in growth medium minus EthBr to give culture B, a portion of which was inoculated into further growth medium (no EthBr) and grown for another eight generations to give culture C. The suppressiveness of cultures A, B and C was 21, 18 and 20%, respectively. The EthBr treatment thus reduced t,he suppressiveness of K45 from 99% to about 20% and this remained essentially unchanged during subsequent growth in the absence of EthBr for at least 16 generations. The composition of these cultures was obtained by measuring the suppressiveness of individual clones isolated from cultures A, B and C. Data for 20 clones selected at random from each of the three cultures are shown in Figure 2. It is evident that two main classes of cells ultimately result from EthBr treatment of K45, namely petites of very high suppressiveness similar in this property to K45, and petites of very low
340
P. NAGLEY
El’
AL.
IL)
s 6 4 2 0L 0
20
.---
i ;o+*o
Suppresslveness (%)
100
FIG. 2. Analysis of the population of cells resulting from EthBr treatment of clone K46. Portions of cultures A, B and C (see text) were plated onto medium I, to allow isolation of single colonies. 20 oolonies from each culture were selected at random and tested for suppressiveness by crossing individually with strain L2200. (a) Clones from culture A; that is, clones taken immediately after EthBr treatment of K46 (36 pg EthBr/ml for 8 generations). (b) Clones from culture B; that is, clones isolated after 8 generations of cellular growth subsequent to EthBr treatment. (c) Clones from oulture C; that is, clones isolated after 16 generations of cellular growth subsequent to EthBr treatment.
and zero suppressiveness. The petite cells of low suppressiveness (10 to 20%) induced under these conditions, and apparent in culture A (Fig. 4(a)) are unstable and are no longer found after further growth of the EthBr-treated culture in EthBr-free growth medium. We have previously reported that a number of zero suppressive petite clones lack mtDNA (Nagley & Linnane, 1970,1972; cf. Michaelis et al., 1971) and that p- clones purified to eliminate cells of zero suppressiveness contain a relatively constant cellular level of mtDNA approximating that of the parent p+ strain (Nagley 6 Linnane, 1972). In p- clones from strain L411 considered here, this level is about 16% of total cellular DNA. Herein we observe a large reduction in the over-all cellular level of mtDNA in the culture of K45 treated with EthBr at 35 pg/ml (Fig. l(b)), namely to about 3% of total yeast DNA (that is, one-fifth of the initial level of mtDNA). Moreover, large numbers of petite cells of zero suppressiveness are induced under these conditions. The mtDNA level of 3% of total DNA is the average of the mtDNA levels of the cells of the culture, which is thus composed of zero suppressive petite cells lacking mtDNA (p”) and the suppressive cells which retain mtDNA. (ii) Studies on petite clones of a range of initial suppressiveness The results described above have shown that both suppressiveness and mtDNA in one petite clone are susceptible to EthBr. We have also carried out studies on
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petite clones of other suppressiveness values to test the generality of this response to EthBr. Cells of clones E17, K55, E18.2, E18.10 (and K45 for comparison) were grown for eight generations in the presence of various concentrations of EthBr. The suppressive-
I
I
I
I
I
i0
20
30
40
50
[EthBr](pg/ml)
FIQ. 3. Reduction of suppressiveness induoed by EthBr in clones of a range of initial suppressiveness values. Cultures of each clone were grown for 8 generations (18 h) in the presence of various oonoentrations of EthBr and washed samples were crossed with strain L2200 for measurement of suppressiveness. -E-W--, El7 (20%); --n--A--, KS6 (46%); -O-o---, El32 (70%); -A-A-, El8.10 (90%); -.-a---, K46 (99%). The numbers in parentheses are the suppressiveness values exhibited by the untreated clones.
ness of the treated cultures was determined and the data obtained (Fig. 3) show that the suppressiveness in all clones tested is susceptible to reduction by EthBrt. This process generally takes place at lower EthBr concentrations than required for a comparable degree of elimination from K45 (Fig. 3), except in the 90% suppressive clone El&O, which is similar to K45. t The data shown in Fig. 3, and in experiments of similar design where the extents of elimination of various parameters as a function of EthBr concentration are determined (Figs 1, 4 and 6) are difficult to reproduce precisely in quantitative terms. Qualitatively the results are highly reproduoible; the relative response of alones tested is always maintained. The results presented are those of single typical experiments in which the data shown were obtained from all the olones in any one Figure within a single experiment. The uncertainties in measurements of individual parameters are as follows: suppressiveness, &2% (p- zygotic olones); oellular level of mtDNA, + 1% (of total yeast DNA); level of ERY resistance genes, &2% (p+ER’ zygotia clones/total p + zygotic clones).
342
ET AL.
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The dichotomy between petites of very high suppressiveness and pebites of lower suppressiveness as regards reduction of suppressiveness of EthBr is reflected in the extent of inhibition of mtDNA synthesis by EthBr at different concentrations in clones of different initial suppressiveness (Fig. 4). Inhibition of mtDNA synthesis in clones K5272 (8% suppressive) and K55 (45% suppressive) takes place at lower (but’ similar) concentrations of EthBr t,han does inhibition of synthesis of mtDNA in K45. These DNA inhibition studies were carried out under the same growth conditions as
100
00
20
0
5
IO [Ether]
15
20
Cpg/ml)
FIG. 4. Inhibition by EthBr of replication of mtDNA in different clones. Cultures of each clone were grown for 8 generations in the presence of various concentrations of EthBr, and analysed for the percentage of total cellular DNA present as mtDNA. Other details of this type of measurement are given in the legend to Fig. 1. The initial cellular levels of mtDNA in the clones were: L411, 18% of total DNA; K6272, 16%; KSS, 12%; K46, 17%. -O-O--, L411; --A--A-, K.5272; -A-A-, KS; -.-a-, K4B.
the suppressiveness reduction experiments, and it can be seen comparing Figures 3 and 4 that the data for the two classes of suppressive petites are displaced along the abscissa (EthBr concentration) to roughly similar extents in both cases. These results establish that suppressiveness in general is closely associated with mtDNA in petite clones. It will be shown in a later part of this paper that EthBr acts on clone K5272 (8% suppressive) to yield large numbers of p” cells ; data on the reduction of suppressiveness in clone K5272 at different EthBr concentrations are not shown in Figure 3 because the low initial suppressiveness precludes precise measurements of changes in this parameter. It is to be observed in Figure 4 that mtDNA replication in strain L411 is even more susceptible to EthBr inhibition than its derived p- clones. The reasons for these differences between the p+ and p- cells, as well as between various p- cells, are not yet understood.
MITOCHONDRIAL
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(b) Studies cm clones carrying erythromyein resistance genes The preceding experiments have centred around the reduction in suppressiveness by EthBr in p- cells, together with the inhibition of mtDNA synthesis by EthBr. An alternative approach to obtaining information about the mitochondrial genomes of p- yeast cells is to follow the fate of specific base sequences in mtDNA as marked by known genes, such as the ERY resistance genes in p-ER’ cells. (i)
Elimination
of erythromycin resistance genes by ethidium bromide
The elimination by EthBr of ERY resistance genes from the p-ER’ clones K5272 and K55, as well as from the parent p+ERP strain L411, is shown in Figure 5. For
[EthErI
(pg/ml)
FIG. 6. Elimination of ERY resistanoe genes in clones K6272, K66 and Strain L411. Cultures of eech clone were grown for 8 generations (18 h) in the presenceof vsrious concentretiona of EthBr, then washed twioe in sterile water and crossed with L2200 to measure the level of ERY resistance genes. Initial levels of ERY resistance for K6272, KS6 and L411 were measured at SSY’, S&J%, 86%, respectively. -A--A-, K6272; -A--A---, K66; --O-O--, L411.
these experiments cells were grown through eight generations in the presence of various concentrations of EthBr. It is seen from Figure 5 that in all casea the ERY resistance genes can be almost completely eliminated by EthBr, but the sensitivity of these genes at a given EthBr concentration varies considerably amongst the three cell types tested. The p- clones are less sensitive than the p+ strain, but a variation in the sensitivities of p- clones also exists; K5272 is far less sensitive than K56 as regards elimination of ERY resistance genes by EthBr. In order to interpret these differenoes in sensitivities of ERY resistance genes towards EthBr elimination, a study was carried out on the process of elimination of these genes from p-ERr cells, using clone K5272. 3
344
I’.
NAGLEY
ET AL.
K5272 was grown for ten generations in the presence of EthBr (20 pg/ml), washed in water, and grown for a further 20 generations in EthBr-free medium. The level of ERY resistance genes retained in the culture was monitored during the experiment, and the results are presented in Figure 6. It is evident that during the EthBr treatment substantial elimination of ERY resistance genes occurs, to a residual level of about 10% of the initial value. Subsequent growth in the absence of EthBr leads to a very small change in the level of ERY
resistance
genes in the culture.
The same be-
haviour is manifested by L411 treated with Et,hBr under the same conditions (Fig. 6)
+ EthBr -10
0 Number
I IO
I 20
-I
of generations
Fro. 6. Pattern of ohanges in the level of ERY resistance genes in clone K6272 and strain L411 during recovery from EthBr treatment. Cells of each clone were grown for 10 generations (24 h) in the presence of EthBr (20 pg/ml), washed twioe in sterile water, and reinoouleted into fresh growth medium without EthBr, at low cell density. One further sub-culture W&B made after 10 generations growth to yield oells grown 20 generations subsequent to EthBr treatment. The level of ERY resistance genes W&B determined at different stages of the experiment by withdrawing samples and orossing with L2200. Note the logarithmic scale on t,he ordinate. (A) K6272; (0) L411.
although the L411 ERY resistance genes are apparently more than two orders of magnitude more sensitive than those of K5272. Individual clones resulting from EthBr treatment of K5272 were analysed in order first to investigate whether the residual resistance genes were distributed equally among the population, or alternatively, if they were concentrated in a small proportion of stable p-ERr cells. A culture of Kb272 was grown 10 generations in the presence of EthBr (20 pg/ml), and after a further 20 generations growth in the absence of EthBr, it was found to retain a level of 5% ERY resistance genes, and was of 5% suppressiveness. Samples of this tial culture were diluted and plated on culture medium I to allow growth of single colonies, and three plates containing a total of 197 colonies were used. A replica plating crossing technique was used to identify colonies containing p-ER’ cells (see legend to Table 1). The resulta obtained aa shown in Table 1 were that only 15 of the 197 colonies contain P-ER’ cells (that is, 7.5% of the population). As summarized in Table 1, 13 of these 15 clones are almost pure for p-ERr cells, whilst only two are clearly mixed for p-ERr and ER” cells. These results show that extensive (but not quite complete) stabilization of cells with respect to ERY resis-
MITOCHONDRlAL
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346
TABLE 1 Analysis of the pop&&on
Colony type
P-J=’ (PuM
p-ER’, p -ER”, pa (mixed)? p-ER”, p0(mixed)$ P0 (pure)S
of cells derived from clone K527.2 treated with ethidium bromide Number
of colonies 13
($2, (24/32)
yo of total colonies 6.6 1.0 23.1 69.3
K6272 was grown for 10 generations (24 h) in the presence of EthBr (20 pgjml), then allowetl to grow for a further 20 generations in the absenoe of EthBr. Samples of this final culture were plated onto medium I, and 197 colonies from 3 seperate plates were analysed by a replica plating crossing technique BFI follows. Each plate was replioated by the velveteen pad technique onto minimal-glucose plates upon whioh a lawn of strain L2200 (10s cells) had been spread. The diploitl colonies which grew up were in turn replica plated onto a medium II-erythromycin plate. Colonies growing up on this latter plate represented oolonies containing p-ER’ cells from the initial medium I master plete. Control medium II replicas of the minimal gluaose plates served to show that each of the initial petite colonies gave one corresponding diploid colony containing sufficient p+ cells to grow on medium II. t To classify further colonies containing p-ERr cells, each of these was mated individually with L2200 to measure the level of ERY resistanoe genes present. Colonies yielding >SO% p+ zygotes of the type p + ERr are classified aa pure p - ER’. Colonies yielding < 80% of p + zygotes of the type p+ERr are classified mixed for p-ER’ and ERO oells. (The two mixed clones identified in this way both yielded about 36 y. p + ER’ zygotes.) $ To determine the frequency of p” colonies amongst the colonies classified 89 ER”, 32 colonies selected at random were tested individually for their suppressiveness by crossing with L2200 (see Fig. 7(a)). Colonies exhibiting <2% suppressiveness were classified as p”.
t’ance genes did occur during the 20 generations growth after EthBr treatment. This finding permits the properties of p-ERr cells and ER” cells to be examined separately. The suppressiveness of the individual clones in the population of EthBr-treated cells of K5272 waa determined, since this data would yield information concerning the nature of mtDNA in the clone. In particular, a change in suppressiveness from a mother clone to a daughter clone would indicate that a change in mtDNA has occurred (cf. Saunders et al., 1971) and second, zero suppressiveness is highly indicative of a loss of mtDNA (Nagley & Linnane, 1972; Michaelis et aZ., 1971). Thirty-two clones classified as ER” by the replica plating technique (Table 1) were selected at random from the medium I plates and tested individually for their suppressiveness. The results are shown in Figure 7(a) and it can be seen that 24 of the ER” clones are zero suppressive petites (p”), the remainder showing very low suppressiveness values. Thus, since ER” cells account for about 90% of the population (Table l), then it may be concluded that about 70% (24 out of 32 tested) of this population is of the type pot probably lacking mtDNA. This correlates with the loss of mtDNA from cultures of K5272 treated with EthBr (Fig. 4). By contrast the suppressiveness distribution of the 13 pure p-ER’ clones, as is shown in Figure 7(b), reveals that such clones fall into the low to medium range of suppressiveness (from 5 to 66%) and there are no clones of zero suppressiveness. Most clones show the same level of suppressiveness as the parent clone K5272 (namely 5 to 8% suppressive), but a number of large changes have also occurred.
346
P. NAGLEY
EZ’
AL. (0)
lJ
I
I
I
-1
-
I (b)
I IO
Ill I I 20 30 40 Suppressiveness ( % I
I 50
I 60
FIQ. 7. Analysis of the suppressiveness of the population of cella derived from clone K6272 treated with EthBr. K6272 was grown for 10 generations (24 h) in the presenoe of EthBr (20 &ml) and then grown for a further 20 generations in EthBr-free medium, and single colonies obtained from this oulture were olaeeified aa p-ER’, or ER” aa described in Table 1. Selected colonies were tested for suppressiveneas by crossing with L2200 individually. (a) Data for 32 clones selected at random from the 182 classified as ER”; (b) data for the 13 clones classified as containing only p-ER’ cells.
These results indicate that the action of EthBr on the mitochondrid genome of clone K5272 leads to the fragmentation of its mtDNA. This conclusion can be drawn because Srstly, many p-ER” cells have been induced and are present in the culture; these are suppressive cells which retain mtDNA but from which have been deleted resistance. Second, a number of p-ER’ sequences determining erythromycin clones are found which have large alterations in their suppressiveness, indicating changes in mtDNA other than in the ERY resistance gene region. These interpretations taken together with the direct observations of inhibition of mtDNA synthesis by EthBr in p-ER’ cells (Fig. 4) lead us to conclude that the mechanism of elimination of mitochondrial genes ad mtDNA from p- cells occurs by the same mechanism as from p+ cells. (ii) Quantitative aqects of elimination of eythromycin resistance genesand mitodwdial DNA by ethidium bromide This work has demonstrated differences in the sensitivities of ERY resistance genes towards EthBr in p+ER’ ad p-ERr clones (Figs 5 and 6). It was considered that if there were a greater absolute number of copies of ERY resistance genes in cells of clone K5272 as compared with K55, and with I.&l, then elimination of these genes by EthBr would be less efficient in the case of K5272, as has been observed. To clarify this argument by means of a specific example consider a particular grade clone (say
MITOCHONDRIAL
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MUTANTS
347
L411) containing 60 copies of the ERY resistance gene per cell, assuming each of the 50 molecules of mtRNA (see Williamson, 1970; Nagley $ Linnane, 1972) to carry this gene. Let us consider further a stabilized p-ER’ clone derived from L411 containing mtDNA of one tenth the complexity of p+ mtDNA, with each sequence including an ERY resistance gene. Since the stable petite cells contain the same total cellular amount of mtDNA (Nagley t Linnane, 1972) it would then follow that 500 copies of the ERY resistance gene are present in each such petite cell. On treatment of the grande and such a petite with EthBr, the probability of elimination of ERY resistance genes is clearly much greater for the grande than for the petite. This interpretation depends on the differences in the susceptibility of mtDNA towards damage caused by EthBr. The extent of inhibition of mtDNA synthesis (Fig. 4) may be used as an indicator of the susceptibility of mtDNA to EthBr action. To relate the elimination of ERY resistance genes to the susceptibility of mtDNA in clones K5272, K55 and L411, we have taken the data in Figures 4 and 5 and calculated, for each clone at each EthBr concentration, the expression residual ERY resistance genes (“/ initial level) residual mtDNA (% initial level)
5
IO
15
20
[ElhBr] (pg/ml)
FIG. 8. Normalization of the date for elimination of ERY resistance genes with respect to the susceptibility of mtDNA to EthBr in clones K6272, K66 end strain L411. Data from Figures 4 and 5 were taken and for each concentration of EthBr the function residual level of ERY resistence genes ( y0 initial level)/residual was calculated, and plotted aa the ordinate in this Figure. ---n---n---, K6272; --A--A--, K66; -O-O-, L411.
level of mtDNA
( y0 initial
level)
348
P. NAGLEY
This expression is plotted as results show clearly that even the sensitivities to elimination order K5272 < K55 < L4lI. K5272 contains more copies of than L411.
ET
AL.
a function of EthBr concentration in Figure 8. The allowing for the different mtDNA responses to EthBr, by EthBr of ERY resistance genes is clearly in the We therefore interpret these data to indicate that ERY resistance genes than K55, and K55 in turn more
4. Discussion (a) Relationship between supprwsiveness and mtDNA The results obtained in this work concerning the simultaneous reduction in suppressiveness and loss of mtDNA from p- cells treated with EthBr reinforces previous proposals that suppressiveness is determined by the mtDNA of p- cells. Sherman & Ephrussi (1962) showed that suppressiveness was associated only with cytoplasmically inherited petite strains, and not with nuclear determined respiratory deficient mutants in which mtDNA is not changed. More recently it was shown that only petites containing mtDNA exhibit suppressiveness, whereas cells that lack mtDNA do not show suppressiveness (Nagley & Linnane, 1970,1972; Michaelis et al., 1971). Third, Saunders et al. (1971) demonstrated that when a p-E& cell spontaneously lost the base sequence specifying ERY resistance to become p-ERO, a change in suppressiveness to a higher or lower value almost always resulted. These latter findings demonstrated that the particular base sequences retained in the mtDNA of a p- cell play a role in determining the suppressiveness of that cell. So far no evidence has been obtained which links a particular trend in the physical properties of mtDNA (for example base composition) in p- clones to the suppressiveness of such clones (compare data of Mehrotra & Mahler (1968), Bernardi et aZ. (1970), Michaelis et al. (1971), Nagley & Linnane (1972) and data collated by Nagley & Linnane (1973)). The foregoing discussion has emphasized the cytoplasmic nature of suppressiveness and the possible relationships with p- mtDNA. However, many aspects of the replication and maintenance of mtDNA depend on nuclear function. It is well established that the rate of spontaneous mutation of p + to p - depends upon certain nuclear genes (see Ephrussi, 1953; Sherman, 1963). It follows that when a cross is made between a p- and a p+ strain the frequency of petite zygotic clones will depend not only upon “suppressive” interactions stemming from the p- mtDNA, but also upon interactions of nuclear genes in the zygotes which have been generated by combination of the two sets of nuclear genes from the haploids. In crosses using petite cells of very low or zero suppressiveness the nuclear regulation of the frequency of petite zygotes assumes significant importance. For this reason it is very important when measuring the suppressiveness of a petite clone derived from a given p+ strain, say strain A, by crossing with a standard p+ strain B, to determine simultaneously the frequency of petite zygotic clones obtained when a p” clone derived from strain A is crossed with strain B (p’). In order to obtain a reliable measurement of the suppressiveness of the tested petite clone, the frequency of petite zygotic clones must be corrected for that frequency obtained in the cross using the p” cells, as is described in Materials and Methods. This correction is preferable to that solely utilising measurements of the petite frequency in the p+ partner of the cross.
MITOCHONDRIAL
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OF PETITE
MUTANTS
349
(b) Concept of increased gene redundancy in p- cede The available evidence supports a general description of petite mitochondrial genomes consisting of some fragments of the grande mtDNA. Since the total amount of mtDNA in stable petite clones closely approximates that of the grande parent cells (Nagley t Linnane, 1972), then at least some sequences retained in the mitochondrial genomes of such petite cells must be present in higher multiplicity than in the grande cells. The validity of this generalization relies on the p- mtDNA not containing sequences which are not found in p + mtDNA. Evidence that this condition does hold for some p- cells has been obtained by Michaelis et al. (1972) who tested the ability of RNA transcribed in vitro from mtDNA of a grande strain and one particular p- clone to hybridize to both p+ and p- mtDNA. The results indicated that whilst the petite lacked 50 to 60% of the wild-type mitochondrial genome, no additional sequences differing from those in the p+ mtDNA could be detected in the p- mtDNA. The quantitative effects of EthBr action on p-ER’ cells are consistent with the notion that the cells studied contain a higher multiplicity of ERY resistance genes than in the parent p+ER’ strain. Our proposal of gene reiteration is in accord with the results of Cohen et al. (1972) who tested the mtDNA of a number of p- clones for the presence of genes coding for the mitochondrial leucyl-tRNA. It was found that in some clones this gene accounted for a substantially higher proportion of mtDNA than in the parent p+ clone. These findings have raised the possibility of biologically concentrating, and thus purifying, specific base sequences in mtDNA. REFERENCES Bernardi, G., Carnevali, F., Nicolaieff, A., Piperno, G. & Tecce, G. (1968). J. Mol. Biol. 37, 493. Bernardi, G., Fames, M., Piperno, G. & Slonimski, P. P. (1970). J. Mol. Biol. 48, 23. Billheimer, I?. E. & Avers, C. J. (1969). Proc. Nat. Acad. Scsci.,U.S.A. 64, 739. Bolotin, N., Coen, D., Deutsch, J., Dujon, B., Netter, P., Petrochilo, E. & Slonimski, P. P. (1971). Bull. Inet. Pasteur, 69, 215. Borst, P. (1972). Annu. Rev. Biochem. 41, 333. Carnevali, F., Morpurgo, G. & Tecce, G. (1969). Science, 163, 1331. Cohen, M., Casey, J., Rabinowitz, M. & Getz, G. S. (1972). J. Mol. BioZ. 63, 441. Dixon, H., Kellerman, G. M. & Linnane, A. W. (1972). Arch. Biochem. Biophye. 152, 869. Ephrussi, B. (1953). Nu~Zeocy~~l~rn~c ReZations in Micro-organisms, Clarendon Press, Oxford. Ephrussi, B., Margerie-Hottinguer, H. & Roman, H. (1955). Proc. Nut. Acad. Sci., U.S.A. 41, 1065. Gingold, E. B., Saunders, G. W., Lukins, H. B. & Linnane, A. W. (1969). Genetics, 62,735. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D. R. & Marmur, J. (1970). J. Mol. BioZ. 52, 323.
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Mehrotra, B. D. & Mahler, H. R. (1968). Arch. B&hem. Biophya. 128, 685. Michaelis, G., Douglass, S., Tsai, M. & Criddle, R. S. (1971). Biochem. Genet. 5, 487. Michaelis, G., Douglass, S., Tsai, M., Burchiel, K. & Criddle, R. S. (1972). Biochemistry, 11, 2026. Mounolou, J. C., Jakob, H. & Slonimski, P. P. (1966). B&hem. Biophya. Res. Commun. 24, 218. Nagley, P. & Lirmane, A. W. (1970). Biochem. Biophya. Res. Commun. 39, 989. Nagley, P. & Linnane, A. W. (1972). J. Mol. Biol. 66, 181. Nagley, P. & Linnane, A. W. (1973). In Advancee in Molec~Z~r Genetica (Falconer, I. R. & Cove, D., eds), Paul Elek Ltd, London, in the press. Ogur, M., St. John, R. & Nagai, S. (1967). Science, 125, 928. Perlman, P. S. t Mahler, H. R. (1971). Nuture New Biol. 231, 12. Rank, G. H. (1970). Can&. J. Genet. Cytol. 12, 129. Saunders, G. W., Gingold, E. B., Trembath, M. K., Lukins, H. B. & Linuane, A. W. (1971). In Autonomy and Bbgeneais of Mitochondriu and ChZoroplaeta (Boardman, N. K., Linnane, A. W. & Smillie, R. M., eds), p. 185, North Holland Publishing Co, Amsterdam. Sherman, F. (1963). Genetics, 48, 375. Sherman, F. & Ephrussi, B. (1962). Genetics, 47, 675. Slonimski, P. P., Perrodin, G. & Croft, J. H. (1968). B&hem. Biophys. Res. Commun. 30, 232. Wallace, P. G., Huang, M. & Linnane, A. W. (1968). J. Cell BioZ. 37, 207. Williamson, D. H. (1970). In Control of OrguneZZeDevelopment (Miller, P. L., ed.), p. 247, University Press, Cambridge. This work was supported (D65115930).
by a grant from the Australian
Research Grants Committee