Current Genetics
Current Genetics (1982)5:9-15
© Springer-Verlag 1982
Modifications of Chloroplast DNA During Streptomycin Induced Mutagenesis in Euglena gracilis Philippe Heizmann, Younis Hussein*, Paul Nicolas, and Victor Nigon Universit6 Claude Bernard Lyon 1, D6partement de BiologieG6n6rale,43, Boulevarddu 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Summary. The evolution of chloroplast DNA was analysed during streptomycin induced mutagenesis in Euglena gracilis strain bacillaris and strain Z. In addition to a massive reduction of the cellular level of chloroplast DNA, several structural modifications have been observed in early stages of mutagenesis but they are generally eliminated during the later stages. The ribosomal cistrons are regularly rearranged: two of the three tandemly arranged cistrons occuring in wild type chloroplast DNA decrease while the third one is relatively more conserved and amplified during mutagenesis and in bleached mutants.
Key words: Chloroplast DNA - Mutagenesis - Ribosomal cistron - Amplification -Euglena
Introduction Euglena gracilis is particularly suitable for the study of mutations affecting the chloroplast system, due to its apitude to grow either phototrophically or on carbon substrates in the absence of photosynthesis. The efficiency of the photosynthetic machinery is thus optional in Euglena and cell survival is possible even in conditions of major chloroplast alterations. Moreover the chloroplast system of that alga is particularly sensitive to various mutagens whicfi can induce irreversible mass bleaching of the cultures. During the cell division following mutagenic treatments, the quantities of chloroplast DNA per cell decrease rapidly (Uzzo and Lyman 1972) and become undetectable by the classical physiochemical * Present address: Department of Genetics, Faculty of Agriculture, Assiut, Egypt Offprint requests to: P. Heizmann at the above address
techniques of DNA analysis in most stabilized bleached mutants (Schiff and Epstein 1965). Using more recent techniques for analysing DNA we demonstrated however the persistence of chloroplast DNA in bleached mutants, most often in very low amounts. These residual chloroplast DNA seem to maintain unchanged the major part of the wild type sequences; they show however some deletions or rearrangements (Heizmann et al. 1981). In the work presented here we analysed the qualitative and quantitative evolution of chloroplast DNA in cultures of Euglena undergoing the mutational bleaching induced by streptomycin. Our results confirm the massive reduction of the cellular content in chloroplast DNA and show the occurence of many transient structural modifications; most of these modifications seem to be eliminated from the progeny. Ribosomal cistrons however appear to be systematically rearranged: these modifications are particularly evident in the strain bacillaris where the three ribosomal citrons differ from each other by deletions located in the ribosomal spacers (see restriction map Fig. 1, made from data from Hallick 1982, Helling et al. 1979; E1 Gewely et al. 1981). The Eco R 1 restriction pattern of chloroplast DNA from the strain bacillaris differs from that of strain Z chloroplast DNA by the duplication of the multiple band Eco Lz into two different bands Eco Mbac and Eco Obac and by the duplication of the band Eco Pz into two different bands Eco Rbac and Eco Oba c (map Fig. 1 and restriction pattern Fig. 2). (We will use the nomenclatures proposed independently by Gray and Hallick 1977, for the strain Z, and by Helling et al. 1979, for the strain bacillaris.) The modifications induced during chloroplast mutagenesis result in the progressive disappearance of fragments Eco Obac and Eco Sba c and in the preferential conservation of fragments Eco Mbac and Eco Rba c. This preferential conservation results from the amplification of the ribosomal cistrons. O172-8083/82/0005/0009/$ 01.40
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
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Fig. 1. Restriction map for the ribosomal cistrons of strains Z and bacillaris, according to Gray and Hallick (1977) and Hallick (1982) (strain Z), and Helling et al. (1979) and E1-Gewely et al. (1981) (strain bacillaris). The nomenclature used is that proposed independantly by the two groups. The fragments Eco Obac and Eco Mbac from baciUaris are homologous to fragments Eco Llz and Eco L2z from strain Z; fragments Eco Rbac and Eco Sbac axe homologous to fragments Eco P2z and Eco P3z from strain Z. A probe made of fragments Eco L z and Eco Pz will label fragments Eco Bz, Fz, Lz and Pz in strain Z and Eco Bbac, Fbac, Obac, Mbac, Rbac and Sbac in strain bacillaris (Fig. 2). The DNA from the two strains differ essentially from each other by: 1. deletions in bacillaris affecting the Eco R 1 site located between Eco Plz and Eco Bz, and affecting about 450 base pairs in the spacer between Eco Llz and Eco P2z; the Eco R 1 site is conserved in this spacer. 2. the arrangement of sequences located between the "supplementary s16S" gene and the first ribosomal cistron
Materials and Methods
Strains and Culture Conditions Euglena gracilis strain Z (1224-5/25) was obtained from the G6ttingen Institute Algensammlung, the strain baeillaris as well as the mutants W3BUL, WloBSmL, Y1BXD and Y3BUL derived from bacillaris were obtained from Professor Schiff; ZHB was obtained from Professor Rawson and ZN2L from Doctor Calvayrac; W34ZUD, W35ZEmsD and W36ZHD were produced in our laboratory. All cultures were grown on an organotrophic medium using sodium butyrate as carbon source (= NCo medium from Freyssinet et al. 1972).
Streptomycin Induced Mutagenesis Etiolated Z or bacillaris cells were inoculated on NCb organotrophic medium supplemented with streptomycin (500 gg/ml at pH 7.4) and harvested after various length of growth in the presence of the drug. The proportion of bleached cells among the progeny of each aliquot was estimated by plating on Petri dishes.
Analysis of DNA Chloroplast DNA were prepared from isolated chloroplasts prepared by flottation according to Manning and Richards (1971). Pellets of cells or of organelles were resuspended in Tris 50 mM EDTA 50 mM - pH 9.0 and lyzed by addition of SDS (1% final concentration); NaC1 was added to 0,5 M and the mixture was extracted with an equal volume of aqueous phenol-chloroform (lv/lv). The ethanol precipitated DNA were dissolved in Tris
10 m M - EDTA 1 mM - pH 7.4, treated with RNase (50 ~g/ml - 1 h - 37 °C) and then pronase (1 mg/ml - 4 h - 37 °C), reextracted with phenol-chloroform and precipitated. Chloroplast DNA were further purified by one of two cycles of CsC1 equilibrium centrifugation. EcoR1 restriction fragments of chloroplast DNA were cloned by ligation with RSF 2124 plasmid DNA and transformation of E. eoli W4554. (Lomax et al. 1977). Southern blots were prepared according to Southern (1975) and hybridizations with cloned DNA probes or total radioactive RNA were carried out according to Jeffreys and Flavell (1977). Autoradiographs were exposed at - 7 0 °C with Kodak XR1 or XAR5 films and Ilford Fast-Tungstate intensifying screens. Restriction enzymes were purchased from Boehringer and T 4 ligase from Miles Laboratories; the enzymes were used following manufacturers recommandations. DNA were labeled in vitro with 32p nucleotides by nicktranslation (Jeffreys and Flavell 1977); total chloroplast DNA was iodinated by the method of Commerford modified by Meeker et al. (1976); radioactive nucleotides and iodine were from Amersham.
Estimations of Chloroplast DNA by Renatumtion Kinetics Total DNA in Tris 10 mM - EDTA 1 mM - pH 7.4 were sonicated to fragment sizes of 400-500 base pairs checked by eiectrophoresis. After addition of tracer amounts of 1251 labeled chloroplast DNA (0.5 ~ug/ml), they were denatured in boiling water bath for 10 minutes, then made NaC1 0.3 M NaCI Tris 10 m M - pH 7.4 and brought to 63 °C at the beginning of the renaturation. Aliquots were taken at various times; single stranded and double stranded DNA were separated by chromatography on hydroxyapatite. The proportions of both forms of DNA were estimated by scintillation counting. Experimental data were represented as
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
Fig. 2. Modifications of the ribosomal cistrons during streptomycin induced mutagenesis in Euglena gracilis strain baeillaris. An exponentially growing culture was added with streptomycin (500 ~g/ml). Total DNA were extracted from aliquots harvested at various moments after addition of the drug. Their Eco R 1 Southern blots were hybridized with a mixture of fragments Eco L z and Eco Pz cloned in RSF 2124 (specific activity about 80 x 106 CPM/~g). These probes hybridized with ribosomal fragments Eco Bbac, Fbac, Mbac, Obae, Rbac and Sbac in bacillaris (Helling et al. 1979) and Eco Bz, Fz, L z and Pz in strain Z (Hallick 1982), according to restriction map of Fig. 1. The common hybridization band between fragments B and F is due to a spurious hybridization of the vector plasmid. The band under fragment F is probably a partial digest. Slot 0 h: total wild type bacillaris DNA. Slots 12 h, 24 h, 4 g, 8 g, 16g: DNA from cells grown with streptomycin for 12, 24 h and 4, 8 and 16 generations. Slots W3 and WI0: DNA from mutants W3BUL and WIO BSmL derived from bacillaris. Slot Zct: purified chloroplast DNA from strain Z. Amounts of DNA loaded per slot: 0 h, 12 h, 24 h, 4 g: 3 ug. 8 g, 16 g, W3, W l 0 : 1 2 pg. Zct: 0.01 ug. Autoradiographic exposures: 0 h, 12 h, 24 h, 4 g: 2 days. 8 g, 16 g, W3, W10, Zct: 5 days
second order plots according to Wetmur and Davidson (1968), expressing the reciprocal of the fraction of single stranded DNA as a function of time. We verified experimentally that these curves are actual straight lines with regression coefficients higher than 0.98-0.99 and with slopes proportional to the concentration of chloroplast DNA initially present in the reassociation mixture. It is thus possible to calculate the unknown concentration of chloroplast DNA introduced among total DNA in reassociation mixtures by comparing the slope of second order curve with that concerning the renaturation of control chloroplast DNA of known concentration. From the cellular content of total DNA (2.5 to 3.5 pg/cell according to our estimations by perchloric acid extraction) one calculates the quantity of chloroplast DNA per ceil.
l1
Fig. 3. Relative amplification of ribosomal cistrons during mutagenesis. A Southern blot prepared with the same DNA preparations as for Fig. 2 was hybridized with a mixture of cloned Eco R 1 fragments from strain Z chloroplast DNA (fragments Eco H, I, J, L, M, P, Q, R, S, T, U and V) labeled by nick-translation to 80 x 106 CPM/~g
Results 1. Qualitative Evolution o f Chloroplast DNA 1.1. Ribosomal Cistrons in the Strain bacillaris.
The structural e v o l u t i o n o f chloroplast D N A have been followed in a culture o f etiolated Euglena strain bacillaris growing after addition o f s t r e p t o m y c i n . The total D N A extracted f r o m aliquots harvested at various m o m e n t s during mutagenesis were digested with E c o R1 restriction endonuclease; S o u t h e r n blots obtained with these digests were hybridized with E c o R~ fragments o f wild t y p e chloroplast D N A (strain Z) cloned and amplified in the plasmid R S F 2 1 2 4 (Fig. 2 and 3). During these bleaching e x p e r i m e n t s we have regularly observed a m o d i f i c a t i o n o f the ribosomal segments concerning the entire p o p u l a t i o n of chloroplast D N A molecules (Fig. 2). The overall level o f h y b r i d i z a t i o n obtained with ribosomal probes decreases progressively, particularly during the late stages after 8 and 16 generations o f growth w i t h streptomycin. It is remarkable that the h y b r i d i z a t i o n with fragments Eco Oba c and Eco Sba c decreases m u c h more rapidly than that with fragments Eco Mba c and Eco Rba c. The ribosomal restriction pattern evolves progressively f r o m the bacillaris type towards the Z type pattern. The ultimate stages of this
12
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
Fig. 4a-c. Degradations and rearrangements of chloroplast DNA during mutagenesis. The same protocol as in Figs. 3 and 4 was used for a culture of Euglena strain Z. (a) probe of total purified chloroplast DNA (60 xl06 CPM/t~g); (b) probe of fragment Eco Mz (80 x 106 CPM/gg); (c) probe of fragment Eco Lz (70 x 106 CPM/gg). Amounts of DNA per slot: 0 h to 24 h: 2 t~g. 6 g to 18 g: 10/~g. Exposures: 5 days at -70 °C, except for the lower part of a (12 days)
evolution are represented by the Eco R 1 restriction patterns obtained with the DNA from the two stabilized mutants W3BUL and WloBSmL derived from the strain bacillaris; these patterns are apparently not different from that obtained with DNA from the strain Z. Moreover, when Southern blots identical to those used for Fig. 2 are hybridized with a mixture of ribosomal probes (fragments Eco Lz and Eco Pz) and of nonribosomal probes (fragments Eco Hz, Iz, Jz, Mz, Qz, Rz, Sz, Tz, Uz and Vz) there is a similar decrease of the hybridization signal for all chloroplast DNA fragments at the beginning of the mutagenesis process. During the late stages on the contrary, after 8 and 16 generations, ribosomal fragments Eco Mbac and Eco Rba c disappear much less rapidly than non-ribosomal fragments or than the ribosomal fragments Eco Oba c and Eco Sbac. The end of this evolution is clearly visible in the case of the bleached mutant W3BUL where only the ribosomal fragments Eco Mbae, Eco Rbac, Eco Bbac and Eco Fba c are intensively labeled.
merits appear during the course of streptomycin induced mutagenesis (Fig. 4). Such unusual fragments can be observed at the level of fragments Eco Sz and Eco Tz, and Eco Qz and Eco R z during the late stages of mutagenesis (Fig. 4a, slots 12 and 18 generations) when a probe made of total chloroplast DNA is used. When cloned restriction fragments are used as probes they hybridize fragments both smaller and larger than the initial fragments. Smaller fragments appear either as discrete bands (Fig. 4b, slots 6 h, 24 h and 6 generations) or as smears (Fig. 4c, slots 3 to 24 h) and demonstrate intensive degradations occuring rapidly after the addition of streptomycin. Rearrangements are suggested by the formation of fragments larger than the intact fragments (Fig. 4b, slots 3 to 24 h). In the later stages of mutagenesis however, i.e. after 12 or 18 generations, fragments apparently identical to the initial wild type restriction fragments remain in large majority in the case of ribosomal fragments (Eeo Lz, Fig. 4c) and in the case of most nonribosomal fragments (Eco Mz, Fig. 4b).
1.2. Non-Ribosomal Cistrons. In some experiments the overexposure of autoradiographs revealed transient alterations affecting only a limited fraction of the entire chloroplast DNA population. Restriction fragments differing from the initial wild type chloroplast DNA frag-
2. Quantitative Evolution of Chloroplast DNA The second order plots representing the renaturation kinetics of chloroplast DNA are actual straight lines for
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
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Table 1, Quantitative evolution of chloroplast DNA (= ct DNA) during streptomycin induced mutagenesis. The quantities of chloroplast DNA as numbers of molecules per cell were calculated from the slopes of the straight lines from Fig, 5 (column 4); they were also calculated in the hypothesis of simple dilution of chloroplast DNA among the progeny (column 5) Time
Generations Proportion of green colonies
ctDNA ctDNA molecules molecules per cell in hypothesis of simple dilution
0h 3h 6h 24 h 1 week 2 weeks 3 weeks
0 0.125 0.25 1 6 12 18
220 150 94 79 69 32 13
1 0.82 0.57 2 x 104 0 0 0
220 195 176 110 3.5 0.06 0.001
the DNA of wild type etiolated cells and up to 24 hours of streptomycin treatment (Fig. 5a). This means that during the initial stages of mutagenesis chloroplast DNA molecules reassociate as a single component homogeneous population. (Stutz and Vandrey 1972; Rawson and Boerma 1976; Chelm et al. 1977). The slope of each second order straight line allows to calculate the quantities of chloroplast DNA present in each aliquot by simple comparison with the slope of the straight line representing the renaturation of pure chloroplast DNA of
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Fig. 5a and b. Reassociation kinetics of chloroplast DNA during the mutagenesis of Euglena strain Z. Chloroplast DNA (0.5 #g/ml) labeled in vitro with 125I (15 x 106 CPM/vg) was renatured either alone or in the presence of total DNA from cultures growing in the presence of streptomycin. The same preparations as those for Fig. 4 were used. Single and double stranded DNA were separated by hydroxylapatite chromatography. The kinetics were represented by their second order plots according to Wetmur and Davidson (1968) (1/% single stranded DNA as a function of time). Their slopes are proportional to the quantities of chloroplast DNA introduced in the renaturation mixture. The results of calculations have been reported in Table 1). Renaturation of: Labeled chloroplast DNA (0.5 #g/ml) (o). (a) addition of total DNA (480 ~zg/ml) from etiolated cultures growing for 0 h (z~), 3 h (o), 6 h (~), and 24 h (v) with streptomycin. (b) total DNA (1,920 ~g/ml) from cultures growing for 6 (v), 12 (=) and 18 (A) generations with streptomycin
Time (hours)
known concentration (see "Materials and Methods" for explanations). Table 1 shows that during the first 6 to 24 h the content in chloroplast DNA decreases more rapidly than if there was simple dilution of chloroplast genomes among the progeny (column 4). This corroborates the occurence of degradations suggested by the restriction patterns of Fig. 4 and also observed during mutagenesis induced by ultraviolet irradiation (Nicolas et al. 1980). After this early stage chloroplast DNA continues to decrease during 18 generations at least; however its content per cell is maintained at a level much higher than that expected if simple dilution occured. Net syntheses of chloroplast DNA take over again which allow the persistence of chloroplast genomes in bleached cells. The proportion of green colonies among the progeny drops very rapidly during the first hours of streptomycin action. After a few generations of growth in the presence of streptomycin the reassociation kinetics of chloroplast DNA occur in two phases: the second order plots form two straight lines with different slopes, which represent the renaturation of two classes of sequences having different reiteration frequencies. The complexity of the more reiterated class of sequences can be estimated by its span on the ordinates axis: it represents about 8% to 12% of the total complexity of wild type chloroplast DNA. The abundance of these sequences relative to that of the less repeated segments increases regularly during late mutagenesis as illustrated by the increasing difference of slopes for
P. Heizmann et al. : Chloroplast DNA Modifications During Mutagenesis in Euglenagracilis
14 Ref
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Discussion
1 7. Propagation and Stabilization of Modifications Induced During Mu tagenesis
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Fig. 6. Identification of repeated chloroplast DNA sequences in bleached mutants. 125I labeled chloroplast DNA (0.3 ~g/ml; 15 x 106 CPM/gg) were mixed with sonicated total DNA (3,200 gg/ml) from mutant W34ZUD, denatured and reassociated to Cot 35 (mole/liter x seconds). The reassociated fraction was isolated on hydroxylapatite and hybridized to Southern blots of chloroplast DNA strain Z. The renaturation of repeated sequences is terminated at Cot 35 while rare sequences are still essentially single stranded. The Cot calculated for the probe is 0.01 while its Cot1/2 is about 0.4, allowing less than 5% self-renaturation. Ref: chloroplast DNA (Z) hybridized with total labeled chloroplast DNA. W34 Cot 35: chloroplast DNA (Z) hybridized with the fast renaturing sequences of DNA from mutant W34ZUD
the two classes (Fig. 5b). The mode of synthesis of chloroplast DNA which operates after mutagenesis systematically amplifies about 10% of the plastid genome.
3. Identification of the More Reiterated Sequences in Bleached Mu rants The biphasic renaturation kinetics of chloroplast DNA appearing during mutagenesis regularly occured for DNA from stabilized bleached mutants. We identified the nature of the corresponding more reiterated sequences by their hybridization affinity with wild type chloroplast DNA. The reiterated chloroplast DNA sequences from the mutant strain W34ZUD were separated from rare sequences owing to their rapid reassociation: after renaturation at Cot 35 they were obtained as double stranded DNA while rare sequences were still single stranded, and isolated by hydroxylapatite chromatography. They were rehybridized to Southern blots of wild type chloroplast
The present study was performed on total DNA from cultures undergoing streptomycin mutagenesis; it gives a global description of individual events affecting the population of chloroplast DNA molecules, and thus only those concerning a significant fraction of chloroplast DNA were detected. Modifications appeared at the level of fragments Qz and Eco Rz during late mutagenesis (Fig. 4a). Among ten stabilized mutants analysed so far, a majority is affected on at least one of these two fragments (Heizmann et al. (1981). However the modifications observed for Eco M z during early mutagenesis (Fig. 4b) seem to be eliminated in the later stages where only apparently intact Eco M z fragments can be detected. None of the bleached mutants analysed carries any detectable modification of fragment Eco M z. This is also the case of the largest part of the chloroplast genome since Eco Qz and Eco R z are the only modified fragments detected among more than 50% of the chloroplast sequences already explored in bleached mutants. In several respects, chloroplast mutagenesis in Euglena is reminiscent of mitochondrial mutagenesis in yeast. However petite 0 - respiratory mutants contain defective mitochondrial DNA molecules consisting of repetitions of a wild type DNA sequence representing between 0.1% and 80% of the wild type genome (Borst and Grivell 1978); the amounts of mitochondrial DNA are about the same as in wild type cells. On the contrary the quantities of chloroplast DNA are generally very low in non photosynthetic bleached mutants of Euglena (about a 100 fold lower than in wild type cells) and very few modifications are apparent. While rearranged sequences are propagated and amplified during petite mutation induction, wild type forms of DNA sequences remain dominant in Euglena chloroplast in spite of mutational events but their quantities decrease considerably,
2. Amplification of Ribosomal Cistrons The preferential disappearance of some ribosomal fragments (Eco Obac and Eco Sbae) with respect to others (Eco Mbac and Eco Rbac) in the strain bacillaris and the progressive evolution of the restriction patterns from the
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
bacillaris type towards the Z type was regularly observed in all experiments. The first observations that the patterns from W3BUL (derived from bacillaris) are similar with those from Z strains made us suspect that our W 3 BUL strain had been mixed up with Z mutants; we omitted the data relative to W 3 BUL from our first report (Heizmann et al. 1981). The present results were obtained with two strains W 3 BUL and Wt0 BSmL recently provided by Professor Schiff. Several arguments indicate that this evolution corresponds to a quantitative modification and amplification of the ribosomal cistrons: the biphasic renaturation kinetics of chloroplast DNA indicates the occurence of two sequence classes in bleaching cells and in bleached mutants; considered alone this argument is not indisputable since this type of renaturation could be partly artefactual: repetitive components of nuclear contaminants of the chloroplast DNA used as probe would reassociate about as rapidly as amplified ribosomal chloroplast ribosomal DNA (Rawson 1975). Since these repetitive sequences account for 40% of nuclear DNA, nuclear contaminations should represent as much as 25% of the chloroplast preparations to rapidly renature 10% of the probe. Analytical ultracentrifugations of chloroplast DNA samples rule out this possibility since the nuclear band (p = 1.707 g/cm 3) represents always much less than 5% of the chloroplast DNA band (p = 1.686 g/cm3). The amplification of ribosomal cistrons during mutagenesis is more convincingly demonstrated: - by the relative increase of the ribosomal fragments Eco Mbae and Eco Rba c with respect to the non ribosomal fragments and to the ribosomal fragments Eco Obae and Eco Sbac providing real internal standarts. - by the very preferential hybridization of repeated chloroplast DNA from mutant Wa4 ZUD to the ribosomal segments of wild type DNA. -
3. Structure o f Amplified Ribosomal Cistrons The disappearance of fragments Eco Obac and Eco Sba c in the strain bacillaris affects the cistrons 1 and 2 (numbered according to Fig. 1). The corresponding Barn H 1 fragments are: Barn Ebac and Barn Fba e in bacillaris Barn Ez and Barn D z in strain Z. These Barn H a fragments are preferentially reduced with respect to those from cistron 3 in the Barn H 1 restriction patterns from bacillaris cultures during streptomycin induced mutagenesis (not shown), from Z mutants (particularly WZN2L and W36ZHD) and from bacillaris mutants (Y1BXD, Y3BUD) (Heizmann et al. 1981). -
-
15
These observations confirm the occurence of deletions extending from cistrons 1 and 2 but leaving cistron 3 relatively amplified. It is remarkable that the spacer between cistrons 1 and 2 is already deleted in the strain bacillaris, indicating that this region is highly mutable. The detailed determination of the structure of the ribosomal cistrons in bleached mutants will probably yield precious informations on the process of chloroplast DNA replication.
Acknowledgments. We thank Mr. P. M. Malet and Mrs. FaureSchwob for their excellent technical assistance, and Drs. G. Bernardi and J. Doly for helpful discussions and advices.
R e f e r e n c e s
Borst P, Grivell LA (1978) Cell 15:705-723 Chelm BK, Hoben PJ, Hallick RB (1977) Biochemistry 16: 782-785 De Deken-Grenson, M (1960) Arch Biol 71:270-340 E1-Gewely MR, Lomax MI, Lau ET, Helling RB, Farmerie W, Barnett WE (1981) Mol Gen Genet 181:296-305 Freyssinet G, Heizmann P, Verdier G, Trabuchet G, Nigon V (1972) Physiol v~g 10:421-442 Gray P, Hallick RB (1977) Biochemistry 16:1665-1671 Hallick RB (1982) In: Buetow DE, (ed)The biology of Euglena, Vol 4. (in press) Heizmann P, Doly J, Hussein Y, Nicolas P, Nigon V, Bernardi G (1981) Biochim Biophys Acta 653:412-415 HeUing RB, EI-Gewely MR, Lomax MI, Baumgartner JE, Schwartzbach SD, Barnett WE (1979) Mol Gen Genet 174: 1 10 Jeffreys AJ, Flavell RA (1977) Cell 12:429-439 Lomax MI, Helling RB, Necker LI, Schwartzbach SD, Barnett WF (1977) Science 196:202-205 Manning JE, Richards OC (1971) Biochim Biophys Acta 259: 285-296 Meeker RR, Thomas J, Tewari KK (1976) Plant Physiol 58:7176 Nicolas P, Hussein Y, Heizmann P, Nigon V (1980) Mol Gen Genet 178:567-572 Rawson JRY (1975) Acta 402:171-178 Rawson JRY, Boerma C (1976) Proc Natl Acad Sci USA 73: 2401-2404 Schiff JA, Epstein HT (1965) The continuity of the chloroplast in Euglena. In: Locke M (ed) Reproduction: Molecular, subcellular and cellular. Academic Press, New York London, pp 131-189 Southern EM (1975) J Mol Biol 98:503-517 Stutz E, Vandrey JP (1972) Euglena gracilis chloroplast DNA: some structural and functional aspects. In: Forti G, Avron M, Melandri A (eds) Proc. IInd Intern Congr. on Photosynthesis. Junk W, The Hague, puN., pp 2601-2609 Uzzo A, Lyman H (1972) The nature of the chloroplast genome of Euglena gracilis. In: Forti E, Avron M, Melandri A (eds) Proc. IInd Int. Congr. on Photosynthesis, pp 2585-2599 Wetmur JG, Davidson N (1968) J Mol Biol 31:349-370 Communicated by R. J. Schweyen Received December 14, 1981
Current Genetics (1982)5:9-15
© Springer-Verlag 1982
Modifications of Chloroplast DNA During Streptomycin Induced Mutagenesis in Euglena gracilis Philippe Heizmann, Younis Hussein*, Paul Nicolas, and Victor Nigon Universit6 Claude Bernard Lyon 1, D6partement de BiologieG6n6rale,43, Boulevarddu 11 Novembre 1918, F-69622 Villeurbanne Cedex, France
Summary. The evolution of chloroplast DNA was analysed during streptomycin induced mutagenesis in Euglena gracilis strain bacillaris and strain Z. In addition to a massive reduction of the cellular level of chloroplast DNA, several structural modifications have been observed in early stages of mutagenesis but they are generally eliminated during the later stages. The ribosomal cistrons are regularly rearranged: two of the three tandemly arranged cistrons occuring in wild type chloroplast DNA decrease while the third one is relatively more conserved and amplified during mutagenesis and in bleached mutants.
Key words: Chloroplast DNA - Mutagenesis - Ribosomal cistron - Amplification -Euglena
Introduction Euglena gracilis is particularly suitable for the study of mutations affecting the chloroplast system, due to its apitude to grow either phototrophically or on carbon substrates in the absence of photosynthesis. The efficiency of the photosynthetic machinery is thus optional in Euglena and cell survival is possible even in conditions of major chloroplast alterations. Moreover the chloroplast system of that alga is particularly sensitive to various mutagens whicfi can induce irreversible mass bleaching of the cultures. During the cell division following mutagenic treatments, the quantities of chloroplast DNA per cell decrease rapidly (Uzzo and Lyman 1972) and become undetectable by the classical physiochemical * Present address: Department of Genetics, Faculty of Agriculture, Assiut, Egypt Offprint requests to: P. Heizmann at the above address
techniques of DNA analysis in most stabilized bleached mutants (Schiff and Epstein 1965). Using more recent techniques for analysing DNA we demonstrated however the persistence of chloroplast DNA in bleached mutants, most often in very low amounts. These residual chloroplast DNA seem to maintain unchanged the major part of the wild type sequences; they show however some deletions or rearrangements (Heizmann et al. 1981). In the work presented here we analysed the qualitative and quantitative evolution of chloroplast DNA in cultures of Euglena undergoing the mutational bleaching induced by streptomycin. Our results confirm the massive reduction of the cellular content in chloroplast DNA and show the occurence of many transient structural modifications; most of these modifications seem to be eliminated from the progeny. Ribosomal cistrons however appear to be systematically rearranged: these modifications are particularly evident in the strain bacillaris where the three ribosomal citrons differ from each other by deletions located in the ribosomal spacers (see restriction map Fig. 1, made from data from Hallick 1982, Helling et al. 1979; E1 Gewely et al. 1981). The Eco R 1 restriction pattern of chloroplast DNA from the strain bacillaris differs from that of strain Z chloroplast DNA by the duplication of the multiple band Eco Lz into two different bands Eco Mbac and Eco Obac and by the duplication of the band Eco Pz into two different bands Eco Rbac and Eco Oba c (map Fig. 1 and restriction pattern Fig. 2). (We will use the nomenclatures proposed independently by Gray and Hallick 1977, for the strain Z, and by Helling et al. 1979, for the strain bacillaris.) The modifications induced during chloroplast mutagenesis result in the progressive disappearance of fragments Eco Obac and Eco Sba c and in the preferential conservation of fragments Eco Mbac and Eco Rba c. This preferential conservation results from the amplification of the ribosomal cistrons. O172-8083/82/0005/0009/$ 01.40
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
10 Barn
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Fig. 1. Restriction map for the ribosomal cistrons of strains Z and bacillaris, according to Gray and Hallick (1977) and Hallick (1982) (strain Z), and Helling et al. (1979) and E1-Gewely et al. (1981) (strain bacillaris). The nomenclature used is that proposed independantly by the two groups. The fragments Eco Obac and Eco Mbac from baciUaris are homologous to fragments Eco Llz and Eco L2z from strain Z; fragments Eco Rbac and Eco Sbac axe homologous to fragments Eco P2z and Eco P3z from strain Z. A probe made of fragments Eco L z and Eco Pz will label fragments Eco Bz, Fz, Lz and Pz in strain Z and Eco Bbac, Fbac, Obac, Mbac, Rbac and Sbac in strain bacillaris (Fig. 2). The DNA from the two strains differ essentially from each other by: 1. deletions in bacillaris affecting the Eco R 1 site located between Eco Plz and Eco Bz, and affecting about 450 base pairs in the spacer between Eco Llz and Eco P2z; the Eco R 1 site is conserved in this spacer. 2. the arrangement of sequences located between the "supplementary s16S" gene and the first ribosomal cistron
Materials and Methods
Strains and Culture Conditions Euglena gracilis strain Z (1224-5/25) was obtained from the G6ttingen Institute Algensammlung, the strain baeillaris as well as the mutants W3BUL, WloBSmL, Y1BXD and Y3BUL derived from bacillaris were obtained from Professor Schiff; ZHB was obtained from Professor Rawson and ZN2L from Doctor Calvayrac; W34ZUD, W35ZEmsD and W36ZHD were produced in our laboratory. All cultures were grown on an organotrophic medium using sodium butyrate as carbon source (= NCo medium from Freyssinet et al. 1972).
Streptomycin Induced Mutagenesis Etiolated Z or bacillaris cells were inoculated on NCb organotrophic medium supplemented with streptomycin (500 gg/ml at pH 7.4) and harvested after various length of growth in the presence of the drug. The proportion of bleached cells among the progeny of each aliquot was estimated by plating on Petri dishes.
Analysis of DNA Chloroplast DNA were prepared from isolated chloroplasts prepared by flottation according to Manning and Richards (1971). Pellets of cells or of organelles were resuspended in Tris 50 mM EDTA 50 mM - pH 9.0 and lyzed by addition of SDS (1% final concentration); NaC1 was added to 0,5 M and the mixture was extracted with an equal volume of aqueous phenol-chloroform (lv/lv). The ethanol precipitated DNA were dissolved in Tris
10 m M - EDTA 1 mM - pH 7.4, treated with RNase (50 ~g/ml - 1 h - 37 °C) and then pronase (1 mg/ml - 4 h - 37 °C), reextracted with phenol-chloroform and precipitated. Chloroplast DNA were further purified by one of two cycles of CsC1 equilibrium centrifugation. EcoR1 restriction fragments of chloroplast DNA were cloned by ligation with RSF 2124 plasmid DNA and transformation of E. eoli W4554. (Lomax et al. 1977). Southern blots were prepared according to Southern (1975) and hybridizations with cloned DNA probes or total radioactive RNA were carried out according to Jeffreys and Flavell (1977). Autoradiographs were exposed at - 7 0 °C with Kodak XR1 or XAR5 films and Ilford Fast-Tungstate intensifying screens. Restriction enzymes were purchased from Boehringer and T 4 ligase from Miles Laboratories; the enzymes were used following manufacturers recommandations. DNA were labeled in vitro with 32p nucleotides by nicktranslation (Jeffreys and Flavell 1977); total chloroplast DNA was iodinated by the method of Commerford modified by Meeker et al. (1976); radioactive nucleotides and iodine were from Amersham.
Estimations of Chloroplast DNA by Renatumtion Kinetics Total DNA in Tris 10 mM - EDTA 1 mM - pH 7.4 were sonicated to fragment sizes of 400-500 base pairs checked by eiectrophoresis. After addition of tracer amounts of 1251 labeled chloroplast DNA (0.5 ~ug/ml), they were denatured in boiling water bath for 10 minutes, then made NaC1 0.3 M NaCI Tris 10 m M - pH 7.4 and brought to 63 °C at the beginning of the renaturation. Aliquots were taken at various times; single stranded and double stranded DNA were separated by chromatography on hydroxyapatite. The proportions of both forms of DNA were estimated by scintillation counting. Experimental data were represented as
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
Fig. 2. Modifications of the ribosomal cistrons during streptomycin induced mutagenesis in Euglena gracilis strain baeillaris. An exponentially growing culture was added with streptomycin (500 ~g/ml). Total DNA were extracted from aliquots harvested at various moments after addition of the drug. Their Eco R 1 Southern blots were hybridized with a mixture of fragments Eco L z and Eco Pz cloned in RSF 2124 (specific activity about 80 x 106 CPM/~g). These probes hybridized with ribosomal fragments Eco Bbac, Fbac, Mbac, Obae, Rbac and Sbac in bacillaris (Helling et al. 1979) and Eco Bz, Fz, L z and Pz in strain Z (Hallick 1982), according to restriction map of Fig. 1. The common hybridization band between fragments B and F is due to a spurious hybridization of the vector plasmid. The band under fragment F is probably a partial digest. Slot 0 h: total wild type bacillaris DNA. Slots 12 h, 24 h, 4 g, 8 g, 16g: DNA from cells grown with streptomycin for 12, 24 h and 4, 8 and 16 generations. Slots W3 and WI0: DNA from mutants W3BUL and WIO BSmL derived from bacillaris. Slot Zct: purified chloroplast DNA from strain Z. Amounts of DNA loaded per slot: 0 h, 12 h, 24 h, 4 g: 3 ug. 8 g, 16 g, W3, W l 0 : 1 2 pg. Zct: 0.01 ug. Autoradiographic exposures: 0 h, 12 h, 24 h, 4 g: 2 days. 8 g, 16 g, W3, W10, Zct: 5 days
second order plots according to Wetmur and Davidson (1968), expressing the reciprocal of the fraction of single stranded DNA as a function of time. We verified experimentally that these curves are actual straight lines with regression coefficients higher than 0.98-0.99 and with slopes proportional to the concentration of chloroplast DNA initially present in the reassociation mixture. It is thus possible to calculate the unknown concentration of chloroplast DNA introduced among total DNA in reassociation mixtures by comparing the slope of second order curve with that concerning the renaturation of control chloroplast DNA of known concentration. From the cellular content of total DNA (2.5 to 3.5 pg/cell according to our estimations by perchloric acid extraction) one calculates the quantity of chloroplast DNA per ceil.
l1
Fig. 3. Relative amplification of ribosomal cistrons during mutagenesis. A Southern blot prepared with the same DNA preparations as for Fig. 2 was hybridized with a mixture of cloned Eco R 1 fragments from strain Z chloroplast DNA (fragments Eco H, I, J, L, M, P, Q, R, S, T, U and V) labeled by nick-translation to 80 x 106 CPM/~g
Results 1. Qualitative Evolution o f Chloroplast DNA 1.1. Ribosomal Cistrons in the Strain bacillaris.
The structural e v o l u t i o n o f chloroplast D N A have been followed in a culture o f etiolated Euglena strain bacillaris growing after addition o f s t r e p t o m y c i n . The total D N A extracted f r o m aliquots harvested at various m o m e n t s during mutagenesis were digested with E c o R1 restriction endonuclease; S o u t h e r n blots obtained with these digests were hybridized with E c o R~ fragments o f wild t y p e chloroplast D N A (strain Z) cloned and amplified in the plasmid R S F 2 1 2 4 (Fig. 2 and 3). During these bleaching e x p e r i m e n t s we have regularly observed a m o d i f i c a t i o n o f the ribosomal segments concerning the entire p o p u l a t i o n of chloroplast D N A molecules (Fig. 2). The overall level o f h y b r i d i z a t i o n obtained with ribosomal probes decreases progressively, particularly during the late stages after 8 and 16 generations o f growth w i t h streptomycin. It is remarkable that the h y b r i d i z a t i o n with fragments Eco Oba c and Eco Sba c decreases m u c h more rapidly than that with fragments Eco Mba c and Eco Rba c. The ribosomal restriction pattern evolves progressively f r o m the bacillaris type towards the Z type pattern. The ultimate stages of this
12
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
Fig. 4a-c. Degradations and rearrangements of chloroplast DNA during mutagenesis. The same protocol as in Figs. 3 and 4 was used for a culture of Euglena strain Z. (a) probe of total purified chloroplast DNA (60 xl06 CPM/t~g); (b) probe of fragment Eco Mz (80 x 106 CPM/gg); (c) probe of fragment Eco Lz (70 x 106 CPM/gg). Amounts of DNA per slot: 0 h to 24 h: 2 t~g. 6 g to 18 g: 10/~g. Exposures: 5 days at -70 °C, except for the lower part of a (12 days)
evolution are represented by the Eco R 1 restriction patterns obtained with the DNA from the two stabilized mutants W3BUL and WloBSmL derived from the strain bacillaris; these patterns are apparently not different from that obtained with DNA from the strain Z. Moreover, when Southern blots identical to those used for Fig. 2 are hybridized with a mixture of ribosomal probes (fragments Eco Lz and Eco Pz) and of nonribosomal probes (fragments Eco Hz, Iz, Jz, Mz, Qz, Rz, Sz, Tz, Uz and Vz) there is a similar decrease of the hybridization signal for all chloroplast DNA fragments at the beginning of the mutagenesis process. During the late stages on the contrary, after 8 and 16 generations, ribosomal fragments Eco Mbac and Eco Rba c disappear much less rapidly than non-ribosomal fragments or than the ribosomal fragments Eco Oba c and Eco Sbac. The end of this evolution is clearly visible in the case of the bleached mutant W3BUL where only the ribosomal fragments Eco Mbae, Eco Rbac, Eco Bbac and Eco Fba c are intensively labeled.
merits appear during the course of streptomycin induced mutagenesis (Fig. 4). Such unusual fragments can be observed at the level of fragments Eco Sz and Eco Tz, and Eco Qz and Eco R z during the late stages of mutagenesis (Fig. 4a, slots 12 and 18 generations) when a probe made of total chloroplast DNA is used. When cloned restriction fragments are used as probes they hybridize fragments both smaller and larger than the initial fragments. Smaller fragments appear either as discrete bands (Fig. 4b, slots 6 h, 24 h and 6 generations) or as smears (Fig. 4c, slots 3 to 24 h) and demonstrate intensive degradations occuring rapidly after the addition of streptomycin. Rearrangements are suggested by the formation of fragments larger than the intact fragments (Fig. 4b, slots 3 to 24 h). In the later stages of mutagenesis however, i.e. after 12 or 18 generations, fragments apparently identical to the initial wild type restriction fragments remain in large majority in the case of ribosomal fragments (Eeo Lz, Fig. 4c) and in the case of most nonribosomal fragments (Eco Mz, Fig. 4b).
1.2. Non-Ribosomal Cistrons. In some experiments the overexposure of autoradiographs revealed transient alterations affecting only a limited fraction of the entire chloroplast DNA population. Restriction fragments differing from the initial wild type chloroplast DNA frag-
2. Quantitative Evolution of Chloroplast DNA The second order plots representing the renaturation kinetics of chloroplast DNA are actual straight lines for
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
o:tl
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Table 1, Quantitative evolution of chloroplast DNA (= ct DNA) during streptomycin induced mutagenesis. The quantities of chloroplast DNA as numbers of molecules per cell were calculated from the slopes of the straight lines from Fig, 5 (column 4); they were also calculated in the hypothesis of simple dilution of chloroplast DNA among the progeny (column 5) Time
Generations Proportion of green colonies
ctDNA ctDNA molecules molecules per cell in hypothesis of simple dilution
0h 3h 6h 24 h 1 week 2 weeks 3 weeks
0 0.125 0.25 1 6 12 18
220 150 94 79 69 32 13
1 0.82 0.57 2 x 104 0 0 0
220 195 176 110 3.5 0.06 0.001
the DNA of wild type etiolated cells and up to 24 hours of streptomycin treatment (Fig. 5a). This means that during the initial stages of mutagenesis chloroplast DNA molecules reassociate as a single component homogeneous population. (Stutz and Vandrey 1972; Rawson and Boerma 1976; Chelm et al. 1977). The slope of each second order straight line allows to calculate the quantities of chloroplast DNA present in each aliquot by simple comparison with the slope of the straight line representing the renaturation of pure chloroplast DNA of
~
I
13
Fig. 5a and b. Reassociation kinetics of chloroplast DNA during the mutagenesis of Euglena strain Z. Chloroplast DNA (0.5 #g/ml) labeled in vitro with 125I (15 x 106 CPM/vg) was renatured either alone or in the presence of total DNA from cultures growing in the presence of streptomycin. The same preparations as those for Fig. 4 were used. Single and double stranded DNA were separated by hydroxylapatite chromatography. The kinetics were represented by their second order plots according to Wetmur and Davidson (1968) (1/% single stranded DNA as a function of time). Their slopes are proportional to the quantities of chloroplast DNA introduced in the renaturation mixture. The results of calculations have been reported in Table 1). Renaturation of: Labeled chloroplast DNA (0.5 #g/ml) (o). (a) addition of total DNA (480 ~zg/ml) from etiolated cultures growing for 0 h (z~), 3 h (o), 6 h (~), and 24 h (v) with streptomycin. (b) total DNA (1,920 ~g/ml) from cultures growing for 6 (v), 12 (=) and 18 (A) generations with streptomycin
Time (hours)
known concentration (see "Materials and Methods" for explanations). Table 1 shows that during the first 6 to 24 h the content in chloroplast DNA decreases more rapidly than if there was simple dilution of chloroplast genomes among the progeny (column 4). This corroborates the occurence of degradations suggested by the restriction patterns of Fig. 4 and also observed during mutagenesis induced by ultraviolet irradiation (Nicolas et al. 1980). After this early stage chloroplast DNA continues to decrease during 18 generations at least; however its content per cell is maintained at a level much higher than that expected if simple dilution occured. Net syntheses of chloroplast DNA take over again which allow the persistence of chloroplast genomes in bleached cells. The proportion of green colonies among the progeny drops very rapidly during the first hours of streptomycin action. After a few generations of growth in the presence of streptomycin the reassociation kinetics of chloroplast DNA occur in two phases: the second order plots form two straight lines with different slopes, which represent the renaturation of two classes of sequences having different reiteration frequencies. The complexity of the more reiterated class of sequences can be estimated by its span on the ordinates axis: it represents about 8% to 12% of the total complexity of wild type chloroplast DNA. The abundance of these sequences relative to that of the less repeated segments increases regularly during late mutagenesis as illustrated by the increasing difference of slopes for
P. Heizmann et al. : Chloroplast DNA Modifications During Mutagenesis in Euglenagracilis
14 Ref
W34 Cot35
EcoB
DNA Eco R1 digests: they labeled intensively fragments Eco Bz, Fz, Lz and Pz carrying the ribosomal genes and thus identified the repeated sequences as ribosomal sequences in bleached mutants (Fig. 6).
Discussion
1 7. Propagation and Stabilization of Modifications Induced During Mu tagenesis
~:~
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Fig. 6. Identification of repeated chloroplast DNA sequences in bleached mutants. 125I labeled chloroplast DNA (0.3 ~g/ml; 15 x 106 CPM/gg) were mixed with sonicated total DNA (3,200 gg/ml) from mutant W34ZUD, denatured and reassociated to Cot 35 (mole/liter x seconds). The reassociated fraction was isolated on hydroxylapatite and hybridized to Southern blots of chloroplast DNA strain Z. The renaturation of repeated sequences is terminated at Cot 35 while rare sequences are still essentially single stranded. The Cot calculated for the probe is 0.01 while its Cot1/2 is about 0.4, allowing less than 5% self-renaturation. Ref: chloroplast DNA (Z) hybridized with total labeled chloroplast DNA. W34 Cot 35: chloroplast DNA (Z) hybridized with the fast renaturing sequences of DNA from mutant W34ZUD
the two classes (Fig. 5b). The mode of synthesis of chloroplast DNA which operates after mutagenesis systematically amplifies about 10% of the plastid genome.
3. Identification of the More Reiterated Sequences in Bleached Mu rants The biphasic renaturation kinetics of chloroplast DNA appearing during mutagenesis regularly occured for DNA from stabilized bleached mutants. We identified the nature of the corresponding more reiterated sequences by their hybridization affinity with wild type chloroplast DNA. The reiterated chloroplast DNA sequences from the mutant strain W34ZUD were separated from rare sequences owing to their rapid reassociation: after renaturation at Cot 35 they were obtained as double stranded DNA while rare sequences were still single stranded, and isolated by hydroxylapatite chromatography. They were rehybridized to Southern blots of wild type chloroplast
The present study was performed on total DNA from cultures undergoing streptomycin mutagenesis; it gives a global description of individual events affecting the population of chloroplast DNA molecules, and thus only those concerning a significant fraction of chloroplast DNA were detected. Modifications appeared at the level of fragments Qz and Eco Rz during late mutagenesis (Fig. 4a). Among ten stabilized mutants analysed so far, a majority is affected on at least one of these two fragments (Heizmann et al. (1981). However the modifications observed for Eco M z during early mutagenesis (Fig. 4b) seem to be eliminated in the later stages where only apparently intact Eco M z fragments can be detected. None of the bleached mutants analysed carries any detectable modification of fragment Eco M z. This is also the case of the largest part of the chloroplast genome since Eco Qz and Eco R z are the only modified fragments detected among more than 50% of the chloroplast sequences already explored in bleached mutants. In several respects, chloroplast mutagenesis in Euglena is reminiscent of mitochondrial mutagenesis in yeast. However petite 0 - respiratory mutants contain defective mitochondrial DNA molecules consisting of repetitions of a wild type DNA sequence representing between 0.1% and 80% of the wild type genome (Borst and Grivell 1978); the amounts of mitochondrial DNA are about the same as in wild type cells. On the contrary the quantities of chloroplast DNA are generally very low in non photosynthetic bleached mutants of Euglena (about a 100 fold lower than in wild type cells) and very few modifications are apparent. While rearranged sequences are propagated and amplified during petite mutation induction, wild type forms of DNA sequences remain dominant in Euglena chloroplast in spite of mutational events but their quantities decrease considerably,
2. Amplification of Ribosomal Cistrons The preferential disappearance of some ribosomal fragments (Eco Obac and Eco Sbae) with respect to others (Eco Mbac and Eco Rbac) in the strain bacillaris and the progressive evolution of the restriction patterns from the
P. Heizmann et al.: Chloroplast DNA Modifications During Mutagenesis in Euglena gracilis
bacillaris type towards the Z type was regularly observed in all experiments. The first observations that the patterns from W3BUL (derived from bacillaris) are similar with those from Z strains made us suspect that our W 3 BUL strain had been mixed up with Z mutants; we omitted the data relative to W 3 BUL from our first report (Heizmann et al. 1981). The present results were obtained with two strains W 3 BUL and Wt0 BSmL recently provided by Professor Schiff. Several arguments indicate that this evolution corresponds to a quantitative modification and amplification of the ribosomal cistrons: the biphasic renaturation kinetics of chloroplast DNA indicates the occurence of two sequence classes in bleaching cells and in bleached mutants; considered alone this argument is not indisputable since this type of renaturation could be partly artefactual: repetitive components of nuclear contaminants of the chloroplast DNA used as probe would reassociate about as rapidly as amplified ribosomal chloroplast ribosomal DNA (Rawson 1975). Since these repetitive sequences account for 40% of nuclear DNA, nuclear contaminations should represent as much as 25% of the chloroplast preparations to rapidly renature 10% of the probe. Analytical ultracentrifugations of chloroplast DNA samples rule out this possibility since the nuclear band (p = 1.707 g/cm 3) represents always much less than 5% of the chloroplast DNA band (p = 1.686 g/cm3). The amplification of ribosomal cistrons during mutagenesis is more convincingly demonstrated: - by the relative increase of the ribosomal fragments Eco Mbae and Eco Rba c with respect to the non ribosomal fragments and to the ribosomal fragments Eco Obae and Eco Sbac providing real internal standarts. - by the very preferential hybridization of repeated chloroplast DNA from mutant Wa4 ZUD to the ribosomal segments of wild type DNA. -
3. Structure o f Amplified Ribosomal Cistrons The disappearance of fragments Eco Obac and Eco Sba c in the strain bacillaris affects the cistrons 1 and 2 (numbered according to Fig. 1). The corresponding Barn H 1 fragments are: Barn Ebac and Barn Fba e in bacillaris Barn Ez and Barn D z in strain Z. These Barn H a fragments are preferentially reduced with respect to those from cistron 3 in the Barn H 1 restriction patterns from bacillaris cultures during streptomycin induced mutagenesis (not shown), from Z mutants (particularly WZN2L and W36ZHD) and from bacillaris mutants (Y1BXD, Y3BUD) (Heizmann et al. 1981). -
-
15
These observations confirm the occurence of deletions extending from cistrons 1 and 2 but leaving cistron 3 relatively amplified. It is remarkable that the spacer between cistrons 1 and 2 is already deleted in the strain bacillaris, indicating that this region is highly mutable. The detailed determination of the structure of the ribosomal cistrons in bleached mutants will probably yield precious informations on the process of chloroplast DNA replication.
Acknowledgments. We thank Mr. P. M. Malet and Mrs. FaureSchwob for their excellent technical assistance, and Drs. G. Bernardi and J. Doly for helpful discussions and advices.
R e f e r e n c e s
Borst P, Grivell LA (1978) Cell 15:705-723 Chelm BK, Hoben PJ, Hallick RB (1977) Biochemistry 16: 782-785 De Deken-Grenson, M (1960) Arch Biol 71:270-340 E1-Gewely MR, Lomax MI, Lau ET, Helling RB, Farmerie W, Barnett WE (1981) Mol Gen Genet 181:296-305 Freyssinet G, Heizmann P, Verdier G, Trabuchet G, Nigon V (1972) Physiol v~g 10:421-442 Gray P, Hallick RB (1977) Biochemistry 16:1665-1671 Hallick RB (1982) In: Buetow DE, (ed)The biology of Euglena, Vol 4. (in press) Heizmann P, Doly J, Hussein Y, Nicolas P, Nigon V, Bernardi G (1981) Biochim Biophys Acta 653:412-415 HeUing RB, EI-Gewely MR, Lomax MI, Baumgartner JE, Schwartzbach SD, Barnett WE (1979) Mol Gen Genet 174: 1 10 Jeffreys AJ, Flavell RA (1977) Cell 12:429-439 Lomax MI, Helling RB, Necker LI, Schwartzbach SD, Barnett WF (1977) Science 196:202-205 Manning JE, Richards OC (1971) Biochim Biophys Acta 259: 285-296 Meeker RR, Thomas J, Tewari KK (1976) Plant Physiol 58:7176 Nicolas P, Hussein Y, Heizmann P, Nigon V (1980) Mol Gen Genet 178:567-572 Rawson JRY (1975) Acta 402:171-178 Rawson JRY, Boerma C (1976) Proc Natl Acad Sci USA 73: 2401-2404 Schiff JA, Epstein HT (1965) The continuity of the chloroplast in Euglena. In: Locke M (ed) Reproduction: Molecular, subcellular and cellular. Academic Press, New York London, pp 131-189 Southern EM (1975) J Mol Biol 98:503-517 Stutz E, Vandrey JP (1972) Euglena gracilis chloroplast DNA: some structural and functional aspects. In: Forti G, Avron M, Melandri A (eds) Proc. IInd Intern Congr. on Photosynthesis. Junk W, The Hague, puN., pp 2601-2609 Uzzo A, Lyman H (1972) The nature of the chloroplast genome of Euglena gracilis. In: Forti E, Avron M, Melandri A (eds) Proc. IInd Int. Congr. on Photosynthesis, pp 2585-2599 Wetmur JG, Davidson N (1968) J Mol Biol 31:349-370 Communicated by R. J. Schweyen Received December 14, 1981
