Current Genetics

CurrentGenetics (1984) 8:9-13

© Springer-Verlag 1984

Rearrangement of chloroplast ribosomal cistrons by unequal crossing-over in

Euglena gracilis Flamant, Philippe Heizmann, and Victor Nigon

Fr6d6rtc

Universit6 Claude Bernard Lyon I, D6partement de Biotogie G6n6rale et Appliqu6e, 43, Boulevard du 11 Novembre 1918, F-69622 Villeurbanne Cedex, France

Summary. The chloroplast DNA of a wild type photosynthetic variant o f Euglena gracilis (ATCC n ° 10616) with five ribosomal cistrons has been analyzed by restriction mapping. The results complete the electron microscope study o f Koller and Delius (MGG 1 8 8 , 3 0 5 , 1982); they support a model o f formation of the variant DNA b y rearrangement of the wild type ribosomal cistrons through unequal crossing-over. The recombination sites have been determined. The recombination model proposed also explains the formation o f the "Z-S" variant with a single ribosomal cistron (Wurtz and Buetow 1981).

Introduction

Key words: Chloroplast DNA - Ribosomal cistrons Rearrangement - Euglena

- the photosynthetic strain "Z-S" has a single cistron (Wurtz and Buetow 1981).

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The chloroplast DNA o f most Euglena strains carry three ribosomal cistrons arranged as direct tandem repeats plus a supplementary 16S RNA gene (= extra 16S or s l 6 S ) (Jenni and Stutz 1979). In Z strains the three ribosomal repeats are identical while in bacillaris strains short deletions have been demonstrated in some of the spacers (Fig. 1) (Helling et al. 1979). Additional variations have been reported to occur in the ribosomal region o f the plastome of Euglena :

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Fig. 1. Restriction maps of the ribosomal region in strains Z and bacillaris. The nomenclature for strain Z is from Hallick (1983). That for bacillaris is from E1 Gewely et al. (1982). The three repeated units are identical in strain Z, they differ in bacillaris due to the presence of three deletions: two of them (180 bp and 250 bp) are located in the spacer between cistrons 1 and 2; the EcoRI site is conserved; the thkd one (1.3 kbp) between cistron 1 and the extra 16S deletes one EcoRI site. Thus EcoB is fused with EcoP1 in strain bacillaris

10

-the bleached strain Y3BUD carries two cistrons (Ravel-Chapuis et at. manuscript in preparation). - the bacillaris strain ATCC 10616 has five cistrons. Koller and Delius (1982) demonstrated by electron microscopy that the ribosomal region of this strain includes in order: an extra 16 S gene n ° 1 with an inverted repeat sequence "I1"; two ribosomal cistrons n ° 1 and 2; a second extra 16S gene n ° 2 (with its inverted repeat "I1"); three ribosomal cistrons n ° 3, 4 and 5 (Fig. 4). On the basis of molecule length measurements, Koller and Delius also suggested that a sequence of about 10 kbp is missing in the genome of strain ATCC 10616. We built a physical restriction map of the chloroplast genome of this strain in order to refine our knowledge of its structure and to determine the mechanism which produced it. Our results suggest that it arose from wild type chloroplast DNA through a simple rearrangement by unequal crossing-over occuring at the level of the tandemly repeated ribosomal cistrons. The restriction map allowed us to identify one of the recombination sites. Modifications of the ribosomal cistrons appear to be quite frequent in the species Euglena graeilis probably as a result of the direct repeat arrangement of these ribosomal cistrons.

Materials and methods Strains and cultures. The wild type Z strain (1244-5/25) was obtained from the G6ttingen Institute Algensammlung and the bacillaris strain from Professor J. A. Schiff (Brandeis University). Another green photosynthetic strain originates from the American Type Culture Collection (bacillaris ATCC n ° 10616) and was provided to us by Doctor Barbara Koller (EMBL, Heidelberg) after identification by electron microscopy of the rearrangement of its ribosomal cistrons (Koller and Delius 1982). The three strains were grown phototrophically on a mineral medium and aerated with air/CO 2 (95/5) (Freyssinet et al. !972). Extraction and analysis of chloroplast DNA. Chloroplast DNA were extracted from chloroplasts isolated by flotation according to Manning and Richards (1971) and further purified by CsC1 gradient ultracentrifugation. DNA were digested by restriction enzymes purchased from Boehringer (EcoRI, BamHI, XhoI, BgllI and Bali) or from BRL (KpnI) following manufacturers recommandations. Digests were fractionated by 0.7% agarose electrophoresis and transfered to nitrocellulose filters (Southern 1975). Ribosomal fragments EcoP and EcoL (from Z strain chloroplast DNA ) cloned in RSF2124 were nick-translated and hybridized onto Southern blots (Jeffreys and Flavell 1977). The size of restriction fragments was estimated by comparison with that of EcoRI and HindlII digests of lambda phage DNA. Autoradiographs were exposed with Kodak XAR films with use of Ilford intensifying screens when necessary.

F. Flamant et al.: Rearrangement of chloroplast ribosomal cistrons

Results Chloroplast DNA from strains Z, bacillaris and ATCC 10616 were digested with EcoRI, BamHI, Bali, BgllI, XhoI and KpnI restriction enzymes. The digests were analyzed by 0.7% agarose gel electrophoresis. EcoRI, BamHI and KpnI patterns as visualized after ethidium bromide staining and UV illumination are displayed at Fig. 2. Hybridization with nick-translated ribosomal EcoP fragment (and EcoL; results not shown) were performed to identify the ribosomal and neighbouring sequences (Fig. 3).

Identification o f the nature o f strain A TCC 10616 Chloroplast DNA from strain bacillaris differs from that of strain Z essentially by the occurence of deletions in the leader regions of cistrons 1 and 2 (Fig. 1) (Hallick 1983; E1-Gewely et al. 1981). These deletions yield typical differences at the level of restriction patterns. Thus the EcoRI patterns of bacillaris DNA differ from Z patterns by: the doubling of ribosomal fragments (EcoP z -+ EcORbac plus EcOSbac; EcoLz -+ EcOMbac plus EcOObac); the comigration of fragments EcOAbae and EcOBba e on 0.7% agarose gels due to the increased size of EcOBba c produced by the fusion of EcoB and EcoP1 (Fig. 2A, lanes t and 2). The BamHI patterns of bacillaris DNA [two ribosomal bands: BamD-E (5.7 kbp) and BamF (5.2 kbp)] differ from the BamHI patterns of Z DNA [two ribosomal bands BamE (5.6 kbp) and BareD (6.9 kbp)] (Fig. 2B, lanes 1 and 2). The KpnI patterns of baeillaris show a doublet [KpnC 5.7 kbp) and KpnD (5.2 kbp)] while the two ribosomal KpnC fragments are identical in Z and form a single band (Fig. 2C, lanes 1 and 2). Bali and XhoI digests (not shown) provide also clear distinction between the two strains (Fig. 1). All restriction patterns converge to identify without any ambiguity the strain ATCC 10616 as a Z strain rather than as a bacillaris strain (Fig. 2, lanes 3) in contradiction with its supposed origin. In terms of physical map this means that none of the three deletions located in the ribosomal spacers of baeillaris DNA is present in the ATCC 10616 chloroplast genome. All the non-ribosomal fragments present in the patterns from strain Z were also present in the patterns from strain ATCC 10616.

F. Flamant et al. : Rearrangement of chloroplast ribosomal cistrons

11

Fig. 2. Restriction patterns of chloroplast DNA from strain bacillaris (1), Z (2) and ATCC 10616 (3). Digestion with: EcoRI (gel A); BamHI (gel B); KpnI (gel C). Arrows point to additional new fragments present only in ATCC 10616. Points indicate features characteristic of the strain bacillaris: the restriction patterns of DNA from strain ATCC 10616 do not show any of these features

Fig. 3. Identification of the ribosomal fragments from strains Z (1) and ATCC 10616 (2). Southern blots from gels identical to those of Fig. 1 and from others digests (BglII, Bali, XhoI) were hybridized with ribosomal EcoP fragment. The size of the additional fragments in ATCC 10616 is indicated in kbp

12

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Hybridization of restriction patterns by ribosomal probes In addition of the fragments normally found in Z type DNA, the digestion of ATCC 10616 chloroplast DNA produced with each enzyme a new fragment hybridizing the ribosomal probe EcoP. The size of these fragments was estimated in each case (Fig. 3): 4.5 kbp with EcoRI; 4.8 kbp with XhoI; 2.8 kbp with BamHI; 1.9 kbp with Bali; 9.5 kbp with KpnI. With BglII a new fragment of 4.5 kbp (deduced from the physical map of Fig. 4) comigrates with the BglH ribosomal fragment originating from the normal cistrons (Fig. 1); it cannot be clearly demonstrated in ATCC 10616. Since all the non-ribosomal fragments are common between Z and ATCC 10616 strains, it was assumed that the new fragments result from the rearrangement of the ribosomal fragments described by Koller and Delius (1982). A restriction map was built based on their results (Fig. 4A). All the new restriction fragments can be placed between restriction sites preexisting in the region of the ribosomal cistrons of wild type chloroplast DNA (see Fig. 1), without creation nor deletion of any new site. This

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Fig. 4. Physical restriction map and hypothetical mechanism of formation of chloroplast DNA from strain ATCC 10616. A: Additional fragments present only in strain ATCC 10616 were located on the physical map constructed from electron microscopy data by Koller and Delius (1982). All the new fragments fit between restriction sites preexisting in the ribosomal region as shown at Fig. 1. B: Unequal crossing-over between two molecules of chloroplast DNA type Z leads to the formation of one molecule with one single cistron (like Z-S; Wurtz and Buetow (1981) see letter joined. One recombination site is located within the extra 16S gene n° 2

map illustrates the particular composition of the extra 16Sgene n ° 2: the leader region of cistron 3 (separating the cistron 3 from the extra 16S gene n ° 2) carries all the restriction sites characteristic of the wild type leader region of cistron 1 (spanning between cistron 1 and the extra 16S gene) (Fig. 1); it also shows the inverted repeat I1 visualized by electron microscopy (Koller and Delius 1982); the leader region of the extra 16S gene n ° 2 (separating this gene from cistron 2) carries all the restriction sites of a wild type intercistronic spacer (Fig. 1). The extra 16S gene n ° 2 of ATCC 10616 is thus of hybrid nature, resulting probably from the recombination by unequal crossing-over of two wild type Z chloroplast DNA molecules after pairing of an extra 16S gene with a cistronic 16S gene (Fig. 4B). The analysis of the combined restriction patterns obtained with various enzymes does not support the assumption of a 10 kbp deletion in the ATCC 10616 genome (Koller and Delius 1982). Except for the modification of ribosomal fragments identified by hybridiza-

-

F. Flamant et al. : Rearrangement of chloroplast ribosomal cistrons tion we could not detect any difference between the restriction patterns from the DNA from Z and ATCC 10616 strains. The level of sensitivity provided by 0.7% agarose gel electrophoresis would probably have allowed to detect a 10 kbp deletion.

Discussion

Classification of euglena strains and structure of the ribosomal cistrons The restriction patterns presented here show close structural relationships between the chloroplast DNA from strain ATCC 10616 and DNA from strain Z, although the strain ATCC 10616 had been classified as a bacillaris type. Actually the distinction between Z and bacillaris strains is not based on any morphological clear difference. The two strains are supposed to differ on a nutritional basis (Wolken 1961): for instance, they accumulate different levels ofparamylum reserves and show different lag phases of chlorophyll synthesis during greening, when grown under indentical conditions (Freyssinet 1976). However taxonomical errors like those reported by Wurtz and Buetow (1981) seem to be rather common. A classification based on molecular criteria (i.e. the presence or absence of deletions in the ribosomal spacers) could avoid such errors.

Mechanisms of variation in the ribosomal region of Euglena Our results are perfectly consistent with the hypothesis that the rearrangement of the ribosomal cistrons having produced the ATCC 10616 strain occured through unequal crossingover between two wild-type molecules. They indicate that one of the recombination sites is located within the extra 16S gene n ° 2 of the strain ATCC 10616. It is remarkable that the scheme proposed for the formation of chloroplast DNA from ATCC 10616 (Fig. 4B) also explains the complementary formation of a chloroplast DNA molecule bearing a single ribosomal cistron analogous to that of the "Z-S" strain described by Wurtz and Buetow (1981). The digestion of this single cistron DNA would produce like that of the "Z-S" DNA: one single ribosomal BamF fragment - no EcoP nor EcoL, but only one EcoB and one EcoF ribosomal fragments. Thus recombinational events between ribosomal cistrons can be considered as more frequent in the species Euglena gracilis than in any other plant species (Palmer and Thompson 1982). The tandem repeat arrangement of the ribosomal cistrons in Euglena allows molecular

13 exchanges by unequal crossingover and have thus promoted the formation of chloroplast genomes with one, two, three and five cistrons. It must be noted however that differences between Z and bacillaris strains due to the occurence of deletions in the ribosomal spacers cannot be explained by this kind of rearrangement. The configuration as inverted repeats is supposed to stabilize the ribosomal genes (and perhaps the whole genomes) in other plant cells (Palmer 1983); this strengthens the idea that the rather high frequency of recombination observed in Euglena is correlated with the tandem arrangement of its ribosomal cistrons. Under normal growth conditions however, chloroplast DNA populations from Euglena cultures are found to be homogeneous. Some mechanism intimately related with the process of DNA replication must operate to maintain this homogeneity. This mechanism is disturbed by various treatments (streptomycin, heat, U.V. irradiation ...) which induce dramatic reduction of chloroplast DNA levels together with systematic rearrangements and relative amplification of ribosomal cistrons, probably closely analogous to those described for ATCC 10616 (Heizmann et al. 1982).

Acknowledgements. We thank Doctor Barbara Koller (EMBL, Heidelberg) for communication of her results and for gift of strain ATCC 10616, Mr. P. M. Malet and Mrs. C. Bosch for technical assistance. This work was supported by a CNRS grant (ATP 8210).

References

E1-Gewely MR, Lomax MI, Lou 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 V6g 10:421-442 Freyssinet G (1976) Plant Physio157:824-830 Hallick RB (1983) In: Buetow DE (ed) The biology of Euglena, vol 4 (in press) Heizmann P, Hussein Y, Nicolas P, Nigon V (1982) Curt Genet 5:9-15 Helling RB, E1-GewelyMR, Lomax MI, Baumgartner IE, Schwartzbach SD, Barnett WE (1979) Mol Gen Genet 174:1-10 Jeffreys AJ, Flavell RA (1977) Cell 12:429-439 Jenni B, Stutz E (1979) FEBS Letters 102:95-100 Koller B, Delius H (1982) Mol Gen Genet 188:305-308 Palmer JD, Thompson WF (1982) Cell 29:537-550 Palmer JD (1983) Nature 301:92-93 Ravel-Chapuis P, Flamant F, Nicolas P, Heizmann P, Nigon V (1983) (in preparation) Manning JE, Richards OC (1971) Biochem Biophys Acta 259: 285-296 Southern EM (1975) J Mol Biol 98:503-517 Wolken JJ (1961) Euglena. Rutgers University Press Wurtz EA, Buetow DE (1981) Curt Genet 3:181-187 Communicated by R. J. Schweyen Received July 18, 1983