Current Genetics

Current Genetics 3, 181-187 (1981)

© Springer-Verlag 1981

Intraspecific Variation in the Structural Organization and Redundancy of Chloroplast Ribosomal DNA Cistrons in Euglena gracilis Edwin A. Wurtz* and Dennis E. Buetow Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois 61801, USA

Summary. Chloroplast DNAs from six different laboratory collections of Euglena gracilis "strain Z" and var. bacillaris were analyzed with restriction endonucleases EcoRI and Barn HI. The most variable portion of the organelle genome is the region containing the ribosomal cistrons. Intraspecific differences occur in both ribosomal DNA cistron number (one or three) and structural organization among those strains designated as "strain Z" and bacillaris. One culture previously designated as " Z " is most likely bacillaris. Key words: Chloroplast - rDNA cistrons - Intraspecific variation - Euglena gracilis

Introduction The structure and organization of chloroplast (ct) genomes have been widely studied recently (Sager and Schlanger 1976; Gillham 1978; Bedbrook and Kolodner 1979, Buetow 1981). In particular, attention has focused on the number and location of ribosomal DNA (rDNA) cistrons carried by these genomes. In the case of Euglena gracilis, the ct genome was claimed to contain anywhere from one to six rDNA cistrons in a series of reports from different laboratories all of which used the technique of RNA: DNA saturation hybridization (Stutz and Vandrey 1971; Rawson and Haselkorn 1973; Scott 1973). However, recent physical maps of the Euglena ct genome determined with restriction endo-

* Present address: Laboratory of Radiation Ecology, Fisheries Center, WH-10, University of Washington, Seattle, Washington 98105, USA Offprint requests to." Dr. D. E. Buetow

nucleases show three tandemty-arranged rDNA cistrons (Gray and Hallick 1978, 1979; Jenni and Stutz 1978; Rawson et al. 1978). This structural organization differs from that in certain higher plant (Bedbrook and Bogorad 1976; Herrmann et al. 1976; Hobom et al. 1976; Bedbrook et al. 1977) and Chlamydomonas chloroplasts (Rochaix 1978) which contain two inverted rDNA cistrons. The present study was begun when we noted that the EcoRI restriction pattern of a Euglena ct DNA isolated from a culture designated as E. gracilis strain Z (Mielenz et al. 1977) differed from the pattern reported by others for strain Z (Stutz et al. 1976; Gray and Hallick 1977; Raws0n and Boerma 1979) but agreed with a pattern obtained from an unspecifed strain of E. gracilis (Lomax et al. 1977) later identified as E. gracilis variety bacillaris (Helling et al. 1979). The differences in the restriction patterns involved those fragments containing the rDNA cistrons. Therefore, we have analyzed the ct DNA from strains of E. gracilis obtained from different sources with the restriction enzyme EcoRI and Barn HI. The restriction patterns show that there is considerable intraspecific variation in E. gracilis both in structural arrangement and number of ct rDNA cistrons. This is the first report of an intraspecific variation in gene redundancy in the ct genome. Materials and Methods

Strains. The following E. gracillisstrains were used: 1) A strain (thought to be strain Z) obtained from C. Hershberger (Mielenz et al. 1977) who originally obtained it from J.A. Schiff; cloned about 6 times over a 6 year period by C. Hershberger for a fast growing, dark green culture; designated here as strain He. 2) A strain Z obtained from R.B. Hallick (Gray and Hallick 1977, 1978); designated strain Z-Ha. O172-8083/81/0003/0181/$01.40

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E.A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA

3) A strain Z obtained from J.A. Schiff; same as American Type Culture Collection #12894; designated strain Z-ATCC. 4) A strain Z obtained from J.A. Schiff; first though to be the same strain obtained from C. Hershberger above; designated strain Z-S. 5) A strain Z obtained in 1959 by D. Buetow from T.L. Jahn and J.A. Gross, University of California, Los Angeles; designated strain Z-UCLA. 6) A strain (vat. bacillaris) obtained from Jahn and Gross (above); designated strain baeillaris. Once obtained these strains were maintained photoautotrophically with monthly subculturing on a defined medium (Buetow 1965).

100 mM Tris-HC1, 10 mM MgC12, 50 mM NaC1, pH 7.5, for EcoRI (Miles Laboratories) restriction. Sufficient endonuclease activity was added to eliminate partially digested fragments. After incubation at 37 °C for 1 h, restriction fragments were resolved by electrophoresis on 0.7% (w/v) agarose (Sigma) gels (Mielenz et al. 1977). Gels were stained in 1 ~g/ml ethidium bromide in electrophoresis buffer for 30 rain and DNA bands were visualized with ultraviolet light. DNA restriction fragments and their molecular weights are designated in the present study as Helling et al. (1979) did for E. graeilis var. baeillaris. Hind III- or EcoRI-restricted X phage DNA (Beckman) fragments were used as molecular weight markers.

Chloroplast DNA Preparation. Two-2 1 flasks containing 1.5 1

Hybridization of Chloroplast rRNA to Restricted Chloroplast DNA. Ct DNA fragments containing rDNA cistrons were identi-

Hutners's medium (Greenblatt and Schiff 1959) were inoculated with 10 ml each of cells grown to the stationary phase of growth on a complex medium (Mego 1964). Cultures were grown at 28 °C with constant rotation (150-180 rpm) under white light illumination (115 ft. candles) to a density of 2-4 x 106 cells/ml, i.e., 1 - 2 divisions short of the stationary phase. Then the cells were harvested and the chloroplasts isolated by flotation (Gray and Hatlick 1977). The isolated organelles were washed extensively (6-10 times) in NET (0.15 M NaC1, 0.1 M Na4EDTA, 0.05 M Tris, pH 9.0), resuspended in 10 ml NET, and incubated with 500 #g/ml Proteinase K (Beckman) and 1% Sarkosyl for 12-16 h at 0 °C. The ct nucleic acids were extracted with 2 vol of 1:1 phenol:chloroform mixture. The aqueous phase was concentrated 5- to 10-fold in dialysis tubing placed in solid sucrose for several h, then made 0.3 M in sodium acetate, pH 8.0, and nucleic acids were precipitated at -20 °C for 2 - 4 h by the addition of 2 vol cold absolute ethanol. The precipitate was collected by centrifugation at 10,000 g for 10 min and resuspended in 5 - 1 0 ml NET. RNA was digested for 2 h at 37 °C with 50 /~g/ml pancreatic RNase A (Worthington) which was first heated to 100 °C for 10 min to destroy any DNase activity (Rawson et al. 1978). The DNA was precipitrated with cold absolute ethanol. This extraction technique avoids the loss of DNA incurred when purified by CsC1 equilibrium centrifugation (Mielenz et al. 1977).

Preparation and Iodination of Chloroplast Ribosomal RNA. Ct ribosomal RNA (rRNA) was obtained from E. gracilis strain Z-UCLA grown photoautotrophically on defined medium (Buetow 1965) in 18 1 carboys. Cultures were bubbled with 5% CO2-air mixture, illiminated (510 ft-candles) with cool-white fluorescent lamps and harvested at a density of 5 x 10 s cells/ml. Chloroplasts were isolated as before (Kissil and Buetow 1974) and lysed in 1% SDS. RNA was extracted from the lysate with 2 vol of 1:1 phenol: chloroform mixture. The aqueous phase was made 0.3 M in sodium acetate, pH 5.5, and mixed with 2 vol cold absolute ethanol. RNA was precipated for 12-16 h at-20 °C and then collected by centrifugation. The rRNA was purified from total ct RNA and DNA by centrifugation on sucrose-gradients (Avadhanl and Buetow 1972). The 16S and 23S rRNA peaks were eluted from the gradients and combined for hybridization experiments. Separated 16S and 23S rRNAs used in other hybridization experiments were obtained by polyacrylamide gel electrophoresis (Avadhani and Buetow 1972). RNA was labeled with 12SI (Amersham) as McCrea and Hershberger (1976).

Restrietion Endonuelease Analysis. One to 4 gg ct DNA was restricted in a 40 #i vol containing 100 mM Tris-HC1, 10 mM MgC12, pH 7.5, for Bam HI (Miles Laboratories) restriction or

fied by hybridization of 125I-labeled ct rRNA to restriction fragments immobilized on cellulose nitrate filters (Southern 1975). The filters were autoradiographed using X-ray intensifying screens (Swanstrom and Shank 1978).

Results Figure 1 shows the restriction patterns generated by E c o R I digestion o f the ct D N A f r o m 6 strain of Euglena. Bacillaris c o n t a i n e d all the D N A fragments f o u n d in the other strains. Thus, all the strains can be c o m p a r e d to bacillaris. Clearly, b o t h bacillaris and strain He showed the same restriction pattern which is designated here as the "bacillaris p a t t e r n " . Strains Z-Ha, Z - U C L A and Z-ATCC had the same restriction pattern as each other but this pattern differed f r o m the bacillaris pattern. This second pattern is designated here as the " Z - A T C C patt e r n " . Finally, strain Z-S showed a third restriction pattern (the " Z - S p a t t e r n " ) which differed f r o m b o t h the bacillaris and Z-ATCC patterns. Diffferences in these three E c o R I restriction patterns (Fig. 1) were as follows: 1) F r a g m e n t M (3.2 kb) was absent in the Z-S pattern, but present in the bacillaris and Z-ATCC patterns. The relative density o f staining indicates that fragm e n t M had a s t o i c h i o m e t r y of t w o in the Z-ATCC pattern and one in the bacillaris pattern. 2) Fragments O and P ( t w o fragments each with a molecular weight o f 2.95 kb) were b o t h present in the bacillaris pattern b u t only one was present in the Z-S and the Z-ATCC patterns. 3) F r a g m e n t R (2.48 kb) was absent in the Z-S pattern but present in the bacillaris and Z-ATCC patterns. However, the s t o i c h i o m e t r y o f fragment R was one for bacillaris and three for the Z-ATCC pattern (Rawson et al. 1978; Helling et al. 1979). 4) F r a g m e n t S (2.30 kb) was absent in the Z-S and the Z-ATCC patterns b u t present in the bacillaris pattern. 125I-iodinated ct r R N A ( c o m b i n e d 16S and 2 3 S ) h y bridized to E c o R I restriction fragments B and F in all

E. A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA

183

Fig. 1. Co-electrophoresis of EcoRI restriction endonuclease digests of ct DNA from 6 strains of E. gracilis on 0.7% agarosegels subsequently stained with ethidium bromide. Lane 1: X-phage DNA restricted by EeoRI; lane 2: bacilIaris ct DNA; lane 3: strain He; lane 4: strain Z-S; lane 5: strain Z-Ha; lane 6: strain Z-UCLA; lane 7: strain Z-ATCC. Arrows denote DNA bands which differ between the strains

Fig, 2. Autoradiogram of 125I-iodinated ct rRNA (combined 16S and 23S) hybridized to EcoRI restriction fragments of ct DNA from 6 strair~s of E. gracilis as Fig. 1. Letters on the right denote the EcoRI restriction fragments to which the labeled bands correspond. Fragment lengths (kilobases) are in parentheses

6 strains (Fig. 2). The bacillaris pattern strains also showed hybridization to fragments M, O, R and S while the Z-ATCC pattern strains showed additional hybridization to fragments M and R only. Strain Z-S did not show stoichiometric hybridization to any additional fragment. However, it did show slight, non-stoichiometric hybridization to fragments M, O, R and S none of which were visibly detectable in ethidium-bromide-stained Z-S restricted ct DNA (Fig. 1). Figure 2 verifies reports that EcoRI fragments B, F, M, O, R, and S carry the ct rDNA cistrons (Lomax et al 1977; Mielenz et al. 1977; Gray and Hallick 1978; Jenni and Stutz 1978; Rawson et al. 1978; Helling et al. 1979), supports the results in Fig. 1 showing that fragments M, O, R and S are not present in all 6 et DNAs, and shows that the differences in the three restriction patterns involve the rRNA cistrons. Next, BAM HI, which has only one recognition site within each tandemly repeated ct rDNA cistron in a strain Z E. gracilis (Gray and Hallick 1978), was used to elucidate the nature of the differences in the rRNA gone region shown by the various E. gracilis strains in Figs. 1 and 2. Barn HI DNA restriction fragments A and B were

not resolved (Fig. 3) but D, E and F are the fragments of interest. Fragment D was present only in the Z-ATCC pattern strains while fragment E was present in all the strains. As judged by its relative straining intensity, fragment E had an apparent stoichiometry of two in both the bacillaris pattern strains and the Z-ATCC pattern strains. The stoichiometry of fragment E in strain Z-S was not discernable since there were no neighboring bands with which to compare it. Fragment F was present with a stoichiometry of one in the bacillaris pattern strains but was absent from all the other strains. 12sI-iodinated ct rRNA (combined 16S and 23S) hybridized to Bam HI fragment E in all the strains and additionally to fragment F in the bacillaris pattern strains and to fragment D in the Z-ATCC pattern strains (Fig. 4). Ct rRNA did not hybridize to any additional Bam HI fragment from strain Z-S. There was no detectable hybridization in the case of any strain (Fig. 4) to the Barn HI fragments A, B or C seen in Fig. 2. The Barn HI restriction results (Fig. 3) again show three restriction patterns amont the 6 strain ofE. gracilis examined. Also, those strains falling into each to the

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E.A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA

Fig. 3. Co-electrophoresis of Bam HI restriction endonuclease digests of ct DNA from 6 strains ofE. gracilis (as Fig. 1). Lane 1: bacillaris ct DNA; lane 2: strain He; lane 3: strain Z-S; lane 4: strain Z-Ha; lane 5: strain Z-UCLA; lane 6: strain Z-ATCC;Iane 7.' X-phage DNA restricted with Hind III. Fragment D, E and F lengths (kilobases) are in parentheses Fig. 5. Autoradiogram of 12sI-iodinated ct 16S rRNA hybridized to EcoRI restriction fragments of ct DNA. Details as Fig. 2

Fig. 4. Autoradiogram of 12sI-iodinated ct rRNA (combined 16S and 23S) hybridized to the BamHI restriction fragments of ct DNA from 6 strains of E. gracilis as Fig. 3. The film was exposed to the radioactivity for 3.5 h. Details as Fig. 2

Fig. 6. Autoradiogram of 1251-iodinated 23S ct rRNA hybridized to EcoRI restriction fragments of ct DNA. Details as Fig. 2

E. A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA A) "Strain Z" I-I

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three patterns are the same as those falling into the corresponding EcoRI patterns derived from the data in Fig. 1. In further support of the EcoRI results in Figs. t and 2, those Barn HI fragments which contain the rDNA cistrons (Fig. 4) are the ones which differ in the three Bam HI restriction patterns (Fig. 3). Further information on the nature of the intraspecific differences in the ct rRNA gene regions can be obtained with EcoRI which restricts each rDNA cistron at two sites: (1) in the spacer region between the cistrons near the 5'-end of the 16S rRNA transcription region, and (2) near the center of each rDNA cistron just within the 23S rRNA transcription region (Gray and Hallick 1978; Jenni and Stutz 1978; Rawson et al. 1978). When restricted at the latter site, the cistron is divided into a 23S rRNA coding part and a 16S rRNA plus a small amount of 23S rRNA coding part. As expected, 16S rRNA hybridized EcoRI restriction fragment B in all strains, R in both the bacillaris pattern and the Z-ATCC pattern strains, and S in the bacillaris pattern strains only (Fig. 5). Again as expected, 23S rRNA hybridized EcoRI restriction fragment F in all six strains, M in both the bacillaris pattern and the Z-ATCC pattern strains, and O in the bacillaris strains only (Fig. 6). In the case of strain Z-S, low level non-stoichiometric hybridization occured between 16S rRNA and fragments R and S (Fig. 5) and between 23S rRNA and fragments M and O (Fig. 6). Some cross-hybridization occurred (Figs. 5, 6). All strains showed a slight non-stoichiometric hybridization to fragment F (Fig. 5), which most likely resulted from contamination of the 16S rRNA with a small amount of 23S rRNA. The low level of hybridization of 23S rRNA to fragments R and S (Fig. 6) was expected be-

4-1

I 2,.~,b

Fig. 7 A and B. Physical map of the rDNA region of the ct DNA from A) "strain Z" (Gray and Hallick 1978, 1979; Jenni and Stutz 1978; Rawson et al. 1978) and from B) baeillaris (Helling et al. 1979). Within the maps, arrows pointing down indicate the Barn HI restriction sites and the arrows pointing up the EcoRI sites. Numbers at the base of the arrows (1-1, 2-1, etc.) label Nven restriction sites. Arrows between the two maps indicate the approxima{e positions of deletions required to change the map of "strain Z" to that of bacilIaris. Brackets at the end of each arrow indicate the approximate extent of the deletion. Fragment lengths (kilobases) are as Hellinget al. (1979)

EcoRI-B

cause they contain a small part of this rRNA's coding sequence.

Discussion EcoRI and Barn HI restriction patterns indicate that the majority of the ct genome is the same in all 6 strains of E. gracilis studied. However, these patterns also show that at least three different ct genomes eydst in this species of Euglena and that the differences occur in the redundant rDNA cistrons. Figure 7 shows physical maps of the ct rDNA regions of "strain Z" E. gracilis based on the data of Gray and Hallick (1978, 1979), Jenni and Stutz (1978), and Rawson et al. (1978) and of bacillaris based on the data of Helling et al. (1979). The maps suggest an organizational relationship between the ct rDNA regions of the two strains as well as which structural changes in the organelle genome of one strain could lead to the structure of the organelle rDNA region of the other strain. In the following discussion, we shall speak of "deletions" in the ct rDNA region of"strain Z" (Fig. 7A) which could lead to the structure of the ct rDNA region of bacillaris (Fig. 7B). The term "deletion" is used here in an operational sense only and no temporal relationship between the two genomes in necessarily implied. Indeed, it will be clear that "strain Z" ct DNA could be described in terms of insertions in bacillaris ct DNA. Differences in the restriction patterns (Fig. 7) suggest that the bacillaris pattern results from 3 deletions in the "strain Z" pattern. The EcoRI-M fragment (3.20 kb) at the 3'-side of the rDNA region in '"strain Z" has a

186

E. A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA

Strain Z - S chloroplast rDNA cistron I-I

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BomHImE 5.68 kb

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2-1

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EcoR I - B

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0.25 kb deletion yielding an EcoRI fragment of 2.95 kb in baeillaris designated here as EcoRI-O. Another EcoRI fragment of the same size (2.95 kb) i.e., EcoRI-P, is present as a single copy in all 6 strains in Fig. 1. However, it does not contain any rDNA because it did not hybridize with ct rRNA (Figs. 2, 6). A second deletion of 0.18 kb comes from the middle EcoRI-R fragment of 2.48 kb in "strain Z" and results in the EcoRI-S fragment (2.30 kb) in bacillaris. A single deletion event causing a deletion in two adjacent restriction fragments, i.e., EcoRI-M and EcoRIR, does not explain the results. Such a deletion event would remove the restriction site separating the two fragments (Fig. 7). In this case the resulting single restriction fragment would carry a DNA segment coding for both 16S and 23S rRNA. However, this is ruled out because 16S (Fig. 5) and 23 S rRNA (Fig. 6) hybridized to separate fragments. Two deletions of 0.18 kb and 0.25 kb, respectively, also explain the change of the 5.68 kb Barn HI-E fragment in "strain Z" to the 5.25 kb Bam HI-F fragment in bacillaris (Fig. 7). The 0.25 kb deletion in "strain Z" must come between EcoRI site 2 - 2 and Bam HI site 3-1 so that the size of both the Barn HI-E and the EcoRIM fragment is reduced by 0.25 kb. This deletion presumably occurs in the non-rDNA region (Gray and Hallick 1979) between the central and the right-hand rDNA cistron (Fig. 7). The 0.18 kb deletion occurs between EcoRI sites 2-1 and 2 - 2 . It is not known whether or not this deletion occurs in a transcribed region. A third deletion of 1.3 kb reduces the 6.98 kb Barn HI-D fragment in "strain Z" to a 5.68 kb Bam HI-E fragment in bacillaris (Fig. 7). This deletion must remove the EcoRI site 3 - 2 found in "strain Z" (Rawson et al. 1978) because this site is missing in bacillaris (Helling et al. 1979). This study, initiated to examine differences between the ct DNA of E. graeilis "strain Z" cultures used in this and in other laboratories and the ct DNA of strain He, shows that the latter is most likely bacillaris and not Z (also asserted by Helling et al. 1979) because it behaves like bacillaris does in all measurements (Figs. 1-6). However, an unexpected result of this study was the discovery (described below) in strain Z-S of a third

type of ct DNA which contains only one complete rDNA cistron (Fig. 8). This result shows that not only the arrangement (cf. Helling et al. 1979) but also, for the first time, that the number of ct rDNA cistrons can vary in a single species. Following EcoRI digestion of ct DNA from strain Z-S, only fragments B and F hybridized ct rRNA to any great extent (Fig. 2). Following Barn HI digestion, this rRNA hybridized only to fragment E. Since EcoRI fragments B and F are present in one copy each in the ct rDNA region of Euglena. Figs. 1,2, 7, see also (Gray and Hallick 1977, 1978; Helling et al. 1979), only one rDNA cistron is present in strain Z-S. Figure 8 shows the apparent physical map of this rDNA cistron. EcoRI site 3 - 2 (Fig. 7A) must be absent in strain Z-S as in bacillaris (Fig. 7B) because, if present, then ct rRNA would have hybridzed stoichiometrically with another EcoRI fragment, probably R (cf., Fig. 7A). This did not occur (Figs. 2, 5). A small amount of ct rRNA did hybridize to EcoRI fragments M, O, R and S of strain Z-S ct DNA even though these fragments were not seen with staining (Fig. 1, lane 4). Autoradiography of RNA-DNA hybridization to restriction fragments is the more sensitive assay and allows the detection of fragments present with a stoichiometry of less than one. The source of this genome heterogeneity in strain Z-S is unknown and could be due to inter- or intracellular heterogeneity or even to inter- or intrachloroplast heterogeneity in the organelle DNA. Small amounts of heterogeneity have been reported before in the ct genomes of higher plants (Bedbrook and Kolodner 1979; Vacek and Bourque 1980). In regard to our finding of an intraspecific variation in the number of ct rDNA cistrons in E. gracilis, it is interesting to note that the ct DNA of a strain (also designated "Z") of E. gracilis Klebs obtained from the Culture Collection of Algae at the University of Texas (Starr 1978) contrains a supplementary 16S rRNA cistron in addition to the usual three complete ct rDNA cistrons (Jenni and Stutz 1979). Thus, one or three complete rDNA cistrons and one, three or four cistrons for 16S rRNA can be found on the ct DNA ofE. gracilis. This intraspecific variation in the number of rDNA cistrons in the chloroplast is reminiscent of the variable number of nuclear rDNA cistrons in different lines, strains and varieties of a number of higher plant species (Mohan and Flavell 1974; Flavell and Smith 1974; Cullis and Davies 1975; Ramirez and Sinclair 1975; Givens and Phillips 1976; Yampol and Pospelov 1978; Maggini et al. 1980). This study of the structure of ct DNA from several different laboratory collections of E. gracilis strain Z and var. bacillaris indicates that the most variable region of this genome is that portion which contains the rDNA cistrons. Intraspecific differences resulting possibly from deletions (or insertions) occur in rDNA eistron number

E. A. Wurtz and D. E. Buetow: Euglena Chloroplast rDNA

as well as in structural organization. Although no temporal ordering of these differences can be concluded as yet, a study of the ct genome of the many different Euglenoids may provide insight into the evolution of organellar multicopy genomes. Acknowledgements. This study was supported in part by NIH grant no. GM 22431 and NSF grant no. PCM 76-20687. We thank Dr. T Gallagher for preparing the 16S and 23S ribosomal RNAs.

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C o m m u n i c a t e d b y F. Kaudewitz Received February 12, 1981