Plant Physiol. (1982) 69, 67-71
0032-0889/82/69/0067/05$00.50/0
Nucleotide Sequence Homology Exists between the Chloroplast
and Nuclear Ribosomal DNAs of Euglena gracilis'
Received for publication April 21, 1981 and in revised form August 4, 1981
STEPHANIE E. CURTIS AND JAMEs R. Y. RAWSON
Department of Botany, University of Georgia, Athens, Georgia 30602
ABSTRACT
The nuclear and chioroplast ribosomal DNAs from Eugkna were shown
to have specific regions of nucleotide sequence homology. The regions of
homology were identified by hybriization of restriction endonuclease DNA
fragments of cloned chloroplast and nuclear ribosomal DNAs to one
another. The regions of homolo between these two ribosomal DNAs
were In that part of the genes that code for the 3' end of the small rRNAs
(16S and 19S) and near or at the DNA sequences coding for the 5S RNAs.
The nucleotde sequence homology between these regions was estimated
to be approximately 94% by the melting point depression of a hybrid formed
between the two ribosomal DNAs.
Nucleotide sequence homology between chloroplast and nuclear rRNAs has been explored in a variety of plants (1, 12, 26,
28). All of these studies have shown hybridization between the
cytoplasmic rRNA and chloroplast DNA or reciprocally between
the chloroplast rRNA and the nuclear DNA. These results have
been interpreted in three ways: (a) nucleotide sequence homology
exists between the nuclear and chloroplast rDNAs2; (b) the cytoplasmic and/or chloroplast rRNAs used for these studies were
contaminated with one another, or (c) the chloroplast and nuclear
DNAs to which the different rRNAs were hybridized were contaminated with one another.
Final resolution of the question of nucleotide sequence homology between nuclear and chloroplast rDNAs required the use of
absolutely pure preparations of nuclear and chloroplast rDNAs.
Both the nuclear and chloroplast rDNAs from Euglena gracilis
have been cloned and individually isolated as homogenous recombinant DNA molecules (4, 20). These cloned rDNAs were used in
hybridization studies to demonstrate the presence of specific nucleotide sequence homology between chloroplast and nuclear
rDNAs and to localize those regions on restriction endonuclease
maps of the two rDNAs.
MATERIALS AND METHODS
Construction of Recombinant Plasmids. Euglena chloroplast
rDNA was cloned into the Bam HI restriction endonuclease site
of the bacterial plasmid vector pBR313 (20) to yield a recombinant
plasmid pVK52 containing a complete chloroplast rDNA repeat
6.2 kbp in length (Fig. 3). This plasmid was used to construct the
plasmid pECRBl which contains a 0.85-kbp nucleotide sequence
1 Supported by research grants PMC79-01596 and PMC80-07507 from
the National Science Foundation to J. R. Y. R. and by National Institutes
of Health Predoctoral Traineeship 5-T37-GM07103-03 to S. E. C.
2Abbreviations: rDNA, ribosomal DNA; kbp, kilobase pairs.
delineated by Eco RI and Bam HI restriction endonuclease sites
(W. H. Andrews, personal communication). This DNA fragment
represents the spacer sequence between the rDNA repeat unit on
the chloroplast genome and contains part of the 5S rRNA gene
(14, 15). An Eco RI chloroplast DNA fragment (Eco RI-P)
containing the complete 16S rRNA gene, two tRNA genes, and
approximately 6% 23S rRNA was cloned into the vector RSF2124
(14, 15, 20) to yield the recombinant plasmid pVK49. This Eco RI
chloroplast DNA fragment is completely contained in pVK52.
Construction of A Recombinants. The nuclear rDNA was plaque
purified from a library of recombinant DNA molecules consisting
ofthe A phage Charon 9 and Eco RI endonuclease DNA fragments
of E. gracilis DNA (4). The library was screened using '25I-labeled
cytoplasmic rRNA (4). The recombinant phage Ch9-ERD8 DNA
was purified and extensively mapped with restriction endonucleases (4).
Propagation and Isolation of Plasmids and A Recombinant
DNAs. Recombinant plasmids were propagated and isolated from
Escherichia coli as described earlier (17, 19). A Recombinant phage
were propagated in E. coli K802 (4, 19) and isolated as described
by Williams and Blattner (29). Phage DNA was prepared by the
method of B0vre and Szybalski (3).
Restriction Endonuclease Digestion of DNAs and Gel Electrophoresis. All restriction endonucleases were purchased from New
England Biolabs and were used as recommended by the supplier.
Restriction endonuclease DNA fragments were separated on the
basis of mol wt by electrophoresis in agarose slab gels. The gels
were prepared in E buffer (36 mm Tris, 30 mm NaH2PO4, and 1
mM EDTA) and run at room temperature at 1.5 to 2.0 v/cm for
12 to 16 h. The gels were stained and photographed as described
earlier (20). HindIII A DNA digestion products were used as mol
wt markers.
Preparation of Southern Imprints and Hybridization of Nucleic
Acids. DNA was eluted from agarose gels onto strips of Millipore
paper (HA, 0.45 pM) according to Southern (24). The Southern
imprints were preincubated in 6 x SSC (SSC; 0.15 M NaCl and 15
mM Na-citrate), 5 x Denhardt's solution (5), and 0.5% SDS for 2
to 3 h at 60°C. Radioactive DNA in 6 x SSC, 0.5% SDS, and
Denhardt's solution was hybridized to the Southern imprints.
Following hybridization, the Southern imprints were washed three
times for 5 min each, twice for a total of 30 min, and three more
times for 5 min each in an excess of 2 x SSC and 0.5% SDS at
600C. (In 2 x SSC, the Tm of a DNA duplex with a base
composition of 50%o is 94°C.) Autoradiographs were prepared of
the Southern imprints by exposing the filters to film (Dupont
Cronex-2DC) in the presence of intensifier screens at -80°C (27).
In Vitro Labeling of DNA and RNA. DNA was labeled in vitro
with [a-mP]dATP by nick translation (21). Following the reaction,
the volume of the mixture was increased to 0.50 ml with 10 mM
Tris-HCl (pH 8.0) and 1 mm EDTA and 100 ,ug E. coli tRNA
were added. The DNA was precipitated with 0.1 volume of 3 M
sodium acetate plus 2 volumes of ethanol and collected in a
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68
CURTIS AND RAWSON
microfuge tube. The precipitate was washed with 80%1o ethanol and
suspended in 10 mm Tris-HCl (pH 8.0) and 1 mm EDTA.
The cytoplasmic rRNAs (19S and 25S) were individually purified from cytoplasmic ribosomal subunits prepared (4) from the
mutant ZHB which contains a decreased level of chloroplast DNA
(11, 18). The RNAs were labeled in vitro with ['251]iodine (7).
Melting Curves of Hybrids. The Tm values of hybrids formed
between [ P]pVK52 DNA and Euglena chloroplast DNA or Ch9ERD8 DNA were determined by thermal elution from hydroxylaBatite columns. DNA-DNA hybrids were formed between
[ P]pVK52 and either chloroplast DNA or Ch9-ERD8 DNA by
hybridizing a vast excess (50,000:1) of the unlabeled DNA to the
radioactively labeled DNA in 0.12 M sodium phosphate (pH 6.8)
at 60°C for 2 h. The DNA-DNA hybrids were adsorbed onto
hydroxylapatite in 0.12 M sodium phosphate (pH 6.8) at 60°C and
the hydroxylapatite extensively washed with 0.12 M sodium phosphate (pH 6.8) at the same temperature. The temperature of the
column was raised in increments, equilibrated at each temperature
for 5 min, and washed with 0.12 M sodium phosphate (pH 6.8).
After the final wash with 0.12 M sodium phosphate (pH 6.8) at
greater than 97°C, the columns were washed with 0.48 M sodium
phosphate (pH 6.8) to assure that all radioactive DNA had been
melted from the column in the 0.12 M sodium phosphate buffer.
The fraction of [32PJDNA eluted from the column at various
temperatures was determined by TCA precipitation ofthe samples
onto Millipore filters and counting them in a scintillation counter.
Plant Physiol. Vol. 69, 1982
Nucleotide Sequence Homology between Nuclear and Chloroplast rDNAs. The most likely region of nucleotide sequence
homology between the nuclear rDNA and the chloroplast DNA
was the chloroplast rDNA. The possibility of such homology was
tested by hybridizing [32P]Ch9-ERD8 DNA and the two individual
['25I1cytoplasmic rRNAs (25S and 19S) to restriction endonuclease
DNA fragments of the plasmid pVK52. Both Ch9-ERD8 DNA
and the 19S cytoplasmic rRNA hybridized to specific regions of
the chloroplast rDNA. These data are shown in Figure 2 and
summarized in Table I. The 25S cytoplasmic rRNA showed no
sequence homology to the chloroplast rDNA (data not shown).
The 19S cytoplasmic rRNA hybridizes to a region of the
chloroplast rDNA (pVK52) which is most narrowly defined by a
1.4-kbp Eco RI-Hind III DNA fragment (Table I and Fig. 3A).
This region of the chloroplast rDNA encompasses the 3'-end of
the 16S rRNA gene and the 5'-end of the 23S gene. The nuclear
rDNA (Ch9-ERD8) also hybridizes to the l.4-kbp Eco RI-Hind
III fragment as well as to the 0.8-kbp Bam HI-Hind III and 0.8kbp Bam HI-Eco RI fragments. The 0.8-kbp Bam HI-Hind III
fragment contains the 3'-end of the 23S rRNA gene and a part of
RESULTS
Hybridization of Nuclear rDNA to Eugkena Restrction Endonucleases DNA Fragments. Studies on the nuclear rDNA of
Euglena (4) initially suggested the possibility of the existence of
nucleotide sequence homology between the chloroplast and nuclear rDNAs. In these studies, a cloned nuclear rDNA repeat unit
(Ch9-ERD8) was hybridized to Southern imprints of E. gracilis
var. Z DNA to test the arrangement of rDNA in the nuclear
genome. Ch9-ERD8 DNA hybridized to restriction endonuclease
DNA fragments which coincided with those predicted from the
map of the nuclear rDNA insert (4) and, in addition, to several
other DNA fragments (Fig. 1). Since there is no nucleotide
sequence heterogeneity in the nuclear rDNA (4) and Charon 9
DNA does not hybridize to Euglena DNA (data not shown), the
possibility existed that these other DNA fragments might be
explained by sequence homology ofthe nuclear rDNA to organelle
DNA (chloroplast and/or mitochondrial DNA).
FIG. 1. Hybridization of the nuclear rDNA clone (Ch9-ERD8) to
restriction endonuclease DNA fragments of Euglena DNA. E. gracilis var.
Z total DNA was digested with the indicated restriction endonucleases
and the DNA fragments separated by electrophoresis in 1.2% agarose gels.
The nuclear rDNA clone, [mPjCh9-ERD8, was hybridized to Southern
imprints of the fragments, and hybrids were detected by autoradiography.
The intense bands on the autoradiograph are hybrids between the nuclear
rDNA probe and genomic nuclear rDNA sequences. The arrows indicate
faint bands which are hybrids formed between the nuclear rDNA probe
and chloroplast rDNA sequences in the genomic DNA.
FIG. 2. Hybridization of nuclear rDNA probes to fragments of chloroplast rDNA. DNA from the recombinant plasmid pVK52 was digested
with the indicated restriction endonucleases. The DNA fragments were
separated by electrophoresis in 0.8% agarose gels and visualizd by fluorescence of the ethidium bromide bound to the DNA. The nuclear rDNA
['2511 19S and L125IJ25S cytoplasmic rRNAs were
to Southern imprints of the DNA fragments.
Hybrids were detected by autoradiography. I1251125S rRNA did not hy-
clone, [mPJCh9-ERD8,
individually hybridized
bridize to any DNA fragment (data not shown). A small amount of
hybridization of Ch9-ERD8 was detected in the region of the 8.7-kbp Bamn
HI DNA fragment (pBR3 13). This must be due to trapping of the smaller
6.2-kbp DNA fragment by the larger DNA fragments as evidenced by the
fact the Ch9-ERD8 did not hybridize to pBR313 DNA (data not shown).
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CHLOROPLAST AND NUCLEAR rDNA HOMOLOGY
69
Table I. Hybridization of Nuclear rDNA and Cytoplasmic rRNA to Chloroplast rDNA Restriction
Endonuclease Fragments
The recombinant plasmid pVK52 or pECRB I was digested with restriction endonucleases, the DNA fragments
separated by electrophoresis in agarose gels and Southern imprints prepared. Individual ['251Icytoplasmic rRNAs
(19S, 25S, 5S) or [32PJCh9-ERD8 DNA was hybridized to the Southern imprints containing DNA fragments
from pVK52. Either [1"IJcytop1asmic 5S rRNA or ['P]Ch9-ERD8 DNA was hybridized to the Southern imprint
containing DNA fragments from pECRBl. ['25IJ25S cytoplasmic rRNA did not hybridize to any DNA fragments.
pVK52
Plasmid
pECRB1
Enzyme(s)
used for
Eco RI + Hind III +
HI +
Bam HI
Eco RI
Hind III
Bam HI Bam
Hind III
digestion
Eco RI
Bam HI
8.7
10.78b
9 b, d
8.5
9.2b, d
8.41
9.2b, d
6
b c, d
2.8b, c
2.4b,
1.2
I.gb, d
c
1.0
1.7b, c
2.4b, c
1.1
1.0
0.8d
1.7b, d
0.6
0.05e
0.8b, d
1.0
0.8b, d
0.3
* Size of DNA fragments was measured in kbp.
b
DNA fragment hybridized to [32P]Ch9-ERD8 DNA.
c DNA fragment hybridized to
rRNA.
d
DNA fragment hybridized to [1251Icytoplasmic 5S rRNA.
e DNA fragment not detected in our gel system.
['25IJ19S
the 5S rRNA. The 0.8-kbp Bam HI-Eco RI fragment contains the
other part of the 5S RNA gene and approximately 0.7 kbp DNA
of unknown function. Since the 25S cytoplasmic rRNA does not
hybridize to the 23S chloroplast rDNA gene, we concluded that
the region of homology existing between this 0.8-kbp Bam HIHind III DNA fragment and the nuclear rDNA must be in the
region near or at the DNA sequence coding for 5S RNA genes
contained on both of these DNA sequences.
The converse experiments were performed by hybridizing the
cloned chloroplast rDNA (pVK52) to Southern imprints of the
cloned nuclear rDNA (Ch9-ERD8). Two subclones of pVK52
(pVK49 and pECRB1) were also used. The hybridization of
pVK52, pVK49, and pECRBl DNAs to Ch9-ERD8 DNA fragments is summarized in Table II and shown diagrammatically in
Figure 3B. The chloroplast rDNA probes show homology with
regions of the nuclear rDNA map which represent the genes for
19S and 5S rRNAs.
Measurement of Tm of DNA-DNA Hybrid Formed between
Chloroplast and Nuclear rDNAs. The degree of homology that
exists between the chloroplast and nuclear rDNA genes was
determined by measuring the Tm of DNA-DNA hybrids were
formed between the two rDNA sequences. Trace amounts of
[ 2P]pVK52 were hybridized with a vast excess of chloroplast
DNA or Ch9-ERD8 DNA. The hybrids were bound to and
thermally eluted from hybroxylapatite in 0.12 M sodium phosphate
buffer (Fig. 4). The Tm of the hybrid formed between pVK52
(chloroplast rDNA) and the chloroplast DNA and between
pVK52 and the nuclear rDNA were 84°C and 78°C, respectively.
Inasmuch as there is no sequence homology between the two
vectors (pBR313 and Charon 9) used to construct the recombinant
DNA molecules pVK52 and Ch9-ERD8, the only possible region
of homology between these 2 recombinant DNA molecules must
be the two rDNAs. The specificity of the nucleotide sequence
homology between the chloroplast and nuclear rDNAs was determined from the difference in the melting temperatures of these
duplexes. Since a 1°C drop in Tm indicates approximately 1%
base pair mismatch of a reassociated DNA duplex (7), those
regions of the two rDNAs which hybridize must be approximately
94% homologous to one another.
these genes in both algae and higher plants (1, 12, 26, 28). These
experiments were all performed using either cytoplasmic and/or
chloroplast rRNAs as hybridization probes and were subject to
criticism because the possibility of contamination between the two
rRNA species or the two DNAs species had not been excluded.
The most direct means of resolving this problem was to study the
sequence homology of cloned nuclear and chloroplast rDNA
sequences. When the cloned nuclear and chloroplast rDNAs of
Euglena were hybridized to one another, two distinct regions of
nucleotide sequence homology were observed. These two regions
of homology were not contiguous with one another and were each
1 kbp or less in size. One region of homology was shown to exist
between the cytoplasmic 19S rRNA gene and that part of the gene
coding for the 3' end of the chloroplast 16S rRNA. This observation is not surprising in that the 3'-terminus of eukaryotic 18S
rRNA molecules, although lacking the Shine-Dalgarno sequence,
are highly conserved and exhibit strong homology with the 3'terminus of the E. coli 16S rRNA (10, 22). The 3'-terminus of the
chloroplast 16S rDNA in Euglena has shown to be similar to the
3'-terminus of E. coli 16S rRNA (8) and probably functions in
mRNA recognition and binding during formation of an initiation
complex during protein synthesis as it does in E. coli (23, 25).
Undoubtedly much of the nucleotide sequence homology existing
between what is probably the 3'-end of the 19S cytoplasmic rRNA
gene and the 3'-end of the 16S chloroplast rRNA gene is related
to the functional significance of these sequences in the ribosome.
The second region of the homology that exists between the
chloroplast and nuclear rDNAs occurs near or at the DNA
sequences that code for the 5S RNAs. The nucleotide sequence
homology between these regions of the rDNAs is more difficult to
explain. Chloroplast 5S RNAs from several plants have been
sequenced (6) and compared to cytoplasmic 5S RNA (16) from
the same plant. There is considerable difference between the
nucleotide sequence of the chloroplast and cytoplasmic 5S RNAs
from these plants. Approximately 50%o residues are different and
there are few long stretches of nucleotide sequence homology
between the chloroplast and cytoplasmic 5S RNAs of the same
plant. Although there are differences between these 5S RNAs, it
is much less than the expected 75% if the two RNAs were
DISCUSSION
completely unrelated random sequences (6). Despite these fmdings
The existence of nucleotide sequence homology between the in higher plants, the present results from Euglena tentatively
chloroplast and nuclear rDNAs has been suggested in studies of suggest a greater degree of homology between the nuclear and
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Copyright © 1982 American Society of Plant Biologists. All rights reserved.
CURTIS AND RAWSON
70
A. Chloroplost rDNA
Ch9-ERD8
Ch9-ERD8
X5S
L19 5j
5S
Ch9-ERD8
23S
16
X_
E
I
II
I ~tRAsI
F_
u
O
X
o
X
LJ
.c
a
co
5S
Wi
I
I
cD
Plant Physiol. Vol. 69, 1982
Table II. Hybridization of Chloroplast rDNA to Nuclear rDNA
Restriction Endonuclease Fragments
The recombinant phage Ch9-ERD8 DNA, containing the nuclear
rDNA, was digested with restriction endonucleases, the DNA fragments
separated by electrophoresis in agarose gels and Southem imprints prepared. The plasmids pVK52, pVK49, and pECRBl, each radioactively
labeled with [3PJphosphorus, were individually hybridized to the DNA
fragments from Ch9-ERD8.
Restriction Endonuclease DNA Fragmentsa of A Recombinant
Containing Nuclear rDNA (Ch9-ERD8)
En-
zyme(s)
used for
digestion
B. Nuclear rDNA
pECRBI
14pVK49
pVK52
p.pVK52
1
*
Hind III
Eco RI
Bam HI Hind III
+ Eco
RI
Xho I
Hind III
+ Xho I
19.8
16.9
12.5
5.5
5.1
19.8
8.2
6. d
5.2
24.0b, d
13.5
4.0
3.4b, c
1.6
22 b, d
8.2
4.9
4.2
l15b, c, d
8.6
6.8
.
3.5b,
u
0
e~~
Ec3 E
-0
M
°E
X
X__
E oCE x
cr~~~~
1 Kbp
FIG. 3. Restriction endonuclease map of chloroplast and nuclear
rDNAs showing regions of nucleotide sequence homology between the
two rDNAs. The plasmid pVK52 consists of a 6.2-kbp Bam HI Euglena
chloroplast DNA fragment and the vector pBR313. The Bam HI fragment
contains a single gene for the 16S, two different tRNAs, 23S and 5S
rRNAs. The plasmid pVK49 contains a 2.6-kbp Eco RI chloroplast DNA
fragment carrying the complete 16S rRNA gene, two tRNA genes, approximately 6% 23S rRNA, and the vector RSF2124. The Eco RI DNA
fragment carried on pVK49 is completely contained in the Bam HI DNA
fragment of pVK52. The chloroplast restriction endonuclease DNA fragment carrying part of the 5S rRNA gene and contained on pECRBI is
also located on the Bam HI DNA fragment in pVK52. The recombinant
phage Ch9-ERD8 consists of Ch9-arms, a Ch9 stuffer fragment, and a
complete nuclear rDNA repeat containing the 19S, 25S, 5.8S, and 5S
rRNA genes. The minimum region defined by various restriction endonuclease sites to which each of these recombinant DNAs hybridize are
indicated by arrows above those DNA fragments.
chloroplast 5S rRNA sequences. Confirmation of this suggestion
awaits sequencing of these two 5S rRNAs or their respective
c
1.8
0.9
0.4
22.6b, d
8.2
7.8
6 lb, c
1.8
0.le
2.8b, d
2.6
1.5
0.1
o.le
3.4b,
c
1.6
1.5
O.le
* Size of DNA fragments in kbp.
b
DNA fragment hybridized to
DNA.
c DNA fragment hybridized to
d
DNA fragment hybridized to [32P]pECRB 1.
eDNA fragment not detected in our gel system.
I32P]pVK52
[32PJpVK49.
1.0
a 0.8
LLI
-J
I
<
z 0.6
0
0.4
I
E0)
IL 0.2
genes.
Although homology between nuclear and chloroplast rDNA
has been previously reported, the present study is the
first to demonstrate unequivocally that such homology is not due
to impure preparations of rRNA or rDNA species. The sequence
homology between the chloroplast and nuclear rDNAs of Euglena
is generally limited and may be attributed to the functional
properties of these sequences. Earlier studies on Euglena nuclear
and chloroplast DNAs (6) indicated an absence of rDNA sequence
homology. These findings may reflect the limited degree of Euglena nuclear and chloroplast rDNA homology which probably
would have been undetectable given the resolution of the techniques used by Groul et al. (9).
When the possibility of nucleotide sequence homology was
explored in the alga Chlamydomonas, using cloned nuclear and
chloroplast rDNAs, no homology was detected between these two
DNA sequences (13). At this time, it is not possible to determine
whether the variance in our observations using Euglena rDNA
with those made in Chiamydomonas is due to a difference in the
stringency of the hybridization conditions or is actually a true
sequences
60
70
80
90
100
TEMPERATURE (°C)
FIG. 4. Thermal elution profile of the hybrid between nuclear rDNA
and chloroplast rDNA. Radioactive pVK52 DNA was hybridized to a vast
excess of either Ch9-ERD8 (O-O) or chloroplast DNA (@- - -0),
adsorbed to hydroxylapatite columns and thermally eluted. The Tm of the
hybrid formed between [32P]pVKS2 and chloroplast DNA or Ch9-ERD8
DNA was 84°C and 78°C, respectively.
reflection of the difference in the evolution of the nuclear and
chloroplast genomes in these two algae.
Acknowledgments-We wisk to thank Mrs. Kim Dix for aiding in several of the
experiments reported here.
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