Structural and Functional Characterization of the Intergenic Spacer

Plant Cell Physiol. 37(2): 233-238 (1996)
JSPP © 1996
Short Communication
Structural and Functional Characterization of the Intergenic Spacer Region
of the rDNA in Daucus carota
Akihiro Suzuki 1 ' 2 , Shigeyuki Tanifuji1, Yoshibumi Komeda1 and Atsushi Kato'
1
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060 Japan
The intergenic spacer (IGS) region of rDNA in Daucus
carota contains at least eight kinds of repeated sequence.
One sequence may act as a genuine initiation site for a
stable transcript and another may act as a spacer promoter
that may be an entry site for proteins required for transcription.
Key words: Daucus carota — IGS — Promoter — rRNA
gene — trans factor — Transcription.
In higher eukaryotes, genes for ribosomal RNA
(rDNA) are organized in tandem arrays at the nucleolar
organizing regions of chromosomes. Each repeating unit
usually contains one coding sequence for each of the 18S,
5.8S and 25-28S rRNAs, and three spacer sequences. The
intergenic spacer (IGS) region from the 3' end of the gene
for 25-28S rRNA to the 5' end of the gene for 18S rRNA
consists of untranscribed spacer and transcribed spacer
regions. In many organisms, various types of repeating
sequence have been found in tandem arrays in the IGS
region, and variations in the numbers of such repeating sequences usually cause the length polymorphisms of rDNA
repeating units (Appels and Honeycutt 1986). Many studies
on the transcription of rRNA in animals have demonstrated that the IGS region contains promoter sequences
for transcription of rRNA and that some of the repeating
sequences found in IGS region have enhancer functions for
the transcription of the rRNA (Sollner-Webb and Tower
1986). In Drosophila and Xenopus, some of the repeats in
the IGS region were shown to have an enhancer function
for the transcription of the precursor rRNA. They appear
to act as entry sites for transcription factors (Coens and
Dover 1982, Reeder 1983). The protein factors required for
transcription have been analyzed in some species (Jantzen
et al. 1990, McStay et al. 1991, O'Mahony and Rothblum
1991).
In plants, studies on the mechanisms of transcription
of genes for rRNA have been initiated. The structure of the
2
Present address: Laboratory of Cell Genetics, Department of
Cell Biology, National Institute of Agrobiological Resources,
Kannondai, Tsukuba, Ibaraki, 305 Japan.
IGS and the consensus sequence surrounding the initiation site have been reported in several plants (DelcassoTremousaygue et al. 1988, Gerster et al. 1988, Vincentz and
Flavell 1989, Kato et al. 1990, Perry and Palukaitis 1990,
Polanco and Vega 1994, Da Rocha and Bertrand 1995).
In Arabidopsis, a spacer promoter, reflecting the duplication of part of the promoter sequences, was reported
(Gruendler et al. 1991). Furthermore, it was reported that
the upstream repeated sequence containing a spacer promoter increased the rate of transcription from the promoter of a Xenopus ribosomal gene in injected frog oocytes,
eventhough the enhancer activity was very limited in transfected protoplasts of Arabidopsis (Doelling et al. 1993).
Protein fractions that interact with a certain sequence in
IGS regions in a sequence-specific manner have been described for several plants (Echeverria et al. 1992, 1994,
Jackson and Flavell 1992, Nakajima et al. 1992, Schmitz et
al. 1989, Zentgraf and Hemleben 1992, Suzuki et al. 1995).
In Arabidopsis, it was reported that bending of DNA occurred as a result of binding of a specific protein and that
the protein was related to the high mobility group (HMG)
proteins (Christine et al. 1995).
Here, we present the complete nucleotide sequence of
the IGS region of the rDNA from Daucus carota. Figure
1A shows the complete nucleotide sequence of 5,775 bp of
the IGS region of rDNA cloned from Daucus carota. The
borders between the spacer and the coding regions were inferred from sequences reported for other plants. From an
examination of a dot matrix self-comparison of the IGS
region, we found several repeated sequences. A diagrammatic representation of the organization of the IGS region
is shown in Figure IB. At least eight kinds of repeated sequence, designated A, B, C, D, E, F, G and H, respectively, were found in this region. In each repeated sequence,
the extent of the sequence similarity ranged from 68%
to 98% (Fig. 1C). The repeat designated G consisted of a
tandem array of three complete copies and one truncated
copy of a 455 bp sequence. The existence of this repeat sequence was reported previously (Taira et al. 1988). The
difference in the number of iterations of this repeat sequence causes the length heterogeneity of the rDNA repeating unit of Daucus carota (Taira et al. 1988).
The last repeat sequence, repeat H, consisted of two sequences of 228 bp, which were separated by a 1,909 bp se233
234
A. Suzuki et al.
CCCTGCCCCATCAAAAATGCACCAAGTAAACGACGAnrfirTATATArraTAKTrnnAAnGCGATAAAATATTTATAnAArcArAAGTArATATACAAAAr.A 100
TTCrrCrnAGArAAC TACATTTAAAATAATTATAnAAGACAAnTArATCTAAAAAnrtAGnrAAAAAAAftr.TAAnrGTRGATArTrGAnAArGTGTTrnAA
A2
GGrGrGrnrGnh'nrnrTrGTTnnnGrGrTrrTr soo
^MnTAGATncnrrTTTnTnTTTrATrrRnr.rrrArnAAAAAATATCGAAAAATAACAGAAAACTCATGTCCAG
TA
HI
GnnAGGGGGT 1000
1GCCBXGGGCGCCTCCCTCGACGGGCAGTTTC
1500
ACGCGCGCGCGTACGATCGTCGGTCGTCGGICCGGGCGCGCTTAGGCGCCTTTGGGCCGACGGGCACGGCGTCGGGCCTGGGCGCCGCTGCTCGGGCGCI
TfifiGrncrTTGGGGrrrGGAGGrrTnG
CTrTCrrAATAATTrAAAAAAAAAr.ATTTATAAAATArAAAATAAATATTAAAAAAATrTGAAAAAATrrnAAAAAAATAArTAAGAAArTAGATTTTTr 3000
H2
AAnrTTyGATTrGGTTATTrAAArnTrGAAAArGGATAAGrGGAhrTrCAAAAAAGGGGGGTfiTftGTTCCTfTGAArACATTTGTTrAAArTfSrTGGAAAT
KiEITGTCGCCCCTCCCTGGGCAGTGGGGTTGGGCGCTCGGTAGGCGCGCGCGGGGCCGTCGTTGGG
3500
TGAAGGGGGCATGTTCCAACTAAAAACTCCCCTCCCCCrfrcGCTGAGGAGCCCTTGGGCTCTTTTGCTTGTGGGGAACAACGTTGGTGTGATTAGGATCA
TGTGGCGTGCTTCGCGTAGACTTGATGGTATTTTGCCCAAAGACACTCCTATGGGGTGCTCGGCCTAGACCTGATGGTTCGTGCTAGGACATTCTGCGGG
GGAAACTTCCCCCCTCCCGCTGGGAAGATTTTTAAGAGACCTGATGGTTGTGTTCCCAAAGATATTCATGTGGGGTGCCTGGCGTAGACTTGATGGTATT
4500
TCTTCCCTCCCCTGGCGGGGCTGCCAACTCTGGTGTCCTCGGAGTACGCCGGTGCTTGGCGCTGCTAGGqCGCTTAGGAGTCCTCGGGCTCTTTGGGCGT
GTCCTCGGATGACGCCGGTGCTTGGCGCTGCTAGGGbcGCTGAGGAGTCCTCGGGCTCTTTGGCGTTTGGGAAAAACGTTGGCGTGAATAGGGTCAGACA
ACGGAGACCGTCCCGGAGTGGCCAAA'ljrTGCGCATAGCGTTCACGTTGCTTACACTACTGTGGGTTGATTTCTTTTAGACCTTTGGGTTTTGAAGGGTTC
5500
CCGTTGCGCTTTGTGTTGAGTAATGGGTGCTAATGTATCTTTTGCTCATGCTTAGCCTTCGGCCTGAAGGAATGC
Fig. 1A
Intergenic spacer of the rDNA in Daucus carota
HI
//H
235
D1 D2 D3
yp|
H2
Gl
G2
G3
G4
Ikbp
Fig.
IB
Fig. 1 (A) Nucleotide sequence of the IGS region of Daucus rDNA.
The plasmid DERI used in this study was described previously
(Taira et al. 1988). Eight families of repeated sequences are indicated by underlining or boxes (A, B, C, D, E, F, G and H). Boldface A
residues at positions 990 and 3,127 are putative sites of initiation of transcription.
(B) Diagrammatic representation of the IGS
region of Daucus carota rDNA.
The copies of each repeated sequence are boxed and marked with alphabetical letters followed by
numerals, sp, spacer promoter; gp, gene promoter.
(C) Alignment of members of each family of repeated sequences shown in Figure
1A and sequence homology.
Hyphens indicate the same nucleotides, and plus signs indicate gaps introduced to maximize homology.
Two members of the families of repeats A, E and H were directly compared with each other in order to calculate sequence homology. In
other cases, consensus sequences (C.S.) were determined and the sequence homology was calculated for each member by comparing it
with the consensus sequence. X represents an undetermined nucleotide in a consensus sequence.
quence that contained repeats D, E and F. Their sequences
showed a high degree of conservation (98%). Furthermore,
each unit of repeat H contained the TATATAGGG motif which is a sequence that is conserved around the site
of initiation of transcription of plant rRNAs (DelcassoTremousaygue et al. 1988, Gerster et al. 1988, Kato et al.
1990). The sequence of repeat H corresponded to positions
— 217 to +11 relative to the presumed site of initiation of
transcription. Thus, the IGS region of the gene for rRNA
of Daucus carota contains multiple putative promoter
sequences, as does that of Arabidopsis (Gruendler et al.
1991), Oryza (Cordesse et al. 1993), Xenopus (Reeder 1983)
and Drosophila (Coens and Dover 1982). In each of these
rDNAs, it was reported that one of the reported promoter
sequences is the true promoter while the others may act as
enhancer sequences. The sequence HI was designated a
spacer promoter and the sequence H2 was designated a
gene promoter, eventhough the true promoter sequence is
unknown.
In order to determine the 5' site of the initiation of transcription of rRNA in Daucus carota, we carried out SI
nuclease protection analyses using both a fragment that
contained a gene promoter (positions 3,001 to 3,289) and a
fragment with the spacer promoter (positions 864 to 1,099)
as probes. When the fragment containing the gene promoter was used as the probe, a clear protected band was detected. The initiation site for the RNA transcript that gave rise
to this protected fragment corresponded to the A residue at
position 3,127 (Fig. 2). This result agrees with previously
reported results (Delcasso-Tremousaygue et al. 1988, Doelling et al. 1993, Gerster et al. 1988, Kato et al. 1990). By contrast, when the fragment containing a spacer promoter was
used as the probe, several bands were detected. Two major
bands corresponded to the A residues at positions 990 and
986. One of them, that corresponding to the A residue at
position 990, also corresponded to the A residue at position
3,127 of the gene promoter, as deduced from a comparison
of the surrounding sequences. However, these bands were
weak and disappeared when the concentration of SI nuclease was increased. These results indicate that the gene
promoter acts as the dominant cis element for the transcription, together with the possibility that the spacer promoter
also acts as such an element in vivo. Similar results, with
two such promoters being active in vivo, were reported for
the transcription of the rDNA of Arabidopsis thaliana
(Doelling et al. 1993).
In the case of Arabidopsis, the transcripts from both
promoters were clearly detected, it is unclear why we failed
to detect a clear transcript from the spacer promoter but
the following explanation is suggested. The nucleotide sequences of the gene promoter and spacer promoter were
highly conserved and this conservation extended from position — 217 to position + 1 1 . The gene promoter had a long
AT-rich sequence in its upstream region, while the AT-rich
sequence located in the upstream region of the spacer promoter was short. AT-rich sequences upstream of the rDNA
promoter may be important for the transcription of rRNA
in plants (Echeverria et al. 1992). Thus, the difference in
length of AT-rich sequences might be related to the transcriptional activity of rRNA in Daucus carota.
In order to study DNA-protein interactions, we performed gel-retardation assays using fragments derived
from the gene promoter and spacer promoter regions. A
crude nuclear extract was partially purified by chromatogra-
236
A. Suzuki et al.
Repeat-A
Al
A2
A
C—A
G
++
++
ATAAGA+GCTTAAAAATAGAGGAGAAGCACAAGTGTCAGACATAGGGCAGGGTGAGCCCGT-K3TTCCCCCGAGA
C
C
TGA
C
91%
Repeat-B
C. S. GTAG+ATGCXGGCCTTTGTGTTTCGCCCGGGCGC+CGA
Bl •
G-G
95%
B2 C — T C
A
C
G
T
82%
B3
++
AT
C-A
84%
Repeat-C
C. S . ++TGCGCTCGTTGGGGCGCTCCTCGGGCGCTCTTCAGGCGCXTCCTGGGGCGCGCGCGG
Cl
+
-H-A
92%
A
C2
+
98%
C3 GGG
T
G
T
A
+
TTTT— 80%
Repeat-D
C . S. GGGGCGCCTCCCTCGACGGGCAGTTTCCTCTrGATAAAACACACACACGCGCGCGCGTACGTGXATGCGTTTGCGTCGG
Dl
C
CG
D2
CG
D3
+++—+++++++
G
+
A
TGAC
+++
+
+
QG
+
+
GGCCCTGTGGGC+CCTTGGGGCTCCGATGCCTC
—G—CTGT
G
9 3%
+
G
+
92%
G
G
C-G—G
88%
Repeat-E
El GCTCCTCAGGCCGCCCGGACTTGTTGGGCCCTCA+GGC
E2
G-TC-TC
87%
Repeat-F
C. S . GCTCGTCXAGCGCTX+TCAGGCGCG+CGC+G<3GCGCTCGTTT+GGGC
Tl
+
T
+
+
A 89%
+
T
C
A
G
89%
F2
F3 T
G
CA
G+
G
T
83%
F4
C—GG
CC
TCATT
++++—C
68%
Repeat-H
HI
H2
A
A
C
A
A
CTAGATTTTTCAAGCTTCGATTCGGTTATTCAAACGTCGAAAACGGATAAGCGGAACTCCAAAAAAGGGGGGTGTGGCTC
CTCGAACACATTTGTTCAAACTGCTGGAAATGAGGAAAAAGCTCAAATGTCATATATAGGGAGGGGGT
98%
Fig. 1C
phy on a column of heparin-Sepharose. After absorption
of the nuclear extract to the column, protein fractions were
obtained by stepwise elution with a buffer that contained
100, 200, 300, 400 and 500 mM KC1. The protein fraction
that was eluted in the presence of 300 mM KC1 yielded two
retarded bands (Fig. 3). Furthermore, the bands obtained
when the probe DNA that contained the gene promoter
was used were similar to the bands obtained when the probe DNA that contained the spacer promoter was used,
To confirm the sequence specificity of protein binding,
competition analyses were performed. When both the gene
promoter and spacer promoter fragments were used as probes, the intensities of the two retarded bands were strongly
reduced by the addition of the unlabeled gene promoter or
237
Intergenic spacer of the rDNA in Daucus carota
10 11 1213
1
2
3 4 5 6 7 8 9
1011 1213
Fig. 2 Identification of the site of initiation of transcription from the gene promoter (A) and the spacer promoter (B) by SI nuclease
mapping.
The gene promoter probe was the Hindlll-Eael fragment (Hindlll: -126, Eael: +163), labeled at the 5' end of the coding
strand. The spacer promoter probe was the ////idIII-£coT14I fragment (Hindlll: - 1 2 6 , EcolHl: +110), labeled at the 5' end of the
coding strand. Numbering is relative to the each putative initiation site, which is designated + 1 . Probe DNA was allowed to hybridize
with 75 fig of carrot RNA (lanes 6, 7, 8 and 9) or yeast tRNA (lane 5), and then it was treated with 10 units (lane 6), 40 units (lanes 5 and
7), 160 units (lane 8) or 640 units (lane 9) of SI nuclease at 37°C for 30 min. After denaturation, protected fragments were subjected to
electrophoresis with DNA sequencing markers (lanes 1 and 10, G; lanes 2 and 11, G + A ; lanes 3 and 12, C + T ; lanes 4 and 13, C). The arrowhead in (A) indicates the endpoint of the clear protected band and the arrowheads in (B) indicate the endpoints of the faint protected
bands.
spacer promoter fragment as a competitor (lanes 3-6 in
Fig. 3). By contrast, these retarded bands were unaffected
by the addition of a 100-fold excess of another DNA fraggP
Sp 3 1
S p
C
O
gp3 1
o
O
O
* x x x x
JtikMJk
1 2 3
gene
4 5 6 7
promoter
1 2 3 4 5 6 7
spacer
promoter
Fig. 3 Gel-retardation assay using a gene promoter and spacer
promoter as probes with various competitors.
The HindlllHindlll (-285 to -126) fragment, designated the gene promoter
(gp), was used as a probe in the left panel and the Rsal-Hindlll
(—290 to —126) fragment, designated the spacer promoter (sp),
was used as a probe in the right panel. Numbering is relative to
each putative initiation site, designated + 1 . Lane 1, no nuclear extract; lane 2, no competitor; lanes 3 and 4, the same fragment as
the gene promoter probe was used as the competitor; lanes 5 and
6, the same fragment as the spacer promoter probe was used as the
competitor; lane 7, the polylinker segment of pIBI31 (31) was used
as the competitor. Each competitor was present at 10 times (lanes
3 and 5) or 100 times (lanes 4, 6 and 7) the level of the probe.
ment as a competitor (lane 7 in Fig. 3). These results indicated that the DNA-protein(s) interactions were specific
to the DNA sequences that contained the gene promoter
and spacer promoter. Furthermore, it appeared that the
same protein fraction interacted with both the gene promoter and the spacer promoter sequences. The function of the
protein fraction is unknown. However, the proteins in this
fraction might be components of the transcriptional apparatus. Thus, there is a possibility that the spacer promoter acts as the site of entry for the protein factor(s) required
for formation of the initiation complex for transcription
of rRNA, eventhough the transcriptional activity from
the spacer promoter seemed very low. A similar hypothesis
has been proposed for transcription of rRNA in maize
(Schmitz et al. 1989). These hypotheses remain to be confirmed but they should not be ignored when enhancer activity during transcription of plant rRNA is considered.
This study was supported by grants from the Ministry of
Education, Science and Culture of Japan.
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(Received August 22, 1995; Accepted January 10, 1995)