DNA Research 10, 213–220 (2003)
CCLS96.1, a Member of a Multicopy Gene Family, may Encode
a Non-coding RNA Preferentially Transcribed in Reproductive
Organs of Silene latif olia
Ryuji Sugiyama,1,∗,† Yusuke Kazama,2 Yutaka Miyazawa,3 Sachihiro Matsunaga,2,‡
and Shigeyuki Kawano1,2
Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo,
Tokyo 113-0033, Japan,1 Department of Integrated Biosciences, Graduate School of Frontier Sciences,
University of Tokyo, Bldg. FSB-601, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan,2 and Plant
Functions Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan3
(Received 2 December 2002; revised 9 September 2003)
Abstract
Dioecy in the model dioecious plant Silene latifolia is determined genetically by its heteromorphic sex
chromosomes. A bacterial artificial chromosome (BAC) clone, #19B12, was isolated by screening a BAC
library from S. latifolia using polymerase chain reaction (PCR) with a set of sequence tagged site (STS)
primers, ScD05, which are specific to the Y chromosome. A portion of #19B12 was subcloned to construct
plasmid #25-1, with an insert of 7.8 kb. This 7.8-kb fragment encodes ScD05 homolog and an antherspecific gene, CCLS96.1. Northern blot analysis of CCLS96.1 indicated a faint band of 1.8 kb in male
and female flower buds. 5 and 3 rapid amplification of cDNA ends (RACE) indicated that transcripts
of CCLS96.1 are very varied in size. Moreover, semi-quantitative reverse transcription-PCR (RT-PCR)
showed that CCLS96.1 was also expressed in both male and female leaves. RACE produced at least
ten species of transcripts, with 79–97% similarity among them. However, no significant ORFs could be
predicted from their nucleotide sequences, since each has numerous stop codons throughout all three reading
frames. Genomic Southern hybridization showed that the S. latifolia genome contains numerous CCLS96.1
homologs. These results suggest that the transcripts of CCLS96.1 play some role as multiple non-coding
RNAs in S. latifolia.
Key words: CCLS96.1; dioecious plant; multicopy gene family; non-coding RNA; Silene latifolia
1.
Introduction
Most flowering plants are hermaphrodites, with dioecious plants accounting for 5% of them.1 Some dioecious
plants contain heteromorphic sex chromosomes, which
determine the plant’s dioecy. This is the case for Silene
latifolia, a model dioecious plant. Sex-specific differences
between male and female flowers arise subsequent to floral primordium formation by the selective arrest of the
development of pistils in male flowers and of stamens in
∗
†
‡
Communicated by Satoshi Tabata
To whom correspondence should be addressed. Tel. +81-47136-3679, Fax. +81-4-7136-3674, E-mail: [email protected]
Present address: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg.
FSB-601, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Present address: Department of Biotechnology, Graduate
School of Engineering, Osaka University, 2-1 Yamadaoka,
Suita, Osaka 565-0871, Japan
female flowers. The arrest of stamen development in female flowers occurs at a later stage than does the arrest of
carpel development in male flowers. These findings support the genetic evidence that two distinct mechanisms
operate to promote stamen development and to arrest
pistil development in male plants and that the Y chromosome contains dominant genes.2,3
The Y chromosome genes have been studied in more
detail in humans. The human Y chromosome genes are
classified into pseudoautosomal genes and three classes
of non-recombining region of the Y chromosome (NRY)
genes largely on the basis of expression profile and homology to the X chromosome.4 Class 1 genes are single copy, expressed widely in the body, and have likefunctioning X-linked homologs. Class 2 genes are multicopy, expressed only in the testis and are without an
active X-linked homolog. Class 3 genes do not fit either profile for class 1 or 2 and include the SRY sex-
214
A Multicopy Gene, CCLS96.1, in S. latifolia
determining gene.
In S. latifolia, male-specific cDNAs have been isolated using differential screening,5 subtraction methods,6
screening of a cDNA library using microdissected Y chromosomes as a probe,7,8 and fluorescent differential
display.9 However, the majority of these sequences share
homology with the X chromosome and/or the autosomes.
Using single-strand-conformation polymorphism (SSCP)
and flow-sorted X chromosomes, MROS3 is identified
as having X chromosome linkage.10,11 Although three
genes, SlY1, SlY4, and DD44Y, are known to be active
Y-chromosome genes,7,8,12 they also have homologs on
the X chromosome (SlX1, SlX4, and DD44X). These
genes are analogous to the class 1 genes of human NRY
genes. To reveal the structure of the Y chromosome of
S. latifolia, it is necessary to analyze the expression profile, the copy numbers and localization of these sequences
in detail.
In this work, we screened an S. latifolia BAC library to characterize Y-chromosome-specific STS markers of S. latifolia, identifying a BAC clone, #19B12, from
which the Y-chromosome-specific STS ScD05 was successfully amplified.13 A 7.8-kb portion of the #19B12
sequence, including ScD05, encodes an anther-specific
gene, CCLS96.1.6 Therefore, we investigated sequences
of a series of CCLS96.1 transcripts using RACE and RTPCR. We discuss the possibility that CCLS96.1 is one of
a multi-copy gene family encoding non-coding RNAs.
2.
Materials and Methods
2.1. Plant material
An inbred line of Silene latifolia was propagated by
eight generations of brother-sister mating. Plants were
grown in flowerpots in a regulated chamber at 23◦ C with
a 16-hr day and 8-hr night light cycle. Plant tissues were
harvested and frozen in liquid nitrogen, and stored at
−80◦ C until isolation of DNA and RNA.
2.2. Subcloning and DNA sequencing
Library screening was performed by PCR using Ex Taq
polymerase (TAKARA BIO INC., Otsu, Japan). The
PCR parameters were 94◦ C for 5 min, followed by 30 cycles of 94◦ C for 1 min, 66◦ C for 1 min and 72◦ C for
1 min. The primers used for the screening were as follows: ScD05F, 5 -TGA GCG GAC ACG GGT GGG
GC-3 ; and ScD05R, 5 -TGA GCG GAC ATT GTG
AGG TTA CCT CC-3 .13 A BAC clone was isolated
using the CONCERT High Purity Plasmid Purification
System (Invitrogen, CA, USA). The isolated BAC clone
was subcloned into the Xho I sites of pBluescript II
SK+ (Stratagene, CA, USA) to obtain plasmid #25-1.
Plasmid #25-1 DNA was isolated using the alkaline lysis method. Nucleotide sequences were determined using an ABI PRISM 3100 automated sequencer (Applied
[Vol. 10,
Biosystems, CA, USA) with the BigDye terminator cycle
sequencing FS ready reaction kit (Applied Biosystems).
Sequence data were assembled using the program Sequencher version 4.0.5 (Gene Codes, MI, USA).
2.3. Northern blot analysis
Total RNAs were extracted from leaves and flower
buds using TRIZOL reagent (Invitrogen). Ten µg of total RNA was denatured with glyoxal and subjected to
gel electrophoresis as described previously.14 The RNA
was transferred to Hybond-XL membrane (Amersham
Biosciences, Buckinghamshire, UK). The membranes
were hybridized with 442 bp of the 5 region of CCLS96.1
as a probe. Cytoplasmic rRNA genes15 were used as
a control. Probes were labeled with [α-32 P]dCTP and
[α-32 P]dGTP using Megaprime DNA labeling systems
(Amersham Biosciences), and hybridized in Church and
Gilbert buffer.16 The membranes were then washed twice
in 2 × SSC (20 × SSC = 3 M NaCl and 300 mM trisodium
citrate), 0.1% SDS for 15 min at room temperature, once
in 1 × SSC, 0.1% SDS for 15 min at 65◦ C, and once in
0.1 × SSC, 0.1% SDS for 15 min at 65◦ C. The hybridization signals were detected using BAS2000 (Fuji Film,
Tokyo, Japan) and analyzed using BAStation software
(Fuji Film) as described previously.17
2.4. RT-PCR
The level of CCLS96.1 transcripts was estimated by
semi-quantitative RT-PCR. Poly(A)+ was isolated using
the PolyA Ttract mRNA isolation system (Promega, WI,
USA), and 100 ng of poly(A)+ RNA was used to generate cDNA with the First-strand cDNA synthesis kit
(Amersham Biosciences) using Not I-d(T)18 as a primer.
The synthesized first-strand cDNA was used as a template for PCR. Primers for CCLS96.1 were as follows:
CCLS96.1RT-F, 5 -GAG TTT CAT TTG GGA GTT
TGC-3 (position 2009–2029 in the 7.8-kb fragment);
CCLS96.1 RT-R, 5 -GTT AGT TGC ATC ATG GCA
TG-3 (position 2599–2617 in the 7.8-kb fragment). An
actin gene was used as a control. Actin primers were as
follows: SLactin01, 5 -TTA CCG TAA AGG TCC TTC
CTG AT-3 ; Slactin02, 5 -AGC TTC GTG TTG CTC
CTG AAG A-3 . The PCR parameters were 94◦ C for
5 min, followed by 11, 13, 15, and 17 cycles of 94◦ C
for 30 sec, 50◦ C (for CCLS96.1) or 56◦ C (for actin) for
30 sec, and 72◦ C for 45 sec. Following electrophoresis
(1.5% agarose, 1 × TAE, 50 V, 1.5 hr), PCR products
were transferred onto the Biodyne B membrane (PALL,
Portsmouth, UK). The membranes were hybridized with
CCLS96.1 and an actin fragment (DDBJ accession number AB094079), using the ECL direct nucleic acid labeling and detection systems (Amersham Biosciences), according to the manufacturer’s instructions. Signals were
quantified using the program MATRIX (QuantaVision
Canada, PQ, Canada). Relative amounts of CCLS96.1
No. 5]
R. Sugiyama et al.
215
Figure 1. Schematic diagram of the 7.8-kb fragment encoding CCLS96.1. The 7.8-kb fragment (DDBJ accession number AB094078)
and the 8011-bp Y chromosome specific fragment (Silene latifolia ORF285 gene)18 are compared schematically. The black arrow
indicates CCLS96.1. White arrows indicate putative open reading frames (ORF505 and ORF285). Gray arrows indicate the inverted
repeat sequences. Gray boxes indicate homologous regions between the 7.8-kb fragment and the Y chromosome fragment. White box
indicates the Y chromosome STS marker, ScD05 homolog. The Y chromosome fragment contains ORF285, that exists as a single
copy in the S. latifolia genome, and CCLS96.1 homolog in its 3 end (94% identity). An asterisk indicates a fragment used as a probe
in Northern blot analysis.
and actin transcripts were estimated from signal intensities after 15 and 17 cycles.
2.5. RACE
The 5 - and 3 -ends of the CCLS96.1 transcript were
cloned using the SMART RACE cDNA Amplification
Kit (BD Biosciences Clontech, CA, USA). Gene-specific
primers used in 5 RACE were as follows: 5 CCLS01,
5 -GTA CCT TGT GGT GTG ATA ACT CCT TGG C3 ; and 5 CCLS02, 5 -CAT GGA TTA AGG CCT AGT
CAA GCG TAC C-3 . In 3 RACE the primers were
as follows: 3 CCLS01, 5 -GCC AAG GAG TTA TCA
CAC CAC AAG GTA C-3 ; and 3 CCLS02, 5 -GGT
ACG CTT GAC TAG GCC TTA ATC CAT G-3 . PCR
was performed using Z Taq polymerase (TAKARA BIO
INC.). The PCR parameters were 96◦ C for 1 sec, followed by 30 cycles of 98◦ C for 1 sec, 62◦ C for 10 sec, and
72◦ C for 15 sec. For assembly of RACE products, the
CCLS96.1 middle region was amplified from 5 RACE
cDNA using Ex Taq polymerase (TAKARA BIO INC.)
and following primers: RACE-contig-F, 5 -TTT GTG
CCC ATT CCC TTT GA-3 ; and CCLS96.1RT-R. The
PCR parameters were 94◦ C for 5 min, followed by 30 cycles of 94◦ C for 1 min, 59◦ C for 1 min, and 72◦ C for
1 min. The PCR products were cloned into the pCR2.1TOPO vector (Invitrogen) for sequence analysis.
2.6. Southern blot analysis
Genomic DNA was extracted from leaves using the
Nucleon PhytoPure kit (Amersham Biosciences). For
Southern blot analysis, genomic DNA was digested
overnight at 37◦ C with HindIII (TAKARA BIO INC.),
and 10 µg of the digests were electrophoresed on 1%
agarose gels and blotted onto the Biodyne B membrane
(PALL). The membrane was hybridized with 442 bp of
the 5 region of CCLS96.1 using the ECL direct nucleic acid labeling and detection systems (Amersham
Biosciences), according to the manufacturer’s instructions.
3.
3.1.
Results and Discussion
Sequence of the 7.8-kb fragment subcloned from the
BAC clone #19B12
A BAC clone, #19B12, was isolated by screening a
BAC library from S. latifolia using PCR with a set of
STS primers, ScD05,13 which are specific to the Y chromosome. BAC clone #19B12 was subcloned into the
Xho I sites of pBluescript II SK+ . A subclone, #25-1,
was isolated from the Xho I subclone library by screening using PCR with the ScD05 primers. The 7.8-kb insert in plasmid #25-1 was sequenced by primer walking
(DDBJ accession number AB094078). The ScD05 homolog in #19B12 has two deletions, as compared to the
Y-chromosome-specific ScD05 (DDBJ accession number
AB094082), indicating that there are at least two homologs of this family located at different loci.
The 7.8-kb fragment contains an ORF that encodes a putative protein of 505 amino acids (ORF505,
Fig. 1). ORF505 has 21% identity (44% similarity)
over 160 amino acids with an ORF from Arabidopsis
thaliana (accession number BAB09350, contains similarity to retroelement pol polyprotein gene id:MXI10.7).
The expression of ORF505 could not be confirmed by RTPCR (data not shown). The 7.8-kb fragment contains another homologous sequence with 92% identity over 577 bp
with an anther-specific gene, CCLS96.16 (black arrow in
Fig. 1), identified by chemical cross-linking subtraction.6
The fact that the ScD05 homolog in the 7.8-kb fragment has two deletions suggests that there are homologs
to ScD05 in the genome of S. latifolia, in addition to the
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A Multicopy Gene, CCLS96.1, in S. latifolia
[Vol. 10,
Figure 2. Transcripts of CCLS96.1 reveled by Northern blot analyses. Total RNAs (10 µg) isolated from leaves (L) and buds (B) of
male and female plants were subjected to electrophoresis on a 1.5% agarose gel, blotted and hybridized with 442 bp of the 5 region
of CCLS96.1 from the 7.8-kb fragment. 26S rRNA was used as a positive control.
Figure 3. Semi-quantitative RT-PCR to examine differential expression of CCLS96.1 using mRNA from leaves and buds of male and
female plants. cDNAs synthesized from mRNA of leaves and buds were used as templates. RT-PCR products were blotted onto a
membrane and hybridized with same probe using northern blot analysis. An actin gene fragment was used as a control. The relative
intensity of each signal was measured by densitometry and is listed in Table 1.
Y-chromosome-specific ScD05. To avoid amplification of
these homologs, we attempted to isolate the ScD05 homolog encoded in the 7.8-kb fragment from either male
or female genomic DNAs by PCR using nested primers.
However, 96 of the ScD05 homologs amplified by PCR
with these primers were found to contain sequences differing from the ScD05 encoded in the 7.8-kb fragment
(data not shown). It is surprising that no identical sequences were found in the nested PCR products. This
implies that there are numerous ScD05 homologs in the
genome of S. latifolia.13 It would be very difficult, using
PCR and sequencing, to directly identify the ScD05 homolog in the 7.8-kb fragment from either the male or the
female genomes.
We previously isolated an 8011-bp Y chromosome
DNA fragment encoding ORF285 from S. latifolia
(Fig. 1).18 This Y chromosome fragment contains homologous regions with the 7.8-kb fragment and a part of
CCLS96.1 (Fig. 1). The 5 flanking region (812 bp) and
the part (259 bp) of this CCLS96.1 on the Y chromosome
were homologous with those of the 7.8-kb fragment (92%
identity, Fig. 1). There was an additional short homologous region with the 5 end region of the 7.8-kb fragment
(245 bp, 84% identity, Fig. 1).
3.2. Expression of CCLS96.1 in leaves and flower buds
We performed Northern blot analysis of CCLS96.1 using as a probe its 5 flanking region sequence that was
homologous between the 7.8-kb fragment and the Y chromosome fragment (Asterisk in Fig. 1). A 1.8-kb transcript was observed not only in male flower buds but
also in female flower buds (Fig. 2). The hybridization
signal of CCLS96.1 in the Northern blot analysis was
so week that semi-quantitative RT-PCR was performed
to analyze the expression patterns of CCLS96.1 (Fig. 3).
CCLS96.1 was expressed also in male and female leaves.
Relative amounts of RT-PCR products of CCLS96.1 were
estimated from the signal intensities after 15 and 17 cycles, and compared with those of the actin gene as a pos-
No. 5]
R. Sugiyama et al.
217
Table 1. Relative amounts of CCLS96.1 transcripts revealed by semi-quantitative RT-PCR.
Male
Female
CCLS96.1 / Actin
Buds / Leaves
15 cycles
17 cycles
15 cycles
17 cycles
Leaves
0.15
0.26
1
1
Buds
0.40
0.89
2.67
3.42
Leaves
1.24
1.07
1
1
Buds
0.08
0.20
0.06
0.19
Signals were quantified using the program MATRIX (QuantaVision Canada, PQ, Canada).
itive control (Table 1). In the male plant, the expression
of CCLS96.1 in flower buds was 2.67- to 3.42-fold higher
than that in leaves. In contrast, in the female plant,
the expression of CCLS96.1 in flower buds was 0.06- to
0.19-fold less than that in leaves. This suggests that the
CCLS96.1 homologs play some role in the male flower
bud.
Barbacar et al.6 have reported that CCLS96.1 is an
anther-specific gene. However, using a region different from theirs as a probe for Northern analysis and
as primers for RT-PCR, we have shown that CCLS96.1
homologs are expressed in leaves and buds (Figs. 2 and
3). Since the chemical cross-linked subtraction (CCLS)
that Barbacar et al performed successfully isolates malespecific fragments, it is possible to isolate only an antherspecific expressed fragment from numerous CCLS96.1 homologs.
3.3. Sequence analysis of RT-PCR and RACE products
CCLS96.1 has been previously reported to be an
anther-specific gene.6 However, since no significant ORF
was found in CCLS96.1, it was thought that only partial transcripts of CCLS96.1 had been identified. To
obtain a complete sequence of CCLS96.1, RACE was
performed using primers designed from the sequence of
CCLS96.1 in the 7.8-kb fragment. Sequences of the 5
and 3 RACE products were aligned with those of the
RT-PCR products of CCLS96.1 to extrapolate complete
sequences of the CCLS96.1 transcripts (Fig. 4). The Sequencher version 4.0.5 program compiled ten species of
putative CCLS96.1 transcript sequences. Representative
sequences are aligned in Fig. 4. The presence of insertions, deletions and substitutions suggests that they are
not truncated forms of one transcript. The transcripts of
CCLS96.1 homologs would be of various sizes and have
sequence diversity. The sequence that completely corresponds to that of CCLS96.1 in the 7.8-kb fragment could
not be amplified.
3.4. Genomic distribution of CCLS96.1
To investigate the genomic distribution of the
CCLS96.1 homologs, Southern blot analysis was performed using 442 bp of the 5 region of CCLS96.1 as
a probe (Fig. 5). Several prominent bands and numerous smeared bands were labeled in the lower and upper
parts of the HindIII digests of both male and female genomic DNAs, respectively. This is a typical Southern
hybridization pattern of a multi-allelic gene family or numerous repetitive sequences. Since there was no significant difference between male and female genomes, there
are probably similar numbers of CCLS96.1 homologs in
the male and female genomes of S. latifolia.
The 8011-bp Y chromosome DNA fragment contains
a homologous region with CCLS96.1 at position 7793–
8011 (219 bp, 94% identity). The Y chromosome thus
likely contains at least one CCLS96.1 homolog. However, sequences identical with this CCLS96.1 gene could
not be found among the RACE and RT-PCR products.
Guttman and Charlesworth10 reported that the X-linked
gene MROS3 is a pseudogene on the Y chromosome. It is
possible that the CCLS96.1 homolog has become a pseudogene on the Y chromosome.
3.5. CCLS96.1 has numerous stop codons
All sequences of the putative CCLS96.1 transcripts
have numerous stop codons in all three reading frames.
For example, CCLS96.1-J1 has 41, 35, and 33 stop codons
in each of its three reading frames of 1869 bp (Fig. 6).
These frequencies are almost equal to the frequency at
which a stop codon will result from a random choice
of three bases. The longest ORF in the CCLS96.1 homologs had 99 deduced amino acids, in CCLS96.1-J3.
This longest ORF is not conserved among the CCLS96.1
homologs.
These sequences were highly homologous (81%–97%
identity), while their deduced amino acid sequences were
not identical. These transcripts are probably not translated and likely function as non-coding RNAs (ncRNA).
Although multi-allelic gene families of ncRNAs have not
218
A Multicopy Gene, CCLS96.1, in S. latifolia
[Vol. 10,
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88AH.+#&"?()-.666II8686II<"III6I688<86I866I86666I666<68686I6<I86II6686I666<6<<66666<66<6II8<666<6I86I6886I<8<<I8II866I6I88I86I<I8
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88AH.+#&"?(+%(I8<<6I<I<<8<I6<I8<6I866I6I<6"6866<<6I6<6866686I66I6I6I6866<""""""""""I8I6868I68666"<<666I<668II<IIIII<<6IIII866IIII
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88AH.+#&"?&.%&"8666IIIII6I<6IIIII<66"""""""""""""""""""""6IIIII866IIIII"""""""""I<<6IIIII<""66I""""""66<6<II666I<II6<66II888"6I88
88AH.+#&"?',&+I8666IIIII6I<<IIIII<8<III6I<6666I6666<6686I6IIIII<I6IIIII8666666III866IIIII6I86IIIIIIII6666<I866""<II6<66III8866I8I
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88AH.+#&"?&&%+)"""""I68I66<8I6868I686I<6I<86I<<IIII""I<II<I<68<<6<6<<6I66III6<6II688I888<II<8<I6I<86II6I<<I<8<I<I86III6I6I<6I<IIII
88AH.+#&"?'.))6I<86IIII6<6II6868I666I66I<86I<66III""6II<<I""I<<6<6<8"I66III666II668I88866I<8II<I<8"II<668II888<6"668666<I66<6I68I
88AH.+#&"?(-,)"""""II6866I8I6868I686I<6I<86I<<<IIIII<III6I<68<<6<6<8<I66III6<6II688I888<II<8<I6I<8"6I<I68II88886"6888<6<6I6<686II
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88AH.+#&"?&&&,'686I8I8IIIII6868<86"III86<6<8I86III<I<I66I6III<II<8IIII8I888I6I""II88<I8I"""68II88<I6I"III<I68III6I""""""I<86<666I<
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88AH.+#&"?(.-'6III8I66I<I8II666IIII6<<<I6<II86I<8686866<6I<866I<86I66668I6I6I6II<I86IIII<<6IIIII8666"I<<<66866I66666<668688I866I<
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88AH.+#&"?&&*.%6<<IIII6<666I66<II<"""II<8<III6II<6I6I6I668<I6686886686I8I6<IIII<<66<<6<<<66I686II66<III8686I86866I6<III6<III6I8II8
88AH.+#&"?'&*%(<<88<I8868668<868<<66888<I<8<I8II8I888866I88<888<IIII88I8II868866I88<888<<6II8I888666668<8I8<<6II886<8886<8<86IIII8
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88AH.+#&"?&&-&(<8II8<8III86II66I6<I6I6<66II<6I6<II66I<8888666<8866II<I8I""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
88AH.+#&"?'&,('688I66III8I68I6I666I688886III<I66I"66I8666866<<66<IIII68I66<<<<66I8IIIII6<8I6<66<6I6I6I88II6<6II6<6II6<<6<I6<6II6<6
88AH.+#&"?("""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
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88AH.+#&"?&&.'-"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
88AH.+#&"?'&-)+6I6<6II68I8III66I8III8868666II68686II66I8III886I66II6II<II866<III6II6<II866<I66<III6II6III66<III"""""""""""""""""""
88AH.+#&"?("""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
88AH.+#&"?)"""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""""
Figure 4. Sequence analysis and alignment of CCLS96.1 cDNAs. Four complete sequences of CCLS96.1 homologs (J1 to J4) were
identified from RACE products. Alignment was constructed with ClustalW Version 1.8 software and shading was done using
BOXSHADE Version 3.21. Identical nucleotides are boxed in black. Similar nucleotides are boxed in gray. Primers used for PCR
and RACE are shown by arrows. DDBJ accession numbers: CCLS96.1-J1 (AB114184), CCLS96.1-J2 (AB114185), CCLS96.1-J3
(AB114186), CCLS96.1-J4 (AB114187).
No. 5]
R. Sugiyama et al.
219
Figure 5. Southern blot analysis using CCLS96.1 as a probe. Genomic DNAs from two parent plants (males F0 , lane 1; females F0 ,
lane 6), and F1 progeny plants (male, lanes 2–5; female, lanes 7–10) were digested with HindIII and hybridized with 442 bp of the
CCLS96.1 homolog from the 7.8-kb fragment as a probe. Size markers are on the left.
Figure 6. Positions and numbers of stop codons of the CCLS96.1 homologs. Vertical bars indicate positions of stop codons (TAA, TAG,
and TGA) in the possible reading frames of CCLS96.1-J1 to -J4. Each sequence is aligned at its position. Numbers in parentheses
indicate the total number of stop codons in each reading frame. “AAAAA” indicates ploy(A) tails. Scale bar is displayed.
220
A Multicopy Gene, CCLS96.1, in S. latifolia
been reported previously, it is reasonable to assume
that the divergent CCLS96.1 transcripts are produced
from the multiple CCLS96.1 homolog genes distributed
throughout the chromosomes.
To date, more than 100 ncRNAs have been identified,
some of them are partly characterized.19 In general, however, it is difficult to identify an expressed sequence as
a non-coding RNA. For example, endo40 was first identified as an ncRNA due to a lack of significant ORFs.20
Later, it was demonstrated that endo40 is translated into
two small peptides, and the RNA itself is important for
its function.21 It is possible that the CCLS96.1 homologs
play some role as a small peptide.
Acknowledgements: This work was supported by
Grants-in-aid for Scientific Research to S. K. (No.
15013215) from the Japanese Ministry of Education,
Science, and Culture. It was also supported by a
grant-in-aid from the Special Postdoctoral Researchers
Program of RIKEN to Y. M.
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