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Biochimica et Biophysica Acta 1491 (2000) 267^272
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Short sequence-paper
Molecular cloning of a cDNA encoding a novel Ca2‡ -dependent nuclease
of Arabidopsis that is similar to staphylococcal nuclease1
Kyoichi Isono a , Kimiyuki Satoh b , Hirokazu Kobayashi
a
a;
*
Laboratory of Plant Cell Technology, Graduate School of Nutritional and Environmental Sciences, University of Shizuoka, 52-1 Yada,
Shizuoka 422-8526, Japan
b
Department of Biology, Faculty of Science, Okayama University, 3-1-1 Tsushimanaka, Okayama 700-8530, Japan
Received 13 September 1999; received in revised form 21 December 1999; accepted 12 January 2000
Abstract
We have isolated a cDNA from Arabidopsis thaliana for a protein consisting of 323 amino acids with similarity to an
extracellular nuclease from Staphylococcus. Nuclease assay using toluidine blue-DNA plates has demonstrated that the gene
product has nuclease activity dependent on Ca2‡ and inhibited by Zn2‡ , designated CAN (Ca2‡ -dependent nuclease).
Differing from the staphylococcal nuclease, CAN has neither a signal peptide nor any long hydrophobic regions, suggesting
that it is not a secreted protein. ß 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ca2‡ -dependent nuclease; Staphylococcal nuclease; Zn2‡ -inhibition; (Arabidopsis)
Genes homologous to a well-characterized extracellular nuclease from Staphylococcus aureus (EC
3.1.31.1) [1,2] have been identi¢ed from some bacteria. The homologous genes, nuc [3] and parB [4,5] in
the plasmid pSa from Shigella £exneri and in the
RP4 plasmid from Escherichia coli, respectively, encode nucleases. Since they have characteristic signal
peptides like that in the staphylococcal enzyme, they
Abbreviations: CAN, Ca2‡ -dependent nuclease; bp, base
pair(s); RT-PCR, reverse transcription-polymerase chain reaction; EDTA, ethylenediaminetetraacetic acid; EGTA, ethyleneglycol-bis-(L-aminoethyl ether)N,NP-tetraacetic acid; TB-D, toluidine blue-DNA
* Corresponding author. Fax: +81-54-264-5584;
E-mail: [email protected]
1
The nucleotide sequence data reported will appear in the
EMBL, GenBank, and DDBJ under the accession number
D84226.
may be secreted. In the higher plant Corydalis sempervirens, cDNA for a homolog of staphylococcal
nuclease has recently been found [6], although its
function has not been characterized. In eukaryotes,
no other homologs have been identi¢ed, and thus the
biological function of the plant homolog is of great
interest. Here we describe cDNA for staphylococcal
nuclease homolog from Arabidopsis thaliana, the
plant most intensively analyzed at the molecular level
[7], and further demonstrate that the gene product is
a nuclease.
We had originally intended to identify cDNAs for
c factors of plastid RNA polymerase from Arabidopsis. A PCR fragment of 440 bp was ampli¢ed with
genomic DNA, using primers corresponding to domains that recognize the 310 and 335 promoter
regions and that are conserved among bacterial
RNA polymerase c factors [8]. The nucleotide sequence of the PCR product di¡ers from that ex-
0167-4781 / 00 / $ ^ see front matter ß 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 4 7 8 1 ( 0 0 ) 0 0 0 0 7 - 5
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K. Isono et al. / Biochimica et Biophysica Acta 1491 (2000) 267^272
Fig. 1. Nucleotide and deduced amino acid sequences of the cloned cDNA. The nucleotides are numbered from the ¢rst nucleotide of
the initiation codon. The arrows indicate synthetic primers used for RT^PCR. The sequence underlined is the recognition site of
EcoRI. Total cellular DNA was extracted with cetyltriethylammonium bromide (CTAB) [26]. Two oligonucleotide primers, 5PTAAGTCGAC(A/G)TA(T/C)GCIACITGGTGGAT(C/T)(A/C)GICAIGC-3P and 5P-TAAGCGGCCGCTTCIAT(C/T)TGICGIATICGTTC-3P, which correspond to amino acid sequences for 335 and 310 sequence-binding domains, respectively, of the principal c-factors from several bacteria were synthesized. The primers and Arabidopsis cellular DNA (0.1 Wg) were used for PCR (94³C for 1 min;
50³C for 2 min; slope for 2 min; and 72³C for 2 min; 30 cycles followed by a 7-min extension at 72³C). Subsequently, 1/30 of the
volume of the product was subjected to secondary PCR (94³C for 1 min; 55³C for 1 min; and 72³C for 2 min; 30 cycles followed by
a 7-min extension at 72³C). The PCR product was random-labeled with [K-32 P]dCTP (NEN, 3000 Ci/mmol) and used for screening of
a V ZAP cDNA library (2U106 pfu) from Arabidopsis leaves as described [27]. The cDNA inserts of positive clones were sequenced
with a 373A DNA sequencer (Applied Biosystems).
Fig. 2. Amino acid sequence comparison of the Arabidopsis cDNA product (Arabid) with the pSa nuclease (pSa NUC), staphylococcal
nuclease (sta NUC), the parB product (PARB), and a functionally uncharacterized protein from Corydalis (Coryd). Amino acids similar to those of Arabid are enclosed with linked lines. The three asterisks mark amino acids in the active sites of the staphylococcal nuclease and the two dots represent residues capable of binding to Ca2‡ .
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K. Isono et al. / Biochimica et Biophysica Acta 1491 (2000) 267^272
269
Fig. 3. Hydropathy plots of the ¢ve homologous proteins. Hydrophobicity scores were calculated following the Kyte^Doolittle scale
[28]. The regions of the amino acid sequence above the central horizontal line are predicted to be hydrophobic, and the regions below
the line to be hydrophilic.
pected for c factor (s) that isolated so far [9]. In our
further screening of a leaf cDNA library with the
DNA fragment as a probe, 8 clones hybridized
with the probe, and they encoded the same sequence.
Fig. 1 shows the nucleotide sequence of the cDNA
and its deduced amino acid sequence. The cDNA is
judged to be full-length, encoding a polypeptide of
323 amino acids. The C-terminal 114 amino acids of
the product were 40^51% similar to a secreted nuclease from Staphylococcus aureus [2], Nuc nuclease [3],
and ParB nuclease [4], and 82% similar (62% similarity through the overall stretch) to a gene product
from the plant Corydalis [6] (Fig. 2), whose function
is not characterized. Amino acid residues, Arg35,
Glu43, and Arg87 at the active site of the staphylococcal nuclease, and Asp21 and Asp40 at the Ca2‡ binding sites [10,11], are conserved among all these
proteins. On the other hand, hydropathy plots of
these proteins have identi¢ed signi¢cant di¡erences
between the bacterial nucleases and the two plant
homologs (Fig. 3). The staphylococcal, Nuc, and
ParB nucleases are hydrophobic and have characteristic signal peptides located at N-terminal regions,
whereas neither of the plant homologs have such
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K. Isono et al. / Biochimica et Biophysica Acta 1491 (2000) 267^272
Fig. 4. Molecular analyses of the gene and the transcript. (A)
Genomic Southern hybridization. Southern blot analysis was
performed as described [29]. A 32 P-labeled EcoRI^XhoI fragment (1.1 kbp) of the cDNA clone was probed with DNA-digests using BamHI (lane 1), EcoRI (lane 2), or PstI (lane 3),
and the probed bands were detected under high-stringency conditions. (B) RT^PCR. Total RNA from leaves was extracted
with 4 M guanidinium thiocyanate [30]. First-strand cDNAs
were synthesized from the leaf RNA with a T-Primed FirstStrand kit (Pharmacia) according to the manufacturer's instructions, and treated with RNase A. Total cellular DNA (0.1 Wg)
(lane 1) and one-third volume of the cDNA solution (lane 2)
were subjected to PCR with a set of sequence-speci¢c primers
(see Fig. 1). The PCR products were detected in 1.5% agarose
gels stained with ethidium bromide. M, molecular markers.
hydrophobic regions. This suggests that the plant
homologs are not secreted.
In genomic Southern blotting under high-stringency conditions, two bands hybridizing with a fragment of the whole cloned cDNA were detected in
BamHI, EcoRI, and PstI digests of total DNA
from Arabidopsis (Fig. 4A). This suggests that two
copies of the gene may exist in the haploid nuclear
genome. Expression of the gene was evaluated by
reverse transcription^polymerase chain reaction
(RT^PCR), because of di¤culty in detecting its transcripts by Northern analysis. The RT^PCR generated a 0.5 kbp product ampli¢ed with cDNA-speci¢c
primers (lane 2 in Fig. 4B), while a 0.8 kbp product
containing an intron was detected when genomic
DNA and the same primers were used (lane 1 in
Fig. 4B).
The enzymatic activity of the cDNA product was
tested on toluidine blue-DNA (TB-D) plates to clarify whether or not the cDNA product would function as a nuclease (Fig. 5). This assay has been employed for identi¢cation of nuclease activities of Nuc
nuclease [3], a thermostable nuclease from Staphylococcus hyicus [12], and the staphylococcal extracellular nuclease [2,13], the activity of which generated a
`pink halo' on the TB-D plate. As shown in Fig. 5,
pink halos were detected around extracts of E. coli
Fig. 5. The TB-D plate assay of nuclease activities. To make an expression construct of the cloned cDNA under the control of the
lac promoter, the 5P region upstream from the EcoRI site (see Fig. 1) in the cDNA to the EcoRI site in the vector was removed by
EcoRI digestion and subsequently self-ligated. The construct was introduced into E. coli HB101. HB101 cells (OD600 = 1.0) grown in 3
ml of LB medium in the presence of ampicillin were pelleted and resuspended with 0.5 ml of LB medium. The cells were then sonicated and 3 Wl of the supernatant (line C) was spotted on TB-D plates without any additive (row 1) and with 1 mM EGTA (row 2),
1 mM EDTA (row 3), 10 mM CaCl2 (row 4), 10 mM MgCl2 (row 5), and 5 mM ZnCl2 (row 6), respectively. Sample solution containing 1 Wg of Bovine pancreas DNase I (line A) as a positive control, and 3 Wl of extract prepared from HB101 harboring pBluescript SK alone (line B) as a negative control were also spotted on the same plates. Pink halos around spots were observed after incubation for 10 h at room temperature.
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K. Isono et al. / Biochimica et Biophysica Acta 1491 (2000) 267^272
cells expressing the cDNA (grid C1), as well as
around Ca2‡ /Mg2‡ -dependent bovine pancreas
DNase I as a positive control (grid A1), while no
pink halo was produced around extracts of cells containing the empty vector (grid B1). Therefore, the
cloned cDNA encodes a nuclease. This is the ¢rst
report that a plant nuclease homolog is functional.
We further tested e¡ects of the divalent cations,
Ca2‡ , Mg2‡ , and Zn2‡ on the activity of the nuclease. The presence of EGTA (grid C2) and EDTA
(grid C3) inhibited nuclease activity, as well as
DNase I activity (grids A2 and A3). The addition
of Ca2‡ led to an increase in the size of the pink
halos (grid C4), indicating an enhancement of nuclease activity, and strongly suggesting that the activity
of the nuclease depends on the concentration of Ca2‡
ions. We designated it CAN (Ca2‡ -dependent nuclease). In contrast, the presence of Zn2‡ inhibited
CAN activity (grid C6). These properties are the
same as those of the staphylococcal nuclease [14]
and ParB [5]. On the other hand, Mg2‡ ions did
not have any signi¢cant e¡ect (grid C5). This property is similar to that of ParB, but not the staphylococcal nuclease whose activity is enhanced by
the presence of Mg2‡ .
We acknowledge the possibility that the nuclease
assay may re£ect endogenous nuclease activity of the
E. coli host cells rather than CAN activity; it is conceivable that CAN might play a role as either an
activator or an inducer of the endogenous E. coli
nuclease. However, this assay has been successfully
employed to prove the activities of Nuc nuclease [3],
a thermostable nuclease from Staphylococcus hyicus
[12], and the staphylococcal extracellular nuclease
[2,13]. Furthermore, CAN has signi¢cant homology
to these nucleases. We thus consider it unlikely that
the detected activity resulted from the endogenous
E. coli nuclease.
It is most likely that CAN is an intracellular nuclease in Arabidopsis. Intracellular nucleases play
critical roles in diverse cellular events such as DNA
recombination and repair [15,16], RNA processing
[17^19], and apoptosis [20^22]. The concentration
of cytosolic free calcium is usually maintained at
less than 0.2 mM in plant cells [23]. However, some
physiological stimuli such as light, hormones, temperature, and drought stress can considerably elevate
the Ca2‡ concentration in plant cells (reviewed in
271
[23,24]). The Kd value of staphylococcal nuclease
for Ca2‡ has been reported to be 0.111 þ 0.02 mM
[25]. Therefore, it is easily speculated that change in
cytosolic Ca2‡ concentrations activates CAN, the
plant homolog of staphylococcal nuclease. Transgenic Arabidopsis lines expressing altered CAN genes
should be prepared for further insights into the biological function of intracellular nucleases.
We thank Dr. Ken-ichi Tomizawa for valuable
discussions. This work was supported by Grants-inAid for Scienti¢c Research for Priority Areas (no.
04273101) and General Research (no. 06404003)
from the Ministry of Education, Science and Culture
of Japan, and also by grants from the Human Frontier Science Program, the New Energy and Industrial
Technology Development Organization (NEDO)/the
Research Institute of Innovative Technology for the
Earth (RITE), the Toray Science Foundation, and
the Salt Science Research Foundation.
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