Embed Size (px)
Citation preview
Full Length Research Paper
Cloning and Characterization of a Novel Hsp100/Clp Gene(osClpD) from Oryza sativa
GUO-AN SHEN, YONG-ZHEN PANG, CHANG-FA LIN, CHUN WEI, XIAO-YIN QIAN, LI-ZHI JIANG, XI-LING DU,KE-GUI LI, KOTB ATTIA and JIN-SHUI YANG*
State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200433, People’s Republic of China
(Received 27 January 2003)
A novel osClpD gene, encoding a highly conservativeClpD subfamily member, was first isolated and charac-terized from Oryza sativa. The full-length cDNA ofosClpD gene was 3140 bp and contained a 2884 bp openreading frame encoding a 938 amino acid protein.The phylogenetic tree and blast search showed thatOSClpD belonged to the ClpD subfamily of theHsp100/Clp family, and contained all protein motifscharacteristic for the ClpD subfamily of Hsp100/Clpproteins. The real-time quantitative PCR analysis provedthat it was inducible by water deficit and temperaturestress in vegetative tissues.
Keywords: osClpD; Oryza sativa; Hsp100/Clp; mRNA
Genbank accession No. AY166599.1
INTRODUCTION
Proteolysis is involved in a wide range of processesduring the biogenesis and maintenance of all organ-isms. Proteases in chloroplast are considered a vitalhomeostatic factor that influences metabolic functionssuch as photosynthesis under both optimal andadverse growth conditions (Adam and Clarke, 2002).One class that has attracted much attention recently isthe caseinolytic protease (Clp), which is ubiquitousamong photosynthetic organisms and has beenidentified in cyanobacteria and in plants. Thephysiological role of the Clp proteins chloroplasts isto degrade misfolded or unassembled proteins anATP-dependent manner (Liu and Jagendorf, 1984;Halperin and Adam, 1996; Majeran et al., 2000).
Clp protein is composed of the proteolytic subunitClpP and ATP-dependent regulatory ATPase sub-unit. The regulatory subunit belongs to a growingfamily of proteins known as HSP100/Clp, which aredivided into two major classes: class I and class II.The class I proteins with five subfamilies (A, B, C, Dand E) contain two nucleotide-binding domains(NBDs) flanked by amino-terminal, middle(or spacer) and carboxy-terminal regions. The classII proteins with four subfamilies (M, N, X and Y) areshorter in length, containing only one NBD and aconservative C-terminal region (Schirmer et al.,1996).
ClpD has so far only been identified in higherplant Arabidopsis thaliana as a desiccation inducibletranscript and located in the stroma of chloroplasts(Kiyosue et al., 1993; Nakashima et al., 1997; Weaveret al., 1999). Although closely related, ClpD differswith ClpC on specific signature sequences(Van Houten and Snowden, 1993) and on itsdifferential expression characteristics (Nakabayashiet al., 1999). A. thaliana ClpD (ATClpD) haspreviously been named as ERD1 (Kiyosue et al.,1993; Nakashima et al., 1997; Nakabayashi et al., 1999)and SAG15 (Lohman et al., 1994). Despite thesestudies, no other ClpD gene has been isolated andcharacterized in the other organism. In this paper, thecloning and partial characterizations of a rice cDNA,encoding a protein similar to A. thaliana ClpD, weredescribed. We also demonstrate that expression ofrice ClpD gene, termed osClpD, are inducible byabiotic stress.
ISSN 1042-5179 print/ISSN 1029-2365 online q 2003 Taylor & Francis Ltd
DOI: 10.1080/1085566031000141153
*Corresponding author. Tel.: þ86-21-65643715. Fax: þ86-21-65648376. E-mail: [email protected]
DNA Sequence, August 2003 Vol. 14 (4), pp. 285–293
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
MATERIALS AND METHODS
Materials
The Oryza sativa cultivar, Zhenxian 97A, was used inthe experiments. First of all, all the seeds weresurface-sterilized with 20% javel water and thengerminated on wet filter paper at 288C with a 16-hphotoperiod. To clarify whether the expression ofthe gene encoding the osClpD mRNA is stress-related, rice plants of Zhenxian97A were treated byvarious conditions involving drought, cold shockand heat shock. For heat-shock experiments, trayscontaining 3-leaf-old seedlings were subjected to408C for 1, 6, 12 and 24 h. For cold-shock experiments,trays containing 3-leaf-old seedlings were subjectedto 48C for 1, 6, 12 and 24 h. For water stresstreatments, 3-leaf-old seedlings were transferred tothe dry Whatman paper for 1, 6, 12 and 24 h. At theindicated time after treatments, leaves were cut witha razor blade and placed directly into liquid nitrogenbefore RNA extraction. Controls were kept at 288C for1, 6, 12 and 24 h.
Shanghai Sangon Biotechnological Company,China, carried out primer synthesis and DNAsequencing. The pGEM T-easy vector was purchasedfrom Promega, USA. The DNase, Taqplus DNApolymerase, and dNTPs were purchased fromShanghai Sangon Biotechnological Company, China.Superscripte II Rnase H2 reverse transcriptase waspurchased from Invitrogen life technologies, USA.
Methods
Total RNA Preparation and Reverse Transcript
One gram of rice tissue was ground to a fine powderin liquid nitrogen. The powder was dispersed in10 ml Trizol (Gibco, USA) and centrifuged atmaximum speed for 5 min at 48C. Followingphenol/chloroform/isoamyl-alcohol (25/24/1,v/v/v) extractions for three times, nucleic acidswere precipitated with ethanol and collected bycentrifugation. Total RNA was digested using RNA-free DNase according to the manufacturer’s instruc-tions (Sangon, China). An aliquot of 120 ng RNAwas reverse transcribed with a cDNA synthesisprimer 50-TTTTTTTTTTTTTTTTT-30.
Cloning and Sequencing of Full-length cDNA ofosClpD
Recently, an EST fragment (750 bp), encoding partialrice Clp protein, had been screened with suppressionsubtractive hybridization technique in our lab. Usingthis EST as query we searched GeneBank and got arice genomic scaffold with a gap (AAAA01002101.1).The open reading frame showed that it encoded aClp protein, but some sequence of this protein was
absent and unknown in the middle region due to theexistence of the gap.
A pairs of primer Clp51: (50-GTGCGTAGTTCGTT-CGTTCGTTG-30) and Clp31: (50-CGATAACATTCCG-CAACTATGG-30) were designed and synthesizedaccording to the corresponding genomic nucleic acidsequence of osClpD gene. The first strand cDNAwere subject to the amplification of the osClpD gene.The PCR was carried out in a total volume of 50mlcontaining 2ml cDNA, 10 pmol each of primers Clp31and Clp51, 10 mmol dNTPs, 1X PCR reaction bufferand 5U Taqplus polymerase. To overcome nonspecificamplification, a hot-start and touchdown polymerasechain reaction (PCR) procedure was performedusing PTC-100 Peltier Thermal Cycler (MJ ResearchIncorporated, USA). Mixed all the componentsexcept Taqplus DNA polymerase, heated the solutionto 948C, supplemented Taqplus DNA polymerase,and processed to touch-down thermal cycling(D’Aquila et al., 1991; Chou, 1992). This touchdownPCR method was optimized as follows: 948C for 5 min,5 cycles at 948C for 45 s, 568C for 30 s, 728C for 3 min,followed by 30 cycles at 948C for 45 s, 488C for 30 s, 728Cfor 3 min, and then 728C for 5 min. The PCR productwas purified and cloned into pGEM-T easy vector,followed by sequencing.
Sequence Analysis
Comparison of the predicted OSClpD amino acidsequence with the database was made usingthe BLAST 2.0 program from NCBI (http://www.ncbi.nlm.nih.gov/BLAST/). The alignment and thephylogenetic tree analysis were done using pro-grams Vector NTI and ClustalX1.8, respectively. Thetheoretical pI and molecular mass were predicted byCompute pI/Mw tool (http://cn.expasy.org/tools/pi_tool.html). Predictions for chloroplast localizationwere made using TargetP V1.0 (Nielsen et al., 1997;Emanuelsson et al., 1999; 2000) and Predotar version
FIGURE 1 osClpD PCR product on 0.8% Agrose gel. Lane 1: Molwt. calibration: lambda Hind III digsted; Lane 2: osClpD geneamplification product.
GUO-AN SHEN et al.286
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
osClpD GENE FROM ORYZA SATIVA 287
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
0.5 (http://www.inra.fr/predotar/). The coiled-coilsupersecondary structure was predicted using theCOILS program (Lupas et al., 1991).
Quantitative PCR of osClpD Gene Expression
A real-time quantitative PCR analysis was used toinvestigate the kinetics of accumulation of osClpDmRNAs in response to various stresses. Primers andprobe were designed by using Primer Expresssoftware. Nucleotide sequences of forward primer(Clp-F) and reverse primer (Clp-R) and the Taq-Manprobe (Clp-P) of osClpD gene were as follows: Clp-F:50-TTTGCGAAGAAGGCTATGACAA-30, Clp-R: 50-T-TCGCTGATCACATCCTCAATC-30, Clp-P: 50-CCAC-TGA GGAGAGCCGTCACCC AC-30. The rice actingene (Genebank accession number X16280.1) served asendogenous reference for expression normalization.The forward primer (Actin-F) and reverse primer(Actin-R) and the Taq-Man probe (Actin-P) of actingenes were followed: Actin-F: 50-ATTGGTGCTGAG-CGTTT-30, Actin-R: 50-CCCGCAGCTTCCATTCCTA-30, Actin-P: 50-CCCTGAGGTCC TCTTCCAGCCTT-CCTT-30. The probes were labeled with FAM andTAMRA fluorochromes and synthesized by TaKaRaBiotechnology (Dalian, China). The quantitativePCRs were done according to the manual of theAppliedBiosystem Company. Relative quantitation
of rice osClpD gene expression was performed usingthe 22DDC
T method. The fold differences of every timepoint, normalized to an endogenous reference (actin)and relative to a calibrator (untreated control), weregiven by the arithmetic formulas: 22DDC
T (Livak andSchmittgen, 2001). The real-time quantitative assayswere carried out using the ABI PRISM 7900 HTSystem and the TaqMan PCR Core Reagent kit(PE, Biosystems). The ABI PRISM 7900 instrumentwas operated under the following thermal cyclingconditions: 508C for 2 min, 958C for 10 min followed by40 cycles at 948C for 30 s, 598C for 30 s and 728C for 30 s.The PCR product was 90 bp in length. One microliterspecimen was added to 40 ul of master mix for afinal reaction mixture, which consisted of 5 mMMgCl2, 0.2 mM each dATP, dCTP, dGTP and0.4 mM dUTP, 0.4 mM forward and reverse primers,0.25 mM TaqMan probe, 1.25U Taq polymerase, 0.5UAmpErase uracil N-glycosylase and 1X TaqManBuffer A.
RESULTS
Isolation and Characterization of the osClpDcDNA
Using the primers (CLP31 and CLP51) designedaccording to the partial genomic sequence,
FIGURE 2 The complete nucleotide and predicted amino acid sequence of osClpD full-length cDNA. The amino acid residues are shownin the single letter code. The conservative one N-terminus region (Clp-N) and two NBDs are underlined. The characteristic signaturesequences are shown in gray boxes. The Walker consensus distinctions in NBDs domain are shown in white boxes. The positions of theprimers and probe are indicated with arrows.
GUO-AN SHEN et al.288
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
a cDNA fragment about 3 kb was obtained byamplification of the first strand cDNA (Fig. 1). Thesequence of the cDNA indicated that it containeda 2884 bp open reading frame encoding 938 aminoacids (Fig. 2). The calculated molecular mass ofthe protein was 101 kDa (pI 6.5), which wassimilar to those of other Hsp100/Clp proteins(Schirmer et al., 1996). The full-length cDNA ofosClpD contained a 50 untranslated region of105 bp and a 30 untranslated region of 220 bp(Fig. 2).
Characterization of the OSClpD Protein
The comparison between the predicted aminoacid sequence of OSClpD with the database(non-redundant GenBank þ EMBL þ DDBJ þ PDB)gave maximum scores to A. thaliana ClpD protein(ATClpD), there were 75% similarity and 61%identity between OSClpD and ATClpD (Fig. 3).
The blast search results revealed that the conserva-tive region of OSClpD also had a high degree ofsequence homology with other 95 Clp familyproteins, which contained signature sequences thatidentify ClpC or B proteins (data not shown).
The representatives of the class I of the Hsp/Clpfamily, at the level of the full-length protein, weresubjected to the generation of the phylogenetic tree.It was obvious that the class I of Clp familywas divided into four subfamilies ClpA, B, Cand D. The results revealed that rice OSClpD andA. thaliana ATClpD were more related to each otherthan to any other Clp subfamily proteins; mean-while, the OSClpD belonged to the ClpD subfamily,which contained only two members known besidesthe ATClpD (Fig. 4).
Hsp100/Clp proteins were characterized byfive highly conservative signature sequence withthe different consensus (Schirmer et al., 1996).A signature sequence comparison between
osClpD GENE FROM ORYZA SATIVA 289
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
OSClpD and other Hsp100/Clp subfamily wereshown in Fig. 5. OSClpD contained aminoterminus, middle and carboxyl terminus signaturesequences that identified ClpD proteins (Schirmeret al., 1996). Furthermore, it contained two NBDs(Fig. 2).
The middle region (or spacer) of the ClpAwas veryshort (54 aa), but that of the ClpC and D wereof intermediate length (101–118 aa) and that of ClpBwas long (172 – 207 aa) (Schirmer et al., 1996).The length (142 aa) of OSClpD in the middle regionwas characteristic of ClpD subfamily, but it waslonger than the length (101 – 118 aa) originallyestimated. None of the characteristic signaturesequence II in the middle region presented in othersubfamily was found in the OSClpD, a similarobservation was reported in A. thaliana ClpD(Schirmer et al., 1996). The signature sequence II
presented in ClpB and C subfamily shared a highlysimilarity with the so-called UVR domain. Thisdomain in Escherichia coli UvrB gene couldinteract with the homologous domain in E. coliUvrC gene throughout a putative coiled coil structure(Van Houten and Snowden, 1993; Moolenaar et al.,1995). A. thaliana ClpD showed no propensityfor coiled-coil formation in the middle region (Nieto-Sotelo et al., 1999). Using the COILS program (Lupaset al., 1991), we confirmed that OSClpD had no coiled-coil structures in the middle region (Fig. 6). Thisindicated that ClpD subfamily had different super-secondary structure from other subfamily and theabsence of this signature sequence was typicalcharacter of ClpD subfamily. The significances ofthese differences were not clear.
Plant ClpC and D homologues include proteinswith amino-terminal transit peptides for chloroplast
FIGURE 3 The comparison of the amino acid sequence of the predicted OSClpD protein with another member of the ClpD subfamilyfrom A. thaliana (D17582). The completely identical amino acids were indicated with red foreground and yellow background. Conservativeamino acids were indicated with blue foreground and light blue background.
GUO-AN SHEN et al.290
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
targeting (Boston et al., 1996). By using the Predotarprogram we predicted that osClpD gene was notchloroplastic or mitochondrial. By using the TargetPprogram we predict that osClpD gene encoded aprecursor OSClpD protein with a 83-amino acidchloroplast transit peptide. Rice OSClpD hadthe characteristic organelle-targeting sequencesfound in the ClpC and D subfamilies.
Taken together, all these features of this proteinallowed us to classify it as a member of the ClpDsubfamily. The transit sequence encoded at the 50 endof the transcript was similar to that of ATClpD(Fig. 3), meanwhile, the import competence ofthe precursor provided support that it was full-length. It had been named osClpD for rice ClpDsubfamily protein.
Accumulation of osClpD mRNA in Response toStress Treatments
Time courses of the mRNA levels of osClpDgenes under stress were analyzed by real-timequantitative PCR analysis in the sterile line (Zhenxian97A), from which the osClpD gene was cloned. Thestress/control ratios of osClpD mRNA at each timepoint were showed in Fig. 7, from which a strongchange in the amount of osClpD transcripts could beobserved during the stress period (24 h).
After 24 h heat treatment the seedlings showedapproximately a diameter of 0.5 cm dead region fromthe leaf tip, but no wilted leaf were observed. In heatshock leaves, transcript level continued to increasefrom 1 h (F ¼ 5.169, P , 0.05) after onset of thetreatment. The mRNA level of osClpD gene increased
FIGURE 4 Phylogenetic tree shows the relationships among Clpmembers. The tree was obtained by using the neighbor-joiningmethod with the ClustalX 1.8. The accession numbers of theproteins are indicated in parentheses. The proteins analyzed were:E. coli ClpA (M31045), Rhodobacter blasticus ClpA (P05444), Triticumaestivum (AF097363), Zea mays Hsp101 (AF077337), O. sativaHsp100 (AF332981), Zea mays (AF083327), E. coli ClpB (M29364),Haemophilus influenzae (HI0859), Synechococcus sp. PCC.7942(U20646), Dichelobacter nodosus ClpB (M32229), Trypanosomabrucei ClpB (M92325), Leishmania major Hsp100 (Z38058),A. thaliana Hsp101 (U13949), Glycine max (L35272), Plasmodiumberghei (U46549), Heterosigma carterae (Z25810), Pisum sativum(L09547), Brassica napus (X75328), Synechococcus sp. (U16134),Synechocystis sp. PCC6803 (D64000), Bacillus subtilis (U02604),Serpulina hyodysenteriae (X73140), Bos taurus (L34677), Lycopersiconesculentum ClpC (P31541), Lycopersicon esculentum (P31542),Mycobacterium leprae (P24428), Lactococcus lactis (Q06716),A. thaliana ClpD (ATClpD, D17582), O. sativa ClpD (OSClpD,AY166599.1).
FIGURE 5 The signature sequence comparison between OSClpD and different subfamily of the Hsp100/Clp protein family. A, B, C, Dand OS indicate ClpA, ClpB, ClpC, ClpD and OSClpD, respectively.
FIGURE 6 Probability of forming coiled-coil supersecondarystructures in OSClpD. (A) The y-axis indicates the probability offorming a coiled-coil with a maximum value of 1.0. In the x-axis, thesmall scale represents a segment of 100 aa. The COILS program wasrun with MTIDK matrix, with weights (at positions a, d ¼ 2.5 and atpositions b, c, e, f, g ¼ 1.0) and a window of 28 aa. M, middle region.
osClpD GENE FROM ORYZA SATIVA 291
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
to 340% of the control at the 12 h (F ¼ 8.243, P , 0.05,Fig. 7A).
To examine the effect of cold shock on osClpD geneexpression, plants were subjected to 48C. After 24 hcold treatment, the seedlings were markedly wilted.The mRNA level of osClpD within 6 h exceeded thecontrol level, reaching 207% of the control. The mRNAof this gene reached its highest relative level of expres-sion (318% of the control) within 24 h (F ¼ 49.997,P , 0.0005) after plants were shifted to 48C (Fig. 7B).
We also investigated the effect of dehydrationon osClpD transcript levels (Fig. 7C). By withholdingwater for 1 day, the water content of the rice leaves wasdramatically reduced, a point at which they appearedheavily wilted. An increase in the amounts of osClpDtranscript, that remained at a high level between 6 h(F ¼ 10.132, P , 0.005) and 12 h (F ¼ 14.381,P , 0.005) indicating that gene expression was alteredby dehydration. The maximum level of accumulationwas at 12 h (194% of the control).
These results showed that the osClpD gene was up-regulated under these stress with different profiles.This suggested that osClpD gene was involved indehydration, cold and heat tolerance in rice.
DISCUSSION
Although many members of the Hsp/Clp familyfrom plant have been cloned, up to now, there is no
report about the subfamily ClpD from rice and theother cereal plants. In this paper, we have identified,isolated and characterized a novel member of theHsp100/Clp family, osClpD gene. The OSClpD ishomologous to the Hsp100/Clp family with all theprotein motifs characteristic of ClpD subfamily ofclass I group (Schirmer et al., 1996). OSClpD displaysa very high degree of similarity to A. thalianaATClpD, the only member of the ClpD subfamily.The cloning of osClpD gene makes the existence of aseparate ClpD subfamily more evident. Whateverthe eventual function, the striking conservative ofthis gene throughout monocots and dicots stronglysuggests that the ClpD subfamily proteins playan important role. On the basis of analysis, itcould be postulated that osClpD was localized inthe chloroplast. However, the localization either inthe chloroplast or in the mitochondria needs to beverified using immunological or biochemicaltechniques.
Previous papers report that the ATClpD is inducednot only by water stress, such as dehydration andhigh salinity, but also by natural senescence anddark-induced etiolation (Nakashima et al., 1997).Meanwhile, the GUS (beta-glucuronidase) reportergene driven by the 0.9 kb promoter of the atClpDgene was induced by dehydration and high saltstress at significant levels in the transgenic plants(Weaver et al., 1999). In this paper, the real-timequantitative PCR analysis revealed that osClpD is
FIGURE 7 A comparative ratio analysis by real-time quantitative PCR amplification of osClpD under different stresses. Fold difference(y-axis) represents the relative expression of osClpD gene under stress compared to the untreated control. Signal strength of the control wastaken as unity. The data representing the averages of triplicate quantitative PCR amplifications were normalized to the expression of actingene in each treatment. Lines above bars indicate standard deviation. *P , 0.05, **P , 0.005, ***P , 0.0005. (A) The accumulation of osClpDmRNA in 3-day-old seedlings in response to the heat shock of 408C. (B) The accumulation of osClpD mRNA in 3-day-old seedlings inresponse to the cold shock of 48C. (C) The accumulation of osClpD mRNA in 3-day-old seedlings in response to the dehydration treatment.
GUO-AN SHEN et al.292
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
induced under water stress and temperature treat-ment (Fig. 7A–C). The promoter region (524 bpupstream from the start codon in the scaffold fromAAAA01002101.1) is used to predict the possiblebinding site for transcript factor, using MatInspectorprogram (http://www.genomatix.de/free_login.html). No typical stress promoter element wasfound among the expected promoter region. Oneexplanation is that the signal pathway of riceOsClpD is independent of HSE, DRE, ABRE, etc.The other explanation is that the promoter regionknown is too short to predict. The array of stressesthat induce osClpD gene expression suggests that thisprotein may play an important role during stresses.However, the expression results showed fromATClpD and in this paper, make it possible for usto research further, intending to get more detailsabout the expression pattern and the promoter fromthis important crop model plant.
Acknowledgements
The authors thank Dr Zhihua Liao for his help inprocessing experiment data. This work wassupported by the Major State Basic ResearchDevelopment Program of China (Grant No.2001CB108805).
References
Adam, Z. and Clarke, A.K. (2002) “Cutting edge of chloroplastproteolysis”, Trends in Plant Science 7, 451–456.
Boston, R.S., Viitanen, P.V. and Vierling, E. (1996) “Molecularchaperones and protein folding in plants”, Plant MolecularBiology 32, 191–222.
Chou, Q. (1992) “Minimizing deletion mutagenesis artifact duringTaq DNA polymerase PCR by E. Coli SSB”, Nucleic AcidsResearch 20, 4371.
D’Aquila, R.T., Bechtel, L.J., Videler, J.A., Eron, J.J., Gorczyca, P.and Kaplan, J.C. (1991) “Maximizing sensitivity andspecificity of PCR by preamplification heating”, NucleicAcids Research 19, 3749.
Emanuelsson, O., Nielsen, H. and Von Heijne, G. (1999) “ChloroP,a neural network-based method for predicting chloroplasttransit peptides and their cleavage sites”, Protein Science 8,978–984.
Emanuelsson, O., Nielsen, H., Brunak, S. and Von Heijne, G. (2000)“Predicting subcellular localization of proteins based on their
N-terminal amino acid sequence”, Journal of Molecular Biology300, 1005–1016.
Halperin, T. and Adam, Z. (1996) “Degradation of mistargetedOEE33 in the chloroplast stroma”, Plant Molecular Biology 30,925–933.
Kiyosue, T., Yamaguchi-Shinozaki, K. and Shinozaki, K. (1993)“Characterization of cDNA for a dehydration-inducible genethat encodes a CLPA, B-like protein in Arabidopsis thaliana L.”,Biochemical and Biophysical Research Communications 196,1214–1220.
Liu, X.Q. and Jagendorf, A.T. (1984) “ATP-dependent proteolysisin pea chloroplasts”, FEBS Letters 166, 248–252.
Livak, K.J. and Schmittgen, T.D. (2001) “Analysis of relativegene expression data using real-time quantitative PCRand the 22DDCT method”, Method 25, 402–408.
Lohman, K., Gan, S., John, M. and Amasino, R.M. (1994)“Molecular analysis of natural leaf senescence in Arabidopsisthaliana”, Plant Physiology 92, 322–328.
Lupas, A., Van Dyke, M. and Stock, J. (1991) “Predicting coiledcoils from protein sequences”, Science 252, 1162–1164.
Majeran, W., Wollman, F.A. and Vallon, O. (2000) “Evidence for arole of ClpP in the degradation of the chloroplast cytochromeb6f complex”, Plant Cell 12, 137–150.
Moolenaar, G.F., Franken, K.L., Dijkstra, D.M., Thomas-Oates, J.E.,Visse, R., Van De Putte, P. and Goosen, N. (1995) “The C-terminal region of the UvrB protein of Escherichia coli containsan important determinant for UvrC binding to the preincisioncomplex but not the catalytic site for 30-incision”, Journal ofBiology Chemistry 270, 30508–30515.
Nakashima, K., Kiyosue, T., Yamaguchi-Shinozaki, K. andShinozaki, K. (1997) “A nuclear gene, erd1, encoding achloroplast-targeted Clp protease regulatory subunit homo-log is not only induced by water stress but also development-ally up-regulated during senescence in Arabidopsis thaliana”,Plant Journal 12, 851–861.
Nakabayashi, K., Ito, M., Kiosue, T., Shinozaki, K. andWatanabe, A. (1999) “Identification of clp genes expressedin senescing Arabidopsis leaves”, Plant Cell Physiology 40,504–514.
Nielsen, H., Engelbrecht, J., Brunak, S. and Von Heijne, G. (1997)“Identification of prokaryotic and eukaryotic signal peptidesand prediction of their cleavage sites”, Protein Engineering 10,1–6.
Nieto-Sotelo, J., Kannan, K.B., Martunez, L.M. and Segal, C. (1999)“Characterization of a maize heat-shock protein 101 gene,HSP101, encoding a ClpB/Hsp100 protein homologue”, Gene230, 187–195.
Schirmer, E.C., Glover, J.R., Singer, M.A. and Lindquist, S.(1996) “HSP100/Clp proteins: a common mechanismexplains diverse functions”, Trends in Biochemical Science 21,289–296.
Van Houten, B. and Snowden, A. (1993) “Mechanism of action ofthe Escherichia coli UvrABC nuclease: clues to the damagerecognition problem”, Bioessays 15, 51–59.
Weaver, L.M., Froehlich, J.E. and Amasino, R.M. (1999)“Chloroplast-targeted ERD1 protein declines but its mRNAincreases during senescence in Arabidopsis L.”, Plant Physio-logy 119, 1209–1216.
osClpD GENE FROM ORYZA SATIVA 293
Mito
chon
dria
l DN
A D
ownl
oade
d fr
om in
form
ahea
lthca
re.c
om b
y N
yu M
edic
al C
ente
r on
11/
09/1
4Fo
r pe
rson
al u
se o
nly.
