Journal of Experimental Botany, Vol. 51, No. 353, pp. 1979±1990, December 2000
Carrot cells contain two top1 genes having the coding capacity for two distinct DNA topoisomerases I1 A. Balestrazzi2, A. Chini2, G. Bernacchia3, A. Bracci3, G. Luccarini4, R. Cella2 and D. Carbonera2,5 2
Department of Genetics and Microbiology `A. Buzzati-Traverso', University of Pavia, Via Abbiategrasso 207, 27100 Pavia, Italy 3 Department of Biology, University of Ferrara, Corso Porta Mare 2, I-44100 Ferrara, Italy 4 Istituto di Mutagenesi e Differenziamento, C N R, via Svezia 2u105, 6100 Pisa, Italy Received 2 May 2000; Accepted 7 July 2000
Abstract
Introduction
Five DNA topoisomerase I cDNA clones were isolated from a carrot (Daucus carota) cDNA library and two classes of nucleotide sequences were found. One component of the first class, pTop9, perfectly matches the open reading frame of pTop28, a truncated top1 cDNA previously described, and extended it by 594 nucleotides (top1a). A member of the second class, pTop11, contains an open reading frame 2727 bp long (top1b) with a coding capacity for a second putative DNA topoisomerase I of 101 kDa. Both pTop9 and pTop11 clones are full length cDNAs. The two deduced amino acid sequences share a relevant similarity (89%) only at the C-terminal domain, whereas the similarity is reduced to 32% in the N-terminal region. Southern blot analysis and PCR amplification of genomic DNAs from carrot pure lines suggested the presence of two distinct loci. Northern blot analysis revealed the presence of two distinct transcripts of 3.0 and 3.2 kb in both cycling and starved cell populations. Three fusion peptides corresponding to the N-terminal domain of the a and b forms and from the common C-terminal domain of carrot topoisomerases I were overexpressed in E. coli cells and used to raise antibodies in rabbit. Immunolocalization seems to suggest the presence of two topoisomerases I in carrot nuclei.
Changes in DNA topology occur during many cellular processes, including replication, transcription and recombination. Topological problems related to DNA metabolism are solved by a class of enzymes, named DNA topoisomerases, able transiently to break and reseal phosphodiester bonds via the formation of a covalent DNA-protein intermediate. Topoisomerases can be classi®ed into two main types depending on their reaction mechanisms: type I topoisomerases cleave only one DNA strand in the duplex, while type II topoisomerases cleave both strands (Bates and Maxwell, 1993). The eukaryotic topoisomerase I is a member of the type IB subfamily since it forms a covalent intermediate between a tyrosyl group of the protein and the 39-end of the broken DNA strand (Wang, 1996). DNA topoisomerase I may play a major role in transcription; the enzyme is physically associated with actively transcribed regions of chromatin where it reduces torsional stress generated during transcription (Fleischmann et al., 1984). However, more recent studies proposed a direct involvement of the enzyme in the modulation of gene expression. The identi®cation of human topoisomerase I as a cofactor for the activation and repression of the basal transcription of class II genes (Merino et al., 1993), argues for a direct role in facilitating binding of other transcription factors. The discovery of mammalian topoisomerase I as a speci®c protein kinase that phosphorylates pre-mRNA splicing factors of the SR family strengthens the opinion that, in addition to its function in
Key words: cDNA cloning, cell proliferation, Daucus carota, heterologous expression, topoisomerase I. 1 5
The nucleotide sequence reported will appear in the GenBank Data Library under the accession number AJ223326. To whom correspondence should be addressed. Fax: q39 382 528496. E-mail:
[email protected]
ß Society for Experimental Biology 2000
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solving torsional stress problems, the enzyme might modulate gene activity (Rossi et al., 1996). The top1 gene is constitutively expressed, but its transcriptional activity is dependent on a vast array of endogenous and environmental signals, among which are those inducing cell proliferation. The top1 mRNA steadystate level greatly increases when cells are induced to divide. The promoter region of human and mouse top1 genes (Kunze et al., 1990; Heiland et al., 1993) contains both elements acting as binding sites for ubiquitous transcriptional factors and sequences corresponding to binding sites for factors responding to exogenous stimuli such as NF-kB (Lenardo and Baltimore, 1989), `mycrelated' factors (Evan and Littlewood, 1993) and factors involved in cAMP-mediated responses (Ziff, 1990). Genetic analyses of mutant strains of Saccharomyces cerevisiae and Schizosaccharomyces pombe defective in the top1 gene have provided evidence that topoisomerase I activity is non-essential for cell viability (Thrash et al., 1984; Goto and Wang, 1985). The situation seems to be different in multicellular organisms such as Drosophila melanogaster and mouse, in which the presence of the enzyme is essential for embryo development. In Drosophila, topoisomerase I activity is required beyond the blastocyst stage of embryo development (Lee et al., 1993), while mouse top1 homozygous mutants fail to reach the 16-cell stage (Morham et al., 1996). The ®nding that human topoisomerase I has an intrinsic protein kinase activity, in addition to its known DNA relaxing activity, leads to the question of whether its essentiality in a multicellular organism might be correlated to this other role. Notably, in lower eukaryotes such as yeasts, topoisomerase I only possesses DNA-relaxing activity (Labourier et al., 1998). In contrast to other eukaryotes, no information on the essentiality of the plant enzyme have yet been gathered. Current knowledge of DNA topoisomerase I from higher plants essentially concerns the biochemical properties of this enzyme. A type I DNA-relaxing activity was isolated for the ®rst time from wheat germ (Dynan et al., 1981) and then from several different plant species, among which Daucus carota (Carbonera et al., 1988, 1990), Nicotiana tabacum (Heath-Pagliuso et al., 1990), Zea mays (Carballo et al., 1991), Pisum sativum (Chiatante et al., 1991, 1993) and Brassica oleracea var. italica (Kieber et al., 1992a). The proteins vary in size from the 70 kDa of the tobacco enzyme (Cole et al., 1992) to the 200 kDa of the protein from Brassica oleracea var. botrytis (Fukata and Fukasawa, 1982). To date only three genes encoding DNA topoisomerases I have been isolated and characterized, namely from Arabidopsis thaliana (Kieber et al., 1992b), Daucus carota (Balestrazzi et al., 1996) and Pisum sativum (Reddy et al., 1998). Recently, the nucleotide sequence for a DNA topoisomerase I-like protein has been identi®ed on
chromosome 4 of A. thaliana, within the remit of the Arabidopsis sequencing project (GenBank Accession No. CAA16524); however, differently from previous genes, the predicted product of this gene showed similarity to a prokaryotic topoisomerase I. In previous work, two partially overlapping cDNAs encoding carrot DNA topoisomerase I have been isolated from a poly(A)q-primed library, using an Arabidopsis thaliana probe, and from a cDNA library spanning the 59 region of the top1 transcript. Northern analysis of hypocotyl tissues induced to proliferate by the addition of 2,4-D showed that the top1 mRNA accumulation parallels the proliferation of provascular cells. In this paper, evidence for the presence of two distinct top1 loci in the nuclear genome of carrot is provided. This is the ®rst documented case in higher plants on the cloning of two nuclear genes with eukaryotic features and containing intact open reading frames for two putative eukaryotic topoisomerases I. Studies on their expression patterns, both at the transcript and protein level, are reported.
Materials and methods Plant material The domesticated cell lines E4 (cv. Lunga di Amsterdam) and Hypo2 (var. Berlikum) from Daucus carota were maintained as described (Cella et al., 1983). Cell suspensions were subcultured by diluting cells with B5 fresh medium (1 : 10) every 10 d. Pure lines (281A and 281C) and their sexual hybrid (F1) were kindly provided by Zaadunie (Enkhuizen, The Netherlands) (Giorgetti et al., 1995).
Library screening, cDNA subcloning and sequencing A carrot cDNA library constructed in the lZAP XR vector (Stratagene) was screened with the 2.2 kb cDNA insert of pTop28 clone as probe (Balestrazzi et al., 1996) under the following hybridization conditions: 50% (vuv) formamide, 5 3 SSC (150 mM NaCl, 15 mM Na3-citrate pH 7.6), 0.5% SDS, 5 3 Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 100 mg ml 1 salmon sperm DNA at 42 8C for 16 h. Final washes were under high stringency (twice with 1 3 SSC, 0.1% SDS at 65 8C for 10 min). Subcloning was performed using standard procedures (Sambrook et al., 1989) and sequence data were analysed with the Mac DNAsis Pro V 3.0 software (Hitachi America, Ltd., San Bruno, CA for Pharmacia-LKB Biotechnology).
Preparation of gene-specific probes The 189 bp fragment spanning the 59 untranslated region of top1a cDNA was obtained by digesting pTop29G6 clone (Balestrazzi et al., 1996) with XhoI and TaqI. The 564 bp probe, spanning the 59 untranslated region of top1b cDNA and 469 bp from the coding region, was produced by digestion of pTop11u1100 subclone with EcoRI and XbaI. The 193 bp fragment (from nt 607 to nt 800 of top1b cDNA) was isolated by PCR using speci®c oligonucleotide primers and standard procedures (50 s at 94 8C, 50 s at 46 8C, 30 s at 72 8C; 35 cycles).
Daucus carota, DNA topoisomerase I, top1 gene expression Southern analysis Genomic DNA was isolated from carrot cells (E4 line) (Rogers and Bendich, 1988). Aliquots of 10 mg were digested with EcoRV, BamHI, XhoI, EcoRI, HindIII, SpeI, and AccI, respectively, separated on 0.8% agarose gel and transferred to a nylon membrane (HYBOND-N; Amersham). Probes were labelled with wa-32Px-dCTP using the Prime-a-Gene system kit (Promega). Filters were hybridized as previously described. Final washes were performed at 50 8C with 2 3 SSC, 0.1% SDS for 10 min. PCR analysis of carrot genomic DNA Carrot genomic DNA (200 ng) was used as template for standard PCR reactions performed in order to amplify a 358 bp top1a fragment (corresponding to the coding region from nt 946 to nt 1189, interrupted by a 136 bp intron) and a 193 bp top1b fragment (from nt 607 to nt 800), respectively. PCR conditions were as follows: 50 s at 94 8C, 50 s at 64 8C, 50 s at 72 8C for top1a and 50 s at 94 8C, 50 s at 46 8C, 30 s at 72 8C for top1b. DNA fragments were isolated from agarose gels and subsequently sequenced to con®rm their origin. Cell starvation experiments Stationary-phase Hypo2 cells (14 d after subculture) were ®ltered and washed four times with 2,4-D and sucrose-free B5 medium. Cells were resuspended at the same cell density as before and maintained in this medium for 48 h. As a control, stationary-phase cells were handled the same way of starved cells, but resuspended and maintained in complete fresh medium for 48 h. Both cell suspensions were then subcultured into complete fresh medium (1 : 5) and incubated for 50 h. At the times indicated, w3Hx-thymidine incorporation into DNA was measured in triplicate. Determination of w3Hx-thymidine incorporation Samples (1 ml) were collected at the reported intervals and incubated with 5 mCi w3Hx-thymidine (25 Ci mmol 1) for 1 h at 30 8C. The reaction was stopped by adding 1 ml of cold 10% trichloroacetic acid (TCA) and the precipitate was washed three times with 5% TCA, once with 95% (vuv) ethanol and once with acetone. Dried ®lters were incubated at 55 8C for 30 min with 0.2 ml soluene (Packard) and subsequently 4 ml of Instagel (Packard) were added to each sample. Counting was performed in a Packard gamma counter. Northern blot analysis Poly(A)q RNA was extracted from carrot cell suspension cultures as previously described (Balestrazzi et al., 1996). For Northern blot analysis, poly(A)q RNAs (1±4 mg) were run on 0.6% agarose denaturing formaldehyde gels and then blotted to nylon membranes (Appligene Oncor Positive Membrane), according to the manufacturer's instructions. Filters were hybridized and washed as indicated for the library screening. Densitometric analysis was performed using a Bio-Pro®le apparatus (Vilber Lourmat). Expression of peptides from carrot topoisomerases Ia and b in E. coli cells In order to produce polyclonal antibodies against carrot topoisomerases I, two different cDNA fragments (224 bp, from
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nt 398 to nt 622 of top1a sequence and 398 bp, from nt 460 to nt 858 of top1b sequence) coding for peptides A (ser42 to asp116) and B (gln112 to pro244), respectively, were ampli®ed by standard PCR technique using the following speci®c oligonucleotide primers: A1 (59-CGGAATTCAGTCATAAACTATCAT-39) and A2 (59-CGGAATTCGTCCTCAAGTTCTGATG-39) for the 224 bp top1a-fragment; B1 (59-CGGAATTCCAAAATTCTCAGCAGAA-39) and B2 (59-CGGAATTCGGGTCTTTTATTTATTG-39) for the 398 bp top1b fragment. The ampli®ed products were cloned into the EcoRI site of the pRSETB vector (Invitrogen) for bacterial expression. A cDNA fragment (180 bp, from nt 2626 to nt 2806 of top1b sequence) coding for the C-terminal peptide C (lys834 to leu894) was obtained as previously described using oligonucleotide primers C1 (59-CGGAATTCAAGAAGATTGCTCAAAC-39) and C2 (59-CGGAATTCGAGACTGTTGAACATCT-39) and cloned into the same vector. The E. coli strain BL21(DE3) (Novagen) was transformed with the recombinant plasmids. Cultures (100 ml) were grown to an OD600 of 0.5±1 and following induction with 1 mM IPTG (isopropyl-b-D-thiogalactoside), incubation was further prolonged for 3 h. Cells were then collected by centrifugation and pellets resuspended in 1±2 ml of buffer B (8 M urea, 0.1 M NaH2PO4.H2O, 10 mM TRIS-HCl pH 8.0). Fusion peptides A, B and C were puri®ed from cell extracts by af®nity cromatography using the Ni-NTA agarose (Qiagen, m-Medical) according to the supplier's instructions and ®nally injected into rabbits as described previously (Harlow and Lane, 1988). Nuclear extract preparation
Protoplasts, obtained from carrot cell suspension cultures (Hypo2 line), were resuspended in buffer A (400 mM sucrose, 25 mM TRIS-HCl pH 7.6, 10 mM MgCl2, 0.3% (vuv) Triton X-100, 5 mM b-mercaptoethanol, and 0.5 mM PMSF (phenylmethylsulphonyl ¯uoride)) and subsequently disrupted with a Potter homogenizer. Nuclei were pelleted, washed with buffer B (400 mM sucrose, 50 mM TRIS-HCl pH 7.6, 5 mM MgCl2, 20% (vuv) glycerol, 5 mM b-mercaptoethanol, and 0.5 mM PMSF) and ®nally resuspended in lysis buffer (25 mM HEPES, 40 mM KCl, 0.5 mM EDTA, 20% (vuv) glycerol, 5 mM MgCl2, 2 mM DTT, 1 mM PMSF, 10 mM 1,10-phenanthroline, and 1 mM pepstatin). Nuclear extracts were then precipitated with 4 M (NH4)2SO4 and dialysed for 20 h against lysis buffer without MgCl2. Protein concentration was determined according to Bradford (Bradford, 1976). Isolation of chloroplasts, mitochondria and extract preparation
Leaves from 28-d-old carrot plants were harvested and chloroplast isolation and protein extract preparation were performed as described previously (Siedlecki et al., 1983) with the following modi®cation: 1 mM PMSF, 10 mM 1,10-phenanthroline and 1 mM pepstatin were added to buffer E. Mitochondria were isolated from roots of 28-d-old carrot plants according to the procedure described earlier (Leaver et al., 1983). Crude extracts were essentially prepared according to Ricard et al. (Ricard et al., 1983). Cytochrome c oxidase activity was evaluated as reported earlier (Hawkesford et al., 1989). Antisera preparation and Western blotting analysis
Rabbit immunization and polyclonal antibodies puri®cation on a protein A-Sepharose column were carried out (Harlow and Lane, 1988). Western blot analysis was performed as
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described previously (Towbin et al., 1979) and immunodetection according to ECL Western blotting protocol (Amersham).
Results Isolation and characterization of an additional top1 cDNA clone
In a previous study (Balestrazzi et al., 1996), two partially overlapping cDNAs, pTop28 and pTop29G6, encoding a DNA topoisomerase I from Daucus carota have been isolated from a poly(A)q-primed library, using an Arabidopsis thaliana probe, and from a gene-speci®c primed cDNA library, respectively. The overall top1 nucleotide sequence contains an open reading frame (ORF) of 2370 bp corresponding to a protein of 90 kDa. In order to obtain a full-length top1 cDNA to use in expression systems, another cDNA library derived from poly(A)q RNA isolated from the domesticated carrot cell line E4 was screened with pTop28 under high stringency conditions. The resulting ®ve positive clones were found to belong to two classes of top1 sequences. One member of the ®rst class, pTop9, perfectly matches the ORF of pTop28 and pTop29G6, while a member of the second class (pTop11) contains an ORF 2727 bp long with a coding capacity for a polypeptide of 906 amino acids and a calculated molecular mass of 101 kDa. This cDNA coding for a putative variant of topoisomerase I was designated top1b, while pTop9 was named top1a. Both pTop9 and pTop11 clones are full length cDNAs. Typical features of eukaryotic genes are present in the top1b cDNA sequence (GenBank Accession No. AJ223326). The ®rst ATG is surrounded by a putative signal for the translation start point (59-GAAATGG-39) with high homology to the consensus sequence (59AuGNNATGG-39) proposed as optimal for eukaryotes (Kozak, 1984). The 39 untranslated region, 231 bp long, contains three polyadenylation motifs (ATACAT, ATACAT, AATCAA), located upstream at a 21 bp long poly(A)q tract (Dean et al., 1986). Alignments of the top1b nucleotide sequence revealed a 66% similarity with top1a, 51% with A. thaliana and 62% with P. sativum. At the amino acid level, the deduced topoisomerase Ib shares a 56% similarity with topoisomerase Ia from D. carota and 57% and 55% with A. thaliana (Kieber et al., 1992b) and P. sativum (Reddy et al., 1998) type I enzymes, respectively. The similarity of carrot topoisomerase Ib to the type I proteins from Saccharomyces cerevisiae (37%), Schizosaccharomyces pombe (39%) and Homo sapiens (31%) is considerably lower. Sequence comparison among the four plant topoisomerases I suggests the presence of a domain organization of the protein, similar to that proposed previously (Stewart et al., 1996) for human topoisomerase I.
Topoisomerase I sequences can be divided into four domains (Fig. 1): the N-terminal, the central, the linker, and the C-terminal domains. Sequence comparison reveals the presence of two conserved regions in all plant type I enzymes: the C-terminal domain (residues Lys834 to Phe909 in carrot topoisomerase Ib) surrounding the tyrosine of the active site, and the central domain (residues Ser336 to Ile792 in carrot topoisomerase Ib). The C-terminal domain of the type Ib protein shares a 89% similarity with topoisomerase Ia and 84% and 67% with the enzymes from A. thaliana and P. sativum, respectively; at the level of the central domain the similarity was slightly lower: 71% with topoisomerase Ia, 69% with Arabidopsis and 68% with pea type I proteins. Similar to other members of the eukaryotic topoisomerase I family, the plant type I enzymes are characterized by the occurrence of a very unconserved NH2-terminal region; the N-terminal domain of carrot topoisomerase Ib (residues Met1 to Phe335) shares a 38% similarity with the corresponding domains from A. thaliana and P. sativum, but only 32% with the N-terminal region from topoisomerase Ia. Remarkably, taking into account the domain organization of the protein, topoisomerase Ib seems to be more similar to the other two plant enzymes than to carrot topoisomerase Ia. In fact, both the length and the sequence of the N-terminal and linker domains of the two carrot enzymes are highly divergent. Genomic organization of top1a and top1b genes
Southern blot analysis was performed with top1a and top1b-speci®c probes. Genomic DNA from the domesticated carrot cell line E4 was digested with EcoRV, BamHI, XhoI, EcoRI, HindIII, SpeI, and AccI and probed with either the 189 bp-top1a or the 564 bp-top1b cDNA fragments. Distinct DNA band patterns were detected, except for the 2.8 kb-AccI fragment recognized by both probes (Fig. 2). In order to determine whether top1a and top1b represent two distinct loci or two allelic forms of the same locus, six plant samples of the two pure lines (281A and 281C) and their sexual hybrid (F1) were analysed. PCR analysis performed with gene-speci®c primers revealed the presence of both the expected a and b ampli®ed products (358 bp for top1a and 193 bp for top1b) in the genomic DNA of all plants from both parents and F1 progeny (Fig. 3), even if the ampli®cation ef®ciency was not identical in all samples. The larger size of the top1a fragment is due to the presence of a 134 bp intron that interrupts the coding region (from nt 962 to nt 1189) of the a gene. Both 358 bp and 193 bp DNA fragments were isolated and sequenced and con®rmed to derive from the top1a and the top1b gene, respectively.
Daucus carota, DNA topoisomerase I, top1 gene expression
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Fig. 1. Protein sequence alignment of plant DNA topoisomerases I. Dc alpha, Daucus carota top1a (Genbank Accession No. U60440); Dc beta, Daucus carota top1b; At, Arabidopsis thaliana (Genbank Accession No. X5754) and Ps, Pisum sativum (Genbank Accession No. Y14558). The boxed areas identify conserved amino acid sequences. Domain boundaries are indicated by arrow heads.
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Fig. 2. Southern analysis of carrot genomic DNA. Genomic DNA (10 mg) was digested with EcoRV (lane 1), BamHI (lane 2), XhoI (lane 3), EcoRI (lane 4), HindIII (lane 5), SpeI (lane 6), and AccI (lane 7), separated on 0.8% agarose gel and blotted onto a Hybond-N membrane. Hybridization was carried out under moderate stringency, using the 189 bp top1a (A) and the 564 bp top1b (B) as probes. The positions of molecular weight markers, in kilobases, are shown.
All these results are consistent with the presence of two distinct top1 loci in the carrot genome. To obviate the possibility that one of these loci could corresponded to a processed pseudogene, PCR ampli®cation experiments using carrot genomic DNA (line E4) were performed to detect the presence of intervening sequences (data not shown). Primers were synthesized on the basis of the cDNA sequence of either top1a or top1b and designed to amplify different parts of each gene. Two distinct introns of about 700 and 134 bp for top1a and two of about 800 and 250 bp for top1b were detected at the genomic level.
Fig. 3. PCR analysis of genomic DNAs from carrot pure lines. Genomic DNAs from single plants of the pure lines 281A (lanes 1, 2, 3, 4) and 281C (lanes 5, 6, 7, 8) and their F1 hybrid (lanes 9, 10, 11, 12) were analysed with speci®c primers for top1a (A) and top1b (B), respectively. Arrow heads indicate the position of the ampli®ed 358 bp-top1a and 193 bp top1b fragments. The positions of molecular weight markers, in kilobases, are shown.
Expression patterns of the top1a and b genes
The expression of both top1a and top1b genes was investigated by Northern blot analysis. Poly(A)q RNAs were isolated from cycling cultured cells and separated by means of a 0.6% agarose denaturing formaldehyde gel, instead of the 1.5% normally used; only this low percentage allowed the separation of the two top1 transcripts. Hybridization was performed under high stringency conditions using two speci®c probes: the 189 bp fragment spanning the 59 untranslated region of top1a cDNA and the 193 bp fragment from the 59 coding region of top1b cDNA. Each top1 probe hybridized with a speci®c transcript of different size: 3.2 kb for the top1b and 3.0 kb for the top1a, respectively (Fig. 4). The ratio between top1b and top1a transcript levels was about 2.5 in proliferating suspension cells, as determined by densitometric analysis. As a starting point for future investigations on the regulation of top1 gene expression, the top1a and top1b mRNA accumulation during re-activation of quiescent carrot cells into division was monitored. Stationary phase cells (14 d after subculture) were maintained in
Fig. 4. Northern blot analysis of carrot top1a and top1b gene expression. Poly (A)q RNA (3 mg) from suspension dividing cells was hybridized with both the 189 bp top1a fragment (lane 1) and the 193 bp top1b (lane 2), as probes. The positions of molecular weights markers, in kilobases, are indicated on the left.
a quiescent state by a 48 h incubation in sucrose-free and 2,4-D-free medium and then stimulated to re-enter the cell cycle by subculturing into complete medium (Fig. 5). Measurements of DNA synthesis by 3H-thymidine incorporation showed that the starvation condition forced the majority of cells into a quiescent state (Fig. 5A). The re-addition of sucrose and 2,4-D to the growth medium at time 0 induced the reinitiation of cell cycling in a fairly synchronous manner. This result was con®rmed by ¯ow cytometric analysis of nuclei from the starved population (not shown). Cells were arrested predominantly at G1
Daucus carota, DNA topoisomerase I, top1 gene expression
Fig. 5. Expression of top1a and top1b genes in cultured carrot cells. (A) Time-course of the w3Hx-thymidine incorporation into DNA during the block-release experiment. 14 d-old cells were either deprived of exogenous 2,4-D and sucrose (m) or replaced with complete fresh medium (k). After 48 h, cells were subcultured in complete fresh medium and allowed to re-enter cell cycle; (B) Northern blot of poly (A)q RNA (2 mg lane 1) isolated from stationary phase, 48 h starved cells and cells collected at time zero, after 5, 7, 9, 11, and 14 h from starvation release, probed with the 193 bp top1b, carrot pTop28, histone H4 cDNA from A. thaliana and carrot 0.6 kb ubi-CEP cDNA. The 1.0 kb parsley poUbi cDNA was used as a constitutive marker. Arrow indicates the release time point.
phase: about 70% of the nuclei had a 2C or G1 DNA content, while 25% a 4C DNA content. As a control, stationary phase cells were handled the same way as starved cells, but resuspended and incubated for 48 h in complete medium and then subcultured at time zero into fresh medium. A fresh medium-dependent activation of the DNA synthesis, although of limited extent, was
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observed during the ®rst 48 h. However, when these cells were subcultured into fresh medium, in contrast to what was observed for starved cells, the outline of the DNA synthesis suggested that an appreciable synchrony in cell division was not obtained. On the other hand, not only in this report but also in other reports (Okamura et al., 1973; Nishi et al., 1977), the direct subculturing of carrot stationary phase cells to fresh medium did not represent a reliable method for obtaining an appreciable synchrony level (not shown). The steady-state transcript levels of molecular marker genes in the starved population during the resting state and during re-entry into the cell cycle were examined in parallel to 3H-thymidine incorporation (Fig. 1A, B). Transcript accumulation of histone H4, an S-phase molecular marker (Reichheld et al., 1995), and ubi-CEP (ubiquitin-carboxyl extension protein), a marker induced in the transition G1uS in carrot cells (Balestrazzi et al., 1998) were used as internal controls, whereas the 1.0 kb parsley polyubiquitin (poUbi) cDNA was utilized as a constitutive marker (Kawalleck et al., 1993). The accumulation of both H4 and ubi-CEP transcripts was restricted to dividing cells, since hybridization signals were lacking, as expected, in both stationary phase cells and starved cells. However, 5 h following the exit from the resting state, both transcripts were detectable and their levels paralleled DNA synthesis, reaching the highest level at 14 h, the last time point examined. As expected, the level of the poUbi transcript remained constant throughout the experiment. Due to the low steady-state level of top1a gene expression, the same blot membrane was hybridized with pTop28 (2.2 kb), a probe which recognizes both top1 transcripts, whereas for top1b the 193 bp speci®c fragment was used. All transcript levels have been quanti®ed by densitometric analysis. Both stationary phase and starved cells appeared to express top1b, although at a very low level; when starved cells were induced to enter the cell cycle at time zero, the b transcript level started increasing gradually 5 h after stimulation, following the kinetics shown for the other two proliferation markers, up to 10-fold (at 14 h) the amount quanti®ed in starved cells. Both stationary phase and starved populations highly expressed top1a and in these cells the quantitation of transcript levels, as measured by densitometric analysis, showed a ratio between b and a of 0.53, because of a 1.8-fold higher a mRNA abundance in resting cells. Surprisingly, the top1a transcript level decreased during the ®rst 5 h from the induction of cell division and then started accumulating, but the level was markedly lower to that seen for top1b. At 14 h the ratio between b and a was 2.3, then re¯ecting the higher accumulation of the b mRNA in these cells.
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Immunodetection of carrot topoisomerase Ia and b in nuclear extracts
With the goal of producing speci®c antibodies against topoisomerase Ia and b to investigate the presence of both enzymes in carrot cell extracts, three cDNA fragments corresponding to the sequence from nt 398 to nt 622 of top1a, from nt 460 to nt 858 and from nt 2626 to nt 2806 of top1b were cloned into the E. coli expression vector pRSETB. The expected translation products were the following: peptides A and B from the N-terminal regions of topoisomerase Ia (Ser42 to Asp116) and Ib (Gln112 to Pro244), and the peptide C from the C-terminal region, highly conserved in both enzymes, of topoisomerase Ib (Lys834 to Leu894). The constructs were introduced into E. coli: all three trasformants exhibited high levels of expression of the fusion proteins 3 h after IPTG induction. Recombinant peptides were expressed in insoluble forms and accumulated in the inclusion bodies. Their molecular masses on SDS gels were larger than expected (peptide A: 13.2 kDa; peptide B: 19.33 kDa and peptide C: 11.4 kDa), probably because of the presence of a 6 3 His-tail at the N-terminal region of each fusion protein (not shown). Peptides A, B and C were puri®ed from cell extracts by af®nity chromatography using NiNTA agarose and used to generate polyclonal antibodies in rabbits. The IgG fraction of each antiserum was eluted from a protein A-Sepharose and utilized for immunodetection of topoisomerase Ia or b in carrot nuclear extracts (Fig. 6, lanes 1±6). The IgGs against the N-terminal a and b regions did not cross-react with the same polypeptides: the two patterns were reproducible and non-overlapping. Antibodies against the a peptide failed to recognize the presence of a full-length protein; however smaller peptides (82, 72, 69, and 57 kDa, respectively) were
Fig. 6. Western blot analysis of proteins from carrot nuclei and chloroplasts. Nuclear and chloroplast proteins (12 mg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Nuclear extract was immunodetected with IgGs to the peptide A from the N-region of topoisomerase Ia (lane 1), the peptide B from the N-region of topoisomerase Ib (lane 3) and the peptide C from the C-region of topoisomerase Ib (lane 5). Lanes 2, 4 and 6: nuclear extract analysed with pre-immune sera. Chloroplast extract was tested with IgGs against peptide A (lane 7) and against peptide C (lane 8). The same extract was analysed using anti-spinach RuBP carboxylase large subunit serum (lane 9). Detection was carried out using the chemiluminescent reaction system (Amersham). Molecular mass markers are shown.
detected in nuclear extracts (Fig. 6, lane 1). Antibodies against the b peptide were able to react with a 118 kDa protein (lane 3). As recently suggested (Stewart et al., 1996), the presence of negatively charged residues in the N-terminal region retards the migration of the full-length topoisomerase I in SDS gels and this could explain the discrepancy between the observed and the expected molecular mass of the carrot Ib enzyme (101 kDa). Two other faint bands were sometimes observed (102 and 86 kDa): they are probably proteolytic degradation products retaining the epitope(s) localized in the N-terminal domain of the protein (not shown). IgGs against the C-terminal region con®rmed the presence of a 118 kDa protein in the nuclear extract (lane 5). Unfortunately, the antibodies against the catalytic site failed to detect a 90 kDa protein or smaller bands that could be attributed to topoisomerase Ia. In summary, these data indicate that the top1b gene product is present in carrot cell nuclei. No full-length topoisomerase Ia (90 kDa) was identi®ed in nuclear extracts where the largest protein found was 82 kDa in size. This might suggest that topoisomerase Ia is more sensitive to proteolytic degradation than the b isoform. To exclude an organellar localization of the DNA topoisomerase Ia, chloroplasts and mitochondria were puri®ed from carrot leaves and roots, respectively. Extracts from Percoll-puri®ed chloroplasts were analysed by Western blots. As shown in Fig. 6, IgGs against the a peptide (lane 7) and against the C-terminal domain of topoisomerase Ib (lane 8) failed to detect any proteins. The absence of detectable nuclear contamination in the chloroplast extract was con®rmed by the absence of any recognition using IgGs against the C-terminal region (compare lanes 5 and 8). The same results were obtained by testing a 8-fold higher amount of the chloroplast extract (not shown). In contrast, a protein of about 50 kDa was detected speci®cally with a polyclonal antiserum raised against the large subunit of spinach ribulose-1,5-biphosphate carboxylase, an enzyme localized in the chloroplast (lane 9), whereas no proteins were revealed by the nonimmune serum (not shown). A similar analysis was performed using extracts from isolated carrot mitochondria by using two different concentrations of the extract (1 3 or 4 3 ). No proteins cross-reacting with IgGs against the a peptide and against the C-terminal region of the b isoform were consistently observed (not shown). In order to con®rm the mitochondrial origin of the tested extract, cytochrome c oxidase, a typical mitochondrial marker, was recovered from solubilized mitochondrial membranes. The speci®c activity of the enzyme, expressed as rate of oxidation of reduced Cyt c, was 0.24 mmol min 1 mg 1. In conclusion, these studies strongly indicate that topoisomerase Ia is a nuclear protein.
Daucus carota, DNA topoisomerase I, top1 gene expression
Discussion In a previous study, a cDNA coding for a carrot DNA topoisomerase I (top1a) was cloned (Balestrazzi et al., 1996). Here the identi®cation of a second class of top1 cDNA (top1b) encoding a putative variant form of carrot DNA topoisomerase I is reported. This seems to be similar to the situation observed in mammalian cells where the presence of two forms of topoisomerase II, deriving from different genetic loci, has been described (Tan et al., 1992) and where a second topoisomerase III gene (hTOP3b) has been recently isolated and characterized (Ng et al., 1999). When carrot genomic DNAs from two pure lines and from their sexual hybrid were analysed by PCR, the occurrence of two distinct top1 loci was demonstrated, differently from the authors' previous work describing a single-copy gene (Balestrazzi et al., 1996). In human and mouse genomes additional top1 loci corresponding to processed pseudogenes which produce only truncated (Yang et al., 1990) or antisense (Zhou et al., 1992) transcripts were detected. In contrast to mammalian genomes, the presence of processed pseudogenes represents an unusual feature in higher plants. The mechanisms by which they arose involve the reverse transcription of the corresponding mRNA and then the integration into the genome (Vanin, 1985). However, differently from animals, plants do not possess a germ line and as a result only retrosequences from genes expressed in the apical meristem might be transmitted to the progeny. Until now, only a few cases of plant retropseudogenes have been described; the potato actin pseudogene is an example of a cDNA copy of the normal potato gene which originated from reverse transcription of its cellular mRNA (Drouin and Dover, 1987). The identi®cation of a top1 retropseudogene in carrot cells could represent another rare case. However, three lines of evidence suggest that the two carrot cDNAs correspond to two functional genes: the presence of intervening sequences at the genomic level, the presence of complete open reading frames and the presence of transcriptional activities. Both carrot top1 genes carry introns and, interestingly the size and position of the intervening sequence at the 59 end is also conserved in the A. thaliana gene (Kieber et al., 1992b). In cycling cultured cells, two transcripts of 3.2 and 3.0 kb corresponding to top1b and top1a, respectively, were detected. Top1b mRNA represents the most abundant species in proliferating cells. To investigate whether the transition from a resting to a proliferating state of cells was accompanied by an early increase of both top1a and b mRNA steady-state levels, cells deprived of sucrose and 2,4-D were induced to re-enter the cell cycle by the addition of nutrients.
1987
These data suggest that in carrot both top1 genes are expressed in stationary phase and starved cells, but the ratio between b and a transcript levels is quite different from the value detected in cycling cells, because of a 1.8-fold higher a mRNA accumulation. Based on the absence of histone H4 and ubi-CEP transcripts in both resting populations, the presence of top1a and b mRNAs in these cells is not due to DNA replication. Moreover, the temporary accumulation of top1b transcript during the reinitiation of DNA synthesis seems to be similar to that seen for histone H4 and ubi-CEP markers, the only difference observed was due to the relative amount of top1b transcript which was signi®cantly lower. These results support the authors' previous suggestions that an enhanced pattern of transcription related to the proliferative state of carrot cells may overlap the top1 basal expression (Balestrazzi et al., 1996). This is true for top1b. In the case of the top1a, further investigation will be needed, since it remains unclear whether the oscillation of the transcript found when cells resume division can be considered signi®cant. The presence of two distinct isoforms of DNA topoisomerase I has been reported in Xenopus laevis (Richard and Bogenhagen, 1989, 1991) and calf thymus (Kudinov et al., 1992). In the ®rst case, a 165 kDa protein was puri®ed from Xenopus ovaries and found to be restricted to oocyte cells where it is degraded during oocyte maturation, while a 110 kDa protein extracted from liver tissue represented the predominant somatic form of the enzyme. Topoisomerase I is sensitive to proteolytic degradation and gives rise to smaller forms of the native enzyme retaining the relaxing activity during puri®cation. However, in the case of calf thymus cells, the authors suggested that the two isolated topoisomerase I forms are distinct proteins, since they possess a different sensitivity to the speci®c topoisomerase inhibitor camptothecin and differ in their proteolytic digestion patterns (Kudinov et al., 1992). Thus, it appears that there are distinct species of topoisomerase IB in eukaryotic cells. In the case of carrot, sequence comparison suggested the presence of two distinct top1 gene products which signi®cantly differ at the N-terminal domain: this region is known to mediate the interaction between topoisomerase I and several different proteins such as nucleolin (Bharti et al., 1996), SV40 large T antigen (Haluska et al., 1998), p53 (Gobert et al., 1996), TATA-binding proteins (Merino et al., 1993), and is also required for the maintenance of topoisomerase I kinase activity. In addition, several NLS (nuclear localization signals) consensus sequences are located in the NH2-terminal region of human and yeast topoisomerases I, suggesting that this very unconserved domain is essential for the nuclear localization of the enzyme (Alsner et al., 1992). Remarkably, of the two carrot proteins, the top1b gene product appears to be more similar in its structural
1988
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organization to Arabidopsis and pea topoisomerases I. For this reason, this study refers to it as to the `classical' carrot top1 enzyme. The a polypeptide differs from the other plant enzymes mainly in the length of the very unconserved N-terminal domain, then raising the question about the possible physiological role of this protein. In carrot nuclear extract, antibodies against the recombinant peptide from the N-terminal region of topoisomerase Ib recognizes a 118 kDa protein which seems to correspond to the b protein. This consideration is strengthened by the fact that IgGs raised against the carboxyl terminal sequence of topoisomerase Ib react with the same protein. The absence of recognition of a 90 kDa protein, corresponding to topoisomerase Ia, would indicate that the top1a gene product is much more sensitive to proteolytic degradation than the b form. When the domain structure of human DNA topoisomerase I was examined using limited proteolytic digestion experiments, both the N-terminal and the linker domains were sensitive to proteolysis (Stewart et al., 1996). Moreover, proteolytic digestion at the level of the linker region caused the release of the conserved C-terminal domain spanning approximately 10 kDa. It is then possible that the 82 kDa polypeptide detected by the anti-a IgGs derives from such a degradation mechanism, since it does not cross-react with antibodies against the carboxyl terminal sequence of topoisomerase Ib. On the other hand, the lack of crossreactivity between the IgGs against the N-terminal region of topoisomerase Ia and those against the N-terminal region of the b protein, seems to exclude the idea that the four a polypeptides are proteolytic fragments of the 118 kDa b protein. Since two chloroplast topoisomerases I from pea (Mukherjee et al., 1994) and cauli¯ower (Fukata et al., 1991) and one from wheat embryo mitochondria (Echeverria et al., 1986) have been classi®ed as eukaryotic type I enzymes, the presence of both topoisomerases Ia and b in carrot organelles was investigated. Western blot results con®rmed the absence of proteins cross-reacting with IgGs against the a and the b isoforms both in chloroplasts and in mitochondria. Up to now, in pea, a single top1 gene coding for a nuclear topoisomerase I has been cloned (Reddy et al., 1998); the search for the gene encoding the organellar enzyme is still an open question. On the other hand, the presence of a second nuclear gene coding for a topoisomerase I has been recently described in A. thaliana (GenBank Accession No. CAA16524). Hence the nuclear genome of Arabidopsis exhibits the coding capacity for both a eukaryotic- and a prokaryotic-like type I protein. This seems to be different from the situation observed in D. carota, where two distinct top1 loci coding for two nuclear topoisomerases I have been identi®ed. The availability of the two full length-recombinant proteins would have allowed a better characterization of
the two enzymes. However, attempts to produce both carrot topoisomerases by overexpressing the corresponding full-length cDNAs in E. coli cells were unsuccessful. The expression of the eukaryotic DNA topoisomerase I in a prokaryotic system results in toxicity to the cells due to the effect of the enzyme on DNA supercoiling (Fernandez-Beros and Tse-Dihn, 1992). It is not possible to exclude that the presence of carrot topoisomerase I affects host cell viability, although a full-length cDNA for the pea enzyme has been recently overexpressed in E. coli cells (Reddy et al., 1998). In conclusion, two top1 loci are present in the nuclear genome of carrot cells. Both genes are transcribed in resting and cycling cells and both proteins seem to be present in carrot nuclei. The availability of transgenic plants carrying antisense top1a and b constructs will probably help to elucidate the physiological role of the two isoforms during plant development. Moreover, transgenic plants containing the inactivated top1b gene may represent a useful tool to study in vivo the possible function of top1a gene.
Acknowledgements We thank JA Bryant (Exeter University, UK) for critically reading the manuscript. We greatfully acknowledge Dr M Fabbi (Istituto Zoopro®lattico Sperimentale della Lombardia e dell'Emilia-Romagna, Sezione di Pavia) for raising antibodies in rabbit. We would like to thank Dr G Mazzini (Centro Grandi Strumenti, University of Pavia) for ¯ow cytometry analysis. This work was supported by MURST.
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