Meristem-specific gene expression directed by the promoter of the S-phase-specific gene, cyc07, in transgenic Arabidopsis

Plant Molecular Biology 24: 863-878, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 863

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene, cyc07, in transgenic Arabidopsis

Masaki Ito 1, Tsutomu Sato 2, Hiroo Fukuda 2 and Atsushi Komamine 2 1Nagoya University BioScience Center, Chikusa-ku Nagoya, 464 Japan," 2 Biological Institute, Faculty of Science, Tohoku University, Sendai, 980 Japan

Received 10 September 1993; accepted in revised form 15 December 1993

Key words." cell cycle, gene expression, meristem, promoter analysis, transgenic Arabidopsis

Abstract

A genomic clone for the cyc07 gene, which is expressed specifically at the S phase during the cell cycle in synchronous cultures of periwinkle (Catharanthus roseus) cells, was isolated. Determination of the nucleotide sequence of the clone revealed that the cyc07 gene consists of seven exons separated by six introns. Genomic Southern analysis indicated that the cyc07 gene is present as a single copy per haploid genome in periwinkle. Expression of related genes was detected in a wide range of other plants. Transgenic Arabidopsis plants were generated that expressed the gene for fi-glucuronidase (GUS) under the control of the promoter of the cyc07 gene. The tissue-specific pattern of expression directed by the promoter was investigated by analysis of GUS activity. Histochemical tests demonstrated that 589 bp of the 5 '-upstream sequence of the cyc07 gene could direct specifical expression of the GUS reporter gene in meristematic tissues in transgenic plants. The spatial pattern of expression directed by the promoter was closely correlated with meristematic activity and cell proliferation, suggesting an associa- tion between the function of the cyc07 gene and cell proliferation.

Introduction

The cell cycle in higher plants is strictly regulated in the developmental process. Elucidation of the mechanisms that govern control of the cell cycle is essential if we are to understand the mechanisms of plant development. The progression of the eukaryotic cell cycle is considered to be regulated by the sequential expression of specific genes. A candidate of such specific genes is often equated with cell-cycle-dependent genes, which

display variability in their rates of transcription during the cell cycle; most other genes are expressed at a constant level throughout the cell cycle [31 ]. The isolation of cell-cycle-dependent genes, which have important functions in the progression and/or regulation of the cell cycle, is one of the main subjects in this area. In yeast systems, a lot of cell-cycle-dependent genes have been identified and their important functions in the cell cycle have been revealed [ 31 ].

Studies of cell-cycle control have mainly in-

The nucleotide sequence data reported will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession numbers D26058.

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volved mammalian and yeast systems, and plant cells have been used to a very limited extent. Re- cently, significant effort has been directed towards the isolation of plant homologues of cell-cycle control genes in yeast [7], for example proliferating cell nuclear antigen (PCNA) [24, 46, 47], p34 cd°2 [6, 8, 9, 15, 20], and cyclins [13, 14]. Alternatively, in attempts to identify novel genes that are associated with cell proliferation, meristem-specific genes have been isolated and characterized [41].

In the present study, an attempt was made initially to isolate novel cell-cycle-dependent genes during the plant cell cycle. Cultures of periwinkle (Catharanthus roseus) cells in suspension can be synchronized by two different methods. In one system, the synchrony is induced by the double phosphate starvation method [2], and in the other system it is induced by auxin starvation and subsequent addition of auxin [36]. In both systems, cells are arrested exclusively in the G1 phase of the cell cycle during the starvation period. Addi- tion of phosphate or auxin induces the synchronous progression of the cell cycle. These two synchronous cell division systems were employed for isolation of a cDNA that corresponds to a cell- cycle-dependent gene, namely cyc07, by differential screening [23]. The deduced amino acid sequence of cyc07 exhibited no significant similarity to any published protein sequences, indicating that eye07 was a previously unknown gene. The cyc07 gene is expressed specifically in the S phase during the cell cycle in cultures of periwinkle cells in which synchronous division of cells is induced by either of the two different methods [17]. Fur- thermore, the m R N A corresponding to cyc07 accumulates preferentially in actively cycling cells in both suspension-cultured cells and intact plantlets [ 17].

Recently, the members of a family of yeast (Saccharomyces cerevisiae) genes that are homologous to cyc07 were identified [ 18]. This gene family is constituted by two closely related genes, PLCI and PLC2. Site-directed disruption of either PLC1 or PLC2 resulted in slower growth, and double PLC1 and PLC2 disruption resulted in a lethal phenotype. Thus, the protein encoded

by the gene family has an essential function in proliferation of yeast cells, and it appears that the amount of protein expressed from this gene family has an important role in the progression of the cell cycle. The striking similarity in terms of deduced amino acid sequence between genes from evolutionarily distant species, cyc07 from periwinkle and its yeast homologues, suggests a conserved function in cell proliferation [ 18].

In this communication, the promoter function of the cyc07 gene is characterized. The spatial pattern of expression of cyc07 in terms of levels of m R N A is related to meristematic activity and cell proliferation [ 17]. As a first step, the function of the promoter of the cyc07 gene was analyzed by monitoring its tissue-specific expression in transgenic plantlets. In this study, it was revealed that a fragment of ca. 0.6 kb of the 5' regulatory region in the periwinkle cyc07 gene confers meristem- and proliferative tissue-specific expression on the gene for/~-glucuronidase (GUS) in transgenic Arabidopsis. The observed spatial pattern of GUS activity and its relation to meristematic activity and cell proliferation in transgenic plants should reflect the pattern of distribution of the cyc07 m R N A in plantlets, and it suggests that the product of cyc07 gene plays a specific role in cell proliferation in higher plants.

A gene homologous to cyc07 and its yeast homologues has recently been isolated from mammalian cells [22]. The gene, named fie-l, was cloned as a gene for a v-fos transformation effector from rat cells. This report includes details of the evolutionarily conserved structure of the protein sequences of cyc07 from C. roseus and fte-1 from rat. The possible function of these conserved genes is discussed.

Materials and methods

Plant materials

Cultures of cells of Catharanthus roseus L. (G) Don. (strain B) were maintained at 27 ° C, in the dark, in Murashige-Skoog medium [34] that contained 3 ~ sucrose and 2 .2#M 2,4-dichloro-

phenoxyacetic acid (2,4-D). Cells were subcul- tured every 7 days. In a previous study [25], gene expression during the cell cycle was examined in this strain because of the high degree of synchronous division that can be induced by the double phosphate starvation method [2].

The rice (Oryza sativa L. cv. Konansou) cells in suspension culture were maintained in N6 medium [5] supplemented with 2,4-D (8.8 #M), sucrose (3~o), casein hydrolysate (300mg/1) and L-proline (10 mM), as described [40]. The carrot (Daucus carota L. cv. Kurodagosun) cells in suspension culture were maintained as described [10]. The cells of Phytolacca americana L. were maintained in Murashige-Skoog medium that contained 2,4-D (5 #M) and sucrose (3~o). The tobacco (Nicotiana tabacum L. cv. Bright Yellow) BY-2 cells [21], was maintained as described [39]. All cells were cultured in the dark at 27 °C. Cells at the exponential phase of growth were harvested and utilized for isolation of poly(A) + RNA.

Isolation of poly(A) + RNA

Total RNA was isolated from cultured cells in suspension by the method described [44]. Poly(A) + RNA was prepared from total RNA by chromatography on oligo(dT) cellulose (Pharma- cia, Uppsala, Sweden).

Northern blot analysis

Ten #g of poly(A) ÷ RNA isolated from cells of various plant species were separated by electrophoresis in 1.0~o agarose gels that contained 2~o formaldehyde by a modified version of the method described in [28] and then blotted onto Biodyne A membranes (Pall Ultrafine Filtration Corpora- tion, New York). The membranes were probed with the 32P-labeled cDNA insert of pCYC07F6. The conditions for hybridization and washing were those given in the instructions from the manufacturer of the nylon membranes, with some modifications. To reduce the stringency of hy-

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bridization, filters were incubated at 42 °C in the solution containing 25 ~o formamide. Low-stringency washes were performed in 2 x SSC and 0.1~o SDS at 50 °C three times for 20 min each.

Genomic Southern blot analysis

High-molecular-weight genomic DNA from periwinkle was prepared from the 6-day-old cultures of strain B by the method described in [35]. It was digested with Eco RI, Hind III, Xho I, or Kpn I, subjected to electrophoresis on a 0.9~o agarose gel and then blotted onto a Biodyne A membrane. The membrane was hybridized with the 32p-labeled 1.1 kb cDNA insert ofpCYC07F6 which contains nearly full-length cDNA of cyc07. The conditions for hybridization and washing were those given in the instructions from the manufacturer of the nylon membranes.

Isolation of a genomic clone of cyc07

Since the genomic Southern blot analysis revealed that a single 4.8 kb Eco RI genomic fragment hybridized with the cDNA, a genomic library was constructed from periwinkle total DNA that had been digested completely with Eco RI. High- molecular-weight genomic DNA from periwinkle cells was subjected to complete digestion with Eco RI, size-fractionation by agarose gel electrophoresis, and ligation of fragments with Eco RI- cut 2gtl 1 vector. The 4.8 kb Eco RI genomic fragment was isolated by screening the library with a pCYC07F6 cDNA insert as a probe. The 4.8 kb cyc07 genomic fragment was subcloned into the unique Eco RI site ofpBluescript KS (Stratagene, La Jolla, CA) to create pBS07ECG.

DNA sequence anaIys~

In order to determine the nucleotide sequence of the genomic clone, a series of unidirectional deletion derivatives was constructed using a Double- Stranded Nested Deletion Kit (Pharmacia LKB

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Biotechnology, Uppsala, Sweden). The nucleotide sequence was determined by the dideoxy chain-termination method [43].

Determination of the transcription initiation site by a primer extension method

Poly(A) + RNA was isolated from exponentially growing cells of strain B as described above. A 30-meric primer (5 '-GCTCCTGAGCTGCT- GCTGCTGCTGTTGTGCTGA-3' ) , which is complementary to the 5'-untranslated region, was synthesized. The 5' end of the primer was labeled with 32p and hybridized with 4 #g of poly(A) + RNA for 16 h at 25 °C, 30 °C, or 35 °C in 90 #1 of a solution of 3 M NaC1, 0.5 M HEPES, and 1 mM EDTA. The RNA and primer were then ethanol-precipitated, and redissolved in the solution for primer extension. The 30-meric primer was extended for 90 rain at 42 °C in a 20 #1 reaction mixture containing 1 mM each dATP, dCTP, dGTP, dTTP, and 10 units of avian my- eloblastosis virus reverse transcriptase (Seika- gaku Kogyo, Tokyo, Japan). The reaction product was ethanol-precipitated, redissolved in 7 #1 of sequencing dye mixture, and analyzed by gel electrophoresis under denaturing conditions and subsequent autoradiography. 32P-labeled DNA fragments, generated in the dideoxy-sequencing reaction from M13mp18 DNA, were run in par- allel on the gel.

Construction of chimeric plasmids including the pro- motet of the cyc07 gene

A 2.5 kb Hind III-Bam HI fragment was isolated from pCYC07ECG. This fragment contained 2.1 kb of the 5'-flanking sequence and three exons. This fragment was introduced into Barn HI-Hind III-cut pBI221 (Clontech, Palo Alto, CA). This construct was cut again with Bam HI, filled in with Klenow fragment of DNA polymerase I in the presence of all four nucleotides to create blunt-ended DNA, and religated to produce an in-frame fusion with the GUS re-

porter gene. A binary vector pKM424 contains neomycin phosphotransferase II gene linked to the nopaline synthase (NOS) promoter and NOS terminater, which have been utilized to produced pLAN421 [49]. The chimeric gene was cloned into the unique Eco RI site of the binary vector pKM424 to create pLYC721. To produce the 5' deletion clone, pLYC721 was digested with Cla I, the restriction site of which is located at position -589 in the genomic fragment, to remove a 1.5 kb Cla I fragment. The larger Cla I fragment was self-ligated to create pLYC706 which contains 589 bp of the 5'-flanking sequence of cyc07.

Transformation of Arabidopsis plants

The chimeric cycO7-GUS fusions in the binary vector were transferred into Agrobacterium tumefaciens strain EHA101 [42] by a freeze-thaw method [ 16 ]. Transformation of A rabidopsis with Agrobacterium was carried out according to [1] with some modifications. All media used for the transformation contained MS basal salts base [34], 10 rag/1 thiamine, 5 rag/1 nicotinic acid, 0.1 g/1 myo-inositol, 2 mg/1 glycine, and 10 rag/1 pyridoxine. The germination medium (GM) contained 10g/1 sucrose; callus-inducing medium (CIM) contained 20 g/1 glucose, 0.05 mg/1 kine- tin, and 0.5 mg/l 2,4-D; shoot-inducing medium (SrIM) contained 20 g/1 glucose, 1.0 mg/1 N6-(2 -

isopentenyl)adenine (2ipA), and 0.15rag/1 in- doleacetic acid; root-inducing medium (RIM) contained 10 g/1 sucrose and 1 mg/1 indole-3- butyric acid; Agrobacterium infection medium (AIM) contains 1 ~o glucose. Seeds of Arabidopsis thaliana ecotype Wassilewskija were sterilized by immersion in a solution of 3 ~o sodium hypochlo- rite and 0.01~o SDS for 5 rain and washed 5 times with sterile water. The seeds were germi- nated on GM plates. Hypocotyls of 14-day-old seedlings were dissected and placed on plates of CIM and incubated for 6 days. For Agrobacte- rium infection, the explants were transferred to a petri dish containing 5 ml of AIM solution plus 1 ml of a concentrated bacterial suspension and gently shaken for 20 rnin. The explants were

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blotted on sterile paper and incubated on CIM plates. After cocultivation for 3 days, the explants were washed in a petri dish with 5 ml of AIM solution that contained 500 mg/1 carbenicillin by shaking gently for 60 rain. This treatment was repeated 5 times to remove the bacteria. Then the explants were blotted on sterile paper, transferred onto SrIM plates containing 50 mg/1 kanamycin and 50rag/1 carbenicillin. The explants were transferred onto fresh SrIM plates at weekly in- tervals. After a week, carbenicillin in the SrIM plates was replaced by 300 rag/1 vancomycin. Shoots that were generated from the calli within 3 to 4 weeks after infection were excised and transferred to RIM plates. Most of the trans- planted shoots generated roots within 2-3 weeks. The plantlets with roots were transferred to small pots containing GM. After a one-week incuba- tion, the plantlets were transferred to small pots containing vermiculite. After one month, each plant had set several hundred seeds.

Fig. 1. Evidence for the fact that periwinkle cyc07 is a single- copy gene. A genomic DNA blot of periwinkle DNA digested with Eco RI, Xho I, Kpn I, or Hind III, as indicated, was hybridized with the 1.1 kb cyc07 cDNA as probe.

Callus induction from transgenic Arabidopsis plants

Stems were cut from the transgenic Arabidopsis plants. They were placed on solid medium com-

E P C B K K P E

(E) B K K P (E)

pCYCO7F6

0.5kb

Fig. 2. Restriction maps of the genomic (pBS07ECG) and cDNA (pCYC07F6) clones of periwinkle cyc07. Boxed areas correspond to exons. The coding region (filled boxes) and the 5' and 3' untranslated regions (hatched boxes) are indicated. Restric- tion enzyme abbreviations: E, Eco RI; P, Pst I; C, Cla I; B, Bam HI; K, Kpn I. The complete nucleotide sequence of the 4.8 kb Eco RI genomic fragment will be appeared in DDBJ, EMBL, GenBank Nucleotide Sequence Databases with the accession number D26058.

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posed of MS basal salts plus 1 ~o sucrose at 27 ° C in the presence or absence of auxin. Either naphthalene-l-acetic acid (NAA) (lmg/1) or 2,4-D (1 rag/l) was added to the medium to induce callus formation.

Histochemical GUS assay

Whole plantlets or detached organs were incubated directly in 100mM phosphate buffer (pH 7.0) containing 1 mM EDTA and 2 mM 5-bromo-4-chloro-3-indolyl-/~-D-glucuronide (X- Gluc) at 37 ° C for 12 h. They were then washed several times in 100mM phosphate buffer (pH 7.0) and fixed in 100 mM phosphate buffer that contained 1 ~o glutaraldehyde at 4 ° C for 3 h. The tissues were cleared in 70~o ethanol in order to remove the chlorophyll.

tected in genomic Southern blot analysis as a single band, was chosen to be isolated. A 2gtl l genomic library was constructed from size- fractionated periwinkle genomic fragments that had been digested completely with Eco RI. The cyc07 cDNA was used as a probe to screen the genomic library. A large number of positive clones were obtained, and ten positive clones were analyzed. All ten clones carried a 4.8 kb fragment and gave the same restriction pattern. The 4.8 kb fragment was subcloned into the plasmid vector, pBluescript, to create pBS07ECG.

Results

Isolation of the cyc07 genomic fragment

To determine the copy number of the cyc07 gene, total genomic DNA was extracted from suspension-cultured periwinkle cells for genomic South- ern blot analysis. As shown in Fig. 1, the entire 1.1 kb cDNA fragment ofcyc07 from pCYC07F6, which contains nearly full-length cDNA, hybridized with a single 4.8 kb band of Eco RI-cut genomic DNA. Similarly, the single band corresponding to a large genomic fragment of 8-10 kb was detected in genomic DNA digested with either Hind III or Xho I. When genomic DNA was digested with Kpn I, for which there are two recognition sites in the 1.1 kb cDNA, three fragments were detected. The smallest 1.8 kb fragment can be expected to correspond to the inter- nal Kpn I fragment of the cDNA, assuming the presence of an intron in this genomic fragment. Thus, the present results indicate that cyc07 is present as a single-copy gene in the periwinkle genome.

For cloning of the entire genomic sequence of cyc07, the 4.8 kb Eco RI fragment, which was de-

Fig. 3. Primer-extension assay for mapping the 5' end of the cyc07 mRNA. The 32P-labeled 30-meric primer was hybridized with poly(A) + RNA, which had been isolated from exponentially growing periwinkle cells, at 25 °C (lane 5), 30 °C (lane 6), or 35 °C (lane 7) and then the primer was extended by reverse transcriptase. The longest extended product is indicated by an arrowhead. The sequencing ladders of M 13rap 18 DNA were used to determine the size of the extended product (lane 1, A; lane 2, C; lane 3, G; lanes 4 and 8, T).

Analysis of the structure of the cyc07 gene

To precisely characterize the entire genomic structure, the complete nucleotide sequence of the cloned 4.8kb EcoRI genomic fragment in pB S07ECG was determined. All of the sequences homologous to the cyc07 cDNA lay within the 4.8 kb Eco RI genomic fragment. The gene consists of seven exons separated by six introns (Fig. 2). The nucleotide sequences of the seven exons match that of cDNA clone exactly. The consensus nucleotide sequences for splice sites (GT and AG) were found at the splice junctions (Fig. 4). The initiation codon and termination codon were found in the first exon and the last exon, respectively (Fig. 2).

In order to locate the transcription startpoint of the gene, primer extension experiments were carried out with an oligonucleotide primer that cor- responded to a region within the 5' non-coding

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sequence of the cDNA (Fig. 3). The longest and major extended product was an 89 base fragment. Thus, transcription starts at the thymine residue that is located 96 bp upstream from the initiation codon (Fig. 4). The length of the transcriptional units, after removal of the introns, is about 1.1 kb, which agrees welt with the actual mRNA size of 1.2 kb if a poly(A) tail of about 100 bp is as- sumed.

The 4.5 kb genomic fragment was found to contain a 2.1 kb 5'-flanking sequence. Good can- didates for a TATA box and a CAAT box were found at nucleotide positions -28 and -68, respectively (Fig. 4). In addition, two direct repeat sequences of 13 nucleotides were found in the 5'-upstream sequence. One is the nucleotide sequence, AATATTGAT(G)GTAG, starting at positions -371 and -344, the other is AATATATCTGGCC, starting at positions -200 and -235 (Fig. 4).

ClaI

-595 /~u~I~TICGATTTTCTTTTCCCAGACTTA~A~Y~GTGT~ATTTGT-~u~TC~-C~AG -536 CATTTTTACTTATAATTATATAATAAAACCTACTCAAAAAAACGTAGTTTCACTAATTGA -476 GTATATTATGTTAGGTGAAAGCGAGT~GTGATACAAATGGCCCTAAAAGTGGGTGACGC -416 CTAA~ACAGAGTTTTAAAAGGTAATATTGATGTA~TTTCTTAGCAAGAAATATTGAGG

repeat 1 repeat 1

-356 TAGGTTTTTTTATTAAAAATTAAAT~GTATTTCCATGCTACTAAGACCTTCCTAAGGAC -296 TTGAAACTTCGAGATTGATTCTGAGGAGAT~AGATTGGAATATATCTGGCCATACCTAG

repeat 2

-236AAAAAAATAATAATAATATATCTGGCCCATTGGATAAAAAACC~CACTTGAATAGAAAC repeat 2

-176 CCACCAAATCCAGACTTAAGAGAATGAAACATATTGGGCCCTCAAATTAACTCTTCATAA

-ll6 CTAATGTATGG~TTTC~TATCTA~CCATAACATATAGGAGCAGAC CCAAT TTCTC

-58 CAAACCTATCTTTATCTATACCTAC~CAG TATAT~ ACCTAGGGTTTTATATGCATT +1

1 ~GTTTTCTCTGATTGAGGCCGTT~TGA~TCTTCATAGC~TCTCTCTTTCTCTCT ist exon

61 CAGCACAACAGCAGCAGCAGCTCAGGAGCTCC~CCATGGCCGTCGG ~GATTATATCC M A V G

120 TCTTTGCCTATAAACCATTCAACCATTGTATGATTCTGATTTGCTGAGACTCATTCCTTT

180 TTGTTTTCTATTTGATGC~ TAAGAACAAGA~ATATCG~GGGTAAGAAAGGAGGAAA K N K R I S K G K K G G K

2nd exen

239 GAAG~G~ ~AAGTATTTTAATGTTTTCTGTTTTATAAAATTCTGATCTTTGGTGGAT K K A

BamHI

278 TTTGTTCTTTTGATGTTTGATTTGAGTGT~TTTTTATTTCATTAT~ AGCGIGATCC A D P 3rd exon

Fig. 4. Nucleotide sequence of the 5' upstream region and part of the coding region of the cyc07 gene. A bent arrow indicates the location of the mNor transcription initiation site, which is numbered + 1. Sequences homologous to the TATA box and CCAAT box ~ e indicated by ~rows. The locations of two repeated sequences (repeat 1 and repeat 2) we indicated. The regions corresponding to exons are underlined, and the deduced amino acid sequence is indicated below the nucleotide sequence.

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Sequence homologous to cyc07 in other higher plants

To demons t ra te the existence of homologous genes in other higher plants, poly(A) + R N A was isolated f rom actively growing cultured cells in suspension. Cultured cells of tobacco, Phytolacca, carrot, and rice were harves ted during exponen-

tial growth, and poly(A) + R N A was extracted. Nor thern hybridizat ion under low-stringency condit ions revealed the presence of m R N A that cross-hybridized to full-length c D N A for cyc07 (pCYC07F6) . The size of the detected m R N A s f rom all plants examined was est imated to be 1.2 kb, which is identical to the size of the cyc07 m R N A in periwinkle (Fig. 5).

Expression of the cyc07 promoter-GUS chimeric gene in transgenic roots of Arabidopsis

Fig. 5. The expression of genes homologous to cyc07 in various species of higher plant. Poly(A) + RNA was isolated from exponentially growing cells of tobacco (lane 1), Phytolacca (lane 2), carrot (lane 3), and rice (lane 4). RNAs were hybridized with cyc07 cDNA as a probe under low-stringency conditions.

In our previous work, levels ofcyc07 m R N A were

examined in periwinkle seedlings by nor thern blot analysis. The cycO7mRNA was preferentially detected in the port ion that contained a root tip,

Clal Clal Sac1 1(2100) [ + 1 ~

NPT II i w ~ ' r / / / / , , / A c / / / / / / / / / ' V / / / / / / / / / A N O ~ . • - 07-p L ,

ATG (ATG)

(pLYC721; 18.5kb)

Clal Sac1

07-p . . . . . .

ATG (ATG)

(pLYCT06; 17.1kb)

Fig. 6. Construction of plasmids used for transformation ofArabidopsis, pLYC721 contains a 2.1 kb 5' promoter region and three exons, separated by two introns, fused in-frame with the GUS gene. The 5'-flanking region of pLYC721 was deleted up to the Cla I site to produce pLYC706, which contains 589 bp of the 5'-flanking sequence. The shaded areas indicate the coding sequence of cyc07 and the GUS gene. Introns are shown as black boxes• The transcription initiation site was indicated ( + 1). Abbrevia- tions: NPT II, gene for neomycin phosphotransferase; 07-P, cyc07 promoter; NOS-t, nos terminater; LB, left border; RB, right border.

suggesting a correlation between the expression of cyc07 and meristematic activity [ 17]. In order to determine which particular tissues within the organ are actually expressing the cyc07 gene and, moreover, whether or not the spatial pattern of such expression is regulated by the 5'-upstream promoter region, a translational fusion between the upstream region of cyc07 and the GUS reporter gene was constructed. A 2.5 kb fragment containing the 2.1 kb 5'-flanking region and three exons of cyc07 was fused in frame with the GUS reporter gene to create pLYC721 (Fig. 6). In addition, pLYC721 was digested with Cla I to de- lete the upstream region, producing pLYC706 which contained a 589 bp 5'-flanking sequence and three exons (Fig. 6). Both clones can produce a hybrid protein that consists of the N-terminal 22 amino acids of cyc07 fused with the GUS protein. Both promoter-GUS fusion constructs were introduced into Arabidopsis plants by Agrobacterium-mediated transformation. Spatial patterns of activity of the cyc07 gene promoter in transgenic organs were examined by histochemical analysis. To determine whether or not the 5 '-upstream regions of different lengths could direct the expression associated with cell proliferation, GUS activity was analyzed in roots of the transgenic Arabidopsis plants, using the chro- mogenic substrate X-Gluc. For both constructs, although intensity of staining varied among indi- vidual transformants, high GUS activity was found to be located in the root tip (Fig. 7b). These observations are in good agreement with the results of northern blot hybridization analysis of periwinkle seedlings.

Expression of the chimeric gene during the development of transgenic Arabidopsis

Since 589 bp of the 5'-flanking sequence of the cyc07 gene could direct meristem-specific expression, just as the 2.1 kb promoter sequence did, the developmentally associated changes in expression of the chimeric gene were analyzed using the 589 bp 5'-flanking fragment.

Positive staining for GUS in root tips persisted

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during plant development (Fig. 7c). Meristem- specific expression was apparent in lateral roots (Fig. 7d). Specific GUS activity was also found in the region of lateral-root initiation (Fig. 7e). GUS activity was detected even in the lateral-root primordia before visible evidence of root formation was apparent (Fig. 7f). The aerial parts of seedlings, including cotyledons, did not show any evidence of detectable GUS activity for three days after the germination (Fig. 7a). Seven days after the beginning of germination, the apical buds be- came apparent and had very strong GUS activity (Fig. 7g, Fig. 7h). The region expressing GUS activity was strictly limited to the shoot apex, indicating a close correlation between meristematic activity and promoter-directed gene expression. Similarly, high GUS activity was detected at sites of axillary-bud initiation (Fig. 7i). In adult plants, GUS activity was found in the anther (Fig. 7k) and the immature embryo inside the immature seed pod (Fig. 7j), tissues in which active cell-cycling is considered to occur. However, no detectable activity was found in either mature organs, such as roots, leaves, and mature seeds, which are known to be composed of resting cells without mitotic activity.

Induction of the promoter activity by treatment with auxin

cyc07 m R N A accumulates preferentially in actively proliferating cells in asynchronously dividing cultures of periwinkle cells in the presence of auxin [23]. Furthermore, when auxin is absent from the culture medium and cells are arrested at the G1 phase of the cell cycle, cyc07 m R N A is barely detectable [ 17]. Thus, accumulation of the m R N A is closely associated with the proliferation of cells that is induced by auxin.

To examine the correlation between the activity of the cyc07 promoter and cell proliferation more directly, excised stems were treated with an auxin, such as 2,4-D or NAA, to induce formation of callus, and GUS activity was measured. The stems from transgenic plant transformed with pLYC706 showed enhanced GUS activity upon

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treatment with 2,4-D or NAA (Fig. 8). By contrast, GUS activity was not induced when excised stems were cultured in the absence of auxin (Fig. 8). Therefore, the 589 bp 5'-flanking sequence of cyc07 was able to direct auxin-induced GUS activity.

Discussion

Fig. 8. Induction of GUS activity by auxin treatment. Stems were excised from transgenic Arabidopsis plants that had been transformed with pLYC706 and treated with auxin for induction of callus. A, stem cultured without auxin; b, stem cultured with 2,4-D (1 mg/ml); c, stem cultured with NAA (1 mg/ml).

The S-phase-specific gene, cyc07, was initially isolated as a cDNA clone by differential screening of a cDNA library prepared from cells in syn- chronously dividing cultures of periwinkle cells [23]. Northern blot analysis showed a close correlation between the expression of cyc07 and cell proliferation, suggesting that the protein encoded by cyc07 has a function that is associated with cell proliferation. Furthermore, the expression of cyc07 in the S phase was completely suppressed by addition of an inhibitor of DNA synthesis, aphidicolin, an indication of the functional cou- pling of expression of the gene and DNA replication [17]. Two genes, a histone gene [29] and the gene for PCNA [24], are known as S-phase- specific genes in higher plants. Both genes are expressed in actively cycling cells [26, 46] and their promoters direct the meristem-specific expression of a reporter gene [3,27]. However, these two S-phase-specific genes differ in that expression of the histone gene is dependent on the DNA replication [29] while expression of the PCNA gene is independent of it [24]. Thus, in terms of the pattern of its expression, the cyc07 gene resembles the histone gene. The protein encoded by cyc07 is also similar to histones in terms of the large number of basic amino acids and the size of the polypeptide, although no significant similarity in protein sequence has been found. Genes encoding histones have been cloned from

Fig. 7. Histochemical localization of GUS activity in transgenicArabidopsis plants. TransgenicArabidopsis plants transformed with pLYC706 were analyzed histochemically.for localization of GUS activity. (a) Aerial part of a 3-day-old seedling; (b) root of a 3-day-old seedling; (c) roots of an adult plant; (d) lateral roots; (e) early stage of lateral root initiation; (f) lateral-root primordia; (g), (h) apical meristematic region of a 7-day-old seedling; (i) auxiliary bud; (j) immature embryo inside the immature seed pod; (k) anther.

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several species of plants [12]. In situ RNA hybridization showed that cells expressing histone H2A were concentrated near the apex of plantlets, and the distribution of the expression was consistent with that expected of cycling cells [26]. Recently, it was reported that transcription directed by the promoter of histone H4 gene is dependent on DNA replication, although a low level of expression is independent of DNA replication [291.

Genomic Southern blot analysis demonstrated that cyc07 is a single-copy gene in the haploid genome of periwinkle. A genomic fragment was isolated and the complete structure of the gene was determined. The cyc07 gene is composed of seven exons and six introns. The promoter activity of the 5'-flanking sequence of the cyc07 gene was analyzed in an effort to clarify the regulatory mode of expression of the cyc07 gene. A translational fusion gene was constructed that contained the 5'-flanking sequence, three exons and two introns of cyc07 fused in frame with the GUS reporter gene. Using the chimeric gene and transgenic plants, it was demonstrate that a fragment containing the 589 bp 5'-flanking region of the cyc07 gene from periwinkle could drive the tissue- specific expression of the GUS reporter gene in Arabidopisis. The genomic fragment containing the 589 bp 5'-flanking sequence can direct gene expression in meristematic tissues such as root tips, lateral-root primordia, and apical and axillary buds. High GUS activity was also found in immature embryos and inside anthers, while in- significant GUS activity was detected in adult tissues, such as leaf, stem, and root.

Histochemical localization of GU S activity, directed by the promoter of the cyc07 gene, is quite similar to that of genes for histones or PCNA. Several genes, including those for histones and PCNA, are attributed to meristem-specific genes in higher plants, for example, genes for deoxyuri- dine triphosphatase [41] and elongation factor 1 [50], as well as genes with unknown functions [33, 45]. The results presented here clearly demonstrate that the cyc07 gene is one of the meristem-specific genes. The promoters of the three known cell-cycle-dependent genes in plants,

the genes for PCNA, and histones and the cyc07 gene can direct gene expression in a meristem- specific manner, thus giving a possible indication that genes with important functions in cell cycle progression can be contained in meristem-specific genes identified so far. In fact, a plant homolog of p34 cdc2 gene can be attributed into a group of meristem-specific gene [6, 30], and it encodes a key component for the progression of the eukaryotic cell cycle [37].

The spatial pattern of GUS activity driven by the fragment containing 589-bp of the promoter of the cyc07 gene was closely associated with cell proliferation, and it was consistent with the expected distribution of cells in the S phase. At present, however, the possibility cannot be ruled out that the two introns contained in the chimeric gene contribute to the pattern of expression of the GUS gene, since it is known that introns sometimes affect gene expression [38, 48]. At least, however, the 589 bp 5' upstream sequence and the N-terminal coding sequence including two introns provide sufficient regulatory information for expression of the hybrid gene specifically in meristematic cells. There are two direct repeat sequences of 13 nucleotides within the 589 bp 5'- flanking sequence. It will be of interest to determine whether and which of these repeated sequences contribute to the regulated expression that is driven by the promoter. The promoter regions of histone genes have been found to contain several conserved elements. For example, the octanucleotide CGCGG(C)ATC is highly conserved in all plant histone genes [12]. However, no sequences analogous to such conserved elements, including the octanucleotide, can be found in the 589 bp upstream sequence of the cyc07 gene. Further study is required for identification of the cis-acting elements in the 589 bp upstream fragment, that contribute to regulatory expression of the reporter gene. Although meristem-specific expression may reflect the fact that meristematic tissues contain a number of cells in the S phase during the cell cycle, it is still unclear whether the 589 bp upstream fragment directs the S-phase- specific expression of the gene. The promoter region and cis-acting element that direct the

S-phase-specific expression of the gene remain to be identified.

The spatial pattern of GU S activity directed by the promoter could provide some information about the possible function of the cyc07 gene. The close correlation between the GUS activity and meristematic activity suggests a specific function for this gene in cell proliferation in plants, possibly in molecular events that occur during the S phase.

In the seedlings oftransgenic Arabidopsis, GUS activity in root tips was detected from the beginning of germination. By contrast, in shoot apex, it was not detected even three days after germination. The northern blot analysis indicated that, in periwinkle seedlings, the portion containing the shoot apex and cotyledons did not show evidence of enhanced expression, whereas the portion containing root tips contained significantly larger amounts of cycO? mRNA than other portions [17]. Thus, the pattern of GUS activity in the transgenic plants was in good agreement with the results of northern blot analysis. The histone promoter-GUS chimeric gene was sometimes expressed at high levels in cotyledons, which are not known as organs with significant mitotic activity. Such expression of GUS may possibly have resulted from DNA synthesis during cell polyploidization [3, 29]. It has been reported that the DNA endoreduplication during cell polyploidization frequently occurs very early in the cotyledons of Arabidopsis seedlings [4]. No GUS activity was directed by the cyc07 promoter in cotyledons of seedlings of transgenic Arabidopsis, and northern blot analysis did not reveal significant amounts of cyc07 mRNA in periwinkle cotyledons. Therefore, the expression of cyc07 appears to be associated with DNA replication in the cell cycle but not with DNA synthesis during cell polyploidization. Thus, the function of cyc07 may not be directly associated with DNA synthesis itself, because the DNA endoreduplication appears to be completed in the absence of the expression of the cyc07 gene.

Recently, two genes with sequences closely related to the cyc07 gene were isolated, namely, MFT1 from a budding yeast [ 11 ] and fte-1 from

875

rat [22]. The MFTI gene was isolated as a gene involved in the import of a fusion protein into yeast mitochondria [ 11 ]. The reported nucleotide sequence of the MFT1 gene is identical to that of the PLC2 gene, which we isolated independently as a yeast homologue ofcyc07 [ 18]. However, we found that the MFT1 gene, which is involved in protein import into mitochondria, corresponds to a locus adjacent to PLC2, that is, the MFT1 gene is different from the PLC2 gene [19]. Thus, the function of cyc07 and its yeast homologs (PLC1 and PLC2) is not related to the protein import into mitochondria, and the precise function of these genes remains to be identified. Kho and Zarbl reported the isolation of an effector gene for transformation that is induced by the v-fos oncogene from rat [22]. The gene, designated fie-1 (v-fos transformation effector gene), appears to be a mammalian homologue of cyc07. Revertants (untransformed variants) were isolated from cells transformed with the v-fos oncogene, by mu- tagenic insertion of the transfected plasmid into the genome. The gene disrupted by insertion of the plasmid was identified and named fte-1. The cDNA clone corresponding to the gene was isolated and sequenced [22]. The amino acid sequence deduced from the nucleotide sequence of the fte-1 cDNA was found to have significant similarity to that of the reported yeast gene MFT1, which is identical to that of PLC2. Thus, it fol- lows that the cyc07 gene from C. roseus, its yeast homologues (PLC1 and PLC2), and thefte-1 gene from rat are homologous to one another, and their evolutionary origin can be traced to a common ancestor. A comparison of the deduced amino acid sequences of fte-1 and cyc07 is shown in Fig. 9. The predicted size of the polypeptide encoded by cyc07 (245 amino acids) is similar to that encoded by fte-1 (264 amino acids). Of 235 amino acids in the N-terminal region, 160 amino acids are identical in the two deduced amino acid sequences. The extent of the similarity is about 68~o if two gaps are introduced. When conser- vative substitutions are included, the extent of the similarity is 94~o. The striking similarity in protein sequences suggests an important function for these genes in maintaining the biological systems.

876

c9c07

fte-I

cyc07 60'

fte-i 61"

CyC07 120'

fte-I 120"

i' MAVGKNKRISK-GKKGGKKKAADPFAKKDWYDIKAPSVFSVRNVGKTLVTRTQGTKIASE

i" MAVGKNKRLTKGGKKGAKKKVVDPFSKKDWYDVKAPAMFNIRNIGKTLVTRTQGTKIASD

GLEHRVFEISLADLQGDEDHSFRKIRLRAEDIQGKNVLTNFWGMDFTTDKLRSLVRKWQS

. . . . . . . . . . . . . . .

GLKGRVFEVSLADLQ-NDEVAFRKFKLITEDVQGKNCLTNFHGMDLTRDKMCSMVKKWQT

LIEAHVDVKTTDSYTLRMFCIGFTKKRANQQKRTCYAQSSQIRQIRRKMREIMVNQAQSC

MIEAHVDVKTTDGYLLRLFCVGFTKKRNNQIRKTSYAQHQQVRQIRKKMMEIMTREVQTN

cyc07 180'

fte~l 180"

DLKDLVQKFIPESIGREIEKATSSIYPLQNVFIRKVKILKAPKFDLGKLMEVHGDYNEDR

DLKEVVNKLIPDSIGKDIEKACQSIYPLHDVFVRKVKMLKKPKFELGKLMELHGEGGSSG

cyc07 240' YWYQVG

fte-I 240" KTTGDETGAKVERADGYEPPVQESV

Fig. 9. Comparison ofproteinsequencesdeduced ~om periwinkle ~c07andratfie-lgenes. Starsand dotsindicateidenticaland biologicallysimilaramino acids, respectively.

The function of the fte-1 gene is unclear, however, Kho and Zarbl discussed the possibility that fie-1 gene expression may have an effect on cell growth [22]. We propose the possibility that these evolutionarily well conserved genes play a crucial role in the progression of the eukaryotic cell cycle, based on the S-phase-specific expression of the cyc07 gene during the plant cell cycle [17], the essential function of the PLC gene family (PLC1 and PLC2) in proliferation of yeast cells [ 18], and the less tumorigenic phenotype that results from disruption of the fte-1 gene [22], as well as the finding that the promoter of the cyc07 gene directs meristem-specific expression of a reporter gene.

Acknowledgements

This research was supported by Grant-in-Aid for Scientific Research (02454007) from Ministry of Education, Science and Culture, Japan.

References

1. Akama K, Shiraishi H, Ohta S, Nakamura K, Okada K, Shimura Y Efficient transformation of Arabidopsis thaliana: comparison of the efficiencies with various organs, plant eeotypes and Agrobacterium strains. Plant Cell Rep, in press (1993).

2. Amino S, Fujimura T, Komamine A: Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus, Physiol Plant 59:393-396 (1983).

3. Atanassova R, Chaubet N, Gigot C: A 126 bp fragment of a plant histone gene promoter confers preferential expression in meristems oftransgenic Arabidopsis. Plant Cell 2:291-300 (1992).

4. Brown SC, Bergounioux C, Tallet S, Marie D: Flow cytometry of nuclei for ploidy and cell cycle analysis. In: Negrutiu I, Gharti-Chhetri G (eds) A Laboratory Guide for Cellular and Molecular Plant Biology, pp. 326-345. Birkhauser, Basel (1991).

5. Chu CC, Wang CC, Sun CS, Hsu C, Yin KC, Chu CY, Bi FY: Establishment of an efficient medium for anther culture of rice through comparative experiments on the nitrogen sources. Sci Sin 18:659-668 (1975).

6. Colasanti J, Tyers M, Sundaresan V: Isolation and characterization of cDNA clones encoding a functional p34 ~dCa homologue from Zea mays. Proc Natl Acad Sci USA 88:3377-3381 (1991).

7. DoonanJH: Cycling plant cells. Plant J h 129-132 (1991).

8. Feller HS, Jacobs TW: Cell division in higher plants: a cdc2 gene, its 34-kDa product, and histone H1 kinase activity in pea. Proc Natl Acad Sci USA 87:5397-5401 (1990).

9. Ferreira PCG, Hemerly AS, Villarroel R, Van Montagu M, Inze D: The Arabidopsis functional homolog of the p34 cdc2 protein kinase. Plant Cell 3:531-540 (1991).

10. FujimuraT, Komamine A: Effects of various growth regulators on the embryogenesis in a carrot suspension culture. Plant Sci Lett 5:359-364 (1975).

11. Garrett JM, Singh KK, VonderHaar RA, Emr SD: Mitochondrial protein import: isolation and characteriza-

tion of the Saccharomyces cerevisiae MFT1 gene. Mol Gen Genet 225:483-491 (1991).

12. Gigot C: Histone genes in higher plant. In Kahl G (ed) Architecture of Eukaryotic Genes, pp. 1367-1373. VCH Verlagsgesellschaft, Weinheim (1988).

13. Hata S, Kouchi H, Suzuka I, Ishii T: Isolation and characterization of cDNA clone for plant cyclin. EMBO J 10: 2681-2688 (1991).

14. Hemerly A, Bergounioux C, Van Montagu M, Inze D, Ferreira P: Genes regulating the plant cell cycle: isolation of a mitotic-like cyclin form Ambidopsis thaliana. Proc Natl Acad Sci USA 89:3295-3299 (1992).

15. Hirt H, Pay A, Gyorgyey J, Bako L, Nemeth K, Bogre L, Schweyen RJ, Heberle-Bors E, Dudits D: Complementa- tion of a yeast cell cycle mutant by alfalfa cDNA encoding a protein kinase homologous to p34 °de2. Proc Natl Acad Sci USA 88:1636-1640 (1991).

16. Holsters M, de Waele D, Depicker A, Messens E, Van Montagu M, Schell J: Transfection and transformation ofA. tumefaciens. Mol Gen Genet 163:181-187 (1978).

17. Ito M, Kodama H, Komamine A: Identification ofanovel S-phase-specific gene during the cell cycle in synchronous cultures of Catharanthus roseus cells. Plant J 1:141-148 (1991).

18. Ito M, Yasui A, Komamine A: A gene family homologous to the S-phase specific gene in higher plants is essential for cell proliferation in Saccharomyces cerevisiae. FEBS Lett 301:29-33 (1992).

19. Ito M, Yasni A, Komamine A: Precise mapping and molecular characterization of the MFT1 gene involved in import of a fusion protein into mitochondria in Saccha- romyces cerevisiae. FEBS Lett 320:125-129 (1993).

20. John PCL, Sek FJ, Lee MG: A homolog of the cell cycle control protein p34 cdc2 participates in the division cycle of Chlamydomonas, and a similar protein is detectable in higher plants and remote taxa. Plant Cell 1:1185-1193 (1989).

21. Kato K, Shiozawa Y, Yamada A, Nishida K, Noguchim M: A jar fermentation culture of Nicotiana tabacum L. suspensions. Agric Biol Chem 36:899-902 (1972).

22. Kho C-J, Zarbl H: Fte-1, a v-fos transformation effector gene, encodes the mammalian homologue of a yeast gene involved in protein import into mitochondria. Proc Natl Acad Sci USA 89:2200-2204 (1992).

23. Kodama H, Ito M, Hattori T, Nakamura K, Komamine A: Isolation of genes that are preferentially expressed at the G~/S boundary during the cell cycle in synchronized cultures of Catharanthus roseus cells. Plant Physiol 95: 406-411 (1991).

24. Kodama H, Ito M, Ohishi N, Suzuka I, Komamine A: Molecular cloning of the gene for plant proliferating-cell nuclear antigen and expression &this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells. Eur J Biochem 197:495-503 (1991).

25. Kodama H, Kawakami N, Watanabe A, Komamine A: Phase-specific polypeptides and poly(A) + RNAs during

877

the cell cycle in synchronous cultures of Catharanthus roseus cells. Plant Physiol 89:910-917 (1989).

26. Koning AJ, Tanimoto EY, Kiehne K, Rost T, Comai L: Cell-specific expression of plant histone H2A genes. Plant Cell 3:657-665 (1991).

27. Kosugi S, Suzuka I, Ohashi Y, Murakami T, Arai Y: Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants. Nucl Acids Res 19:1571-1576 (1991).

28. Lehrach H, Diamond D, Wozney JM, Boedtker H: RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16:4743-4751 (1977).

29. Lepetit M, Ehling M, Chaubet N, Gigot C: A plant histone gene promoter can direct both replication-dependent and -independent gene expression in transgenic plants. Mol Gen Genet 231:276-285 (1992).

30. Martinez MC, Jorgensen J-E, Lawton MA, Lamb CJ, Doerner PW: Spatial pattern of cdc2 expression in relation to meristem activity and cell proliferation during plant development. Proc Natl Acad Sci USA 89:7360-7364 (1992).

31. McKinney JD, Heintz N: Transcriptional regulation in the eukaryotic cell cycle. Trends Biochem Sci 16: 430- 435 (1991).

32. Medford JI: Vegetative apical meristems. Plant Cell 4: 1029-1039 (1991).

33. Medford JI, Elmer JS, Klee HJ: Molecular cloning and characterization of genes expressed in shoot apical meristems. Plant Cell 3:359-370 (1991).

34. Murashige T, Skoog F: A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473-497 (1962).

35. Murray MG, Thompson WF: Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8: 4321- 4325 (1980).

36. Nishida T, Ohnishi N, Kodama H, Komamine A: Estab- lishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells. Plant Cell, Tissue Organ Culture 2 8 : 3 7 - 4 3 (1992).

37. Nurse P: Universal control mechanism regulating onset of M-phase. Nature 344:503-508 (1990).

38. Ohta S, Mira S, Hattori T, Nakamura K: Construction and expression in tobacco of a/~-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805-813 (1990).

39. Okada K, Takebe I, Nagata T: Expression and integra- tion of genes introduced into highly synchronized plant protoplasts. Mol Gen Genet 205:398-403 (1986).

40. Ozawa K, Komamine A: Establishment of a system of high-frequency embryogenesis from long-term cell suspension cultures of rice (Oryza sativa L.). Theor Appl Genet 77:205-211 (1989).

41. Pri-HadashA, Hareven D, Lifschitz E: A meristem-

878

related gene from tomato encodes a dUTPase: Analysis of expression in vegetative and floral meristems. Plant Cell 4:149-159 (1992).

42. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, KmaufVC: Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75:685-694 (1988).

43. Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 (1977).

44. Schmidt GW, Bartlett SG, Grossman AR, Cashmore AR, Chua N: Biosynthetic pathway of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex. J Cell Biol 91:468-478 (1981).

45. Smith JA, Krauss MR, Borkird C, Sung ZR: A nuclear protein associated with cell divisions in plants. Planta 174:462-472 (1988).

46. Suzuka I, Daidoji H, Matsuoka M, Kadowaki K,

Takasaki Y, Nakane PK, Moriuchi T: Gene for proliferating-cell nuclear antigen (DNA polymerase /5 auxiliary protein) is present in both mammalian and higher plant genomes. Proc Natl Acad Sci USA 86:3189-3193 (1989).

47. Suzuka I, Hata S, Matsuoka M, Kosugi S, Hashimoto J: Highly conserved structure of proliferating cell nuclear antigen (DNA polymerase /5 auxiliary protein) gene in plants. Eur J Biochem 195:571-575 (1991).

48. Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K: Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and efficient splicing of the intron. Nucl Acids Res 18:6767-6770 (1990).

49. Uematsu C, Murase M, Ichikawa H, ImamuraJ: Agro- bacterium-mediated transformation and regeneration of kiwi fruit. Plant Cell Rep 10:286-290 (1991).

50. Ursin VM, IrvineJM, Hiatt WR, Shewmaker CK: De- velopmental analysis of elongation factor-lc~ expression in transgenic tobacco. Plant Cell 3:583-591 (1991).

Documents

Meristem-specific gene expression directed by the promoter of the S-phase-specific gene, cyc07, in transgenic Arabidopsis