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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

Full-length cDNAs are essential for functional analysis of plant genes. We constructed high-content, full-length cDNA libraries from Arabidopsis thaliana plants based on chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment, to select full-length cDNA. More than 90% of the total clones obtained were of full length; recombinant clones were obtained with high efficiency (2.2 × 106/9 μg starting mRNA). Sequence analysis of 111 randomly picked clones indicated that 32 isolated cDNA groups were derived from novel genes in the A. thaliana genome.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Arabidopsis Genome Initiative (AGI) has recently been launched to complete sequencing of the Arabidopsis thaliana genome by 2004 (Bevan et al. 1997). Several large cDNA projects are in progress to determine the expressed genes (ESTs) of A. thaliana (Cooke et al. 1996;Höfte et al. 1993;Newman et al. 1994). These sequences serve as a key source for gene mapping and are thus essential for the positional candidate gene approach. However, despite the great amount of biological information produced by large-scale cDNA sequencing (Cooke et al. 1996;Höfte et al. 1993;Newman et al. 1994), limited information is available on the derived protein sequences for functional analysis of cDNAs because of the partial sequence data. Therefore, information on the complete cDNA sequence is essential when investigating biological function.

Several approaches to establish a system for constructing full-length cDNA libraries have been reported (CLONTECHniques 1996;Ederly et al. 1995;Maruyama & Sugano 1994). However, some drawbacks remained, including the need for PCR amplification, low yield, the use of RNA ligase, and the need for very large amounts of starting mRNA (100 μg).

Recently, Carninci et al. (1996, 1997) have reported high-efficiency selection of mouse full-length cDNA by biotinylated CAP trapper. This system is based on the chemical addition of a biotin group to the CAP structure. This step is followed by digestion by RNase I, a ribonuclease that can cleave single-stranded RNA at any site, followed by selection of full-length cDNA. The library produced by this method contained a very high proportion of mouse full-length cDNAs and produced an excellent yield without involving PCR amplification (Carninci et al. 1996). To construct this library, we used trehalose-thermoactivated reverse transcriptase (Carninci et al. 1998), which can synthesize long cDNAs at very high efficiency.

In this study, we applied the biotinylated CAP trapper method to A. thaliana and succeeded in cloning full-length cDNAs at high efficiency. As far as we know, this is the first report of the construction of a full-length cDNA library from A. thaliana without using PCR amplification.

Results and discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

Construction of full-length cDNA library

About 180 ng of double-strand (ds) cDNA was obtained from 9 μg of starting mRNA that had been prepared from leaves and stem tissues of normally grown A. thaliana plants. Afterwards, the ds cDNA was cleaved with SstI and XhoI and ligated to a Lambda ZAPII vector. The packaged DNA produced more than 2.2 × 106 plaques. More than 95% of the plaques were recombinant phages.

The average size of the insert DNA was about 1.2 kbp . This value is similar to that reported on A. thaliana cDNA by Kotani et al. (1997) but smaller than that of mouse cDNA, approximately 1.6 kbp (Carninci et al. 1996). These results indicate that the average size of A. thaliana cDNAs is generally smaller than that of mouse cDNAs.

The largest cDNA out of 161 clones was longer than 3.5 kbp. Gel electrophoresis of first- and second-strand cDNAs also showed that the synthesized cDNAs extended up to approximately 7.5 kbp in size, which is close to the size of the largest plant cDNA we know (data not shown). These results indicate that plant cDNAs of up to approximately 7.5 kbp can be synthesized by the biotinylated CAP trapper method using trehalose-thermoactivated reverse transcriptase. The presence of full-length cDNA of ferredoxin-dependent glutamate synthase (GenBank accession number Y09667), whose full-length cDNA size is 5178 bp, was confirmed by PCR analysis of the cDNA library using a 5′-end primer (from nucleotide 1 to nucleotide 20) and a 3′-end primer (from nucleotide 4940 to nucleotide 4959) of the ferredoxin-dependent glutamate synthase cDNA (data not shown). However, the presence of full-length cDNA of acetyl-CoA carboxylase (accession number D34630;Yanai et al. 1995), whose full-length cDNA size is 7332 bp, was not confirmed by PCR analysis of the cDNA library using a 5′-end primer (from nucleotide 408 to nucleotide 427) and a 3′-end primer (from nucleotide 7153 to nucleotide 7172) of the acetyl-CoA carboxylase cDNA (data not shown). This is because the mRNA used for construction of the cDNA library in this study did not contain that of the acetyl-CoA carboxylase (data not shown).

Full-length cDNA representation of random mRNA

To analyze the cDNA population, 111 randomly selected recombinant clones were sequenced from their 5′ ends (Tables 2a) and Table 2b and Table 2c and Table 2d. As shown in Table 1a and Table 1b and Table 1c, these clones can be classified into four categories. Among the 111 clones, 31 represent cDNAs already identified in A. thaliana. Of these, 29 were grouped as full-length clones, since all were between 84 bases shorter and 79 bases longer than the putative full-length cDNA sequence in databases (Table 1a) and contained a sequence with a length between +15 and +218 bases upstream of the first ATG codon (Table 1a). Two clones containing A. thaliana cDNA sequences carried truncated cDNAs (FL-30 and FL-31, Table 1b), which encoded A. thaliana plasma membrane intrinsic protein 2a (GenBank accession number X75883;Kammerloher et al. 1994) and heat shock cognate protein hsc70–1 (accession number X77199), and are shortened by more than 124 and 567 nucleotides, respectively. These are clear examples of contamination by short-ended clones. Another group of 22 clones represents the homologues to the reported cDNAs; these were putatively classified as full-length (Table 1a and Table 1b and Table 1c). Another two clones (FL-54 and FL-55, Table 1a) that have sequence similarity with genes for a receptor serine/threonine kinase, PR5K (accession number U48698;Wang et al. 1996), and a protein kinase C inhibitor (accession number Z29643;Simpson et al. 1994) may represent the shortened clones (Table 1a and Table 1b and Table 1c).

Table Table.  Characterization of cDNAs isolated from A. thaliana full-length cDNA library conscructed bythe biotinylated cap trapperaThe gene was reported first in this study (New) or is identical to reported sequences (Existing) in the GenBank DNA database.Thumbnail image of
Table Table.  Characterization of cDNAs isolated from A. thaliana full-length cDNA library conscructed bythe biotinylated cap trapperaThe gene was reported first in this study (New) or is identical to reported sequences (Existing) in the GenBank DNA database.Thumbnail image of
Table Table.  Characterization of cDNAs isolated from A. thaliana full-length cDNA library conscructed bythe biotinylated cap trapperaThe gene was reported first in this study (New) or is identical to reported sequences (Existing) in the GenBank DNA database.Thumbnail image of
Table Table.  Characterization of cDNAs isolated from A. thaliana full-length cDNA library conscructed bythe biotinylated cap trapperaThe gene was reported first in this study (New) or is identical to reported sequences (Existing) in the GenBank DNA database.Thumbnail image of
Table 1.  Summary of one-pass sequencing data for cDNAs that show homology with known sequencesThumbnail image of
Table 1.  Summary of one-pass sequencing data for cDNAs that show homology with known sequencesThumbnail image of
Table 1.  Summary of one-pass sequencing data for cDNAs that show homology with known sequencesThumbnail image of

The content of full-length cDNAs in the library, as determined by evaluating random sequencing data of only A. thaliana genes, is approxaimtely 94% (Tables 1a and b and c). This percentage is consistent with a report of a previously characterized mouse cDNA library (Carninci et al. 1996).

A nucleotide, G, has been observed in the 5′ end of the many cDNA clones analysed (Table 1). This may be due to the addition of C to the 5′ end of the first-strand cDNA in a non-template directed manner by reverse transcriptase (CLONTECHniques 1996; P. Carninci et al. unpublished observations). Thus, due to this ‘tailing’ activity, the sequence Gm (where m = 1–3) at the 5′ end of our clones may not be a part of the original mRNA, but an artefact of cloning.

Full-length cDNA representation of CHLH

To examine the representation of full-length cDNA in the library, we also focused on a long transcript, Mg Chelatase subunit (CHL H) cDNA, whose full-length size is 4428 bp (Gibson et al. 1996). Seventy-eight per cent of clones hybridized to the 3′-end probes of CHL H cDNA also showed a positive signal to the 5′-end probes (Table 3). Hybridization analysis of another A. thaliana cDNA library (Abe et al. 1997) constructed by simply omitting the selection of full-length cDNA showed much lower correspondence of 3′- and 5′-end probes; for the CHL H probe, correspondence was only 18% (Table 3). Sequences of the 5′ end of the CHL H cDNA were found to be longer in all clones by 9 or 10 nucleotides than the cloned sequence deposited in the database. These clones probably contained the sequence from the first transcribed nucleotide (Table 1a and Table 1b and Table 1c). These results indicate that the library constructed by the biotinylated CAP trapper contained a very high proportion of A. thaliana full-length cDNA.

Table 3.  Examination of the representation of full-length CHLH cNDA in the cDNA libraries
 Number of positive clones hybridized to the 3′ end probe of CHLH cDNANumber of positive clones hybridized to the 5′ end probe of CHLH cDNAPercentage of full-length cDNA (%)
Library constructed by the1038078
biotinylated CAP trapper
Library constructed by simply92416818
omitting the selection of full-
length cDNA

Characterization of the cDNAs in the library

Ninety-six cDNA groups were isolated from 111 randomly sequenced clones (Table 2a and Table 2b and Table 2c and Table 2d). No sequences identical to those of the 32 cDNA groups of 35 selected cDNA clones were found in the database. This indicates that the cDNA groups are derived from novel genes (Table 2a and Table 2b and Table 2c and Table 2d). There are two possible reasons for discovery of many novel cDNAs in our cDNA library. One reason might be due to stressed plant materials for the preparation of the cDNA library. Another reason might be that partial sequences of cDNAs previously available in the EST libraries did not contain 5′ sequences of full-length cDNA clones. Based on our results, we are planning to prepare full-length cDNA libraries from various organs of unstressed or stressed A. thaliana plants to find novel cDNAs with full-length.

Based on a search for homology in the GenBank database using the BLAST program, seven cDNAs (FL-32, FL-33, FL-49, FL-51, FL-55, FL-76 and FL-96) were found to have sequence similarity to genes for a cysteine tRNA synthetase (accession number M59381;Hou et al. 1991), a pyruvate kinase (accession number X53688), Hordeum vulgare ABA-induced protein HVA22 (accession number L19119;Shen et al. 1993), Brassica napus myrosinase-associated protein (accession number U39289;Taipalensuu et al. 1996), a protein kinase C inhibitor (accession number Z29643;Simpson et al. 1994), an ATP sulfurylase (APS2) (accession number U59737;Murillo & Leustek 1995), and A. thaliana transcriptional activator CBF1 (accession number U77378;Stockinger et al. 1997).

Only 65% of the cDNA groups have sequence identity with the ESTs found in the database (Table 2a and Table 2b and Table 2c and Table 2d), even though about 36 000 entries of ESTs from A. thaliana have been registered in the GenBank database as of October 1997 (Cooke et al. 1996;Höfte et al. 1993;Newman et al. 1994).

Among the cDNA clones isolated in this study were some drought-stress inducible genes, such as cor6.6 (FL-4; accession number X55053;Gilmour et al. 1992) and ERD15 (FL-9; accession number D30719;Kiyosue et al. 1994). This might be because the plants were stressed before the plant samples were frozen.

Conclusions and perspectives

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

Many EST projects for A. thaliana are currently under way (Cooke et al. 1996;Höfte et al. 1993;Newman et al. 1994), and approximately 36 000 entries of ESTs from A. thaliana have been registered in the GenBank database as of October 1997. However, because of the partial sequence data, there is only limited information available on the derived amino acid sequences. In this paper, we report high-content full-length cDNA libraries from A. thaliana plants based on the use of thermoactivated reverse transcriptase and chemical introduction of a biotin group into the diol residue of the CAP structure of eukaryotic mRNA, followed by RNase I treatment to select full-length cDNA. These cDNAs will be useful materials for biochemical and molecular analyses of functions of their encoding proteins in vitro and in vivo using transgenic plants because these cDNAs were synthesized without involving PCR amplification, which causes misincorporation. The precise gene structure can be accurately described only by sequence analysis of full-length cDNA. By identifying and defining the structure of the A. thaliana genes, this information will also be applicable to other plants. Therefore, aliquots of this cDNA library will be distributed to academic researchers through the Arabidopsis Biological Resource Center at The Ohio State University.

Among the cDNA clones isolated were nine cDNAs for ribulose 1,5-biphosphate carboxylase small subunit (rbcS) (FL-20, FL-21, FL-22, FL-23, FL-24, FL-25, FL-26, FL-27 and FL-28) and six cDNAs for chlorophyll a/b binding protein (cab) (FL-6, FL-7, FL-39, FL-40, FL-41 and FL-42). Therefore, to enhance the frequency of cDNAs expressed at low levels, we are planning to isolate RNA from all plant tissues and plants subjected to various stress conditions, mix them equally, and use them as starting materials for construction of cDNA libraries.

Expression profiles are powerful tools for understanding gene expression in diverse tissues and conditions (Okubo et al. 1992). Full-length cDNAs will also be useful materials for expression profiles because the cDNA population in each full-length cDNA library should faithfully represent the mRNA population in the materials used for construction of that library. We are also planning to construct some full-length cDNA libraries from diverse plant tissues and plants subjected to various stress conditions and to compare the expression profiles in these libraries.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant materials

Arabidopsis thaliana plants (ecotype Columbia) were grown in soil for 8 weeks under continuous light at 22°C. Leaves and stems of the plants were frozen in liquid nitrogen and stored at – 80°C until RNA preparation.

RNA preparation

Total RNA was prepared from 10 g of frozen plant tissues by modification of the method of Chirgwin et al. (1979). The tissues were ground with a mortar and pestle in the presence of liquid nitrogen. The powder was then mixed with 5 volumes of solution D (4 m guanidium thiocyanate, 25 mm sodium citrate (pH 7.0), 0.5% sodium N-lauroylsarcosine, 0.1 m 2-mercaptoethanol). The cellular debris was pelleted out (14 000 g for 10 min at 4°C). About 2.4 ml of the supernatant was layered on top of 1.1 ml of 5.7 m CsCl cushion solution (5.7 m CsCl, 0.1 m EDTA) to create a step gradient and centrifuged for 2 h in a TLA-100.3 rotor (Beckman, CA, USA) at 240 000 g and20°C. The RNA pellet was dissolved in 10 mm Tris–HCl (pH 7.5), 5 mm EDTA (pH 8.0). The supernatant was extracted with successive, equal volumes of phenol-chloroform and chloroform. The upper phase was collected and mixed with 1/3 volume of 8 m LiCl. The RNA was precipitated at 4°C overnight and centrifuged at 14 000 g for 30 min. The pellet was washed with 70% ethanol and dissolved in DEPC-treated water. Poly(A)+ RNA was isolated by using Oligo(dT)-Latex (OligotexTM-dT30-Super; Roche, Tokyo, Japan) as described by the manufacturer.

Construction of cDNA library

A full-length cDNA library was constructed essentially as reported previously (Carninci et al. 1996, 1997) by biotinylated CAP trapper using trehalose-thermoactivated reverse transcriptase (Carninci et al. 1998). Figure 1 shows the flowchart protocol. The following sections describe the details of the method.

image

Figure 1. Overall strategy for full-length cDNA library construction by the modified biotinylated CAP trapper.

(a) A diol group is present on each of the CAP structure and the 3′ end of the mRNA.

(b) Biotinylation reaction of diol groups (shown only for the CAP structure).

(c) Flowchart of the construction of a cDNA library by improved biotinylated CAP trapper.

Download figure to PowerPoint

First-strand cDNA preparation

Nine micrograms of mRNA was denatured at 65°C for 10 min in a final volume of 52.4 μl, together with 8.4 μg of the first-strand XhoI primer-adaptor 5′ (GA)10ACTAAGTCTCGAG (T)16MN (where N is any nucleotide and M is G, A, or C), and 22.4 μl of 80% glycerol. Subsequently, a mixture of 36.3 μl of 5 × first-strand buffer (SuperscriptTMII Reverse Transcriptase, GIBCO BRL, MD, USA), 18.2 μl of 0.1 m DTT, 11.8 μl of dNTP mixture (dATP, dTTP, and dGTP at a final concentration of 0.6 mm each and 5-methyl-dCTP at a final concentration of 0.3 mm), 4.5 μl of 2.5 μg μl–1 BSA, 59.2 μl of saturated trehalose (Sigma Chemical Co., MO, USA), 1.8 μl of RNase inhibitor (WAKO, Osaka, Japan), 1 μl of [α-32P]dGTP (3000 Ci mmol–1, 10 mCi ml–1; Amersham, Buckinghamshire, UK), and 20 μl of 200 units μl–1 Superscript™II reverse transcriptase (GIBCO BRL, MD, USA) was added to the reaction sample to carry out the reverse transcription reaction. The primer was annealed on a thermal cycler: samples were preheated at 45°C in an MJ Research thermal cycler and then mixed. The annealing profile included an initial negative ramp of 10°C per min until 35°C was reached. After further incubation at 35°C for 5 min, the sample was incubated at 45°C for 5 min and the temperature was raised with a ramp of 1°C 5 sec–1 until 55°C was reached, and then raised with a ramp of 0.5°C min–1 until 60°C was reached. The reaction mixture was then incubated at 55°C for 2 min and subsequently at 60°C for 2 min, and this cycle was then repeated 10 times.

Biotinylation of diol groups of RNA

The biotinylation of RNA diol groups (CAP and 3′ termini of RNA) is a two-step reaction that involves diol group oxidation followed by the coupling of the oxidized RNA termini with the biotin hydrazide long arm, also called biocytin hydrazide (Vector Laboratories, CA, USA). The biotinylation reaction was performed after the first-strand cDNA synthesis in this study. To the sample, 4 μl of 0.5 m EDTA, 2 μl of 10% SDS, and 2 μl of 10 mg ml–1 proteinase K were added, and the mixture was incubated at 45°C for 15 min. The sample was extracted with phenol-chloroform and chloroform, followed by ethanol precipitation. The pellet was washed with 80% ethanol and resuspended in 47 μl of RNase-free water. The sample was oxidized in 50 μl of 66 mm sodium acetate buffer (pH 4.5) containing 5 mm NaIO4 (ICN Biomedicals, CA, USA) as the oxidizing agent. The oxidation was carried out on ice in the dark for 45 min. The oxidized mRNA was subsequently precipitated by the addition of 0.5 μl of 10% SDS, 11 μl of 5 m NaCl, and 61 μl of isopropanol. After a 30 min incubation at 4°C in the dark, the oxidized mRNA/cDNA hybrid was precipitated by centrifugation for 15 min at 4°C. The pellet was washed with 80% ethanol and then resuspended in 50 μl of RNase-free water. Next, 5 μl of 1 m sodium acetate (pH 6.1), 5 μl of 10% SDS, and 150 μl of 10 mm biotin hydrazide long arm (freshly dissolved in water) were added to the sample. The oxidized diol group on the RNA was then biotinylated by overnight incubation at room temperature (22–26°C) in the dark. Finally, nucleic acids were precipitated again by the addition of 5 μl of 5 m NaCl, 75 μl of 1 m sodium acetate (pH 6.1) and 2.5 volumes of ethanol. After a 30 min incubation at – 80°C, the biotinylated sample was precipitated by 20 min centrifugation at 4°C. The pellet was washed twice with 80% ethanol and finally dissolved in 70 μl of RNase-free water.

RNase protection of full-length cDNA

RNase digestion of the first-strand cDNA reaction was performed with RNase I (RNase ONE, Promega, WI, USA). Twenty microliters of 10 units μl–1 RNase I and 10 μl of RNase I buffer were added to the sample of the first-strand reaction, and the sample was incubated at 37°C for 15 min, followed by the addition of 2.5 μl of 40 μg μl–1 DNA-free tRNA (Sigma Chemical Co., MO, USA).

Blocking of magnetic beads and capturing of full-length cDNA

The first-strand full-length cDNA/mRNA hybrid was captured on magnetic porous glass particles coated with streptavidin (CPG, NJ, USA). Before binding of the nucleic acids, 500 μl of beads (1% suspension; 1 mg of beads can bind 800 pmol of a biotinylated 25-mer oligonucleotide) was blocked by adding 2.5 μl of 40 μg μl–1 DNA-free tRNA and incubating on ice for 30 min with occasional gentle vortexing. Just before nucleic acid capture, the beads were separated using a magnetic stand, and the supernatant was removed by pipetting. All subsequent capture, washing and release procedures were performed with the help of a magnetic stand. After the blocking step, the beads were washed three times with 500 μl of 2 m NaCl and 50 mm EDTA (pH 8.0) and finally resuspended in 400 μl of 2 m NaCl and 50 mm EDTA (pH 8.0). Finally, cDNA (approximately 0.5 μg) was captured at room temperature for 30 min with continuous gentle mixing in the presence of 100 μg of tRNA as a carrier to prevent bead sedimentation. After removal of unbound cDNA, the beads were washed twice with 2 m NaCl and 50 mm EDTA, followed by 1 washing with 0.4% SDS and 50 μg ml–1 yeast tRNA; 1 washing with 10 mm Tris–HCl (pH 7.5), 0.2 mm EDTA, 10 mm NaCl, 20% glycerol, one washing with nuclease-free water containing 50 μg ml–1 yeast tRNA; and, finally, 1 washing with 1 X RNase H buffer (20 mm Tris–HCl (pH 7.5), 10 mm MgCl2, 20 mm KCl, 0.1 mm EDTA, 0.1 mm (DTT). To release full-length cDNA from the beads, the sample was incubated with three units of RNase H in 100 μl of RNase H buffer at 37°C for 30 min with continuous mixing, followed by the addition of 0.1% SDS and 10 mm EDTA and additional incubation at 65°C for 10 min. To remove the cDNA fraction which was not removed from the beads because of incomplete RNase H treatment, additional alkaline hydrolysis was performed in the presence of 100 μl of Tris-formate buffer (pH 9.0) (obtained by combining 100 mm Tris base with 16.6 mm formic acid, 0.016 mm EDTA, and 0.1% SDS, all at their final concentrations) at 65°C for 10 min. The cycle of alkaline elution was repeated four times. The alkaline-treated fraction was removed and processed with the RNase H-released fraction. One microgram of glycogen was added to the single-stranded cDNA sample. The cDNA was ethanol-precipitated under standard conditions (Sambrook et al. 1989), dissolved in 50 μl of water, and subjected to G100 Sephadex column chromatography. The radioactive fractions were collected in siliconized Eppendorf tubes, 1 μg of glycogen was added, and the cDNA was ethanol-precipitated and dissolved in 31 μl of water.

Oligo(dG) tailing of single-stranded cDNA

Five microliters of terminal deoxynucleotidyl transferase buffer (× 10, TOYOBO, Tokyo, Japan), 5 μl of 1 mm dGTP, 5 μl of 10 mm CoCl2, and 4 μl of 8 units μl–1 terminal deoxynucleotidyl transferase (Takara Shuzo Co., Kyoto, Japan) were added to the cDNA sample and incubated at 37°C for 30 min. Finally, 1 μl of 0.5 m EDTA, 1 μl of 10% SDS, and 1 μl of 10 μg μl–1 proteinase K were added, and the sample was incubated at 45°C for 15 min. The sample was then extracted with phenol-chloroform and chloroform and then precipitated with ethanol.

Second-strand cDNA synthesis

The oligo(dG)-tailed first-strand cDNA was incubated in a final volume of 60 μl containing the following reagents: 3 μl of second-strand low buffer (200 mm Tris–HCl (pH 8.75), 100 mm KCl, 100 mm (NH4)2SO4, 20 mm MgSO4, 1% Triton X-100, 1 mg ml–1 (BSA), 3 μl of second-strand high buffer (200 mm Tris–HCl (pH 9.2), 600 mm KCl, 20 mm MgCl2), 600 ng of second-strand SacI primer-adaptor (sequence: 5′-(GA)9GAGCTCACTAGTC11), 0.25 mm dNTPs, 0.5 mmβ-NADH, 15 units of ExTaq DNA polymerase (Takara Shuzo Co., Kyoto, Japan), 150 units of Ampligase, a thermostable DNA ligase (Epicentre Technologies, WI, USA), and three units of Hybridase, a thermostable RNase H (Epicentre Technologies, WI, USA). The reaction was carried out in an MJ Research thermal cycler. The incubation cycling of the second-strand synthesis included an initial incubation at 55°C for 5 min. Enzymes were added when samples reached 55°C for a ‘hot start’. Subsequently, the primer was allowed to anneal with a negative ramp of 0.3°C min–1 from 55° to 35°C, and then the reaction mixture was incubated at 35°C for 15 min and subsequently at 72°C for 15 min. The annealing-extension cycle was then repeated three times. Finally, 1 μl of 0.5 m EDTA, 1 μl of 10% SDS, and 1 μl of 10 μg μl–1 proteinase K were added, and the sample was incubated at 45°C for 15 min. The sample was then extracted with phenol-chloroform and precipitated with ethanol.

Restriction digestion and cloning

The cDNA was next restricted with SstI and XhoI under standard conditions. At the end of the reaction we added 1 μl of 0.5 m EDTA, 1 μl of 10% SDS, and 1 μl of 10 μg μl–1 proteinase K to the sample and allowed it to incubate at 45°C for 15 min. The cDNA was then extracted with phenol/chloroform and chloroform followed by passage through a SizeSep™ 400 Spun Column (Pharmacia Biotech, Tokyo, Japan). The cDNA was collected and then ethanol-precipitated. Next, 180 ng of cDNA was ligated overnight at 12°C to Lambda Zap II vector, digested with SstI and XhoI using 200 units of T4 DNA ligase (New England Biolabs, MA, USA) in a volume of 5 μl. The ligated cDNA was packaged using MaxPlax packaging extract (Epicentre Technologies, WI, USA) (Gunther et al. 1993).

DNA extraction and sequencing

Plasmid DNA was extracted with a Kurabo DNA extraction instrument NA 100, treated with RNase A, and then purified by precipitation with polyethylene glycol. DNA sequences were determined using the dye terminator cycle sequencing method with a DNA sequencer (model 373 A; Applied Biosystems, CA, USA). DNA clones were subjected to single-pass sequencing from the 5′ ends of the cDNA. The primer was 5′-CAGGAAACAGCTATGAC. The sequences were analyzed with GENETYX software (Software Development, Tokyo, Japan) on a Macintosh computer, and sequence homologies were examined with the GenBank/EMBL database using the BLAST program.

Measuring the cDNA size

Phage clones picked randomly from the library were added to 0.5 ml of SM buffer (100 mm NaCl, 8 mm MgSO4, 50 mm Tris–HCl (pH 7.5), 0.01% gelatin) supplemented with 10 μl of chloroform and subjected to PCR amplification. The primers were 5′-GTTTTCCCAGTCACGAC and 5′-CAGGAAACAGCTATGAC. PCR was carried out in a 100 μl reaction mixture containing 1 ×Ex Taq buffer (Takara Shuzo Co., Kyoto, Japan), 0.2 mm each of dNTPs, 100 pmol of each primer, 5 μl of template, and 2.5 units of Takara Ex Taq polymerase (Takara Shuzo Co., Kyoto, Japan). The mixture was subjected to 94°C for 30 sec, to 35 cycles of 94°C for 60 sec, 55°C for 150 sec, and 72°C for 300 sec, and then to 72°C for 600 sec. The amplified cDNA inserts were electrophoresed and the size of the cDNA was measured.

Plaque hybridization

Plaque hybridization was performed using established techniques (Sambrook et al. 1989). Fragments from the 5′ end (from nucleotide 110 to nucleotide 620) and the 3′ end (from nucleotide 3722 to nucleotide 4300) of A. thaliana Mg chelatase (CHL H) cDNA (Gibson et al. 1996) were prepared by PCR amplification and used as DNA probes. The DNA fragments were labelled by random priming using the BcaBEST DNA labelling kit (Takara Shuzo Co., Kyoto, Japan) according to the manufacturer’s protocols. Filters from the library containing approximately 250 000 phage plaques were hybridized with the probes from the 3′ end of the CHL H cDNA. The DNA probes were then stripped by boiling in a 0.5% SDS solution, and the filters were rehybridized with the probes from the 5′ end of the CHL H cDNA.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work was supported in part by a grant for Genome Research from RIKEN, the Program for Promotion of Basic Research Activities for Innovative Biosciences, the Special Coordination Fund of the Science and Technology Agency, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan to K.S. It was also supported in part by Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology Corporation, Special Coordination Funds and a Research Grant for the Genome Exploration Research Project from the Science and Technology Agency of the Japanese Government, and a Grant-in-Aid for Scientific Research on Priority Areas and Human Genome Program from the Ministry of Education and Culture, Japan, to Y.H. M.S. was supported by a fellowship from the Science and Technology Agency of Japan. We thank Mr Takahashi, Mr Ogawa, Ms Kanahara, and Ms Furukawa for their skilled technical assistance.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results and discussion
  5. Conclusions and perspectives
  6. Experimental procedures
  7. Acknowledgements
  8. References
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