Molecular cloning and DNA sequence of the Arabidopsis thaliana alcohol dehydrogenase gene ( gene structure / enzyme

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Author	Chang, Caren Meyerowitz, Elliot M.
Abstract	Arabidopsis thaliana provides an excellent experimental plant system for molecular genetics because of its remarkably small genome size, near absence of dispersed middle repetitive DNA, and short life cycle. We have cloned and determined the nucleotide sequence of a single-copy gene from A. thaliana likely to be the gene encoding alcohol dehydrogenase (ADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1). The gene was isolated from a random recombinant library by cross-hybridization with a maize AdhM gene probe. The DNA sequence contains an open reading frame capable of encoding a polypeptide the same length as maize ADH1 and ADH2 (379 amino acids) and having '80% homology with both maize enzymes. This open reading frame is interrupted by six introns whose positions are conserved with six of the nine intron positions present in both maize genes. The 5' and 3' untranslated regions are, respectively, 58 and 204 base pairs long. Sequences important for eukaryotic gene expression such as the TATA box, polyadenylylation signal, and intron splicesite sequences are found in the expected locations. The gene hybridizes to a specific anaerobically induced RNA in Arabidopsis whose appearance correlates with the anaerobic induction of Arabidopsis ADH protein. Alcohol dehydrogenase (ADH; alcohol:NAD' oxidoreductase, EC 1.1.1.1) is an easily assayed enzyme whose activity has been observed in numerous higher plants including Arabidopsis, maize, pearl millet, sunflower, wheat, and pea (1, 2). Most plants have two or three isozymes ofADH, which exist as both heteroand homodimers in various organs (1). The enzyme is presumably required by plants for NADH metabolism, via reduction of acetaldehyde to ethanol, during periods of anaerobic stress. High levels ofADH activity are found in dry seeds (3, 4) and in anaerobically treated seeds (5, 6), roots (5), and shoots (7). The most extensive study of a plant ADH system has been in maize (8) from which both Adh genes, AdhI and Adh2, have been cloned and sequenced (9-11). The coding sequences of these genes are 82% homologous, interrupted by nine identically positioned introns that differ in sequence and length. ADH1 and ADH2 belong to a small group of proteins in maize primary root that are selectively translated in response to anaerobiosis (12); the increased levels of ADH are due to induction of Adh mRNA (9-11, 13). Arabidopsis ADH is similar to the maize ADHs, although genetic experiments indicate only one Adh locus in Arabidopsis (14). Examination ofArabidopsis ADH in crude extracts has shown that the enzyme behaves as a homodimer ofMr 87,000 (14), close to the maize ADH Mr of '80,000 (15). ADH is induced anaerobically in Arabidopsis (16) as in maize. ADH is also induced in both maize root and Arabidopsis callus by the synthetic auxin 2,4-dichlorophenoxyacetic acid (16, 17), and for Arabidopsis this has been shown to be the result of de novo synthesis of a poly(A) mRNA (16). Arabidopsis ADH has potential as a biochemical marker for genetic transformation of Arabidopsis: null mutations exist, and ADH is easily induced and assayed. We present here the molecular cloning and characterization of an inducible single-copy gene from Arabidopsis likely to be the gene encoding ADH. MATERIALS AND METHODS Arabidopsis Strains. The Landsberg erecta strain of A. thaliana was obtained from F. J. Braaksma (Department of Genetics, Biology Centre, Haren, The Netherlands); the Bensheim strain was obtained from A. R. Kranz (Botanisches Institut, J. W. Goethe-Universitit, Frankfurt am Main, Federal Republic of Germany). General Nucleic Acid Methods. Arabidopsis DNA was prepared from whole plants as described in ref. 18. Library construction followed the procedure in ref. 19. The clone nomenclature system is described in ref. 20 with the following additions: f for X EMBL4 (21); b for pMT21, a 1.9-kilobasepair (kbp) pBR322 derivative (H. V. Huang, personal communication); j and k, respectively, for pSP65 and pSP64 (Promega Biotec, Madison, WI) containing the SP6 RNA polymerase promoter. Radiolabeled DNA probes were produced by nick-translation (22). Hybridizations with the maize probe were in 50% formamide/5 x SSPE (5 x SSPE is 5 mM Na2EDTA/40 mM NaOH/50 mM NaH2PO4'H20/900 mM NaCl) at 370C with washes in 0.05x SSPE at 370C. Genome blot hybridizations were in 50% formamide/5 x SSPE at 430C, with washes in lx SSPE at room temperature. All DNA manipulations were carried out as described in ref. 23. TheDNA sequence was determined by the method ofMaxam and Gilbert (24). Anaerobic Treatment. Seeds were treated for 1-2 days at 40C in Petri dishes containing distilled H20-soaked filter paper (Whatman 3). The seeds were germinated on fresh filter paper on 0.7% agar plates at 250C with constant illumination (7000 lx). After 3-5 days, seedlings were transferred into 1-2 ml of distilled H20 (60-100 seedlings per ml). Untreated seedlings were left on plates. At various times, seedlings were placed on filter paper in a Buchner funnel with suction to remove excess water. They were then used for eitherRNA or protein preparations. RNA Analysis. Seedlings (60-100) were homogenized for 2 min in a 0.5-ml microtube fitted onto a mini-BeadBeater (Biospec Products). Each tube contained 20 1ul of 50% phenol/0.2 M Tris, pH 7.7/10 mM NaCl/75 mg of 0.5-mm zirconium oxide beads (Biospec Products). After homogenization, an additional 20 1.l of buffer, 10 A.l of 10% NaDodSO4, and 2 Al of yeast tRNA (10 mg/ml) were mixed in by Abbreviations: ADH, alcohol dehydrogenase; kbp, kilobase pair(s). 1408 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 83 (1986) 1409 vortexing. The contents were extracted three times with an equal volume of phenol/chloroform/isoamyl alcohol (50:49.5:0.5; vol/vol), twice with chloroform/isoamyl alcohol (99:1; vol/vol), once with ether, and then precipitated with ethanol. For blot analysis, the RNA was glyoxylated (25), subjected to electrophoresis in agarose gels and transferred to nitrocellulose (23). Radiolabeled RNA probes were synthesized by using SP6 RNA polymerase (26). RNA induction was quantitated by liquid scintillation counting of bands excised from the blots. Protein Analysis. Seedlings (60-100 per 0.5-ml microtube) were homogenized for 2 min in the mini-BeadBeater. Each tube contained 5.0 Ald of 70 mM TrisHCl, pH 7.2/25% (vol/vol) glycerol/0.8% (vol/vol) 2-mercaptoethanol/0.25% (wt/vol) bromophenol blue/75 mg of0.5-mm zirconium oxide beads. A hole was punched in the bottom of each tube with a 26-gauge needle and the homogenates were spun into 1.5-mi microtubes. The resulting supernatants were loaded onto nondenaturing polyacrylamide gels consisting of 7.3% (wt/vol) acrylamide/0.2% (wt/vol) N,N'-methylenebisacrylamide/70 mM Tris HCl, pH 7.2/0.8% (wt/vol) ammonium persulfate/0.08% (vol/vol) N,N,N',N'-tetramethylethylenediamine. The gels were run at 10 mA at 22°C with recirculated 8.25 mM Tris/30 mM diethylbarbituric acid electrode buffer (pH 7.4). Gels were stained for ADH activity at 37°C in 0.1 M Tris*HCl, pH 7.6/1.5 mM NAD+/0.25 mM nitroblue tetrazolium/0.26 mM phenazine methosulfate/0.5% (vol/vol) ethanol. Ethanol was omitted in substrate-dependence controls. 5' and 3' RNA Mapping. The 5' end was mapped by primer extension (27) using induced RNA and a synthetic oligonucleotide primer complementary to the RNA sequence at the 3' end of the believed first exon. The 3' end was mapped by ribonuclease protection (28) using a gel-isolated in vitrosynthesized complementary strand RNA probe, -645 bases long, and having one terminus within the last intron. RESULTS Isolation of Arabidopsis DNA Clones. Four genome equivalents of an Arabidopsis DNA library (described in ref. 18) were screened for cross-hybridization with a maize Adhl gene fragment probe (kindly provided by M. Freeling). Four positive clones were detected; restriction digests revealed that all four were identical, each containing the same 4.9-kbp EcoRI restriction fragment of Arabidopsis DNA (the At3001 fragment; Fig. la) and a 6.6-kbp EcoRI stuffer fragment ofthe X vector. To obtain larger clone segments, a new library was constructed consisting of Landsberg erecta strain DNA that had been partially digested with Mbo I and ligated into BamHIdigested XEMBL4. After amplification of the library, four genome equivalents were screened with nick-translated bAt3001 (a plasmid containing the At3001 fragment). Three positive clones were detected; two contained 15.6-kbp inserts that were identically oriented and indistinguishable by restriction mapping (XfAt3lO2), and the third contained a 17.2-kbp insert in the opposite orientation (AfAt3lOl) (Fig. la). Genome blots verified that these clones represent the sequence organization present in genomic DNA. Seven different restriction digests ofArabidopsis DNA were probed with nick-translated bAt300l andjAt3Oll (a plasmid containing the 2.5-kbp Sac I/HindIII At3Oll fragment of XfAt3lO2, Fig. lb). The hybridized restriction fragments completely agreed with the map produced by the X clones, suggesting that the region of DNA spanned by the clones exists in a single copy in the Arabidopsis genome (data not shown). The maize Adhl gene fragment cross-hybridized with each of the X clones as expected; the region of hybridization is a R S -L ).L Bg Bg H Bg HB H X XbAt3001 XfAt3lOI Sc Sc S R R RJR R R S 11 X H X, H (XXBg) (XBg)
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