Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana

GENE A N I N T E R N A T I O N A L JOURNAL O N GENES AND OENOME5

ELSEVIER

Gene174(1996)217-220

Cloning and characterization of the gene encoding IMP dehydrogenase from Arabidopsis thaliana F r a n k R. C o l l a r t a, J e r z y O s i p i u k a, J o n a t h a n

T r e n t ~, G a r y J. O l s e n 1 b, E l i e z e r H u b e r m a n

~'*

a Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, IL 60439, USA b Department of Microbiology, University of Illinois, Champaign-Urbana, Champaign-Urbana, IL 61801, USA Received 31 October 1995; revised 4 December 1995; accepted 5 January 1996

Abstract

We have cloned and characterized the gene encoding inosine monophosphate dehydrogenase (IMPDH) from Arabidopsis thaliana (At). The transcription unit of the At gene spans approximately 1900 bp and specifies a protein of 503 amino acids with a calculated relative molecular mass (Mr) of 54 190. The gene is comprised of a minimum of four introns and five exons with all donor and acceptor splice sequences conforming to previously proposed consensus sequences. The deduced IMPDH amino-acid sequence from At shows a remarkable similarity to other eukaryotic IMPDH sequences, with a 48% identity to human Type II enzyme. Allowing for conservative substitutions, the enzyme is 69% similar to human Type II IMPDH. The putative active-site sequence of At IMPDH conforms to the IMP dehydrogenase/guanosine monophosphate reductase motif and contains an essential active-site cysteine residue. Keywords: Gene isolation; DNA sequence; Protein homology; Plant

I. Introduction

Inosine m o n o p h o s p h a t e dehydrogenase ( I M P D H ; EC 1.1.1.205) catalyzes the rate-limiting step in the de novo biosynthesis of guanine nucleotides (Weber, 1983) and has an essential role in providing necessary precursors for D N A and RNA biosynthesis and in signal transduction pathways that mediate cell differentiation ( H u b e r m a n et al., 1995) and transformation (Jenkins et al., 1993). This essential nature of this enzyme is reflected in the diverse application of I M P D H inhibitors in the areas of cancer chemotherapy (Tricot et al., 1990), immunosuppression (Dayton et al., 1994), arthritis (Goldblum, 1993), and parasitic diseases (Hupe et al., 1986).

i Tel. + 1 708 2440616. * Corresponding author. Tel. + 1 708 2523819; Fax + 1 708 2523387; e-mail: [email protected] Abbreviations: aa, amino acid(s); At, Arabidopsis thaliana; bp, base pair(s); cDNA, DNA complementary to RNA; Ec, Escherichia coli; IMPDH, inosine monophosphate dehydrogenase; IMPDH, gene encoding IMPDH; kb, kilobase(s) or 1000 bp; Mr, relative molecular mass; nt, nucleotide(s); ORF, open reading frame; PCR, polymerase chain reaction.

Genomic and c D N A sequences specific for I M P D H have been cloned from eukaryotes ( H u b e r m a n et al., 1995) and bacteria (Tiedeman and Smith, 1985). The bacterial and eukaryotic forms of I M P D H are similar in size and show a high degree of amino-acid sequence conservation but differ with respect to kinetic constants and sensitivity to various inhibitors (Hupe et al., 1986). These observations suggest that, although there has been a structural and functional selection for conservation of amino acid (aa) sequence, some fundamental differences remain between the different forms of I M P D H . We have cloned and characterized the I M P D H gene from Arabidopsis thaliana, which is widely used as a model organism for plant molecular genetics. Characterization of At I M P D H will be useful in understanding the role of this enzyme in plant biology. In many tropical legumes, fixed nitrogen is exported from the nodules primarily as the ureides, allantoin and allantoic acid, which are formed by a pathway involving de novo purine synthesis. The suggestion has been made that I M P oxidation via I M P D H might be the predominant pathway leading to xanthine and the formation of ureides in vivo (Atkins et al., 1985). In view of the relative abundance of this enzyme (approximately 0.7% in cowpea nodules; Atkins et al., 1985), it is likely that this

0378-1119/96/$15.00 Copyright © 1996 Published by Elsevier Science B.V. All rights reserved PH S 0 3 7 8 - 1 1 1 9 ( 9 6 ) 0 0 0 4 5 - 5

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FR. Collart et al./Gene 174 (1996) 217-220

enzyme has an essential role in nitrogen fixation in some plants.

2. Results and discussion

2.1. Cloning and sequencing of the 1 M P D H gene from At Primers homologous to conserved regions of known I M P D H coding sequences were used to obtain a 1.4-kb amplified fragment from genomic DNA of At. This size was as predicted on the basis of the location of the primer annealing sites and the presence of introns in this gene. Preliminary sequence analysis indicated homology with previously cloned I M P D H coding sequences. The amplified fragment was used as a probe to extract homologous clones from an At genomic DNA library. Genomic clones of 3.0 kb were obtained, consistent with the results observed by analysis of Southern blots with the amplified probe. Sequence analysis of the genomic clones showed homology with known I M P D H coding regions. On the basis of this homology and established conventions for intron donor and acceptor splice junction sequences (Luehrsen et al., 1994), an aa sequence for I M P D H was deduced from the genomic clones (Fig. 1). The coding region spans approximately 1900 bp and specifies a protein of 503 aa with a calculated Mr of 54 190. The observation of in-frame and out-of-frame stop codons, coupled with the lack of acceptable splice sites, supports the selection of the putative A U G start codon. In addition, another A U G nt sequence is not present in the 160 nt preceding the designated start codon. Further evidence in support of the predicted initiation codon is the observation that the first 30 aa residues of the At I M P D H sequence are 60% similar to the human Type II I M P D H and that another inframe Met codon is not observed until codon 60. A search of the Eukaryotic Promoter Database did not identify any plant-specific promoter elements; however, a TATA box is located 134 nt upstream of the putative A U G start codon. The I M P D H gene from At is comprised of a minimum of four introns and five exons (Table 1). The location of the splice junction sites was confirmed by partial sequencing of an RT-PCR product obtained from At total RNA. The four introns in the coding region of the gene ranged in size from 89 to 139 bp, which is typical for plants and greater than the ~ 6 4 nt minimum length required for efficient splicing of plant and animal introns (Luehrsen et al., 1994). Consistent with previously characterized plant introns (Luehrsen et al., 1994), these introns are (A+T)-rich, with a range of 67 74% A + T in the genomic DNA sequence. The intron donor and acceptor splice sequences (Table 1) conform to previously proposed consensus sequences (Mount, 1982). The deduced At protein sequence is similar to that reported for other eukaryotic forms of I M P D H with

respect to size and aa composition. The putative termination codon is located proximal to the HindIII site, precluding analysis of the 3' noncoding regions in the clones isolated in this study. The assignment of the stop codon was based on the sequence information obtained from the RT-PCR product and homology with known I M P D H proteins, Characterization of At I M P D H will be useful in understanding the role of this enzyme in nitrogen fixation and de novo purine biosynthesis in plants. This organism represents a deep branch in the evolutionary tree and, as such, will be useful for future studies to determine kinetic constants and sensitivity to various inhibitors. 2.2. Conservation of amino-acid sequence The deduced I M P D H aa sequence from At shows a remarkable similarity to other eukaryotic I M P D H sequences. Using the BESTFIT analysis, the protein sequence shows a 48% identity to human Type II I M P D H (Fig. 2). Allowing for conservative substitutions, 1 M P D H from At is 69% similar to human Type II 1MPDH. A reduced, but still significant, homology is observed with prokaryotic I M P D H sequences, where the At peptide shows a 34% identity (57% similarity) with the I M P D H peptides from Escherichia coli (Ec) and Bacillus subtilis. The At I M P D H enzyme is also one of the smallest of the eukaryotic enzymes; in comparison with the human sequence (Fig. 2), a reduced number of residues are observed at the N-terminus. A cysteine residue at codon 327 in human Type II I M P D H (codon 328 in Ec) has been proposed as essential for the catalytic activity of human (Antonino et al., 1994) and Ec (Andrews and Guest, 1988) I M P D H . A consensus sequence that includes this active-site cysteine has been proposed as a signature motif for I M P D H and guanosine monophosphate reductase enzymes (Bairoch, 1995). All I M P D H sequences thus far characterized conform to the consensus sequence. The deduced aa sequence from At conforms to the proposed I M P D H signature motif and contains a putative active-site cysteine at codon 322. Furthermore, in the active-site region, this sequence displays a remarkable degree of aa conservation, with 18 of the 21 residues identical in the plant and human Type II sequence.

3. Conclusions

We have cloned and characterized the IMPDH gene from At. The At IMPDH gene is comprised of a minimum of four introns and five exons with all splice sequences conforming to previously proposed consensus sequences. The putative active-site of the deduced sequence con-

F.R. Collart et al./Gene 174 (1996) 217-220

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Fig. 1. Molecular organization and sequence of the IMPDH gene from At (Genbank accession No. L34684). All noncoding sequences are represented in lowercase and plain letters; the putative TATA box is boldface and underlined. All transcribed sequences are in capital and bold letters, with the deduced protein sequence represented in single letter code underneath the corresponding nt sequence. The putative translation stop codon (UAA) is indicated by an asterisk. Methods: Polymerase chain reaction (PCR) primers homologous to I M P D H were constructed on the basis of the alignment of conserved segments of I M P D H coding regions from various organisms. At degenerate nt positions, inosine was selected on the basis of its ability to base pair with multiple deoxynucleotides (Ohtsuka et al., 1985). Primer ARC5A, 5'-GAGICIIGIATGGIATIGCAATGGC-3', is homologous to the N-terminal coding region and corresponds to nt 270-295 of the human Type II coding sequence (GenBank accession No. J04208). Primer ARC5B was a truncated version (nt 1-19) of primer ARC5A. Primer ARC3A, 5'-CATIGCATCIAIIGAICCCATICCICGGTA-3', is homologous to the complement of the C-terminal coding region and corresponds to nt 1278-1307 of the human Type II coding sequence. Primer ARC3B was a truncated version (nt 1-19) of primer ARC3A. Amplification reactions (50/A) contained 100 ng of genomic DNA, 200 ng of each primer, 0.5 m M deoxynucleotides, 2 m M MgC12, and 1.5 units of Taq polymerase (Life Technologies Inc.). Reaction buffer was added as recommended by the vendor, and the solutions were subjected to 30 cycles of PCR (denature, 60 s at 94°C; anneal, 90 s at 37°C; and extend, 80 s at 70°C). The amplification product was used to screen an At genomic library (DNA kindly provided by Dr. Brian Keith, University of Chicago, Chicago, IL, USA). Putative I M P D H clones were identified and characterized according to the procedures described by Collart and Huberman (1988). Initial alignment and characterization of sequences used programs of the G C G (Genetics Computer Group, Madison, WI, USA) Sequence Analysis package (Devereux et al., 1984) for VAX/VMS computers. Analysis also used the electronic-mail-based and Internet BLAST (Altschul et al., 1990) servers at the National Center for Biotechnology Information.

220

FR. Collart et al./Gene 174 (1996) 217 220

Table 1 Summary of splice junction sequences in the IMPDH gene from Arabidopsis Exon No.

Size (bp)

5'-Splice a donor

3'-Splice acceptor

Codon(s)

I II III IV

97 109 79 89

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tattggtttaaatgcagGAA tgatttggacctgttagGCT ttttgtggtaataacagAAC tttggtttttgtgacagGCT

T 135 Q335/A336 T 394/N395 E4S]/A49z

aSplice sequences were confirmed by sequence analysis of an amplification product obtained by reverse transcription of At total RNA. The amplification product spanned segments from nt 115 to 2056 in the genomic sequence (Fig. 1), implying a minimum mRNA length of 1565 nt. Conditions for PCR amplification and reverse transcription were as described by Jiang et al. (1994) using total cellular RNA from cultured At cells. The RNA was kindly provided by Dr. Janet Braam (Rice University, Houston, TX, USA).

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Fig. 2. Alignment of IMPDH sequences from At (plant) and human (Type II sequence, GenBank accession No. J04208). Sequences were aligned by using the BESTFIT program of the GCG Sequence Analysis package. The active-site region is boxed, with the putative active-site cysteine indicated in boldface.

forms to the IMPDH signature motif and contains a putative active site cysteine.

Acknowledgement This work was supported by the U.S. Department of Energy, Office of Health and Environmental Research, under Contract No. W-31-109-Eng-38.

References Altschul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J.J. (1990) Basic local alignment search tool. Mol. Biol. 215, 403 410. Andrews, S.C. and Guest, J.R. (1988) Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12. Biochem. J. 255, 35 43. Antonino, L.C., Straub, K. and Wu, J.C. (1994) Probing the active site of human IMP dehydrogenase using halogenated purine riboside 5'-monophosphates and covalent modification reagents. Biochemistry 33 (1994) 1760-1765. Atkins, C.A., Shelp, B.J. and Storer, P.J. (1985) Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). Arch. Biochem. Biophys. 236, 807 814. Bairoch, A. (1995) Prosite Dictionary: Release 12.2, University of Geneva, Geneva, Switzerland. Collart, F.R. and Huberman, E. (1988) Cloning and sequence analysis of the human and Chinese hamster inosine-5'-monophosphatedehydrogenase cDNAs. J. Biol. Chem. 263, 15769 15772. Dayton, J.S., Lindsten, T., Thompson, C.B. and Mitchell, B.S. (1994) Effects of human T lymphocyte activation on inosine monophosphate dehydrogenase expression. J. Immunol. 152, 984 991. Devereux, J., Haeberli, P. and Smithies, O. (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387 395. Goldblum, R. (1993) Therapy of rheumatoid arthritis with mycophenolate mofetih Clin. Exp. Rheumatol. 11, S117 Sl19. Huberman, E., Glesne, D. and Collart F.R. (1995) Regulation and role of inosine-5'-monophosphate dehydrogenase in cell replication, malignant transformation, and differentiation. In: Sahota, A. and Taylor, M. (Eds.), Purine and Pyrimidine Metabolism in Man, VIII. Plenum Press, New York, pp. 741 746. Hupe, D.J., Azzolina, B.A. and Behrens, N.D. (1986) IMP dehydrogenase from the intracellular parasitic protozoan Eimeria tenella and its inhibition by mycophenolic acid. J. Biol. Chem. 261, 8363 8369. Jenkins, D.C., Stables, J.N., Wilkinson, J., Topley, P., Holmes, L.S., Linstead, D.J. and Rapson, E.B. (1993) A novel cell-based assay for the evaluation of anti-ras compounds. Br. J. Cancer 68, 856 861. Jiang, H., Lin, J., Su, Z., Collart, F.R., Huberman, E. and Fisher, P.B. (1994) Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAFI/CIP1 expression in the absence of p53. Oncogene 9, 3397 3406. Luehrsen, K.R., Taha, S. and Walbot, V. (1994) Nuclear pre-mRNA processing in higher plants. Prog. Nucleic Acid Res. Mol. Biol. 47, 149 193. Mount, S.M. (1982) A catalogue of splice junction sequences. Nucleic Acids Res. 10, 459 472. Ohtsuka, E., Matsuki, S., Ikehara, M., Takahashi, Y. and Matsubara, K. (1985) An alternative approach to deoxynucleotides as hybridization probes by insertion of deoxyinosine at ambiguous codon positions. J. Biol. Chem. 260 (1985) 2605-2608. Tiedeman, A.A. and Smith, J.M. (1985) Nucleotide sequence of the guaB locus encoding IMP dehydrogenase of Escherichia coli K12. Nucleic Acids Res. 13 (1985) 1303-1316. Tricot, G., Jayaram, H.N., Weber, G. and Hoffman, R. (1990) Tiazofurin: biological effects and clinical uses. Int. J. Cell Cloning 8, 161 170. Weber, G. (1983) Biochemical strategy of cancer cells and design of chemotherapy. Cancer Res. 43 (1983) 3466-3492.