Skip to main content
SpringerLink
Log in

Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

We have isolated an alfalfa leaf cDNA clone that encodes aspartate aminotransferase (AAT, EC 2.6.1.1) by direct complementation of an Escherichia coli aspartate auxotroph with a plasmid cDNA library. DNA sequence analysis of the recombinant plasmid, pMU1, revealed that a 1514 by cDNA was inserted in the correct orientation and in-frame with the start of the lacZ coding sequence in the vector, pUC18. The resulting fusion protein is predicted to be 424 amino acids in length with a molecular weight of 46387 Daltons. The cDNA-encoded protein has a characteristic pyridoxal phosphate attachment site motif and has substantial amino acid sequence homology to both animal and bacterial AATs. Plasmid pMUl encodes an AAT with a Km for aspartate of 3.3 mM, a Km for 2-oxoglutarate of 0.28 mM, and a pH optimum between 8.0 and 8.5. Several lines of evidence including Western blot analysis, the isoelectric point of the encoded protein, and the effect of pH on the activity of the fusion protein, suggest that the cDNA encodes the isozyme AAT-1 rather than AAT-2. Northern blot analysis showed that the aat-1 clone hybridized to a 1.6 kb transcript present in alfalfa leaves, roots and nodules. The relative concentrations of aat-1 mRNA in these tissues were 1: 2: 5, respectively. Thus, transcription of aat-1 appears to be induced during nodule development. Southern blot analysis suggested that AAT-1 in alfalfa is encoded by either a single-copy gene or a small, multigene family.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Alfano JR, Kahn MK (1990) Isolation and characterization of three amino acid aminotransferase genes from Rhizobium meliloti. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New York, p 507

    Google Scholar 

  • Appels MA, Haaker H (1991) Glutamate oxaloacetate transaminase in pea root nodules: participation in a malate/aspartate shuttle between plant and bacteroid. Plant Physiol 95:740–747

    Google Scholar 

  • Bennett MD, Smith JB (1976) Nuclear DNA amounts in angiosperms. Phil Trans Roy Soc Lond B 274:227–274

    Google Scholar 

  • Berg CM, Wang M, Vartak NB, Liu L (1988) Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12. Gene 65:195–202

    Google Scholar 

  • Bilofsky HS, Burks C, Fickett JW, Goad WB, Lewitter FI, Rindone WP, Swindell CD, Tung CS (1986) The GenBankTM genetic sequence data bank. Nucleic Acids Res 14:1–4

    Google Scholar 

  • Bousquet-Lemercier B, Pol S, Pave-Preux M, Hanoune J, Barouki R (1990) Properties of human liver cytosolic aspartate aminotransferase mRNAs generated by alternative polyadenylation site selection. Biochemistry 29:5293–5299

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Google Scholar 

  • Cathala G, Savouret J-F, Mendez B, West BL, Karin M, Martial JA, Baxter JD (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2:239–335

    Google Scholar 

  • Delauney AJ, Verma DPS (1990) A soybean gene encoding δ-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet 221:299–305

    Google Scholar 

  • Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395

    Google Scholar 

  • Diaz F, Jouve N (1986) Structure of the isozymes of the AAT-2 and AAT-3 systems of aspartate aminotransferase in wheat, rye and triticale. Euphytica 35:129–135

    Google Scholar 

  • Farnham MW, Miller SS, Griffith SM, Vance CP (1990) Aspartate aminotransferase in alfalfa root nodules II Immunological distinction between two forms of the enzyme. Plant Physiol 93:603–610

    Google Scholar 

  • Forest JC, Wightman F (1972) Amino acid metabolism in plants III Purification and some properties of a multispecific aminotransferase isolated from bushbean seedlings (Phaseolus vulgaris L). Can J Biochem 50:813–829

    Google Scholar 

  • Fotheringham IG, Dacey SA, Taylor PP, Smith TJ, Hunter MG, Finlay ME, Primrose SB, Parker DM, Edwards RM (1986) The cloning and sequence analysis of the aspC and tyrB genes from Escherichia coli K12 — comparison of the primary structures of the aspartate aminotransferase and aromatic aminotransferase of E. coli with those of the pig aspartate aminotransferase. Biochem J 234:593–604

    Google Scholar 

  • Givan CV (1980) Aminotransferases in higher plants. In: Stumpf PK, Conn EE (eds) The biochemistry of plants, volume 5. Academic Press, New York, pp 329–357

    Google Scholar 

  • Griffith SM, Vance CP (1989) Aspartate aminotransferase in alfalfa root nodules I Purification and partial characterization. Plant Physiol 90:1622–1629

    Google Scholar 

  • Gubler V, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269

    Google Scholar 

  • Hatch MD, Mau SL (1973) Activity, location and role of aspartate aminotransferase and alanine aminotransferase isoenzymes in leaves with C4 photosynthesis. Arch Biochem Biophys 156:195–206

    Google Scholar 

  • Hamm GH, Cameron GN (1986) The EMBL data library. Nucleic Acids Res 14(1):5–10

    Google Scholar 

  • Jaussi R, Cotton B, Juretic N, Christen P, Schuemperli D (1985) The primary structure of the precursor of chicken mitochondrial aspartate aminotransferase: cloning and sequence analysis of cDNA. J Biol Chem 260:16060–16063

    Google Scholar 

  • Kahn ML, Kraus J, Somerville JE (1985) A model of nutrient exchange in the Rhizobium-legume symbiosis. In: Evans HJ, Bottomley PJ, Newton WE (eds) Nitrogen fixation research progress. Martinus Nijhoff, Dordrecht, pp 193–199

    Google Scholar 

  • Kuramitsu S, Okuno S, Ogawa T, Ogawa H, Kagamiyama H (1985) Aspartate aminotransferase of Escherichia coli: nucleotide sequence of the aspC gene. J Biochem 97:1259–1262

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685

    Google Scholar 

  • Mattes U, Jaussi R, Ziak M, Juretic N, Lindenmann JM, Christen P (1989) Structure of cDNA of cytosolic aspartate aminotransferase of chicken and its expression in E. coli. Biochemie 71:411–416

    Google Scholar 

  • Miao G-H, Hirel B, Marsolier MC, Ridge R, Verma DPS (1991) Ammonia-regulated expression of a soybean gene encoding glutamine synthetase in transgenic Lotus corniculatus. Plant Cell 3:11–22

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

    Google Scholar 

  • Obaru K, Nomiyama H, Shimada K, Nagashima F, Morino Y (1986) Cloning and sequence analysis of mRNA for mouse aspartate aminotransferase isoenzymes. J Biol Chem 261:16976–16983

    Google Scholar 

  • Pol S, Bousquet-Lemercier B, Pave-Preux M, Pawlak A, Nalpas B, Berthelot P, Hanoune J, Barouki R (1988) Nucleotide sequence and tissue distribution of the human mitochondrial aspartate aminotransferase mRNA. Biochem Biophys Res Commun 157:1309–1315

    Google Scholar 

  • Reynolds PHS, Boland MJ, Farnden KF (1981) Enzymes of nitrogen metabolism in legume nodules: Partial purification and properties of the aspartate aminotransferase from lupine nodules. Arch Biochem Biophys 209:524–533

    Google Scholar 

  • Rogers SO, Bendich AJ (1985) Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol 5:69–76

    Google Scholar 

  • Ryan E, Bodley F, Fottrell PF (1972) Purification and characterization of aspartate aminotransferases from soybean root nodules and Rhizobium japonicum. Phytochemistry 11:957–963

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning — a laboratory manual. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Scandalios JG, Sorenson JC, Ott LA (1975) Genetic control and intracellular localization of glutamate oxaloacetic transaminase in maize. Biochem Genet 13:759–769

    Google Scholar 

  • Schubert KR (1986) Products of biological nitrogen fixation in higher plants: synthesis, transport and metabolism. Annu Rev Plant Physiol 37:539–574

    Google Scholar 

  • Snustad DP, Hunsperger JP, Chereskin BM, Messing J (1988) Maize glutamine synthetase cDNAs: Isolation by direct selection in Escherichia coli. Genetics 120:1111–1124

    Google Scholar 

  • Tance CP, Farnham MW, Degenhart N, Larson RJ, Miller SS, Barnes DK, Gantt JS (1990) Alfalfa root nodule aspartate aminotransferase (AAT): biochemical importance and genetic control. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New York, pp 693–699

    Google Scholar 

  • Wallsgrove RM, Keys AJ, Lea P, Miflin BJ (1983) Photosynthesis, photorespiration and nitrogen metabolism. Plant Cell Environ 6:301–309

    Google Scholar 

  • Wightman F, Forest JC (1978) Properties of plant aminotransferases. Phytochemistry 17:1455–1471

    Google Scholar 

  • Wych RD, Rains DW (1978) Simultaneous measurement of nitrogen fixation estimated by acetylene-ethylene assay and nitrate absorption by soybeans. Plant Physiol 62:443–448

    Google Scholar 

  • Yagi T, Kagamiyama H, Nozaki M, Soda K (1985) Glutamateaspartate transaminase from microorganisms. Methods Enzymol 113:83–89

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Biological Chemistry, Washington State University, 99164-6340, Pullman, WA, USA

    Michael K. Udvardi

  2. Department of Microbiology, Washington State University, 99164-6340, Pullman, WA, USA

    Michael L. Kahn

Authors
  1. Michael K. Udvardi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael L. Kahn
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by H. Hennecke

Rights and permissions

Reprints and permissions

About this article

Cite this article

Udvardi, M.K., Kahn, M.L. Isolation and analysis of a cDNA clone that encodes an alfalfa (Medicago sativa) aspartate aminotransferase. Molec. Gen. Genet. 231, 97–105 (1991). https://doi.org/10.1007/BF00293827

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00293827

Key words

Search

Navigation