Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA −auxotroph (original) (raw)
Summary
Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants. Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism. To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA −auxotroph. The maize DHPS activity expressed in the complemented E. coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme. The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence. The molecular weight of 35854, predicted from the deduced amino acid sequence, was similar to the 38 000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize. DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues. Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome.
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Authors and Affiliations
- Department of Agronomy and Plant Genetics, and Plant Molecular Genetics Institute, University of Minnesota, 55108, St. Paul, MN, USA
David A. Frisch, Andrew M. Tommey, Burle G. Gengenbach & David A. Somers
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- David A. Frisch
You can also search for this author inPubMed Google Scholar - Andrew M. Tommey
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Communicated by H. Saedler
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Frisch, D.A., Tommey, A.M., Gengenbach, B.G. et al. Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA −auxotroph.Molec. Gen. Genet. 228, 287–293 (1991). https://doi.org/10.1007/BF00282478
- Received: 24 February 1991
- Issue Date: August 1991
- DOI: https://doi.org/10.1007/BF00282478