Cloning of a polycistronic cDNA from tomato encoding gamma-glutamyl kinase and gamma-glutamyl phosphate reductase

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 94; no. 15; pp. 8249 - 8254
• Main Authors: Garcia-Rios, M. (Texas AandM International University, Laredo, TX.), Fujita, T, LaRosa, P.C, Locy, R.D, Clithero, J.M, Bressan, R.A, Csonka, L.N
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 22.07.1997; National Acad Sciences; The National Academy of Sciences of the USA
• Subjects: ADN; ALDEHYDE OXIDOREDUCTASES; Aldehyde Oxidoreductases - genetics; Amino Acid Sequence; AMINO ACID SEQUENCES; Antibodies; ARN MENSAJERO; ARN MESSAGER; Biological Sciences; Blotting, Western; Cell culture techniques; CHEMICAL COMPOSITION; CLONACION; CLONAGE; CLONING; Cloning, Molecular; Codon, Terminator; Codons; COMPLEMENTARY DNA; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; Cultured cells; DNA; DNA, Complementary; Escherichia coli; Escherichia coli - genetics; EXPRESION GENICA; EXPRESSION DES GENES; GENBANK/U27454; GENE EXPRESSION; Genetic loci; Glutamate-5-Semialdehyde Dehydrogenase; LOCI; LOCUS; LYCOPERSICON ESCULENTUM; MESSENGER RNA; MOLECULAR SEQUENCE DATA; NUCLEOTIDE SEQUENCE; Nucleotides; OPEN READING FRAMES; OXIDOREDUCTASES; OXIDORREDUCTASAS; OXYDOREDUCTASE; Phosphotransferases (Carboxyl Group Acceptor) - genetics; RNA; SECUENCIA NUCLEOTIDICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; Solanum lycopersicum - enzymology; Solanum lycopersicum - genetics; Stop codon; Tissue culture techniques; TOMPRO1 LOCUS; TRANSFERASAS; TRANSFERASE; TRANSFERASES
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.15.8249

We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues. GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames