Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Garcia-Rios M

PROVIDER: S-EPMC21589 | biostudies-literature | 1997 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

García-Ríos M M Fujita T T LaRosa P C PC Locy R D RD Clithero J M JM Bressan R A RA Csonka L N LN

Proceedings of the National Academy of Sciences of the United States of America 19970701 15

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. ...[more]

PMID: 9223347

Similar Datasets

Project description:Ralstonia solanacearum RSc2741 has been predicted as a gamma-glutamyl phosphate reductase ProA catalyzing the second reaction of proline formation from glutamate. Here, we experimentally demonstrated that proA mutants were proline auxotrophs that failed to grow in a minimal medium, and supplementary proline, but not glutamate, fully restored the diminished growth, confirming that ProA is responsible for the biosynthesis of proline from glutamate in R. solanacearum. ProA was previously identified as one of the candidates regulating the expression of genes for type three secretion system (T3SS), one of the essential pathogenicity determinants of R. solanacearum. Supplementary proline significantly enhanced the T3SS expression both in vitro and in planta, indicating that proline is a novel inducer of the T3SS expression. Deletion of proA substantially impaired the T3SS expression both in vitro and in planta even under proline-supplemented conditions, indicating that ProA plays additional roles apart from proline biosynthesis in promoting the expression of the T3SS genes. It was further revealed that the involvement of ProA in the T3SS expression was mediated through the pathway of PrhG-HrpB. Both the proA mutants and the wild-type strain grew in the intercellular spaces of tobacco leaves, while their ability to invade and colonize tobacco xylem vessels was substantially impaired, which was about a 1-day delay for proA mutants to successfully invade xylem vessels and was about one order of magnitude less than the wild-type strain to proliferate to the maximum densities in xylem vessels. It thus resulted in substantially impaired virulence of proA mutants toward host tobacco plants. The impaired abilities of proA mutants to invade and colonize xylem vessels were not due to possible proline insufficiency in the rhizosphere soil or inside the plants. All taken together, these results extend novel insights into the understanding of the biological function of ProA and sophisticated regulation of the T3SS and pathogenicity in R. solanacearum.

Project description:The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p)2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p)2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets