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.

Figure 1

Schematic representation of the structure of the tomPRO1–1 cDNA clone. The complete sequence is available from GenBank (accession no. U27454). The open reading frame specifying GK is initiated at position 279 and terminated at the TAA at positions 1,092–1,094; the open reading frame encoding GPR is initiated at position 1,100 and terminated at the TAA at positions 2,342–2,344. There are nine additional ATG codons in the 5′ leader upstream of the GK reading frame, at positions 27–29, 116–118, 182–184, 188–190, 199–201, 210–221, 221–223, 224–226, and 227–229. The rectangles immediately above the nucleotide position scale line indicate two fragments obtained with PCR amplification of DNA from tomato tissue cells; they contained nucleotides 898–1,188 and 898–1,617, and their sequence was identical to that of the corresponding regions of the tomPRO1–1 cDNA clone.

Figure 2

Comparison of the GK specified by the tomPRO1 clone (tomGK) with bacterial GKs. The amino acid sequence of the GK encoded by tomPRO1 was aligned to the indicated bacterial GKs with the pileup program, and the results displayed with the boxshade program. Letters in the black and gray backgrounds denote identical and similar residues, respectively. Sequences of GKs from the various bacteria are available from GenBank under the following accession numbers: E. coli, P07005; Bacillus subtilis, P39820; Corynebacterium glutamicum, U31230; Thermus thermophilus, D29973; Haemophilus influenzae, P431763.

Figure 3

Inhibition of the Δ¹-pyrroline-5-carboxylate synthetase (P5CS) activity of GK/GPR products of the tomPRO1 clone. P5CS activity was assayed in crude extracts of E. coli strains carrying the tomPRO1–1 clone in pKSII+. Assays were carried out in the presence of the indicated concentrations of proline, as described in Materials and Methods. Results are expressed as percent of specific activities in the absence of proline, which was 13 nmol (min)⁻¹⋅(mg total protein)⁻¹.

Figure 4

RNase protection analysis of tomPRO1 transcript. (A) Probe was a 378-nt riboprobe containing nucleotides 1–363 from the antisense strand of the tomPRO1–1 cDNA clone plus an additional 15 non-complementary nucleotides from linkers (Probe I in Fig. 1). Lanes: 1, RNA from normal (S0) cells; 2, RNA from NaCl adapted (S15) cells; 3 and 4, Controls, containing 50 μg yeast RNA with and without RNase, respectively. (B) Probe was a 748 nt riboprobe containing nucleotides 826-1,158 from the tomPRO1 antisense strand plus 15 non-complementary nucleotides from linkers (Probe II in Fig. 1). Lanes: 5, RNA from S15 cells; 6, control containing 50 μg yeast RNA without RNase. Conditions for RNase protection assays are described in text. The protected probes (lanes 1, 2, and 5) were 15 nucleotides shorter than the respective full length probes (lanes 4 and 6), because of loss of the 15 non-complementary nucleotides.

Figure 5

Western blot analysis of proteins of tomato tissues with antibodies against the GPR product of the tomPRO1 locus. Extracts of the indicated tomato tissues were subjected to electrophoresis on 4–20% polyacrylamide gradient SDS gels and probed with polyclonal antibodies against GPR. Antibodies were obtained from eggs of a chicken that was injected with a highly purified preparation of GPR, synthesized in E. coli from the tomPRO1–1 clone as a 44-kDa product. Lanes: 1 (S15 cells), proteins from tissue culture cells (cv. VFNT Cherry) adapted to 15 g/liter NaCl; 2–6, proteins from the indicated tissues of tomato plants (cv. Rutgers); 7, negative control, containing an extract of E. coli strain JM109 (21); and 8, positive control, containing extract from a derivative of strain JM109 carrying the tomPRO1–1 clone on pKSII+. Lanes 1–4 and 6 were loaded with 25 μg protein, lane 5 with 5 μg protein, and lanes 7 and 8 with 0.5 μg protein. The specific immunoreactive 70-kDa protein (lane 1), 60-kDa protein (lanes 2–5), and 44-kDa protein (lane 8) were absent when the extracts were probed with pre-immune antibodies (data not shown).