Cloning and characterization of PRO1, a tomato cDNA encoding the first two enzymes of proline biosynthesis
Abstract
A gene encoding the first enzyme of proline biosynthesis from tomato (Lycopersicon esculentum) was cloned by complementing a $\gamma$-glutamyl kinase (proB) mutation in E. coli with a cDNA library from tomato fruit. The complementing gene, designated PRO1, is present on a 3.0 kilobasepair fragment. When used as a probe in Southern analysis, the fragment hybridized as a single copy gene to chromosomal digests of tomato. Nucleotide sequence analysis of the PRO1 clone revealed two long open reading frames. The ORF at the 5$\sp\prime$ end of the PRO1 clone encodes a product with $>$60% similarity to the $\gamma$-glutamyl kinases of E. coli and yeast and to the $\gamma$-glutamyl kinase region of the P5CS gene from mothbean, whereas the second ORF encodes a product with a 69% similarity to the $\gamma$-glutamyl phosphate reductase of E. coli. and the $\gamma$-glutamyl phosphate reductase region of the P5CS gene from mothbean. The PRO1 gene has a very unusual structure for a nuclear eukaryotic gene. We found an in-frame TAA translation termination codon at the end of the $\gamma$-glutamyl kinase sequence, followed after 5 bases by a potential translation initiation codon ATG at the start of the $\gamma$-glutamyl phosphate reductase information. Utilizing an assay that measures the coupled activities of $\gamma$-glutamyl kinase and $\gamma$-glutamyl phosphate reductase, we determined the kinetic and allosteric properties of the polypeptides encoded by PRO1. The coupled activity was sensitive to feedback inhibition by proline. Both protein products synthesized from the PRO1 clone in E. coli. were purified, and polyclonal antibodies against the $\gamma$-glutamyl phosphate reductase polypeptide were raised in chickens. The antibodies were utilized in Western blot analysis of protein extracts from different plant organs. The antibodies recognized at least two proteins, with apparent molecular masses of 60 and 70 kDa. Preliminary studies of PRO1 expression were conducted in vitro using a transcription-translation system. These experiments showed that PRO1 can direct the synthesis of two polypeptides in wheat germ extracts. The unique nature of the tomato PRO1 locus is discussed.
Degree
Ph.D.
Advisors
Csonka, Purdue University.
Subject Area
Molecular biology|Genetics|Microbiology|Botany
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