Identification of a peptide methionine sulphoxide reductase gene in an oleosin promoter from Brassica napus^‡

Abstract

A bidirectional promoter can be defined operationally as a short segment of DNA that regulates divergent transcription. In an attempt to investigate whether the intergenic region between the oleosin and a second open reading frame (ORFII) in Brassica napus (L.) is a divergent promoter, and also to characterize the ORFII, cDNA clones homologous to ORFII were isolated from a leaf cDNA library. A representative cDNA (clone D) of one of the two classes identified was identical, in DNA sequence, to the genomic ORFII. The second representative cDNA (clone O) was 97% identical at the nucleotide level to the genomic ORFII. The predicted amino acid sequence of the cDNA clones each exhibit homology with the peptide methionine sulphoxide reductase (PMSR) of Escherichia coli. The gene structure of ORFII was elucidated and the relative positions of the oleosin, ORFII, and the intergenic promoter region were determined. This confirms that the B. napus oleosin-ORFII intergenic region has divergent promoter activity. Consequently this is the first such plant nuclear divergent promoter identified. RFLP-mapping results showed that all four ORFII genes are linked to four of the six copies of the oleosin genes. This suggested that the bidirectional promoter locus is conserved within the B. napus genome. The ORFII gene product is targeted to the chloroplast, which is consistent with previous data indicating the presence of PMSR activity in the chloroplast. The over-expressed recombinant fusion protein (minus the transitpeptide) showed the capability to reduce peptide methionine sulphoxide residues in vitro, indicating PMSR activity. This study demonstrates that ORFII is transcribed and encodes a plant PMSR, and is the first example of the isolation of a eukaryotic PMSR gene.

Differential Regulation of Plastidial and Cytosolic Isoforms of Peptide Methionine Sulfoxide Reductase in Arabidopsis

“…Studies with the artificial substrate N-acetyl-MetSO have shown PMSR activity in plants (Ferguson and Burke, 1994), where it was particularly strong in photosynthetic tissues (Sanchez et al, 1983). We have previously isolated a pmsr gene from Brassica napus and demonstrated that the corresponding protein had PMSR activity (Sadanandom et al, 1996). In the present study we report the discovery of a complex family of at least five pmsr genes encoding two different protein isoforms in the model plant Arabidopsis.…”

“…An Arabidopsis genomic fragment of 3.4 kb contained a pmsr reading frame within an oleosin gene promoter in a similar orientation to that described for Brassica napus (Sadanandom et al, 1996). A specific DNA probe based on the Arabidopsis genomic sequence was used to screen an Arabidopsis leaf cDNA library.…”

“…No TATA or CAAT boxes were found, but there was a putative GATA box (Teakle and Kay, 1995) 20 nt upstream of the transcription start site. The two exons encoded a 204-residue, PMSR-like protein of 22,705 D. These data are summarized in Figure 1A, and the full (Sadanandom et al, 1996). genomic sequences are filed as GenBank accession nos.…”

“…Various tissues were ground in liquid nitrogen and extracted as in Sadanandom et al (1996). Protein amounts were determined as in Bradford (1976), analyzed by SDS-PAGE on acrylamide gels (Laemmli, 1970), and transferred to PVDF membranes according to the instructions of the manufacturer (electrophoresis manual, Bio-Rad Laboratories, Hercules, CA).…”

“…Membranes were incubated with anti-PMSR primary antibodies at 1:500 dilution and developed using a westernblotting detection system (ECL PLUS, Amersham, Buckinghamshire, UK). Total RNA from various plant tissues was extracted (Prescott and Martin, 1987), and 10-g aliquots were separated on 1.6% (v/v) agarose gels and probed with gene-specific c-pmsr or p-pmsr cDNAs as in Sadanandom et al (1996).…”