Revolver is a new class of transposon-like gene composing the triticeae genome

Abstract

Revolver discovered in the Triticeae plant is a novel class of transposon-like gene and a major component of the large cereal genome. An 89 bp segment of Revolver that is enriched in the genome of rye was isolated by deleting the DNA sequences common to rye and wheat. The entire structure of Revolver was determined by using rye genomic clones, which were screened by the 89 bp probe. Revolver consists of 2929-3041 bp with an inverted repeated sequence on each end and is dispersed through all seven chromosomes of the rye genome. Revolver is transcriptionally active, and the isolated full-length cDNA (726 bp) reveals that Revolver harbors a single gene consisting of three exons (342, 88, and 296 bp) and two introns (750 and 1237 bp), and encodes 139 amino acid residues of protein, which shows similarity to some transcriptional regulators. Revolver variants ranging from 2665 to 4269 bp, in which 5' regions were destructed, indicate structural diversities around the first exon. Revolver does not share identity with any known class I or class II autonomous transposable elements of any living species. DNA blot analysis of Triticeae plants shows that Revolver has existed since the diploid progenitor of wheat, and has been amplified or lost in several species during the evolution of the Triticeae.

Figure 1

The scheme for cloning genome-specific elements by subtracting sequences common to wheat and rye. Genomic subtraction recovered a repetitive sequence, which is enriched in rye, but absent from wheat. Southern blot analysis of the EcoO109I digests confirmed that the rye 89 bp fragment (AB304276) hybridized well to the genome of rye ( Secale cereale, S.vavillovi and S.montanum), but did not hybridize to the wheat genomic DNA. The full structure of the element was determined by sequencing positive genomic clones screened by the initial rye-specific probe.

Figure 2

The transposon-like gene Revolver is dispersed on all seven chromosomes of rye. A FISH experiment was conducted using dual probes of Revolver labeled by biotin-16-dUTP and the subtelomeric 350 bp family labeled by digoxigenin-11-dUTP. Each probe was visualized by avidin-FITC (blue color) and anti-dig-rhodamine (red color), respectively.

Figure 3

Identification of Revolver mRNA. (A) Multiple sizes of transcripts homologous to Revolver were found in rye but no transcript was found in wheat. (B) A full-length Revolver cDNA clone (AB124665–124666), 726 bp long, was identified. Nucleotide sequence comparison between Revolver and the cDNA revealed that Revolver includes a single gene consisting of three exons and two introns. The predicted 139 amino acid sequence is highly conserved in Revolver cDNAs (AB124645, AB124666, AB304271–304275), which are recovered from S. cereale, S. silvestre, D. villosum, and Ae. tauschii, indicating activity in all of these species. The ORF shows similarity to some DNA-binding proteins. The Revolver ORF includes a DDE motif. The initial 89 bp clone (AB304276) obtained by genomic subtraction corresponds to the second exon. Revolver (AB124639, AB124640) shows 60% homology to parts of both ends (5′ side 104 bp, 3′ side 2213 bp) of LARD (4327 bp), however, in which the region extending from exon 1 to the half of intron 1 of Revolver is replaced by a different sequence and the coding region is not present.

Figure 4

Revolver cDNAs isolated by RT–PCR from S. cereale, S. silvestre, S. fragile, D. villosum, Ae. tauschii, and T. monococcum (AB124645, AB304271–304275). Wheat and barley ESTs showing partial homology to Revolver were found in the Triticeae EST database. The predicted Revolver ORF shows similarity to some transcriptional regulators with DNA-binding ability. The ORF is highly conserved among the top four species, indicating that Revolver is active in these species. In wheat and barley, however, only partial sequences encoded in exon 2 are retained.

Figure 5

Structural diversities of Revolver in the rye genome. Several sizes of Revolver variants ranging from 2665 to 4269 bp (Revolver-3–6, AB124641–12464) were obtained from the rye genome. All of the lengthy variants may be non-autonomous elements of Revolver, because they have the downstream region of second intron of Revolver, but they have structural mutation occurred at the 5′ side, in which the coding region especially in the first exon is considerably destructed. Among the Revolver family showing extremely structural diversities, Revolver-1 (3041 bp) in lambda-1 and Revolver-2 (2929 bp) in lambda-2, which indicates the highest coincidence with a full-length cDNA of 728 bp, should have basic structure of Revolver.

Figure 6

Distribution of Revolver in plant genomes as revealed by genomic Southern blotting. Revolver cDNA hybridized strongly to Secale species and moderately to T. monococcum (AA), Ae. speltoides (SS), Ae. tauschii (DD), and T. dicoccum (AABB). In contrast, Revolver was not detected in the common wheat genome. These facts indicate that Revolver has existed since the diploid progenitor of wheat (3000–8000 copies), and it has been amplified in rye (20 000 copies), but it has been lost from bread wheat after the allopolyploidy event.