Abstract
Determining the genomic structure of diapause-associated transcripts (DAT) -2 and -3 led to the isolation of four novel miniature subterminal inverted repeat-like elements (MSITE): Mild-1, -2, -3 and -4. Mild-1a is inserted within the first intron of dia- pause protein-1. Mild-1a is 284 bp in length, has a 14 bp target site duplication and three sets of subterminal inverted repeats. The second element, Mild-2a, is inserted within the 3' terminus of Mild-1a. Mild-2a is 29 bp long with a 3 bp target site duplication and one set of subterminal inverted repeats. Using primers based on Mild-1, genomic clones were developed leading to the isolation of Mild-3a. Mild-3a shares 60% identity with Mild-1a, is 253 bp long, has a 9 bp target site duplication and has one set of subterminal inverted repeats. Mild-4a is inserted within the first intron of DAT-2 and is 227 bp in length with a 12 bp target site duplication. Mild-4a appears to be an intermediate form between a miniature inverted repeat transposable element (MITE) and a MSITE because the 5' inverted repeat is terminal (i.e., adjacent to the target site duplication) as in MITEs, but the 3' inverted repeat is separated (in this case, by 33 bp) from the 3' target site duplication as in MSITEs. The target site duplications of Mild-1, -3 and -4 families share a common conserved core of AATTT. All of the transposable elements are AT rich and are able to form hairpin structures. Within the promoter region of DAT-3 is a 163 bp sequence (Mild-1b) that shares 77% identity to the 3' terminus of Mild-1a. Mild-4a has iden- tity to 25 and 53 bp regions within the promoter of the juvenile hormone esterase B gene. Southern blot analysis revealed the pres- ence of Mild-1 and -3 elements in both Leptinotarsa decemlineata and Leptinotarsa juncta indicating that these elements are ancestral to the L. decemlineata, L. juncta separation.
Highlights
The Colorado potato beetle (CPB) Leptinotarsa decemlineata is the major pest of potato (Ferro, 1985; Hare, 1990; Cloutier et al, 1996) and attacks tomato (Schalk & Stoner, 1979) and eggplant (Cotty & Lashomb, 1982; Hamilton & Lashomb, 1996)
Sequence analysis of genomic clones of the diapause-regulated genes Diapause-associated transcripts (DAT)-2 and -3 led to the discovery of novel miniature subterminal inverted repeat transposable-like elements (MSITEs)
MSITES bear strong structural similarity to miniature inverted repeat transposable elements (MITEs) and may in some cases be an intermediate in the development of new MITEs (Tu & Orphanidis, 2001)
Summary
The Colorado potato beetle (CPB) Leptinotarsa decemlineata is the major pest of potato (Ferro, 1985; Hare, 1990; Cloutier et al, 1996) and attacks tomato (Schalk & Stoner, 1979) and eggplant (Cotty & Lashomb, 1982; Hamilton & Lashomb, 1996). Sequence analysis of genomic clones of the diapause-regulated genes DAT-2 and -3 led to the discovery of novel miniature subterminal inverted repeat transposable-like elements (MSITEs). MSITES bear strong structural similarity to miniature inverted repeat transposable elements (MITEs) and may in some cases be an intermediate in the development of new MITEs (Tu & Orphanidis, 2001). MITEs as a class share a number of structural features such as their small size (< 1600 bp), terminal or subterminal inverted repeats, AT rich composition, lack of open reading frames, and the ability of most to form secondary structures (reviewed, Feschotte et al, 2002a). We report here the isolation of four genomic elements bearing strong similarities to MSITEs. Southern blot analyses revealed that two of these elements are present in high copy number in both L. decemlineata and Leptinotarsa juncta (false potato beetle)
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