Retrotransposons are mobile genetic elements able to propagate in the genome through a cycle of transcription, reverse transcription, and insertion (Boeke and Corces 1989; Finnegan 1990; Grandbastien, 1992). Unlike classical DNA transposons, integrated copies are not excised. Both the structure and mechanism of retrotransposons is extremely similar to retroviruses such as the AIDS virus HIV-1 and the leukemia virus HTLV, with the exception that they lack the env (envelope) domain which enables the viruses to spread from cell to cell. A common evolutionary origin for all retroelements has been inferred from sequence comparisons between the diverse members of this group (Doolittle et al., 1989; Xiong and Eickbush 1992); at least a conserved portion of the reverse transcriptase gene appears to be present in every plant genome inspected, from cycads to cereals (Flavell et al., 1992).
We have isolated, cloned, and sequenced the first complete retrotransposon, and the first transposon of any description, to be found in barley, which we named BARE-1 (Manninen and Schulman, 1993). The BARE-I element (accession Z17327) is structurally similar to copia of Drosophila and Ty1 of yeast (Fig. 1). The transposon is bounded by long terminal repeats (LTRs), which in BARE-1 are 1829 bp, contain 6 bp inverted repeats at their ends and flanked by 4 bp direct repeats in the host DNA. Between the LTRs is an internal domain with a derived amino acid sequence of 1301 residues, with well conserved gag, aspartic proteinase, endonuclease, and reverse transcriptase/RNase H domains. For the plants, full-length retrotransposons have been isolated from only a relatively few species, including Tnt1 from tobacco, Ta-1 from Arabidopsis and Bs1 from maize. An element similar to BARE-1, WIS-2, has been isolated from wheat, but the copy studied contains many mutations (stop codons and frameshifts) and is nonfunctional.
We are now studying the functions and mechanism of action of BARE-1. Such retrotransposons may play an important role in determining genome structure and in generating new variability and mutations. Several approaches are being taken with BARE-1: 1) analysis of the evolution and copy number of the retrotransposon and related sequences in other Hordeum spp. and members of the tribe Triticeae; 2) analysis of the function of the gene products encoded by the BARE-1 open reading frame; 3) isolation of a complete cDNA clone; 3) examination of the role of the BARE-I long terminal repeats (LTRs), which form the ends of the element, as a promoter, terminator, and substrate for integration into the genome.
We have detected transcripts for BARE-I in several tissues as well as in protoplasts. In functional retroelements, the 5' LTR drives transcription and the 3' LTR termination and polyadenylation. In order to analyze BARE-I expression, we have made reporter-gene constructs containing uidA (ß-glucuronidase) or luc (luciferase) fused to the intact LTR and to a series of LTR deletions. These were transferred by electroporation into barley leaf protoplasts, which were then incubated for 12-48 h and assayed for the reporter enzyme. The BARE-I LTR is able to drive reporter-gene expression in barley protoplasts, and the deletion analyses have defined regions critical for promoter function. Partial cDNA clones for BARE-I have now been isolated; this confirms that this retroelement is indeed transcribed in the intact plant.
|Fig. 1. |
Map of cloned BARE-1 retrotransposon (accession Z17327). Selected restriction sites predicted from the sequence are shown to the left. Regions depicted (top to bottom) are: flanking genomic DNA (thick line); LTR (large box) with predicted TATA boxes for transcription initiation (a, b); untranslated leaded (stippled); coding domains (shaded) consisting of gag (281 aa); aspartic proteinase (151 aa), integrase (297 aa), reverse transcriptase-RNaseH (572 aa); insertion into 3' LTR (c); polyadenylation signal (d). Black triangles, stop codons; black diamond, framshift.
We thank Anne-Mari Kakko and Pirjo Aaltonen for excellent technical assistance. The contributions of Ina Manninen in isolating and sequencing BARE-1 are gratefully recognized. This work was supported by a grant from the Ministry of Agriculture and Forestry of Finland.
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