Dr. Alan H. Schulman, Docent

Scientists:

Marko Jääskeläinen

Dr. Ruslan Kalendar

Jaakko Tanskanen

Dr. Carlos Vicient

Technicians:

Anne-Mari Narvanto

Retrotransposons are major genome components. In recent years, retrotransposons have been established to be ubiquitous and active components of the plant genome. A recent review (Andersson et al., 1998) stated that "the functional consequences of retrotransposable DNA sequences in the eukaryotic chromosomes are among the most controversial and intriguing aspects of molecular genetics." Retrotransposons are the major class of transposons in plants, and due to their replicative life cycle should be considered as intracellular viruses.

Barley is an ideal system for study of retrotransposons. Small plant genomes, particularly arabidopsis, do not appear to have or tolerate active families of such elements. Arabidopsis contains perhaps 1% of its genome as retrotransposons, whereas maize and barley have upwards of 50%. Unlike maize, the wild direct ancestors of cultivated barley still grow in the original range in which the species is endemic and was domesticated, and in addition the genus is spread throughout the Americas and Eurasia. This enables us to examine the function and evolution of the element from a climatic and geographic standpoint within the Hordeum genus. Despite the ubiquity of retrotransposons in plants, few active elements are known. Our system, BARE-1 in barley, one of few shown to be active and being studied in depth.

BARE basics. The life cyle of active retotransposons are thought to contain the same basic steps as that of retroviruses except that the completed particle does not been enveloped for passage out of the cell. BARE-1 is the first complete transposon of any kind to be identified in barley. Bounded by 1.8 kb long terminal repeats (LTRs), BARE-1 contains a protein coding domain with a predicted protein product specifying GAG, aspartic proteinase (AP), integrase (IN), reverse transcriptase (RT), and RNaseH functions. These gene products, when aligned with their counterparts from retroviruses and other copia-like retrotransposons, are well-conserved (Manninen and Schulman 1993). For example, the integrase domain contains all invariant retroviral integrase residues, notably all those identified as essential by mutagenesis experiments with the HIV enzyme (van Gent et al., 1992). We have demonstrated that BARE-1 is transcribed in barley tissues, and that the transcripts begin within the BARE-1 LTR downstream of two TATA boxes (Suoniemi et al., 1996b). Thus, BARE-1 is active in somatic cells in its natural host. The LTR was used to drive expression of luc in transiently transformed barley protoplasts. This is dependent on the presence of a TATA box functional in planta as well. We identified regions within the LTR responsible for expression within protoplasts by deletion analyses of LTR-luc constructs. Similarities between promoter regulatory motifs, including ABREs (ABA response elements) were found within the LTR promoter region.

BARE-1 is a major, dispersed component of the genome. We have also examined the BARE-1 chromosomal localization by in situ hybridization (Suoniemi et al., 1996a). The long terminal repeat (LTR) probe displayed a uniform hybridization pattern over the whole of all chromosomes, excepting paracentromeric regions, telomeres, and nucleolar organizer (NOR) regions. The integrase probe showed a similar pattern. The 5' untranslated leader (UTL) probe, expected to be the most rapidly evolving component, labeled chromosomes in a dispersed and non-uniform manner, concentrated in the distal regions, possibly indicating a target site preference. The uniform distribution of BARE-1 makes it suitable as a source of mapping and marker probes.

BARE-1 RT and IN recognition motifs. We have used inverse PCR with LTR-based primers to establish the consensus sequences for the terminal region of the LTR, the external dinucleotides of the cDNA integration intermediate, and the minus- and plus-strand priming sites (Suoniemi et al., 1997). Design of BARE-1 -based vectors as well as functional analysis of integration and reverse transcription requires knowledge of these motifs. We found that these key functional entities are well-conserved in the BARE-1 family including wheat Wis2, but differ from those of other plant retrotransposons. The overall (-)-stand primer-binding site complements tRNA^et closely, and the plus-strand priming site (PPT) is highly conserved in the BARE-1 family, but differs from the PPT for Tnt-1. The terminal nucleotides, 5' GGGAG 3', are conserved in virtually all retrotransposons and retroviruses. This suggests selection and possibly co-evolution for slightly differing cleavage properties of RNase H in each plant, with an absolute requirement for five purines nearest the LTR. In retroviruses, IN is known to recognize the 3'-terminal CA of LTRs and remove the succeeding, external dinucleotide which is added during reverse transcription. The comparison here of genomic BARE-1 copies reveals that the dinucleotide is conserved and symmetric, and would yield 5' overhangs of 5' AC 3' if processing by BARE-1 IN follows the retroviral model. In Tnt-1 by comparison, the external dinucleotide is both highly conserved and asymmetric with respect to the two cDNA ends.

BARE-1 insertion sites. We also used inverse PCR to examine the sequences flanking BARE-1 insertion sites (Suoniemi et al., 1997). The target site duplication was established as 5 bp, indicating that the BARE-1 IN generates a 5 bp staggered cut during the integration reaction. Of the 13 identified integration sites, seven were other BARE-1 elements and three were other retrotransposons, one a Grande-like element previously reported only for maize and its near relatives. Of 17 BARE-1 insertion sites we've positively identified, 65% are retrotransposons. Almost simultaneously with our report, Jeff Bennetzen's lab reported a highly nested insertion pattern for retrotransposons in maize. This pattern has not appeared, however, in analyses of the sequences coming out of the arabidopsis genome project. Whether the nested insertion pattern, perhaps typical of large genomes with many retrotransposons, is the result of targeted integration or only the record of non-deleterious insertions remains open. The question has both practical consequences for plant retrotransposon vector development, mapping applications (see below), as well as basic interest regarding integrase action.

BARE-1 retroelements have clearly been highly successful in replication and integration during the evolution of the barley genome, and are most likely still active in the genome. We are interested in understanding the structure and function of the this retrotransposon and its component parts in the lifecycle of the barley plant in in the evolution of the Hordeum genus.

References and Selected Publications

Andersson, G., Svensson, A.-C., Setterblad, N. and Rask, L. (1998). Retroelements in the human MHC class II region. Trends in Genetics 14, 109-114.

Manninen, I. and Schulman, A.H. (1993). BARE-1, a copia-like retroelement in barley (Hordeum vulgare L.). Plant Molecular Biology 22, 829-846.

Suoniemi, A., Anamthawat-Jónsson, K., Arna, T. and Schulman, A.H. (1996a). Retrotransposon BARE-1 Is a major, dispersed component of the barley (Hordeum vulgare L.) genome. Plant Molecular Biology 30, 1321-1329.

Suoniemi, A., Narvanto, A. and Schulman, A. (1996b). The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays. Plant Molecular Biology 31, 295-306.

Suoniemi, A., Schmidt, D. and Schulman, A.H. (1997). BARE-1 insertion site preferences and evolutionary conservation of RNA and cDNA processing sites. Genetica 100, 219-230.

Suoniemi, A., Tanskanen, J. and Schulman, A.H. (1998). Gypsy-like retrotransposons are widespread in the plant kingdom. Plant J. 13, 699-705.

van Gent, D.C., Oude Groeneger, A.A.M. and Plasterk, R.H.A. (1992). Mutational analysis of the integrase protein of human immunodeficiency virus type 2. Proceedings of the National Academy of Sciences USA 89, 9598-9602.

Last revised:  21 May 1998