Bigfoot: a new family of MITE elements characterized from the Medicago genus Be  ne  dicte Charrier 1,² , Fabrice Foucher 1 , Eva Kondorosi 1 , Yves d'Aubenton-Carafa 2 , Claude Thermes 2 , Adam Kondorosi 1,3 and Pascal Ratet 1, * 1 Institut des Sciences Ve Âge Âtales, UPR40, 2 Centre de Ge Âne Âtique Mole Âculaire, Centre National de la Recherche Scienti®que, Avenue de la Terrasse, 91198 Gif- sur-Yvette CEDEX, France, and 3 Institute of Genetics, Biological and Research Center, Hungarian Academy of Sciences, 61701 Szeged, Hungary Summary We have characterized from the legume plant Medicago a new family of miniature inverted-repeat transposable elements (MITE), called the Bigfoot transposable elements. Two of these insertion elements are present only in a single allele of two different M. sativa genes. Using a PCR strategy we have isolated 19 other Bigfoot elements from the M. sativa and M. truncatula genomes. They differ from the previously characterized MITEs by their sequence, a target site of 9 bp and a partially clustered genomic distribution. In addition, we show that they exhibit a signi®cantly stable secondary structure. These elements may represent up to 0.1% of the genome of the outcrossing Medicago sativa but are present at a reduced copy number in the genome of the autogamous M. truncatula plant, revealing major differences in the genome organization of these two plants. Introduction During her study on maize, Barbara McClintock (McClin- tock, 1947; McClintock, 1948) developed the concept of transposable elements as mobile genetic entities. She named them `controlling elements' as these elements seemed to be able to modify gene expression when they were inserted in, or in the vicinity of, a given locus. Since this pioneering work, numerous transposable elements have been described in plants and other organisms, from bacteria to mammals (Flavell, 1986; Saedler and Gierl, 1996). Nowadays they are extensively used by geneticists and molecular biologists as tools to create tagged muta- tions and to search for mutations of speci®c genes of interest in tagged mutant libraries. In plants, transposable elements transposing through DNA intermediates (Saedler and Gierl, 1996) were studied in detail at the molecular level in maize (Ac/Ds, En/Spm, Mu) and Antirrhinum majus (Tam) and were also used as tags for gene isolation. Recently, the use of these elements as mutagens was extended to other plant species, such as Arabidopsis (Balcell et al., 1991; Sundaresan, 1996; Walbot, 1992). Retrotransposons, representing another class of these elements and transposing through RNA intermedi- ates, have also been described in plants (Wessler et al., 1995). Tnt1, the ®rst plant retrotransposon, was isolated from tobacco (Grandbastien et al., 1989). Recently, a new class of transposable elements called MITEs ( miniature inverted-repeat transposable elements) was described in plants (Wessler et al., 1995). The ®rst MITE described, called Tourist, is responsible for one of the maize waxy B2 alleles and was found to be part of a larger family of elements present in many cereal genomes (Bureau and Wessler, 1992; Bureau and Wessler, 1994a; Rio et al., 1996). The Tourist family is probably present at 10 4 copies per genome of maize, which makes it one of the most abundant transposable elements described so far in plants, after del-2, a line-like element from Lilium (Leeton and Smyth, 1993). Other families of MITEs identi®ed in the genes of both monocotyledonous and dicotyledonous plants (Blume et al., 1997; Bureau and Wessler, 1994b; Bureau et al., 1996; Casacuberta et al., 1998; Pozueta- Romero et al., 1995, 1996) have been identi®ed. They are all characterized by their small size (between 100 and 300 bp), their high copy number per genome, high A + T content, the lack of coding capacity, the presence of short- terminal inverted repeats, and their capacity to form secondary structures (Wessler et al., 1995). Because of their high copy number, this new class of transposable elements has been hypothesised to play an important role in plant genome evolution. These studies have also revealed that some of these elements have probably transposed recently because they are present in only some alleles of the genes analysed. However, because of their short size and the lack of coding capacity, their transposition mechanism is unknown at present. The constant size of the elements in one family may indicate that this character is important for their transposition mechanism (Wessler et al., 1995). In this paper, we report the characterization of a new type of MITE insertion element present in the Medicago sativa ssp. sativa and M. truncatula genomes. Two of these Received 22 December 1998; revised 9 April 1999; accepted 13 April 1999. *For correspondence (fax +331 69823695; e-mail Pascal.Ratet@isv.cnrs-gif.fr). ² Present address: School of Biology, University of Leeds, Leeds LS2 9JT, UK. The Plant Journal (1999) 18(4), 431±441 ã 1999 Blackwell Science Ltd 431