BERGAMO, ITALY

Istituto Sperimentale per la Cerealicoltura

COLOGNE, WEST GERMANY1

Max-Planck-Institut

Molecular analysis of the Bg-rbg transposable element system --H. Hartings, N. Lazzaroni, C. Spilmont, L. Asperti, N. Di Fonzo, R. Thompson1, F. Salamini1, M. Motto The Bg-rbg transposable element system consists of both autonomous (Bg) and nonautonomous (rbg) members. The autonomous Bg elements are able to transpose on their own, whereas rbg elements can transpose only when an active Bg element is present in the same cell.

In earlier work of our laboratory a newly isolated unstable mutation of the waxy locus (wx-m32::Bg) was reported to be caused by the insertion of an autonomous Bg element into the waxy gene (Motto et al., Maydica 34:107, 1989). In order to isolate this element, a genomic library in lambda EMBL4 vector was prepared using plant DNA extracted from heterozygous A69Y wx/wx-m32 plants. The screening of the library with two different pairs, one derived from the central part of the waxy gene and the other derived from the rbg receptor element, has permitted the identification of a clone containing the complete autonomous Bg element.

Detailed restriction endonuclease mapping indicated that the Bg autonomous element has a length of about 5.0 kb and is inserted in an intron, between exon 12 and exon 13, in the C-terminal part of the waxy gene. The DNA sequence of the Bg element was partly determined and revealed a number of interesting features. The Bg element appears to generate an 8-bp duplication of the target site upon integration and contains 17 bp imperfect inverted repeats (CAGGGAAAACTTTATCG----CGATAGAGTAAA CCCTG) at its termini. The inverted repeat starts with the bases CA, like a number of other elements, and exhibits some further homology to these elements. At 815 bp from the 5' end of the element we have found an ATG codon that, considering its surrounding sequence, is consistent with consensus sequence for the translation start reported for plant genes. The ATG codon is followed by an ORF with a length of 736 bp. The end of this ORF coincides with a sequence canonical for a splice-junction site. The rest of the Bg element sequence following this site contains 3 major ORFs, interrupted with obvious non-coding regions. A number of putative splice-junction sites are also present in this part of the sequence suggesting the existence of one or more introns in the coding region of the element. The Bg sequence located upstream of the presumed translation start site at bp 815 does not contain any motifs in common to most plant promoters. Particularly, it lacks a recognizable TATA box. However, an interesting aspect of the Bg element can be revealed if we consider the G+C content of this part of the sequence. A low percentage of G+C (approximately 40%) is found in the first 120 bp of the sequence. Following these 120 bp, a region of 700 bp with a very high percentage (approximately 80%) of G+C can be found. The rest of the Bg sequence starting from the ATG codon at position 815 has a G+C content of approximately 40%. Because it has been reported that a number of mammalian housekeeping genes lack TATA boxes and have instead GC-rich sequences preceding the transcription start sites, the presence of a similar region in the Bg element appears of great interest.

A number of findings indicate several similarities between the Bg element and the Ac transposable element of maize. Both elements generate 8 bp duplications at the target sites. As is the case for the Ac element Bg lacks a recognizable TATA box or any other motif common to most plant promoters. Furthermore, both the Bg element and the Ac element contain a region with a high content in G+C upstream of the translation start site. This region might act as a promoter causing a weak expression on a basal level of the Bg element.

Preliminary sequence data obtained for the rbg element indicated not more than 75% of homology between this element and the autonomous one. Furthermore, a comparison of the restriction maps of these elements indicated the presence of a 700 bp deletion in the 5' part of the rbg element. This deletion involves part of the GC-rich region present in the autonomous Bg element and extends beyond the ATG codon at position 815 of these elements.


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