Molecular analysis of the En/Spm transposable element system

Comparison of the autonomous element and its receptor component: Both the autonomous element En1 (Peterson, 1953, Genetics 38:682-683) and its receptor homologue Spm-I8 (McClintock, 1961, Carnegie Inst. Wash. Yearbook 60:469-476) were molecularly cloned from the wx-844 (Pereira et al., EMBO Journal, in press) and wx-m8 (Schwarz-Sommer et al., 1984, EMBO J. 3:1021-1028) alleles, respectively. The autonomous component En1 is 8.4 kb in size and is inserted in an intron of the wx gene, while the receptor or Inhibitor element Spm-I8 is 2.2 kb in size and is inserted in an exon of the wx gene. Heteroduplex analysis revealed that Spm-I8 is a deletion derivative of the autonomous element. Spm-I8 has retained 1kb and 1.2kb, respectively, from each end of the En element. Sequence analysis of their termini and of their flanking regions at their integration site revealed strong structural homology between the autonomous element En1 with its receptor component Spm-I8 (e.g. 3 bp target site duplication, 13 bp perfect terminal inverted repeat, extended stem and loop structure at the ends, etc.). This proves molecularly the genetic homology (Peterson, 1965, Am. Naturalist 99:391-398) of both the En and Spm system.

Transcription products of En: Two En-specific RNAs, 2.5 and 7 kb in size, were detected in Northern blot analysis of polyA+ RNA from a line that contains an active En element. A cDNA copy of the smaller transcript containing 1.2 kb of its 3'-end was cloned and sequenced. Comparison to the Spm-18 DNA sequence revealed that the 3'-half of the cDNA is homologous to Spm-I8 and that the region of homology is interrupted by intervening sequences. We intend to clone the cDNA into expression vectors to raise antibodies against the putative En protein.

An assay system for the suppressor function of En: In the absence of the autonomous element two chimeric transcripts, 2 kb and 3.2 kb in size, are observed in the wx-m8 line which are probably initiated at the wx promoter and terminate in the Spm-I8 insertion. In the presence of En these transcripts are suppressed, possibly by a trans-acting function of En, inhibiting transcriptional readthrough into Spm-I8. We intend to use this system to characterize the suppressor function of the En/Spm system (Gierl et al., manuscript submitted).

Plant transposable elements generate the DNA sequence diversity needed in evolution: Two germinal and sixteen somatic En-induced reversion events of the Spm-I8 receptor element from the wx-m8 allele were cloned and studied by sequence analysis (Schwarz-Sommer et al., manuscript submitted). Excision of Spm-I8 from the wx gene results in various mutant DNA sequences. This leads to altered gene products, some of which are still capable of restoring the wild type phenotype. Thus the excision of a transposable element generates sequence alterations which can change the amino acid sequence of a protein and hence visitation of genes by transposable elements can be useful in the evolution of new gene functions.

All revertant sequences studied are compatible with a model of the transposition mechanism recently proposed (Saedler and Nevers, EMBO J., in press). Possible "footprint" sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild type (wx+) and the mutant (wx-m8) alleles of the wx gene were compared. These findings show that in evolution transposing elements are also involved in more subtle changes of the genetic material besides the well known gross chromosomal rearrangements such as duplications, deletions, inversions, translocations etc.

Two states of the a-m1 allele differ in the size of the Inhibitor element inserted at the A1 gene: Two states of Inhibitor at the a-m1 allele (McClintock, 1965, Brookhaven Symp. Biol. 18:162-184; McClintock, 1968, Carnegie Inst. Wash. Yearbook 67:20-28) have been cloned and analysed by restriction mapping and Southern hybridization. Within the limits of resolution of the methods applied, the site of the I-homologous insertions is identical in both alleles. However, the sizes of the insertions differ considerably: state 5719A contains a 400 bp insert while that of state 6078 is 2.2 kb. This can be correlated with the residual gene activity present in the mutants in the absence of an autonomous En/Spm. Thus the basic color of state 5719A is medium dark, because the relatively small insert of 400 bp might allow some A1 gene expression. The large 2.2 kb insert of state 6078 allows virtually no gene expression and, accordingly, this state is colorless in the absence of Spm.

Sequence analysis of these insertions will allow identification of sequences involved during En/Spm-directed excision events because the two states also differ in the frequency and timing of spotting. Molecular cloning and sequence analysis of derivatives of these two states as well as others differing in background color will shed light on structures necessarily present for the suppression of residual gene activity when the receptor elements are supplied with the S function of En/Spm.

Rita Berndtgen, Heinrich Cuypers, Alfons Gierl, Ralf Bernd Klosgen, Andy Pereira, Peter A. Peterson1, Heinz Saedler and Zsuzsanna Schwarz-Sommer

1Dept. of Agronomy, Iowa State University, Ames, Iowa


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