Plant-transposable elements and gene tagging.
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Base Sequence↗
Biomedical subjects
A Gierl
Publications and source records attributed to A Gierl.
Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.
The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.
Base Sequence↗
Eukaryotic transposable elements with short terminal inverted repeats.
Transposable elements with short terminal inverted repeats are believed to transpose directly from DNA to DNA via excision and integration. The cis/trans requirements for transposition have recently been characterized for some of these elements. Common features seem to emerge for the mechanisms of excision of these elements, with the mechanisms apparently similar for the different elements.
Animals↗
En/Spm encoded tnpA protein requires a specific target sequence for suppression.
The En/Spm encoded suppressor function has been reconstituted in transgenic tobacco protoplasts. The suppressor affects genes which contain an En/Spm responsive transposable element in the transcribed sequences. The En/Spm encoded protein tnpA binds a defined cis element in the inserted transposon, repressing expression of the adjacent gene. This was shown by monitoring transient expression of a bacterial marker gene (GUS) expressed from a strong plant viral promoter. Suppressible variants of the marker gene were produced by inserting I element sequences into the untranslated sequences of the GUS transcript. Comparison of transient expression of these variants in wildtype tobacco protoplasts with their expression in protoplasts transgenic for tnpA protein demonstrates that tnpA is the suppressor. In addition, the minimal cis element required for suppression has been defined as a dimer consisting of two 12 bp tnpA binding sequences in a particular inverted orientation. One of these dimers occurs in each En/Spm end close to the characteristic 13 bp terminal inverted repeat. TnpA binding sites in different arrangements do not respond as well to tnpA. The implications of this observation are discussed. This system can be used to analyse tnpA-DNA interactions involved in gene regulation further.
Alleles↗
The En/Spm transposable element of Zea mays contains splice sites at the termini generating a novel intron from a dSpm element in the A2 gene.
The A2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable elements rcy and dSpm as gene tags. The A2 gene encodes a putative protein of 395 amino acids and is devoid of introns. Two a2-m1 alleles, containing dSpm insertions of different sizes, were characterized. The dSpm element from the original state allele has perfect termini and undergoes frequent transposition. The element from the class II state allele is no longer competent to transpose. It has retained the 13 bp terminal inverted repeat but has lost all subterminal sites at the 5' end, which are recognized by tnpA protein, the most abundant product of the En/Spm transposable element system. The relatively high A2 gene expression of one a2-m1 allele is due to removal of almost all dSpm sequences by splicing. The slightly altered A2 enzyme is still functional as shown by complementation of an a2 mutant with the corresponding cDNA. The 5' and 3' splice sites are constituted by the termini of the dSpm element; it therefore represents a novel intron of the A2 gene.
Alleles↗
Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins.
An excision assay system for En/Spm was developed in transgenic tobacco. The characteristics of excision and integration are similar to the natural system of Zea mays. In this transgenic model system two En/Spm encoded trans-acting functions, TNPA and TNPD, are required for excision. A biochemical model for transposition is proposed that might also be applicable to other transposable elements.
Cloning, Molecular↗
How maize transposable elements escape negative selection.
The transposable element systems En/Spm and Ac affect gene structure and control the expression of genes. In some cases, the deleterious consequences of insertional mutagenesis are reduced because certain members of these families of elements mimic introns. The potential benefits of such interactions, and a multilevel control of transposition activity, might explain 'survival' of these elements during evolution.
Base Sequence↗
The En/Spm transposable element of Zea mays.
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DNA Transposable Elements↗
Maize transposable elements.
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DNA Transposable Elements↗
TnpA product encoded by the transposable element En-1 of Zea mays is a DNA binding protein.
TnpA protein, the function encoded by the most abundant transcript of En-1 was expressed in Escherichia coli. DNA binding experiments with partially purified tnpA protein revealed that it binds to the subterminal repetitive region of En-1. TnpA protein recognizes a 12-bp-long sequence motif which is reiterated several times at the termini of En-1. Binding is reduced if the cytosine residues of CG dinucleotides and CNG trinucleotides within the motif are methylated. These data suggest a model in which the product of tnpA serves as a regulator of element activity.
Base Sequence↗
The defective En-I102 element encodes a product reducing the mutability of the En/Spm transposable element system of Zea mays.
Genetic and molecular analysis has revealed a specific En-element of deletion derivative (En-I102) which reduces En/Spm-induced mutability. In the presence of En-I102 the excision frequency of both the autonomous En-1 element and the inhibitor element Spm-I5719A is reduced and excision occurs later in development. The 3697 bp long En-I102 element is derived from En-1 by an internal deletion of 4590 bp removing nucleotides 1862-6451. The promoter at the left end and sequences required for polyadenylation are retained in En-I102. It is transcribed to yield predominantly a 1.8 kb poly(A) RNA. cDNA analysis of this transcript indicated that it contains the coding capacity for a 386 amino acid polypeptide. This polypeptide shares homology with En/Spm encoded functions and we suggest that it interferes with transposition at the protein level.
Alleles↗
Influence of transposable elements on the structure and function of the A1 gene of Zea mays.
The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1-kd A1 protein is an NADPH-dependent reductase. Germinal derivatives of the mutable a1-m1 allele with either recessive or wild-type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame-shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild-type gene expression. The presence of a second, promoter-like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1-m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080-bp-long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of a1-m2 alleles to trans-active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1-mpapu, a1-mr 102 and a1-ml.
Alcohol Oxidoreductases↗
Molecular analysis of the En/Spm transposable element system of Zea mays.
The nucleotide sequence of the autonomous transposable element En-1 isolated from the wx-844::En-1 allele has been determined. En-1 is 8287 bp long. The structure of the mosaic gene 1, coding for the major En transcript, has been established. The promoter gene 1 is located in the highly structured left end of the element and the gene spans almost the entire length of En-1. The first intron of gene 1 is 4434 nucleotides long and contains two large open reading frames, 2714 bp and 761 bp in size, which hybridize to minor RNA species in Northern blot experiments.
Alleles↗
Genetic and molecular analysis of the Enhancer (En) transposable element system of Zea mays.
A newly isolated, unstable mutation wx-844::En-1 of Zea mays was proven to be caused by the insertion of the autonomous transposable element En into the Waxy (Wx) gene. Molecular analysis revealed that En-1 is 8.4 kb long, has a 13-bp long perfect inverted repeat at its termini and generates a 3-bp target site duplication. En-1 is integrated into an intron located approximately in the middle of the transcribed region of the Wx gene. Structural evidence is presented indicating that a receptor component (Inhibitor) can arise by internal deletion of an autonomous En element.
Alleles↗
Molecular interactions between the components of the En-I transposable element system of Zea mays.
The sequence of the Inhibitor element Spm-I8 isolated from the wx-m8 allele has been determined. The element is 2242 bp in length. Its ends can be folded into long stem and loop structures. In a line containing the autonomous En element (wx-m8+En) we have detected a 2.5-kb transcript hybridizing to Spm-I8. A cDNA copy of this En-specific transcript containing 1.2 kb of the 3' end was cloned and it DNA sequence was determined. The 3' half of the cDNA is homologous to Spm-I8 and the region of homology is interrupted by intervening sequences. In the absence of an autonomous En element two chimeric transcripts 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 the En element, these transcripts are suppressed, possibly by a trans-acting function of En, inhibiting transcription read-through into Spm-I8.
Alleles↗
Plant transposable elements generate the DNA sequence diversity needed in evolution.
Two germinal and 16 somatic reversion events induced by the Enhancer (En) transposable element system at the wx-8::Spm-I8 allele of Zea mays were cloned and studied by sequence analysis. Excision of the Spm-I8 receptor element 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. Possible 'foot-print' sequences that may have arisen by the excision of transposable elements were observed when intron sequences of the wild-type (wx+) and mutant (wx-m8) alleles of the wx gene were compared. The sequence divergence generated by visitation of a locus by plant transposable elements is discussed with respect to the molecular evolution of the new gene functions.
Base Sequence↗
Sequence comparison of 'states' of a1-m1 suggests a model of Spm (En) action.
Two states of the a1-m1 allele featuring different phenotypes in the absence as well as in the presence of Spm or En have been cloned and sequenced.. The insertion site and orientation of the Inhibitor (I) element within the two alleles is identical. The sizes of the I elements differ, being 2.2 kb in state 6078 and 789 bp in state 5719A-1. The internal deletion in state 5719A-1 affects sequences within one side of the terminal inverted repeats of the I element. This alteration can be correlated with the decreased response of this state to the Mutator function of Spm. A model for the interaction between Spm (En)-encoded functions and the receptor element is discussed explaining the phenotypic differences between the states of the locus.
Alleles↗
The Spm (En) transposable element controls the excision of a 2-kb DNA insert at the wx allele of Zea mays.
The waxy (Wx) locus of Zea mays was cloned from strains carrying the wild-type and wx mutant alleles. The receptor component of the Suppressor-Mutator (Spm) controlling element system in the wx allele was shown to be a 2 kb long insertion within the transcribed region of the Wx gene. The insertion, termed Spm-I8, is excised during somatic reversion events induced by the autonomous controlling element Enhancer (En), which is an equivalent to Spm. Integration of Spm-I8 into the Wx gene generates a 3-bp target site duplication. Spm-I8 has a 13 bp long inverted repeat at its termini. The ends of the element can be further folded to build a large double-stranded structure consisting of five perfectly matching double-stranded regions of 9-13 bp in length, interrupted by single-stranded loops. A comparison of the wild-type and wx alleles revealed two additional insertions 6 (insert-1) and 0.25 (insert-2) kb in length. No En-induced excision of insert-1 and insert-2 could be detected so far. There is remarkable structure and sequence homology between Spm-I8 and the transposable elements Tam1 and Tam2 of Antirrhinum majus at their termini, reflecting a possible evolutionary and/or functional relationship between transposons in different plant species.
Journal Article↗