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Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

ABSTRACT: Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon, Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.

SUBMITTER: Colot V

PROVIDER: S-EPMC109017 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

Colot V V Haedens V V Rossignol J L JL

Molecular and cellular biology 19980701 7

Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly rema ...[more]

PMID: 9632817

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Project description:BACKGROUND: The nonautonomous maize Ds transposons can only move in the presence of the autonomous element Ac. They comprise a heterogeneous group that share 11-bp terminal inverted repeats (TIRs) and some subterminal repeats, but vary greatly in size and composition. Three classes of Ds elements can cause mutations: Ds-del, internal deletions of the 4.6-kb Ac element; Ds1, ~400-bp in size and sharing little homology with Ac, and Ds2, variably-sized elements containing about 0.5 kb from the Ac termini and unrelated internal sequences. Here, we analyze the entire complement of Ds-related sequences in the genome of the inbred B73 and ask whether additional classes of Ds-like (Ds-l) elements, not uncovered genetically, are mobilized by Ac. We also compare the makeup of Ds-related sequences in two maize inbreds of different origin. RESULTS: We found 903 elements with 11-bp Ac/Ds TIRs flanked by 8-bp target site duplications. Three resemble Ac, but carry small rearrangements. The others are much shorter, once extraneous insertions are removed. There are 331 Ds1 and 39 Ds2 elements, many of which are likely mobilized by Ac, and two novel classes of Ds-l elements. Ds-l3 elements lack subterminal homology with Ac, but carry transposase gene fragments, and represent decaying Ac elements. There are 44 such elements in B73. Ds-l4 elements share little similarity with Ac outside of the 11-bp TIR, have a modal length of ~1 kb, and carry filler DNA which, in a few cases, could be matched to gene fragments. Most Ds-related elements in B73 (486/903) fall in this class. None of the Ds-l elements tested responded to Ac. Only half of Ds insertion sites examined are shared between the inbreds B73 and W22. CONCLUSIONS: The majority of Ds-related sequences in maize correspond to Ds-l elements that do not transpose in the presence of Ac. Unlike actively transposing elements, many Ds-l elements are inserted in repetitive DNA, where they probably become methylated and begin to decay. The filler DNA present in most elements is occasionally captured from genes, a rare feature in transposons of the hAT superfamily to which Ds belongs. Maize inbreds of different origin are highly polymorphic in their DNA transposon makeup.

Project description:BackgroundRapid improvements in the development of new sequencing technologies have led to the availability of genome sequences of more than 300 organisms today. Thanks to bioinformatic analyses, prediction of gene models and protein-coding transcripts has become feasible. Various reverse and forward genetics strategies have been followed to determine the functions of these gene models and regulatory sequences. Using T-DNA or transposons as tags, significant progress has been made by using "Knock-in" approaches ("gain-of-function" or "activation tagging") in different plant species but not in perennial plants species, e.g. long-lived trees. Here, large scale gene tagging resources are still lacking.ResultsWe describe the first application of an inducible transposon-based activation tagging system for a perennial plant species, as example a poplar hybrid (P. tremula L. × P. tremuloides Michx.). Four activation-tagged populations comprising a total of 12,083 individuals derived from 23 independent "Activation Tagging Ds" (ATDs) transgenic lines were produced and phenotyped. To date, 29 putative variants have been isolated and new ATDs genomic positions were successfully determined for 24 of those. Sequences obtained were blasted against the publicly available genome sequence of P. trichocarpa v2.0 (Phytozome v7.0; http://www.phytozome.net/poplar) revealing possible transcripts for 17 variants.In a second approach, 300 randomly selected individuals without any obvious phenotypic alterations were screened for ATDs excision. For one third of those transposition of ATDs was confirmed and in about 5% of these cases genes were tagged.ConclusionsThe novel strategy of first genotyping and then phenotyping a tagging population as proposed here is, in particular, applicable for long-lived, difficult to transform plant species. We could demonstrate the power of the ATDs transposon approach and the simplicity to induce ATDs transposition in vitro. Since a transposon is able to pass chromosomal boundaries, only very few primary transposon-carrying transgenic lines are required for the establishment of large transposon tagging populations. In contrast to T-DNA-based activation tagging, which is plagued by a lack of transformation efficiency and its time consuming nature, this for the first time, makes it feasible one day to tag (similarly to Arabidopsis) every gene within a perennial plant genome.

Dataset Information

Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

Publications

Extensive, nonrandom diversity of excision footprints generated by Ds-like transposon Ascot-1 suggests new parallels with V(D)J recombination.

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