Activation of tobacco retrotransposons during tissue culture.

Activation of tobacco retrotransposons during tissue culture.

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Unformatted text preview: The EMBO Journal vol.12 no.6 pp.2521 -2528, 1993 Activation oftobacco retrotransposons during tissue culture Hirohiko Hirochika Department ofMolecularBiology, NationalInstituteofAgrobiological Resources, Tsukuba, Ibaraki305,Japan Communicated by D.J.Finnegan Sequencesofatleastthreenewfamiliesofretrotransposons (Ttol-Tto3) were amplified by PCR from cDNA preparedfromprotoplastsofanestablishedtobaccocell line,basedonthefactthatcertainaminoacidsarehighly conserved in the reverse transcriptases encoded by retrotransposons.StructuralanalysisindicatesthatTtol is5.5kblongandhasfeaturestypicalofretrotransposons. TranscriptionofTtolstartinginthelongterminalrepeat was activeonlyincultured cells.Protoplastformation enhanced thetranscription. The copy number ofTtol increased10-foldinestablishedcelllines;italsoincreased inplantsregeneratedfromtissueculturesandintrans- genicplants.TheseresultsindicatethatTtolisactivated duringtissueculture.Thisisthefirstdemonstrationof activationofaplantretrotransposonby tissueculture. The copy number of Tto2 and a previously isolated transposon,Tntl,alsoincreasedinestablishedcelllines, indicatingthatthesetwo retrotransposons may alsobe activatedbytissueculture.Thesethreeretrotransposons are crypticinnormallypropagatedplants:nodifference inthecopy number was observed between individuals ofthesamecultivarsorevenbetweendifferentcultivars. Keywords:LTR/retrotransposon/reversetranscriptase/tissue culture/transposition Introduction Transposableelementswerefirstfoundinmaizemorethan 40yearsago.Sincethen,manyplanttransposableelements havebeenidentifiedbygeneticanalysisbecausetheyinduce unstable mutations. These elements include the well characterizedAc/DsandSpmelementsofmaize(Fedoroff, 1989)andTamelementsofsnapdragon(Coenetal.,1989). Recentstudieshaveshownthatplantgenomesalsohavea second type oftransposable elements, namely retrotrans- posons (Johnsetal., 1985; Harberd etal., 1987; Voytas andAusubel, 1988;Grandbastienetal.,1989;Smythetal., 1989). Retrotransposons differ both functionally and structurallyfrom thetransposableelements ofmaize and snapdragon. The lattertype excises from one siteinthe genomeand reintegratesinto new sitesinthegenome,thus inducingunstablemutations.Retrotransposonsresemblethe integratedcopiesofretrovirusesandarecharacterizedby thepresenceof longterminalrepeats(LTRs)andaninternal domain encodingproteinsanalogoustothegroup-specific antigen (gag) and the pol polyprotein of retroviruses (BinghamandZachar, 1989;Boeke,1989).LTRs carrythe sequencesrequiredfortranscriptionandtranscribedRNA worksasanintermediatefortranspositionthroughreverse transcription. Because retrotransposons generally induce stablemutations,itisnoteasytodistinguishretrotransposon- induced mutations from othersby geneticanalysis. Most plantretrotransposonshavethereforebeenidentifiedonly recentlythrough molecularapproaches (seereferencesin Grandbastien, 1992)....
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This note was uploaded on 08/01/2009 for the course 231 224-12 taught by Professor Mazik during the Spring '06 term at A.T. Still University.

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Activation of tobacco retrotransposons during tissue culture.

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