Copyright 0 1991 by the Genetics Society of America Identification of a Regulatory Transposon That Controls theMutator Transposable Element System in Maize Paul Chomet,*” Damon Lisch,* KristineJ. Hardeman: Vicki L. Chandler? and Michael Freeling* *Deflartment of Plant Biology, University of Calqornia, Berkeley, Calqornia 94720, and tInstitute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 Manuscript received April 16, 1991 Accepted for publication May 22, 1991 ABSTRACT The Mutator system of maize consists of more than eight different classes of transposable elements each of which can be found in multiple copies.All Mu elements share the -220-bp terminal inverted repeats, whereas each distinct element classis defined by its unique internal sequences.The regulation of instability of this system has been difficult to elucidate due to its multigenic inheritance. Herewe present genetic experimentswhich demonstrate that thereis a single locus,MuRI, which can regulate the transposition of Mu1 elements. We describe the cloning of members of a novel class of Mu elements, MuR, and demonstrate that a member of the class is the regulator of Mutator activity, MuRI. This conclusion is based on several criteria: MuRl activity and a MuR-homologous restriction fragment cosegregate; when MuRl undergoes a duplicative transposition, an additionalMuR restric- tion fragmentis observed, andMuRl activity and the cosegregatingMuR fragment are simultaneously lost within clonal somatic sectors. In addition, the MuR element hybridizes to transcripts in plants with Mutator activity. Our genetic- experiments demonstrate that the MuRl transposon is necessary to specify Mutator activity in our lines. ERTAIN lines of maize, called “Mutator lines,” a 9-bp genomic duplication upon insertion (BARKER C generate a high frequency of mutations (ROB- et al. 1984; CHENet al. 1987; SCHNABLE,PETERSON ERTSON 1978). Mutations arising in Mutator lines and SAEDLER1989). At least six different elements often exhibit somatically unstable phenotypes charac- representing four classes (Mul, Mul.7, Mul-del, Mu3, teristic of transposable element-induced alleles. The Mu7 and Mu8) are capable of transposition (STROM- molecular characterization of Mutator induced muta- MER et al. 1982; TAYLORand WALBOT1987; OISHI tions at Adhl, Brl, A1 and other loci, has established and FREELING1987; HARDEMANand CHANDLER that transposable elements, called Mu transposons, are 1989). The other four classes (Mu4, Mu5, Mu6 and likely to cause the high rate of mutation (STROMMER MuA) have been identified by their DNA homology et al. 1982; TAYLOR,CHANDLER and WALBOT1986; to the common -220-bp inverted terminal repeats O’REILLYet al. 1985). (TALBERT,PATTERSON and CHANDLER1989; QINand Nine different classes of Mu transposons have been ELLINGBOE1990; V. L. CHANDLER,unpublished). At distinguished on the basis of the sequence heteroge- least a few sequences characteristic of each class of neity within the element (BARKERet al. 1984; TAYLOR element are found in most non-Mutator standard and WALBOT1987; OISHIand FREELING1987; TAL- inbred lines of maize(CHANDLER, RIVIN and WALBOT BERT and CHANDLER1988; TALBERT,PATTERSON and 1986; TALBERT,PATTERSON and CHANDLER1989; V. CHANDLER1989; SCHNABLE,PETERSON and SAEDLER L. CHANDLERunpublished data). To date there is no 1989; VARAGONA,FLEENOR and WESSLER1987; QIN evidence that any of these characterized Mu elements and ELLINGBOE1990; V. CHANDLER,unpublished). encode a transposase (LILLIS,SPIELMANN and SIMPSON All classes ofMu elements have in common the -220- 1985; ALLEMANand FREELING1986; see TALBERT, base pair (bp) terminal inverted repeats, but different PATTERSONand CHANDLER1989 for discussion), al- classeshave distinct unrelated internal sequences. though, recently a MuA element has been shown to Memberswithin the same class share astrong se- be homologous to atranscript associated withMutator quence homology throughout the internal regions of activity (QINand ELLINCBOE1990). the elements. Furthermore, the described classes can Unlike the well-defined controlling element systems exist in multiple, heterogeneous copies and all ele- Ac-Ds and Spm, the genetic basis of Mutator activity ments shown to be capable of transposition generate appears to segregate in a non-Mendelian or multigenic manner (ROBERTSON 1978; WALBOT1986). When ’ Present address: Dekalb Plant Genetics, Eastern Point Rd.,Groton, active Mutator stocks are outcrossed to standard non- Connecticut 06340. Mutator lines, the typical result is that 90% of the Genetics 149: 261-270 (September, 1991) 262 P. Chomet et al. progeny retain and 10% lose Mutator activity. How- Progeny segregating for the MuRl element(s) was obtained ever, these percentages are variable and simple men- from the cross: delian ratios reflecting the segregation of only one or +MuRl; al-mum2 Sh2/al sh2 X a1 sh2/al sh2; -MuRl two regulatory genes are rarely observed. Mutator- or inactive progeny are simultaneously altered relative to Mutator-active progeny with.respect to a number +MuRl; al-mum2 Sh2/al sh2 of phenotypesassociated with the Mutator system. X al-mum2 ShPlal sh2; -MuRl. First, the forward mutation rate is reduced, the ele- A Mutator stock, with a typical number of Mu1 elements ments are somatically stable and thehigh copynumber (20-60) was used to obtain new shl mutants. Details of the of Mu elements is not maintained upon outcrossing stock construction were previously described (HARDEMAN (ROBERTSONet al. 1985; BENNETZEN1987; WALBOT and CHANDLER1989). Eight shl mutants isolated at a fre- and WARREN1988), indicating Mu elements are no quency of 1.1 X were obtained (K. J. HARDEMANand longer transposing. Second, certain nucleotides in V. L. CHANDLER,unpublished). The shl mutant relevant to this work was designated shl-A83 and contained a 4.0-kb the ends of Mu transposonsbecome methylated insertion in the shl gene. (CHANDLERand WALBOT1986; BENNETZEN1987; Nucleic acid samples:Maize DNA from leaves was puri- BENNETZEN,BROWN and SPRINGER1988). Third, ex- fied according to CONE(1989). RNA was isolated from trachromosomal Mu circles associated with Mutator leaves and immature ears by the guanidinium thiocyanate activity are not detectable (SUNDARESANand FREEL- method and the sodium dodecyl sulfate (SDS) method (Au- SUBEL et al. 1987). Plasmids containing Mul, Mul.7, Mu4 ING 1987). Finally, a reporter allele that is normally and Mu5 probes were previously described (TALBERT,PAT- suppressed by Mutator activity and expresses a mutant TERSON and CHANDLER1989). A plasmid containing the phenotype, expresses the wild-type phenotype in Mu- Mu3 element was obtained from K. OISHI and a plasmid inactive lines(MARTIENSSEN et al. 1990). In some, but containing Mu8 was obtained from S. WESSLER.Internal fragments of the Mu6 and Mu7 elements were from D. not all cases, the loss of Mutator activity can be re- TURKS.The actin probe was obtained from R. MEACHER versed by crossing to Mutator-active plants (ROBERT- (MEACHERet al. 1983). A KfnI fragment from the 5' end SON et al. 1985; BENNETZEN1987; WALBOT1986). of the A1 gene was obtained from J. BROWN(O'REILLY et ROBERTSONand STINARD(1989) reported an un- al. 1985). usual Mutator stock where the control of Mu1 excision Cloning: An -8.5-kb sequence from the shl-A83 allele was cloned by ligating size-fractionated BclI-digested maize 1: segregated 1 in a testcross, suggesting the presence DNA into theBamHI site of the XBv2 vector (obtained from of a single regulator locus. Using a similar line con- N. MURRAY,University of Edinburgh). After in vitro pack- structed independently inthis laboratory we have aging (HOHN 1979), the phage were plated and screened identified a Mu transposon with regulatory activity (BENTONand DAVIS1977) using a Shl clone, p17.6, ob- and designated it Mu-Regulator1 (MuRI). Wehave tained from C. HANNAH.An 8-kb PstI fragment containing the entire Mu insertion, shl sequences, and -1.8 kb of the demonstrated that this gene is itself a transposon and X vector was subcloned into a pTZl8 vector (United States is necessary to confer activity on different classes of Biochemical). The site of insertion of the element in the shl Mu elements (this report; P. CHOMET,D. LISCHand allele was determined by sequencing the two SstI fragments M. FREELING,unpublished data). (subcloned into a pTZ 18vector) which contained homology We have identified and cloned two members of a to both the Mu-termini and Shl. Both strands were se- quenced by the dideoxy chain termination method (SANGER, new class ofMu elements that have not been described NICKLENand COULSEN1977) using the 17-mer universal before. These elements have the Mu terminal inverted primer (Pharmacia). An internal EcoRI-BamHI fragment of repeats, and hybridize with the Mu5 element's ex- the element at shl-A83 was designated Mu*. tended terminal repeats. However, their internal se- A BclI clone containing most of the MuRl element was quences do not hybridize with those of Mul, Mul.7, obtained by size-fractionating BclI digested DNA from a plant carrying MuRl. This DNA was ligated into theBamHI Mu3, Mu4, Mu5, Mu6, Mu7or Mu8. In this paper we site of XEMBLS (purchased from Stratagene Inc.). The demonstrate that a member of this new class of Mu phage were in vitro packaged, plated and screened using elements is the MuRl transposon, a regulator for the the Mu* probe. A clone carrying the MuRl-diagnostic 1.4- multiclass Mutator system. In addition, we show that kb internal XbaI fragment was restriction mapped further. the MuR elements