Insertion mutagenesis and study of transposable elements using a new unstable virescent seedling allele for isolation of haploid petunia lines

The new unstable virescent seedling ( vis* ) allele of a petunia mutant, that has green leaves but white cotyledons with green revertant spots, was used to identify spontaneously occurring haploid petunia lines with active transposable elements. Endogenous transposons were trapped into the single petunia nitrate reductase structural gene ( nia ) using chlorate selection on haploid protoplasts. In two mutant lines, the dTph1 -like transposable element dTph1–3 was inserted at almost the same position but in opposite orientations in the first exon of the nia gene. In a third mutant, a different transposable element was integrated into the fourth exon. This element, called dTph4 , is 787 bp long and has 13 bp terminal inverted repeats of which 12 bp are identical to those of dTph1 . Insertion of dTph1–3 and dTph4 results in an 8 bp duplication of the target site, as already described for dTph1 . In contrast to dTph1 -like elements, dTph4 is present at low copy number in the petunia genome. This can facilitate its use for gene tagging in petunia. The dTph1–3 and dTph4 elements excise frequently, as transposon footprints were found in most of the insertion mutants. The data demonstrate that haploid petunia is an excellent system for gene tagging and for the study of transposable elements.     

Transposition of the autonomous Fot1 element in the filamentous fungus Fusarium oxysporum

Autonomous mobility of different copies of the Fot1 element was determined for several strains of the fungal plant pathogen Fusarium oxysporum to develop a transposon tagging system. Two Fot1 copies inserted into the third intron of the nitrate reductase structural gene (niaD) were separately introduced into two genetic backgrounds devoid of endogenous Fot1 elements. Mobility of these copies was observed through a phenotypic assay for excision based on the restoration of nitrate reductase activity. Inactivation of the Fot1 transposase open reading frame (frameshift, deletion, or disruption) prevented excision in strains free of Fot1 elements. Molecular analysis of the Nia+ revertant strains showed that the Fot1 element reintegrated frequently into new genomic sites after excision and that it can transpose from the introduced niaD gene into a different chromosome. Sequence analysis of several Fot1 excision sites revealed the so-called footprint left by this transposable element. Three reinserted Fot1 elements were cloned and the DNA sequences flanking the transposon were determined using inverse polymerase chain reaction. In all cases, the transposon was inserted into a TA dinucleotide and created the characteristic TA target site duplication. The availability of autonomous Fot1 copies will now permit the development of an efficient two-component transposon tagging system comprising a trans-activator element supplying transposase and a cis-responsive marked element.     

The controlling element Ds at the Shrunken locus in Zea mays: structure of the unstable sh-m5933 allele and several revertants

We have analyzed the structure of the Shrunken (Sh) locus in a strain containing an unstable recessive mutation, sh-m5933, caused by the transposable controlling element Dissociation (Ds). We have also analyzed nine spontaneous Sh revertant alleles. The sh-m5933 allele contains a 30 kb insertion at the Sh locus, as well as a duplication that includes part of the insertion and the Sh locus sequence on the 5' side of the insertion site. The revertants continue to show Ds-mediated chromosome breakage at the Sh locus, have an intact Sh locus from which the insertion has been excised, and retain the duplication. One of the nine revertant alleles has a 2 kb deletion at the junction between the Sh locus and the insertion sequence in the duplicated segment of the locus. The revertant also shows a temporal change in the pattern of somatic chromosome breakage, implicating the junction sequence as the site of Ds-mediated chromosome breakage.     

A general method to isolate genes tagged by a high copy number transposable element

The Petunia hybrida line W138 contains more than 200 copies of the transposable element dTph1. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTph1 flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerase chain reaction (IPCR) products originating from the mutated plant. It directly yields a probe for the mutated gene which can be used to screen pre-existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.     

Epigenetic interactions among three dTph1 transposons in two homologous chromosomes activate a new excision-repair mechanism in petunia.

Unstable anthocyanin3 (an3) alleles of petunia with insertions of the Activator/Dissociation-like transposon dTph1 fall into two classes that differ in their genetic behavior. Excision of the (single) dTph1 insertion from class 1 an3 alleles results in the formation of a footprint, similar to the "classical" mechanism observed for excisions of maize and snapdragon transposons. By contrast, dTph1 excision and gap repair in class 2 an3 alleles occurs via a newly discovered mechanism that does not generate a footprint at the empty donor site. This novel mechanism depends on the presence of two additional dTph1 elements: one located in cis, 30 bp upstream of the an3 translation start in the same an3 allele, and a homologous copy, which is located in trans in the homologous an3 allele. Absence of the latter dTph1 element causes a heritable suppression of dTph1 excision-repair from the homologous an3 allele by the novel mechanism, which to some extent resembles paramutation. Thus, an epigenetic interaction among three dTph1 copies activates a novel recombination mechanism that eliminates a transposon insertion.     

Stable two-element control of dTph1 transposition in mutator strains of Petunia by an inactive ACT1 introgression from a wild species

The high copy dTph1 transposon system of Petunia (Solanaceae) is one of the most powerful insertion mutagens in plants, but its activity cannot be controlled in the commonly used mutator strains. We analysed the regulation of dTph1 activity by QTL analysis in recombinant inbred lines of the mutator strain W138 and a wild species (P. integrifolia spp. inflata). Two genetic factors were identified that control dTph1 transposition. One corresponded to the ACT1 locus on chromosome I. A second, previously undescribed locus ACT2 mapped on chromosome V. As a 6-cM introgression in W138, the P. i. inflata act1(S6) allele behaved as a single recessive locus that fully eliminated transposition of all dTph1 elements in all stages of plant development and in a heritable fashion. Weak dTph1 activity was restored in act1S6/ACT2S6 double introgression lines, indicating that the P. i. inflata allele at ACT2 conferred a low level of transposition. Thus, the act1S6 allele is useful for simple and predictable control of transposition of the entire dTph1 family when introgressed into an ultra-high copy W138 mutator strain. We demonstrate the use of the ACT1W138/act1S6 allele pair in a two-element dTph1 transposition system by producing 10,000 unique and fixed dTph1 insertions in a population of 1250 co-isogenic lines. This Petunia system produces the highest per plant insertion number of any known two-element system, providing a powerful and logistically simple tool for transposon mutagenesis of qualitative as well as quantitative traits.     

Transposon Display identifies individual transposable elements in high copy number lines

The dTph1 transposable element family of Petunia hybrida line W138 consists of between 100 and 200 members. A strategy that allows simultaneous detection of individual elements is described. Sequences flanking dTph1 elements are amplified by means of a ligation-mediated PCR. The resulting fragments are locus-specific and can be analysed by polyacrylamide gel electrophoresis. One of the applications of Transposon Display is the isolation of dTph1-tagged genes. Fragments that co-segregate with a mutant phenotype can be extracted from the gel and reamplified, providing access to tagged genes, as demonstrated in a reconstruction experiment. Data on the molecular identification of a phenotypic mutant, isolated in a random tagging experiment is also presented. Upon sequencing, the obtained candidate fragment was found to be identical to part of the previously identified Fbp1 gene.     

The 17-kb Tam1 element of Antirrhinum majus induces a 3-bp duplication upon integration into the chalcone synthase gene

The DNA sequence of the termini and the flanking regions of the 17-kb transposable element Tam1 was determined. Tam1 is integrated in the chalcone synthase gene of the niv-53 mutant of Antirrhinum majus. The element has a 13-bp perfect inverted repeat at its termini and appears to induce a 3-bp duplication of the target site upon integration. The DNA sequence of a niv⁺ revertant was analyzed and found to differ from the wild-type sequence by an additional 2 bp that seem to derive from the target site duplication. Stretches of homologous sequences have been found between the ends of Tam1, within each terminus of the element, and between the termini and target site sequences. Structural similarities between the ends of Tam1 and the Spm-18 element of Zea mays reflect a possible horizontal spread of a common progenitor.     

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.     

Genetic analysis and molecular mapping of a pale flower allele at the W4 locus in soybean.

In soybean (Glycine max (L.) Merr.), the w4-mutable line that harbors the w4-m allele was identified in 1983. It was proposed that this line contained an autonomous transposable element at the W4 locus, which is a major locus controlling the biosynthesis of anthocyanin. The w4-m allele can revert to the W4 allele that produces the wild-type phenotype, or sometimes to other alleles that produce intermediate phenotypes. Mutant plants that produce pale flowers were identified among the progeny of a single germinal revertant event from the w4-mutable line. Through genetic analysis, we established that the pale-flower mutation was conditioned by a new allele (w4-p) at the W4 locus. The w4-p allele is dominant to the w4 allele but recessive to the W4 allele, and the w1 allele has an epistatic effect on the w4-p allele. The pale-mutant line (w4-pw4-p) was designated as Genetic Type Collection number T369. An F2 mapping population derived from the cross of Minsoy (W4W4) x T369 (w4-pw4-p) was used to map the W4/w4-p locus, using simple sequence repeat (SSR) markers. The W4 locus was located at one end of molecular linkage group D2, 2.3 cM from the SSR marker Satt386 and close to the nearby telomere.     

Genetic analysis of instability in Petunia hybrida

Summary In crossing experiments with Petunia hybrida, new mutations, some unstable, have been found in descendants of plants having an unstable allele of the anthocyanin gene An1. One of the unstable mutations affecting the new anthocyanin gene An11 was genetically analyzed, and it was subsequently established in which step of anthocyanin synthesis that An11 is involved. The discovery of new, unstable mutations at other loci indicates that in Petunia also a relation exists between unstable mutations and the presence of transposable elements in the genome. It was demonstrated that reverted alleles (an1 ^+/+) originating from unstable An1 alleles are less stable than the original wild-type allele An1, and that reversions do not increase the chances of occurrence of new, stable or unstable mutations at other loci. These results provide additional arguments in favour of the hypothesis posed in an earlier paper that reversions of unstable An1 alleles are not the result of excision of the inserted transposable element, but are due to the repair of secondary mutations induced by the insert in the regulatory region of the locus. Consequently, a reverted allele still contains the inserted element that may again induce mutations leading to inactivation of An1.     

Molecular characterization of rDt, a maize transposon of the “Dotted” controlling element system

Summary We have molecularly cloned the rDt transposon, one component of the classic Dotted two-element system of controlling elements. The rDt transposon was identified as a DNA insertion in each of two independent mutation events of the maize A1 gene, a gene necessary for the biosynthesis of anthocyanin pigment. Both mutant alleles result in a stable, anthocyaninless phenotype in all plant tissues. When the transposon Dotted, (Dt), is present in the genome each allele exhibits a characteristic mutable phenotype (spots of anthocyanin pigmentation). The DNA insertion has been designated rDt, for it responds to or is regulated by the Dt element to allow expression of the otherwise mutated gene, and it had not been named in earlier genetic studies. Sequence analysis revealed the rDt element to be an identical 704 bp insertion within the two mutable alleles, but in opposite orientation and in different exons of the gene. rDt contains an imperfect terminal inverted repeat with similarity to transposable elements of various species. A duplication of 8 bp of the target host site is formed upon integration of the element, and the element is excised from the locus in a germinal revertant. The difference in phenotype of the two unstable alleles, a1 and am-1:Cache, is discussed.     

Mobilization of a Minos Transposon in Drosophila Melanogaster Chromosomes and Chromatid Repair by Heteroduplex Formation

Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in ~75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.     

Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.     

w4-Mutable line in soybean

An unstable mutation for anthocyanin pigmentation in soybean (Giycine max [L.] Merr.) was identified in 1983. The mutability is conditioned by an allele at the w4 locus that is recessive to wild type. The population containing the mutable allele is known as the w4-mutable line. Most plants in the line have chimeric flowers with purple sectors on a near-white background. The mutable allele yields germinal revertants at a rate that varies from 5 to 10% per generation, and the revertant alleles are stable. Approximately 1% of the progenies derived from germinal revertant plants contain mutations at other loci These features, as well as the occurrence of pale flower phenotypes and changes of state, suggest that a transposable element system is producing the unstable phenotype. Several new mutants were isolated in an experiment designed to tag loci. The first three chlorophyll-deficient mutants found (CD-1, CD-2, and CD-3) are inherited as single-gene recessives. Each of the mutants lacks the same two mitochondrial malate dehydrogenase (MDH) bands. No recombination has been detected between the MDH phenotype and the chlorophyll-deficient phenotype. Genetic data indicate that the three mutants are allelic, and additional evidence suggests that each of the CD mutants is the result of a deletion. In the CD-1, CD-2, and CD-3 mutants, the deletions result in the silencing of an MDH locus, atypical chloroplast development, and an altered chlorophyll composition. Additional mutants for root necrosis, partial and near sterility, chlorophyll deficiency, and flower color isolated from the transposon tagging study have provided material for future research.     

Somatic Variegation and Germinal Mutability Reflect the Position of Transposable Element Dissociation within the Maize R Gene

The R gene regulates the timing and tissue-specificity of anthocyanin deposition during maize development. The Ac/Ds system of transposable elements was used to induce insertional mutants of the R-sc:124 allele during two cycles of mutagenesis. Of 43 unstable, spotted-aleurone mutants generated, 42 contain inserts of the Ds6 transposable element differing only in the position and orientation of the element. The remaining mutant, r-sc:m1, contained an insert of a Ds element of the approximate size of the Ds1 transposable element. The patterns of somatic variegation of these mutants, resulting from excision of Ds, define a spectrum of phenotypes ranging from sparse to dense variegation. The sparsely variegated mutants produce few germinal revertants but relatively many stable null derivative alleles; densely variegated mutants produce many germinal revertants and few stable null derivatives. Molecular analysis shows that the sparsely variegated alleles are caused by Ds6 insertions in protein coding regions of R-sc:124 whereas the densely variegated mutants result from insertions in introns or in flanking regions of the gene. The excision rate of Ds6 from R, estimated as the proportion of R genomic DNA restriction fragments lacking the element, was uniform regardless of position, orientation or whether the element was inserted in R-sc:124 or another R allele. The excision rate was greater, however, for the mutable alleles involving the Ds element from r-sc:m1. These data indicate that, although the excision rates are uniform for a given Ds element, the somatic and germinal mutability patterns of alleles associated with that element vary widely and depend primarily on the position of the transposable element within coding or noncoding regions of the gene.     

Insertion and Excision of the Transposable Element Mariner in Drosophila

The transposable element mariner is active in both germline and somatic cells of Drosophila mauritiana. Activity of the element is greatly enhanced in the presence of Mos1, a genetic factor identified as an autonomous copy of mariner. A strain of D. mauritiana containing Mos1 and other copies of mariner was used to initiate a screen for visible mutations. More than 20 mutations were obtained, including alleles of white, yellow and vermilion. Six alleles were characterized at the molecular level, and all were found to contain a mariner element inserted into the affected gene. Four insertions into the white locus were sequenced to determine the exact site of insertion of mariner. There appears to be little sequence specificity requirement for mariner insertion, other than an absolute requirement for the dinucleotide TA, which is duplicated upon insertion. Sequences of phenotypically wild-type germline and somatic revertants obtained from various white alleles, including the previously isolated wpch allele, were obtained using the polymerase chain reaction. Mariner excision is imprecise in both germline and soma, and the most frequent excision events are the same in the two tissues. Mutant derivatives of wpch were also studied, and were found to exhibit a wide range of molecular structures and phenotypes.     

Genetic characterisation of Act1, the activator of a non-autonomous transposable element from Petunia hybrida

The line W138 of Petunia hybrida has variegated flowers because it is homozygous for the mutable an1-W138 allele. Excision of the element, causing instability, depends on the presence of the activatorAct1. The previously characterised non-autonomous element dTph1 excises from the dfrC gene in response to Act1. This implies that both non-autonomous elements belong to the same transposable element family. In a range of distantly related cultivars we could detect a single functional Act1 element. Linkage analysis for 11 of these lines showed that Act1 was located on chromosome I in all cases, indicating that the element might be fixed in the genome. A group of cultivars that did not exhibit Act1 activity could be traced back to a recent common origin (Rose of Heaven). Cultivars within this group presumably harbour the same inactivated Act1 element. Among the lines tested were 7 lines representing the two species (P. axillaris and P. integrifolia) from which P. hybrida originated. None of these exhibited Act1 activity. We assume that Act1 is present in an inactive state in these lines and that it was activated upon interspecific crossing. In general, lines representing the two parental species and P. hybrida cultivars contain between 5 and 25 dTph1 elements. The lines R27 and W138, however, contain significantly more dTph1 elements (> 50) than all other lines.