Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco

A transposon tagging system for heterologous hosts, based on the maize En/Spm transposable element, was developed in transgenic tobacco. In this system, the two En-encoded trans-acting factors necessary for excision are expressed by fusing their cDNAs to the CaMV 35S promoter. The dSpm receptor component is inserted in the 5-untranslated leader of the bar gene. Germinal revertants can therefore be selected by seed germination on L-PPT-containing medium or by spraying seedlings with the herbicide Basta. Using this bar-based excision reporter construct, an average frequency of germinal excision of 10.1% was estimated for dSpm-S, an En/Spm native internal deletion derivative. Insertion of En-foreign sequences in a receptor, such as a DHFR selectable marker gene in dSpm-DHFR, does not abolish its capacity to transpose. However, dSpm-DHFR has a lower frequency of somatic and germinal excision than dSpm-S. Revertants carrying a transposed dSpm-DHFR element can be selected with methotrexate. Germinal excision is frequently associated with reinsertion but, as in maize, dSpm has a tendency to integrate at chromosomal locations linked to the donor site. Concerning the timing of excision, independent germinal transpositions are often found within a single seed capsule. All activity parameters analysed suggest that transposon tagging with this system in heterologous hosts should be feasible.     

The inveterate wanderer: study of Enhancer wandering on chromosome 3 in maize

The transposition of the maize transposable element Enhancer (En) had been focused on one chromosome 3 for several generations. From the a1-m(Au) allele with an autonomous En, a new En reporter allele a1-m(r)3927-1, was isolated that undergoes very infrequent and late excision events, producing one or two small spots in the aleurone. This allele is seriously impaired in its capacity to excise. Coincident with the origin of this allele, an En was located at a site close to the a1 locus. From this initial insertion site, the movement of this En was followed for three to four generations in 974 families with a higher transposition rate of this En (50% of the testcross progeny) than that found in a previous study of En transposition. This is the first case reported where a particular En was followed for more than three generations. The higher rate of wanderings of this En along the same chromosome led to the term vagabond En (En ^vag ). Genetic evidence that En may transpose from a replicated donor site to an unreplicated site is provided. Speculative mechanisms on the origin of a1-m(r)3927-1 and En ^vag are discussed.Journal Paper No. J-15864 of Iowa Agricultural and Iowa Economical Experiment Station Project #3176     

Molecular cloning of the c2 locus of Zea mays, the gene coding for chalcone synthase

Summary The c2 locus of Zea mays, identified as one of the genes affecting anthocyanin biosynthesis, was cloned using the transposable element En (Spm) as a gene tag. The Spm element present at the c2 locus in the autonomously mutating c2-m1 line was isolated using En1 element specific probes. Sequences flanking the element were identified as c2 locus specific and were used to clone the nonautonomous c2-m2 and wild-type alleles. The cloning and analysis of a cDNA complementary to the c2 locus provided evidence that this gene encodes the enzyme chalcone synthase.     

Analysis of a case of somatic instability in a strain of maize from India

A case of somatic instability affecting aleurone colour in a strain of maize from India with flint background was analysed. The somatic instability is localized to theC ¹ (Inhibitor) allele ofC locus on the short arm of chromosome 9. Molecular tests indicated thatAc is not present in the Indian stock and the evidence is consistent with the involvement of theEn (Spm) transposable element in the instability. The presence of theEn (Spm)-like element in the stock would suggest that these elements have been present in the maize genome for a long time. A new allele ofshrunken (sh1) gene with a somewhat unorthodox breeding behaviour is also described.     

Identification ofTnr3, aSuppressor-Mutator/Enhancer-like transposable element from rice

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion. A 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1536 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5-CACTA--- -3; these repeats are similar to those found in members of theEn/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to theEn/Spm family and is thus namedTnr3 (transposable element inrice no.3). In fact,Tnr3 carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of theEN/Spm family. The subterminal repeat sequences inTnr3 are, however, of two kinds, although they share homology with each other.Tnr3 and its relatives were present in multiple copies in rice. Considering the length ofTnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.     

Spm and I element changes with the a-m2 8004 allele in maize

Summary Among the mobile element systems in maize, the En (Spm) system (En — the regulatory element and I the receptive element — a nonfunctional En) has several interesting aspects of control of gene expression (En and Spm are homologous in structure and activity). One of the alleles arising from the Spm group included the a-m2 8004 allele that has a low spotting pattern and unique ringed areas. The interest in this allele is that Spm or En will induce it to co-express the A phenotype and mutability. Several exceptions of the allele were analyzed. Two are Spm changes and two are I changes. The analysis shows that the heritable changes include I changes that are co-expressed in various grades of color and different degrees of mutability. All these changes occur with I at the locus. The Spm changes also include changes in mutability patterns and a mottling pattern.Journal Paper No., J-11792 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

The role of subterminal sites of transposable element Ds of Zea mays in excision

Transposition depends on DNA sequences located at or near the termini of the transposon. In the maize transposable element Ds, these sequences were studied by site-directed mutagenesis followed by a transient excision assay in Petunia protoplasts. The transposase-binding AAACGG motifs found in large numbers in the element are important, but none of them is in itself indispensable, for excision. However, mutation of an isolated motif at the 3 end considerably reduced excisability. The inverted termini were confirmed to be indispensable. Point mutations in regions outside the inverted termini of Ds and not located in the transposase-binding motifs had, in some cases, a pronounced effect on excision frequency. The implications of these findings are discussed.     

Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture

Summary Previous experiments have revealed that the maize transposable element Activator (Ac) may become active during tissue culture. The objective of the present study was to determine whether a second transposable element, Suppressor-mutator (Spm), could also be activated in tissue culture and detected in regenerated maize plants. Approximately 500 R₁ progeny of 143 regenerated plants (derived from 49 embryo cell lines) were crossed as males onto an Spm-responsive tester stock. Spm activity was observed in two R₁ progeny of a single regenerated plant. This plant had been regenerated from Type II (friable embryogenic) callus of an A188 × B73 genetic background after 8 months in culture; the absence of Spm activity in four other plants regenerated from this same callus demonstrates that Spm activity was not present before culturing. Approximately 20 Spm-homologous DNA sequences were detected in each of the inbreds used to initiate the tissue cultures; it is presumed that one of these became active to give rise to Spm activity.     

Structural analysis of Tpn1, a transposable element isolated from Japanese morning glory bearing variegated flowers

The 6.4 kb transposable element Tpn1 belonging to the En/Spm family was found within one of the DFR (dihydroflavonol-4-reductase) genes for anthocyanin biosynthesis in a line of Japanese morning glory (Pharbitis nil) bearing variegated flowers. Sequencing of the Tpn1 element revealed that it is 6412 by long and carries 28-bp perfect terminal inverted repeats. Its subterminal repetitive regions, believed to be the cis-acting sequences for transposition, show striking structural features. Twenty-two copies of the 10-bp sequence motif GACAACGGTT can be found as direct or inverted repeats within 650 by of the 5 end of the element, and 33 copies of the sequence motif lie within 800 by of the 3 terminus. All these 22 copies of the sequence motif near the 5 terminus and 30 copies in the 3 terminal region are arranged as inverted repeats and 3–8 by AT-rich sequences are detected between these inverted repeats. In addition, four copies of 122-bp tandem repeats and six copies of 104-bp tandem repeats are present in the 5 and 3 subterminal repetitive regions, respectively. No large open reading frame characteristic of autonomous elements of the En/Spm family can be detected within the element. The results are discussed with respect to heritable changes in flower variegation in this line of Japanese morning glory.     

Excision and transposition of two Ds transposons from the bronze mutable 4 derivative 6856 allele of Zea mays L

Summary The regulatory mutation bronze mutable 4 Derivative 6856 (bz-m4 D6856) contains a complex 6.7 kb Dissociation (Ds) element tagged with a duplication of low copy bz 3 flanking sequences (Klein et al. 1988). This creates a unique opportunity to study the transposition of a single member of the repetitive family of Ds elements. Eighteen full purple revertants (Bz alleles) of bz-m4 were characterized enzymatically and by genomic mapping. For 17 of the Bz alleles, reversion to a wild-type phenotype was caused by excision of the 6.7 kb Ds transposon. Nine of these Bz alleles retained the transposon somewhere in their genome. In this study we show that like Ac (Schwartz 1989; Dooner and Belachew 1989), the 6.7 kb Ds element can transpose within a short physical distance, both proximal and distal to its original position. Additional bz sequences have been mapped immediately distal to the mutant locus in bz-m4 D6856; genetic evidence suggests these are flanked by two additional Ds elements. The remaining Bz revertant, Bz :107, arose from excision of a more complex 13 kb Ds element.     

Transposition of the En/Spm transposable element system in maize (Zea mays L.): reciprocal crosses of a1-m(Au) and a1-m(r) alleles uncover developmental patterns

Transposition studies of the transposon, En/Spm, have dealt with general aspects of the timing of the excision event with regard to DNA replication and plant development, but without describing details of the process. By following the excision events of an En transposon inserted at the a1 locus [a1-m(Au)], several features of this process can be elucidated. In progenies from reciprocal crosses between the a1-m(Au) allele containing an En insert, and a nonautonomous En allele, [a1-m(r) is a deficiency derivative of En], several features of the En at the a1-m(Au) allele can be observed taking place during ear development and during microsporogenesis. First, it has long been known that the distribution of mutant kernel phenotypes on an ear indicates that En transposes late in most of the events during ear development. Second, the phase change of En (presence and absence of activity) is observed during cob development. Third, discordant kernel phenotypes of two ears, reported herein, resulting from a reciprocal cross with the parental phenotype can be deduced to arise from the transposition of En during microsporogenesis and subsequent fertilization, leading to a discordant genotype between endosperm and embryo. The phase change and discordance lead us to conclude that these events can arise from transposition after host DNA replication. It can also be concluded that the activity of the En inserted in this a1-m(Au) allele is not limited to a specific stage or timing during plant development. Further, this study illustrates the power of genetic analysis in the determination of cellular events. Received: 26 May 1999 / Accepted: 11 November 1999     

The maize transposable element Ac is mobile in the legume Lotus japonicus

To evaluate the prospects for transposon mutagenesis in the autogamous diploid legume Lotus japonicus, the behaviour of the maize transposable element Ac was analysed in the progeny of 38 independent transgenic plants. The conditions for monitoring donor site excision using histochemical localization of -glucuronidase activity or the alternative spectinomycin resistance assay were established, and used to follow Ac mobility through two generations. Somatic excision was monitored as variegated cotyledons in the T₂ generation and germinal excision events were scored in segregating T₃ families as complete -glucuronidase-mediated staining of cotyledons or as a fully green spectinomycin-resistant phenotype. Using these assays an average germinal excision frequency of 12% was estimated in the T₃ offspring from variegated plants. The fidelity of the excision assays was ascertained by comparing the frequency of germinal excision to the frequency of Ac reinsertion at new positions of the genome. Transposition of Ac in 42% of the plants and detection of the characteristic Ac insertion/excision footprints suggests that insertion mutagenesis with the autonomous maize Activator element is feasible in Lotus japonicus. Parameters influencing Ac behaviour, such as dosage, position effects and modification of the element itself, were also investigated comparing homozygous and hemizygous plants from the same family and by analysing different transformants.Abbreviations W white - V variegated - FG fully green - FB fully blue - aadA spectinomycin adenyltransferase     

Mechanism of Ds1 excision from the genome of maize streak virus

Summary We have previously shown that the maize transposable element Ds1 introduced into maize plants by agroinfection can be excised from the genome of geminivirus maize streak virus (MSV). Excision depended strictly on the presence of an active Ac element in the plants. In this study, the excision products or footprints left in the MSV genome after Ds1 excision were extensively characterized and the effects of flanking sequences on Ds1 excision were analysed. Most types of footprints obtained were comparable to those described for Ds1 excision in the maize genome, and could be explained by the models proposed for excision of plant transposable elements. In two revertants, however, some terminal sequences of the Ds1 element were found to have been left behind at the excision site. The finding of this novel type of Ds1 footprint indicated that gene conversion events occurred during and/or after Ds1 excision from the MSV genome. A partial deletion of one copy of the 8 by duplications flanking the Ds1 element had no effect on the frequency or on the types of footprints of Ds1 excision from the MSV genome. Thus, the duplicated 8 by sequences flanking the transposable element are not involved in Ds1 excision. These results, as well as a statistical analysis of the modifications of the bases flanking the Ds1 element after excision, are discussed in terms of excision models.     

Conservation of IS66 homologue of octopine Ti plasmid DNA in Rhizobium fredii plasmid DNA

Summary DNA sequences homologous to the T-DNA region of the octopine Ti plasmid from Agrobacterium tumefaciens are found in various fast-growing Rhizobium fredii strains. The largest fragment (BamHI fragment 2) at the right-boundary region of the core T-DNA hybridizes to more than one plasmid present in R. fredii. However, one smaller fragment (EcoRI fragment 19a) adjacent to the core T-DNA shows homology only with the plasmid carrying the symbiotic nitrogen-fixation genes (pSym). Hybridization data obtained with digested R. fredii USDA193 pSym DNA suggests that the homology is mainly with two HindIII fragments, 1.7 kb and 8.8 kb in size, of the plasmid. The 1.7 kb HindIII fragment also hybridizes to two regions of the virulence plasmid of A. tumefaciens, pAL1819, a deletion plasmid derived from the octopine Ti plasmid, pTiAch5. Hybridization studies with an insertion element IS66 from A. tumefaciens indicate that the 1.7 kb HindIII fragment of R. fredii plasmid, homologous to the T-DNA and the virulence region of Ti plasmid, is itself an IS66 homologue.     

In vitro propagation of the Greek olive cultivar `Chondrolia Chalkidikis'

The effect of basal media (Woody Plant Medium (WPM), Quoirin and Lepoivre (QL) and Olive Medium (OM)) and of various concentrations of cytokinins (6-benzyladenine (BA), zeatin, 6-(,-dimethylallyl-amino) purine (2iP)), solely or in combinations with each other and with gibberellic acid (GA₃), on in vitro shoot proliferation of the greek olive cultivar `Chondrolia Chalkidikis' was investigated. WPM proved to be the most effective one, resulting in better morphological appearance of the microshoots produced. The highest number of new microshoots/explant (1.68), with a 3.0 cm shoot height and a 4.2 proliferation rate, was obtained when this medium was supplemented with 20 M zeatin. The number of microshoots/explant and proliferation rate increased to 1.85 and 6.8, respectively, by using the combination of 5–20 M zeatin with 1 M BA, but shoot height was reduced. 2iP was the least effective of the cytokinins tested. Combination of 20 M zeatin with 10 M GA₃ affected positively shoot proliferation resulting in 1.80 microshoots/explant, 3.0 cm shoot height and 7.0 proliferation rate. However, the same concentration of GA₃ in combination with 1 M BA reduced the number of new microshoots/explant (0.48) as well as the proliferation rate (2.4), although shoot height remained nearly the same (2.7). The effect of indole-3-butyric acid (IBA), -naphthaleneacetic acid (NAA) and putrescine on root induction was also studied. A rooting percentage of up to 70% and 2.3 roots/microshoot were achieved by the combination of 12 M IBA + 3 M NAA, however, abscission of shoot tips and leaves appeared. Both variables were increased, up to 93% and 4.0, respectively, by the addition of 30 M putrescine in the medium, without any undesired side effect. After acclimatization, survival of rooted microshoots was high (90%) while that of non-rooted ones was low (30%).     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Establishment of a gene tagging system in Arabidopsis thaliana based on the maize transposable element Ac

Summary An Ac-derived, two-component transposable element system has been developed and analyzed with respect to its use in Arabidopsis thaliana. This system consists of an immobilized Ac element (Ac clipped wing, Ac_cl) as the source of transactivating transposase and a nonautonomous Ds element, Ds_A, which is inserted into a chimaeric neomycinphosphotransferase gene used as excision marker. After separate introduction of Ac_c1 and Ds_A into Arabidopsis thaliana, progeny analysis of crosses between five different Ac_cl lines and seven different Ds_A lines shows that: (1) different Ac_cl lines differ greatly in their capacity to transactivate Ds_A; (2) different Ds_A lines do not differ significantly with respect to Ds_A transactivation by one Ac_cl line; (3) reintegration of excised Ds_A elements, both at (genetically) linked and unlinked sites, occurs in about 50% of the excision events; and (4) plants with a high rate of somatic excisions can be used as source of new Ds_A transpositions, allowing the creation of a large number of independent Ds_A insertions.     

Molecular cloning of the o2-m5 allele of Zea mays using transposon marking

Summary The deposition of zein protein in maize endosperm is under the control of several regulatory loci. The isolation of DNA sequences corresponding to Opaque-2 (O2), one of such loci, is described in this paper. The mutable allele, o2-m5 was first induced moving the Ac transposable element present at the wx-m7 allele to the O2 locus. Genetic data suggest that a functional Ac element is responsible for the observed somatic mutability of o2-m5. The isolation of genomic clones containing flanking sequences corresponding to the O2 gene was possible by screening an o2-m5 genomic libary with a probe corresponding to internal Ac sequences usually absent in the defective element Ds. Out of 27 clones isolated with homology to the central part of Ac element, only clones 6IP and 21IP generated a 2.5 kb internal fragment size of an active Ac element when digested with PvuII restriction enzyme. A sequence representing a XhoI fragment of 0.9 kb lying, in the 6IP clone, adjacent to the Ac elements, was subcloned and utilized to prove that it corresponded to a part of the O2 gene. To obtain this information we made use of: (1) DNAs from several reversions originating from the unstable (o2mk-(r) allele, which, when digested with SstI, showed a correct 3.4 kb fragment typical of non-inserted alleles of the O2 locus; and (2) recessive alleles of the O2 locus which were devoid of a 2.0 kb mRNA, present on the contrary in the wild type and in other zein regulating mutants different from O2.This paper is dedicated to the memory of R. Marotta, who actively participated in the realization of this work     

P element excision in Drosophila melanogaster and related drosophilids

Summary The frequency of P element excision and the structure of the resulting excision products were determined in three drosophilid species, Drosophila melanogaster, D. virilis, and Chymomyza procnemis. A transient P element mobility assay was conducted in the cells of developing insect embryos, but unlike previous assays, this mobility assay permitted the recovery of excision products from plasmids regardless of whether the excision event was precise or imprecise. Both quantitative and qualitative differences between the products of excision in the various species studied were observed. The frequency with which P element excision products were recovered from D. melanogaster was 10-fold greater than from D. virilis and C. procnemis; however, the proportion of all excision events resulting in the reversion of a P-induced mutant phenotype was the same. Virtually all excision products recovered, including those resulting in a reversion of the mutant phenotype, did not result in the exact restoration of the original target sequence. Sequence analysis suggested that duplex cleavage at the 3 and 5 termini of the P element, or their subsequent modification, occurred asymmetrically and interdependently. P element-encoded transposase was not absolutely required for P element excision.