Newly activated germinal Uq elements in maize are clustered on one linkage group independently of the standard Uq element

Summary Allelism tests between the standard Uq element (Uq1) and five newly activated germinal Uq elements (Uq2, Uq3, UQ4, Uq5, and Uq6) demonstrate that these new Uq elements are independent of Uq1. Gametes that either contain one Uq or various combinations of two different and phenotypically distinguishable Uq elements, have been constructed either with or without the a-ruq reporter allele. Genetic analyses of the progenies of the gametes (using the standard a-ruq tested line as the other parent) have indicated that (i) each Uq element, when present alone, has the capacity to express full activity except when a secondary transposition or loss of activity has occurred; (ii) all five new Uq elements are independent of Uq1 with respect to transposition activity; and (iii) these newly originated Uqs are clustered on one linkage group. Uq2 is allelic to Uq4, and Uq3 is allelic to Uq5, whereas Uq6 is linked to both allelic pairs. A putative linkage map of these Uq elements is presented. In reciprocal crosses there is a striking difference in phenotypic segregation of Uq; when transmitted via the male parent Uq loses full expression capacity.     

Molecular analysis of the Ubiquitous (Uq) transposable element system of Zea mays.

Summary The Uq transposable element of maize is the most widely dispersed among different maize populations and genetic testerstrains. Despite intensive genetic characterization, little is known about its molecular structure. In order to obtain information relevant to this topic, we have cloned and sequenced three ruq receptors. Surprisingly, they are all Ds1-like receptor types of the Ac-Ds transposon family. Based on our molecular data, we present a model to explain the functional differences associated with the differential expression of the Uq and Ac transposon systems.     

A novel class of <Emphasis Type="Italic">Helitron</Emphasis>- related transposable elements in maize contain portions of multiple pseudogenes

We recently described a maize mutant caused by an insertion of a Helitron type transposable element (Lal, S.K., Giroux, M.J., Brendel, V., Vallejos, E. and Hannah, L.C., 2003, Plant Cell, 15: 381–391). Here we describe another Helitron insertion in the barren stalk1 gene of maize. The termini of a 6525 bp insertion in the proximal promoter region of the mutant reference allele of maize barren stalk1 gene (ba1-ref) shares striking similarity to the Helitron insertion we reported in the Shrunken-2 gene. This insertion is embedded with pseudogenes that differ from the pseudogenes discovered in the mutant Shrunken-2 insertion. Using the common terminal ends of the mutant insertions as a query, we discovered other Helitron insertions in maize BAC clones. Based on the comparison of the insertion site and PCR amplified genomic sequences, these elements inserted between AT dinucleotides. These putative non-autonomous Helitroninsertions completely lacked sequences similar to RPA (replication protein A) and DNA Helicases reported in other species. A blastn analysis indicated that both the 5 and 3 termini of Helitrons are repeated in the maize genome. These data provide strong evidence that Helitron type transposable elements are active and may have played an essential role in the evolution and expansion of the maize genome.     

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.     

A candidate autonomous version of the wheat MITE <Emphasis Type="Italic">Hikkoshi</Emphasis> is present in the rice genome

A miniature inverted-repeat transposable element (MITE), designated as Hikkoshi, was previously identified in the null Wx-A1 allele of Turkish bread wheat lines. This MITE is 165 bp in size and has 12-bp terminal inverted repeats (TIRs) flanked by 8-bp target site duplications (TSDs). Southern and PCR analyses demonstrated the presence of multiple copies of Hikkoshi in the wheat genome. Database searches indicated that Hikkoshi MITEs are also present in barley, rice and maize. A 3.4-kb element that has Hikkoshi-like TIRs flanked by 8-bp TSDs has now been identified in the rice genome. This element shows high similarity to the 5 subterminal region of the wheat Hikkoshi MITE and contains a transposase (TPase) coding region. The TPase has two conserved domains, ZnF_TTF and hATC, and its amino acid sequence shows a high degree of homology to TPases encoded by Tip100 transposable elements belonging to the hAT superfamily. We designated the 3.4-kb element as OsHikkoshi. Several wheat clones deposited in EST databases showed sequence similarity to the TPase ORF of OsHikkoshi. The sequence information from the TPase of OsHikkoshi will thus be useful in isolating the autonomous element of the Hikkoshi system from wheat.     

Reactivation of the Mutator transposable element system following gamma irradiation of seed

Summary The bz2-mu1 allele contains a 1.4 kb Mu element insertion in the open reading frame of the bronze-2 locus. This insertion suppresses gene activity. In an active Mutator line, however, the bz2-mu1 allele shows high somatic instability resulting in numerous purple spots of full gene activity against a beige background in the aleurone tissue of the kernel; restoration of gene activity results from excision of the Mu element. In contrast, in plants with an inactive Mutator system, uniformly bronze kernels are found, and the Mu element at bz2-mu1 is stabilized. Accompanying a loss of somatic instability, this Mu element, as well as the Mu elements elsewhere in the genome, have an increased level of DNA modification. Spontaneous reactivation of somatic instability in inactive Mutator lines rarely occurs; however, reactivation can be induced with gamma irradiation. Reactivated plants regain both the spotted kernel phenotype indicative of element excision from the bz2-mu1 reporter allele and diagnostic restriction sites within the Mu elements indicative of a hypomethylated state. The reactivated plants transmit these characters to their progeny. These data support the hypothesis that genomic shock can elicit cryptic transposable element activities in maize. Possible mechanisms for inactivation and reactivation of the Mutator transposable element system are also discussed.     

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     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

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.     

A maize Ds transposable element containing a dihydrofolate reductase gene transposes in Nicotiana tabacum and Arabidopsis thaliana

Summary A mouse dihydrofolate reductase gene (DHFR), encoding an enzyme conferring methotrexate (MTX) resistance, under the control of the cauliflower mosaic virus (CaMV) 35 S promoter, was inserted within a maize nonautonomous Ds transposable element. The presence of at least one element (Ds-DHFR) can easily be monitored using methotrexate selection in plants. This chimeric element is able to transpose at a frequency similar to its unmodified progenitor in transgenic tobacco callus containing an autonomous Ac element. The orientation of the selectable marker cassette in the Ds element does not affect relative excision frequencies. Approximately two-thirds of these elements can be detected after excision while the remaining one-third cannot. The Ds-DHFR element is useful in elucidating the mechanism by which Ac/Ds transposition occurs, and allows for a rapid identification of mutants in which methotrexate resistance cosegregates with a mutant phenotype.     

Germinal reversion of an unstable mutation for anthocyanin pigmentation in soybean

Summary Plants of the w4-mutable line of soybean [Glycine max (L.) Merr.] are chimeral for anthocyanin pigmentation. Mutable plants produce both near-white and purple flowers, as well as flowers of mutable phenotype with purple sectors on near-white petals. It is established here that the mutable trait is conditioned by an unstable recessive allele of the w4 locus that conditions anthocyanin biosynthesis. The gene symbol w4-m is assigned to the mutable allele. Allele w4-m was derived from a stable, wild-type W4 progenitor allele and reverts at high frequency to a stable, wild-type W4 allele. Reversion occurs both early and late during the development of the germ line. Several experiments give estimates of germinal reversion frequency, indicating that approximately 6% of mutable alleles revert to wild-type from one generation to the next. Allele w4-m exhibits many features typical of an allele controlled by a transposable element.     

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     

A possible hot spot for Ac insertion in the maize P gene.

Summary We have analyzed the footprints left by a single Ac transposable element during its intragenic transposition to different positions in the maize P gene. One site appears to have been visited twice by transposons, indicating that it may be an insertion hot spot. Implications of this finding for the origin of the P-vv allele are discussed. Analysis of transposon footprints may prove generally useful for establishing pedigree relationships among gene alleles.     

Cre recombinase expression can result in phenotypic aberrations in plants

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Some plants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct pattern of chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression, aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severity of the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing of cre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severe phenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype was observed when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for the application of recombinases in plants, expression is best limited to specific tissues and a short time frame.[12pt] Abbreviations: bar, the phosphinotricin acetyltransferase gene; CAM, chloramphenicol resistance gene; Ds 5 & Ds 3, borders of the Ds transposable element from maize forming a functional transposable element that embodies the interjacent DNA; gus, the -glucoronidase gene; gus-int, the gus gene interrupted by a plant intron; hpt, the hygromycin phosphotransferase gene; nptII, the neomycin phosphotransferase gene; ORI, bacterial origin for plasmid replication in Escherichia coli of plasmid p15A     

DNA methylation in the Alcohol dehydrogenase-1 gene of maize

Using a battery of methylation-sensitive restriction enzymes, cytosine methylation at 23 sites in a 7.6 kb region surrounding the Alcohol dehydrogenase-1 (Adh1) gene was measured in DNA prepared from immature maize cobs. Both the 5 upstream region and the entire coding region were hypomethylated in the two alleles examined. Methylation in Adh1 is independent of changes in Mutator transposable element methylation. The role of DNA methylation in Adh1 gene regulation is discussed.     

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.     

Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This transposition-deletion system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.     

Attempted “egg transformation” in Zea mays L., using irradiated pollen

Summary Experiments were conducted to determine if egg transformation could be achieved in Zea mays L. as described by Pandey in Nicotiana L. Multiple recessive and multiple dominant marker stocks were employed, as well as a tester and a donor line for the En transposable element. Recipient tester females were pollinated with dominant donor pollen, which was applied in several treatment combinations. The pollination treatments included: 1) pollen irradiated at 20, 30, 40, 80, and 100 Krad; 2) pollen irradiated with the same doses, mixed with non-irradiated recipient pollen; 3) pollen irradiated at 80 Krad, followed by self pollination delayed 18 h; 4) non-irradiated donor pollen mixed with non-irradiated recipient pollen. Zero seed were produced from 100 pollinations with irradiated pollen. There were 258 pollinations made with irradiated donor plus self pollen mixtures, producing over 21,300 seed. Of these seed, 3 were unexpected. One was clearly from pollen contamination, one was clearly derived from a pre-meiotic mutation, and the third occurred as a mutant sector in the seed's endosperm. There were 56 pollinations with non-irradiated pollen mixtures, producing over 5,000 seed. Among these seed, there were 7 unexpected seed. Three of these were clear-cut cases of heterofertilization. Four progeny were dominant for all seed and seedling markers except one endosperm marker. These cases appear to represent spontaneous recessive endosperm mutations. More than 59,000 potential transformation events were screened producing only 6 apparent mutations. It is concluded that if egg transformation occurs in Zea mays, it is a very rare event, and is not likely to be useful in corn improvement.Approved by the Director of the New York State Agricultural Experiment Station for publication as Journal Paper No. 3556     

Characterization of the molecular defect in a null allele of the opaque-2 locus of maize

The molecular defect in an opaque-2 (o2) mutant, previously characterized as a null allele, has been identified as containing an insertion of the transposable element of the Bergamo (Bg) family. Restriction mapping and partial sequence analysis of the Bg in the o2 null allele indicates that this element is distinct from the previously described Bg as well as the defective Bg (rbg) of the o2m(r) allele. It is, however, inserted at the same site in O2 as the rbg of 62m(r) and can transpose when Bg is present. This study shows that, depending on genetic background, this allele may not behave as a stable null which could dramatically influence the conclusions drawn from experiments based on this particular mutant.     

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.