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     

An altered state of a specific en regulatory element induced in a maize tiller

There are numerous states of the regulatory element, Enhancer (En). With specific receptor alleles, such as a2m(r-pa-pu) or a2m(r), specific mutability patterns are expressed. One specific derivative En allele, En-v (En-variable), was originally identified with a coarse pattern of mutability with the a2m(r-pa-pu) allele and giving progeny with varied En expression (standard to reduced within an ear progeny). Derivatives of En-v were subsequently found on numerous occasions to give only a very reduced expression (fewer mutant spots) with the a2m(r-pa-pu) allele in the ears derived from the main stalk of the corn plant. When a comparison is made of the effect of this changed En-v state between tiller ears and main stalk ears of the same plant, the tiller ears show an increased level of En-v expression (coarse pattern), while the main-stalk ears continue to show the very reduced level of En-v expression (low frequency of very late variegation). This increased level of mutability of the tiller ears is maintained when transmitted through the main-stalk ear in the subsequent generation. These results indicate that heritable alterations of controlling elements can be produced by endogenous environmental factors present during normal plant development.     

The a2m(r-Pa-Pu) Allele of the En-Controlling Element System in Maize

The Enhancer (En) controlling element system in maize includes numerous alleles, one of which, a2m(r-pa-pu), was recovered as a derivative of the a2m(1 1511) allele. The behavior and composition of this allele is described. In the presence of the independently inherited regulatory element, En, the a2m(r-pa-pu) allele mutates and expresses in the aleurone purple, pale and colorless sectors within a colorless background. In the absence of En, the pigmentation is uniformly pale. Colored and colorless germinal mutations of the a2m(r-pa-pu) allele were recovered, although the derivative alleles tested did not respond to En. Uniformly colored derivatives, designated A2', arise only from states of a2m(r-pa-pu) that respond to an active En at relatively early stages in plant development, suggesting that somatic and germinal mutations may arise through similar events. Mutations to A2-expressing alleles appear to occur relatively late in plant development, that is, at or near meiosis, since recognizable mutant sectors do not appear on testcross ears. Kernels exhibiting such mutants are randomly distributed over the ear. The resulting A2' alleles express pigmentation ranging in a continual sequential series from dark pale to intense purple.     

Enhancer transposable element induced changes at the A locus in maize: The a-m-1 6078 allele

Summary Transposable (controlling) elements in maize are highly variable in their versatility in inducing changes at any locus. With the Enhancer (En) (Suppressor-Mutator-Spm) controlling-element system,a multitude of changes result from the action of En on specific alleles. One such allele, McClintock's a-m-1 6078 allele, illustrates the diversity of events that can take place that involve three parameters; namely, spotting-timing, spotting-frequency, and background pigment formation with and without En. A series of derivative alleles has been isolated and described according to the three paramters. The multitude of changes that has been isolated illustrates the striking versatility of these transposing elements to initiate changes at the controlled locus.Journal Paper No. J-11135 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 2381     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Construction of an inducible transposon,INAc, to develop a gene tagging system in higher plants

An inducible transposable element, termed INAc (inducible Activator), was constructed for development of a gene tagging system in higher plants. The advantage of such an inducible element is that, unlike the native transposon, its excision can be induced at any time during plant development and the resulting mutants are stable after removal of the inducer. A fusion of the SA inducible promoter (PR-1a) with the Ac transposase gene was inserted together with a hygromycin resistance gene between ca. 400 bp sequences from each end of the maize Ac element, yielding INAc. The INAc element was introduced into tobacco and tomato plants. A high frequency of spontaneous transposition was apparent in primary transformed tomato calli but not in tobacco calli. Treatment of tobacco plants with salicylic acid induced transposition of INAc in both somatic and germinal tissue, with germinal transposition events being revealed by characterization of the progeny of transformed plants whose flowers were exposed to SA. The INAc element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species.     

Characterization of an Ac transposon system based on apt1-m1 (Ac) on the long arm of maize chromosome 9

Activator/Dissociation (Ac/Ds) transposable elements have been used in maize insertional mutagenesis as a complement to Mutator (Mu). In this study, to further improve the efficiency of the Ac/Ds mutagenesis system, we adopted apt1-m1 (Ac) on the long arm of chromosome 9 (9L) as a donor Ac to create an Ac insertion library. This system is based on the negative selection pressure against the donor Ac, and it was highly efficient for isolating new transposition events. We obtained 9,625 transposition events from 1083 F1 ears with an average transposition rate of 8.66?% (rates ranged from 1.11 to 29.73?%). We also adopted a modified PCR-based genome walking strategy to improve the efficiency of the new method for isolating transposon-flanking sequences. This method is more efficient than the Southern-based method that was used in previous studies. A validation step was developed to distinguish transposon tags derived from newly transposed Ac or Ds elements. Using this PCR-based method, we isolated 67 inheritable flanking sequences from the apt1-m1 (Ac) transposition library; of these, 51 were confirmed as tr-Ac-flanking sequences and 11 were tr-Ds-flanking sequences. Similar to other Ac donors from different loci, the apt1-m1 (Ac) system also exhibited a preference for short distance transposition. In this study, we have further improved the Ac mutagenesis system in maize for gene isolation and functional genomics studies.     

Genome rearrangements by nonlinear transposons in maize.

Transposable elements have long been considered as potential agents of large-scale genome reorganization by virtue of their ability to induce chromosomal rearrangements such as deletions, duplications, inversions, and reciprocal translocations. Previous researchers have shown that particular configurations of transposon termini can induce chromosome rearrangements at high frequencies. Here, we have analyzed chromosomal rearrangements derived from an unstable allele of the maize P1 (pericarp color) gene. The progenitor allele contains both a full-length Ac (Activator) transposable element and an Ac terminal fragment termed fAc (fractured Ac) inserted in the second intron of the P1-rr gene. Two rearranged alleles were derived from a classical maize ear twinned sector and were found to contain a large inverted duplication and a corresponding deficiency. The sequences at the junctions of the rearrangement breakpoints indicate that the duplication and deletion structures were produced by a single transposition event involving Ac and fAc termini located on sister chromatids. Because the transposition process we describe involves transposon ends located on different DNA molecules, it is termed nonlinear transposition (NLT). NLT can rapidly break and rejoin chromosomes and thus could have played an important role in generating structural heterogeneity during genome evolution.     

Frequent Loss of the En Transposable Element after Excision and Its Relation to Chromosome Replication in Maize (Zea Mays L.)

A model of En transposition during chromosome replication is presented following a study of somatic events associated with the transposition of En in the endosperm tissue of the maize kernel. Two supporting assays, the excision and the postexcision events, were used in following these events. The excision of the En transposon has been monitored in the starch-producing endosperm tissue by using the wx-844 autonomously mutable allele, and events after excision have been monitored by using various reporter alleles of the En-I (Spm-dSpm) system. The initial observations revealed an unusually large amount of loss of the En transposon following its excision from the wx-844 allele. Subsequent analysis of the somatic events using the a2-m1 reporter allele to monitor the dosage of En suggested that the large amount of loss would result from the transposition of En during chromosome replication. Transposition of En from a replicated segment of the chromosome to another site that has also undergone replication explains most of the somatic events observed.     

gaMS-1: A gametophytic male sterile mutant in maize

Several pollen-specific genes from different species have been isolated and characterized at the molecular level, but the precise role of most of them is unknown. Mutant analysis represents a direct approach to uncovering gene function, but the paucity of available mutants affecting pollen development and/or function and the poor characterization of the known mutants have so far limited the exploitation of this approach. Here we present the cytological characterization ofgametophytic male sterile-1 (gaMS-1), a maize mutant that we identified in a program of transposon insertion mutagenesis for the production of mutations in gametophytically acting genes involved in microsporogenesis.gaMS-1 is expressed during or immediately after the first microspore division and leads to the production of immature, nonfunctional pollen grains. The mutation appears to affect the events leading to the developmental switch that follows the first microspore mitosis.     

Germinal derivatives of the En controlling-element system in maize: Characterization of colored,pale and colorless derivatives a2-m

Summary Several stable germinal derivatives, with varied phenotypic expression have been recovered from testcrosses of four different a2 mutable alleles (a2m-7 8018, a2m-6 8144, a2m-6 8140, and a2m-1 1511) under the control of the En controlling-element system. These are colored, pale, and colorless aleurone types; the former two represent mutations of a2-mA2 type. The phenotypic variation among colored and pale derivatives has been confirmed by quantitative determination of anthocyanin content in the aleurone tissue of the kernels.The data suggest that significant variation exists between phenotypically similar colored derivatives arising from different mutable alleles as well as from the same source allele. Every tested colored derivative is significantly different from the pale, colorless, and colored control (W22 color converted line). Genetic analysis of these colored derivatives suggests that all of them contain one or more En and, hence, they are characterized as permanent changes of a nonresponsive (nr) type. Their frequencies are variable among the testcrosses.Similarly, the pale derivatives, which are clearly different from colored and colorless, also show significant differences in terms of anthocyanin content. Significant differences exist between pales arising from the same allele as well as between pales arising from different alleles. All the tested pales are significantly different from colorless and colored derivatives. Thus, the pales, as well as colored derivatives, represent a differential impairment of anthocyanin synthesis in the aleurone tissue. The genetic analysis confirmed that all the pales, except the pales of a2m-1 1511, lack the capacity to respond to En. These pale derivatives can be with or without En.Qualitative differences among the colored, pale, and colorless types have been investigated by thin-layer chromatographic and spectroscopic techniques. The results suggest that there are no qualitative differences, in terms of anthocyanin pigments, between colored, pale, and colored control. All accumulate the same anthocyanins (namely, cyanidin-3-glucoside and pelargonidin-3-glucoside in appropriate proportions depending upon the Pr and pr constitution).Journal Paper No. J-8431 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Project No. 1884: Support from NSF GB38328.     

Fusion of reverse-oriented Ds termini following abortive transposition in Arabidopsis: implications for the mechanism of Ac/Ds transposition

We studied the products of alternative transposition reactions that utilize reverse-oriented Ds termini as substrates. In this configuration, Ds transposition can generate genome rearrangements including deletions, inversions, and reciprocal translocations. In approximately half of the transposition products recovered in Arabidopsis, the termini of the reversed ends Ds element were ligated together. The sequences at these fused-end junctions suggest that the excised transposon termini form covalently closed hairpin structures. These results shed new light on the mechanism of Ac/Ds transposition.     

Unstable alleles of the singed locus in Drosophila melanogaster with reference to a transposon marked with a visible mutation

Summary The gene clw can be integrated into another site in the X chromosome to form the mutable two gene transposon system sn::Tn-clw. When brought under the common control of the transposon, sn and clw can concomitantly mutate and manifest themselves. Tn-clw was found to move from the sn region to a position of±52 m.u. This transposition was associated with changes in sn::Tn-clw:(1) loss of the instability property by the sn ⁺ allele; (2) appearance of a new lethal allele, clw ⁹, designated as a transposon allele. Based on analysis of the direction and frequency of the sn-clw mutations, the unstable genes of the sn locus were grouped in three pleiades. Interallelic mutations occurred regularly at frequencies of 0.1%–5.6% with changes in the manifestation of the clw mutation specific to each pleiade. Transition from one pleiade to another was rare (5×10^–4), and it was associated with a new phenotypic expression of the sn and clw alleles. The mutational differences between the pleiades are presumably related to differences in the localization of Tn-clw within the sn locus.It was shown that the presence of Tn promotes recombination events in the rightmost portions of the sn-oc interval.     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE STERILE (MS,) AND MALE FERTILE SOYBEANS (GLYCINE MAX (L.) MERR.)

A comparative study of microsporogenesis in fertile and in male sterile (ms₁) soybean plants (Glycine max (L.) Merr.) was conducted by using various microscopic techniques. Once the developmental pattern for fertile microsporogenesis was established, it was compared with the developmental pattern in sterile plants to determine the time of microsporogenesis breakdown. Sterility of the ms₁ mutant is caused by failure of cytokinesis after telophase II. The four nuclei resulting from meiosis become enclosed in a single-celled structure, termed a coenocytic microspore. These microspores develop a pollen-like wall and become engorged with lipid and starch reserves. Coenocytic microspores usually degenerate after engorgement. This study of fertile and sterile (ms₁) microsporogenesis has shown that nuclear and cytoplasmic events must occur at precise times for the successful development of 1n pollen grains from 2n sporogenous cells. Any disruption during this process leads to sterility.     

Chromosome labeling with transposable elements in maize

Transposable elements randomly insert into a targeted locus at a frequency of 10^-6 to 10^-5. The En element has been shown in previous studies to transpose more frequently into closely linked sites. Thus, it is appropriate to place an En element onto each of the 20 chromosome arms in maize to maximize tagging efficiency. This is called chromosome labeling for tagging purposes with transposons. After a chromosome arm has been labeled with a transposon, genes residing in that arm will have a greater chance to be tagged by the transposon. To date, all of the maize chromosome arms have been labeled with at least one of five Encontaining alleles. The elements were linked to the arms using reciprocal translocations. The usage of these arm-labeled lines is discussed in the context of gene tagging.Journal Paper No. 15224 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa; Project No. 3176     

Isolation of an IS1 flanked kanamycin resistance transposon from R1drd19

Summary We have isolated and identified an IS1-flanked transposon from the plasmid R1drd19. This transposon specifies resistance to kanamycin and is 10.4 kb long. It exhibits a frequency of transposition two orders of magnitude lower than that of the smaller, IS1-flanked transposon Tn9. We have named it Tn2350.     

Generation of rat mutants using a coat color-tagged <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposon system

A significant barrier to exploiting the full potential of the rat as a biomedical model is the lack of tools to easily modify its germline. Here we show that a tyrosinase-tagged Sleeping Beauty transposon can be used as a simple, efficient method to generate rat mutants in vivo. By making two lines of transgenic rats, one carrying the transposon and another expressing the transposase in germ cells, we are able to obtain bigenic males in which transposition occurs in the germ cells. We show that transposition leads to the appearance of new coat colors in the offspring. Using such bigenic males, we obtained an average of 1.2 transpositions per gamete and identified 19 intragenic integration events among 96 transposition sites that were sequenced. In addition, gene trapping was confirmed and rats with evidence for transposon-induced dominant ocular anomalies were identified. These data suggest that the modified Sleeping Beauty transposon represents a powerful new tool for producing molecularly defined mutagenesis in the rat.     

Maize Ac/Ds transposon system leads to highly efficient germline transmission of transgenes in medaka (Oryzias latipes)

As the medaka is a popular fish model in genetics, developmental biology and toxicology, the development of an efficient transgenic medaka technique is important for a variety of biological experiments. Here we demonstrated that the maize transposon system, Ac/Ds, greatly improved the transgenesis of microinjected DNA. Using the Ac/Ds system, two types of stable transgenic medaka lines, Tg(hsp70:gfp) and Tg(cyp1a1:gfp), were established with germline transmission rates of 83.3% (10/12) and 100.0% (4/4) from GFP-expressing founders, respectively. The percentages of transgenic progeny ranged between 3.1% and 100.0% in F1 from different transgenic founders. Interestingly, multiple insertions were found from transgenic founders and the cloned insertion sites confirmed the transposition mediated by Ac transposase. In addition, we demonstrated the inducible GFP expression in both GFP transgenic medaka lines. In Tg(hsp70:gfp) whose gfp gene was under the control of a heat shock inducible medaka hsp70 promoter, GFP expression was induced ubiquitously after heat shock. In Tg(cyp1a1:gfp), the gfp gene was driven by medaka cyp1a1 promoter that could be activated by various xenobiotic chemicals including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); indeed, GFP expression was found to be induced in the liver, intestine and kidney by TCDD. Our data presented here demonstrated the highly efficient transgenesis with the aid of the maize Ac/Ds transposon system.     

Cis requirements for transposition of Tc1-like transposons in C. elegans

The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100 bp are sufficient for transposition of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency. Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length of the transposon from 1.9 kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro. Received: 21 April 1999 / Accepted: 10 June 1999     

A segmental deletion series generated by sister-chromatid transposition of Ac transposable elements in maize

Certain configurations of maize Ac/Ds transposon termini can undergo alternative transposition reactions leading to chromosome breakage and various types of stable chromosome rearrangements. Here, we show that a particular allele of the maize p1 gene containing an intact Ac element and a nearby terminally deleted Ac element (fAc) can undergo sister-chromatid transposition (SCT) reactions that generate large flanking deletions. Among 35 deletions characterized, all begin at the Ac termini in the p1 gene and extend to various flanking sites proximal to p1. The deletions range in size from the smallest of 12,567 bp to the largest of >4.6 cM; >80% of the deletions removed the p2 gene, a paralog of p1 located ~60 kb from p1 in the p1-vv allele and its derivatives. Sequencing of representative cases shows that the deletions have precise junctions between the transposon termini and the flanking genomic sequences. These results show that SCT events can efficiently generate interstitial deletions that are useful for in vivo dissection of local genome regions and for the rapid correlation of genetic and physical maps. Finally, we discuss evidence suggesting that deletions induced by alternative transposition reactions can occur at other genomic loci, indicating that this mechanism may have had a significant impact on genome evolution.