Transposable Element System Research Articles

Transpositionally active episomal hAT elements

BackgroundhAT elements and V(D)J recombination may have evolved from a common ancestral transposable element system. Extrachromosomal, circular forms of transposable elements (referred to here as episomal forms) have been reported yet their biological significance remains unknown. V(D)J signal joints, which resemble episomal transposable elements, have been considered non-recombinogenic products of V(D)J recombination and a safe way to dispose of excised chromosomal sequences. V(D)J signal joints can, however, participate in recombination reactions and the purpose of this study was to determine if hobo and Hermes episomal elements are also recombinogenic.ResultsUp to 50% of hobo/Hermes episomes contained two intact, inverted-terminal repeats and 86% of these contained from 1-1000 bp of intercalary DNA. Episomal hobo/Hermes elements were recovered from Musca domestica (a natural host of Hermes), Drosophila melanogaster (a natural host of hobo) and transgenic Drosophila melanogaster and Aedes aegypti (with autonomous Hermes elements). Episomal Hermes elements were recovered from unfertilized eggs of M. domestica and D. melanogaster demonstrating their potential for extrachromosomal, maternal transmission. Reintegration of episomal Hermes elements was observed in vitro and in vivo and the presence of Hermes episomes resulted in lower rates of canonical Hermes transposition in vivo.ConclusionEpisomal hobo/Hermes elements are common products of element excision and can be maternally transmitted. Episomal forms of Hermes are capable of integration and also of influencing the transposition of canonical elements suggesting biological roles for these extrachromosomal elements in element transmission and regulation.

ROSINA (RSI) is part of a CACTA transposable element, TamRSI, and links flower development to transposon activity

ROSINA (RSI) was isolated as a DNA binding factor able to bind to the CArG-box present in the promoter of the MADS-box gene DEFICIENS of Antirrhinum majus. The mosaic nature of RSI and its multi-copy presence in the A. majus genome indicated that RSI could be a part of a mobile genetic element. Here we show that RSI is a part of a CACTA transposable element system of A. majus, named TamRSI, which has evolved and is still evolving within the terminal inverted repeats (TIRs) of this CACTA transposon. Interestingly, RSI is always found in opposite orientation with respect to the transcription of a second gene present within the CACTA transposon, which encodes a putative TRANSPOSASE (TNP). This structural configuration has not yet been described for any member of the CACTA transposons superfamily. Internal deletion derivatives of the TamRSI produce aberrant RSI transcripts (RSI-ATs) that carry parts of the RSI RNA fused to parts of the TNP RNA. In addition, an intriguing seed phenotype shown by RNAi transgenic lines generated to silence RSI, relate TamRSI to epigenetic mechanisms and associate the control of flower development to transposon activity.

A barley activation tagging system

Activation tagging, as the result of random genomic insertion of either promoter or enhancer sequences, can produce novel, dominant mutations by over-expression of endogenous genes. This powerful genomics tool has been used extensively in dicot species such as Arabidopsis, while rice is the only cereal for which an equivalent system exists. In this study we describe an activation tagging system in barley based upon the maize Ac/Ds transposable element system. A modified Ds element (UbiDs) containing two maize polyubiquitin promoters, transposed in families derived from multiple independent UbiDs transformants and generated new Ds insertion events at frequencies ranging from 0% to 52% per family. The majority of transposed UbiDs elements activated high levels of adjacent flanking sequence transcription. Transposon-mediated expression was detected in all barley cell and tissue types analysed suggesting that this system is applicable to all aspects of plant development and biogenesis. In addition to transcriptional activation, this system is also capable of generating insertional knockout mutants and a UbiDs inactivated allele of the granule bound starch synthase I gene (waxy) was recovered that lead to reduced amylose accumulation. The recovery and analysis of dominant over-expression phenotypes generated by this system will provide a novel approach to understanding gene function in large cereal genomes where gene redundancy may mask conventional loss-of-function mutations.

Differentiation of a Miniature Inverted Transposable Element (MITE) System in Asian Rice Cultivars and Its Inference for a Diphyletic Origin of Two Subspecies of Asian Cultivated Rice

AbstractIn the present study, we report a survey on a Miniature Inverted Transposable Element (MITE) system known as mPing in 102 varieties of Asian cultivated rice (Oryza sativa L.). We found that mPing populations could be generalized into two families, mPing‐1 and mPing‐2, according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice, namely O. sativa ssp. japonica and indica. O. sativa japonica has a higher proportion of mPing‐1 as a general trait, whereas O. sativa indica has a higher proportion of mPing‐2. We also examined the mPing system in a doubled haploid (DH) cross‐breeding population of jingxi 17 (japonica) and zhaiyeqing 8 (indica) varieties and observed that the mPing system was not tightly linked to major subspecies‐determining genes. Furthermore, we checked the mPing system in 28 accessions of Asian common wild rice O. rufipogon and found the mPing system in O. rufipogon. The distribution pattern of the mPing system in O. rufipogon indicated a diphyletic origin of the Asian cultivated rice O. sativa species. We did not find the mPing system in another 20 Oryza species. These results substantiated a previous hypothesis that O. rufipogon and O. nivara species were the closest relatives of O. sativa and that the two extant subspecies of O. sativa were evolved independently from corresponding ecotypes of O. rufipogon.(Managing editor: Li‐Hui Zhao)

A modifier of the Bg element of the Bg-rbg transposable element system of maize

A modifier of the Bg autonomous element of the Bg-rbg system of transposable elements has been found in the genotype of the inbred maize strain 346. In the presence of this modifier (termed Mbg), the frequency of reversion of mutable allele o2-lf in combination with the Bg-lf element increases by 7-24 times. An increase in the Mbg dosage by three times increases the o2-lf reversion frequency by a factor of about two. The presence of Mbg and Bg-lf in the same genotype before meiosis is necessary for the expression of the Mbg modifying effect. The possible nature and mechanism of action of the novel modifier are discussed.

Horizontal Transfer of a Plant Transposon

The majority of well-documented cases of horizontal transfer between higher eukaryotes involve the movement of transposable elements between animals. Surprisingly, although plant genomes often contain vast numbers of these mobile genetic elements, no evidence of horizontal transfer of a nuclear-encoded transposon between plant species has been detected to date. The most mutagenic known plant transposable element system is the Mutator system in maize. Mu-like elements (MULEs) are widespread among plants, and previous analysis has suggested that the distribution of various subgroups of MULEs is patchy, consistent with horizontal transfer. We have sequenced portions of MULE transposons from a number of species of the genus Setaria and compared them to each other and to publicly available databases. A subset of these elements is remarkably similar to a small family of MULEs in rice. A comparison of noncoding and synonymous sequences revealed that the observed similarity is not due to selection at the amino acid level. Given the amount of time separating Setaria and rice, the degree of similarity between these elements excludes the possibility of simple vertical transmission of this class of MULEs. This is the first well-documented example of horizontal transfer of any nuclear-encoded genes between higher plants.

Spotting factor (Spf) from the Spotted-dilute System in Maize Is a Member of the En/Spm Controlling Element Family

The Spotted-dilute controlling element system in maize involves an autonomous Spotting factor (Spf), and a receptor at the r1 locus haplotype R1-r(spotted dilute2). Its relationship with other maize transposable element systems is poorly characterized. Through development of a genetic tester that carries receptors for both the Spotted-dilute and the En/Spm controlling element systems, we determined that both receptors respond equally to Spf and En/Spm and that Spf is therefore a member of the En/Spm family of controlling elements.

Control of the Frequency of Reversion of the Mutable Alleles at the opaque2 Locus by the Bg-rbg System of Transposable Elements in Maize

Maize lines differing in the frequency of reversion of the opaque2 (o2) mutable alleles controlled by the Bg-rbg system of transposable elements were studied. In the presence of the Bg regulatory element, these alleles can revert to normal. When reversion occurs prior to the first division of the primary endosperm nucleus, phenotypically normal kernels or whole endosperm revertants (WER) develop. It was shown that the low frequency of formation of whole endosperm revertant may be produced by different genetic mechanisms. The frequency of WER formation was shown to nonlinearly depend on the dose of the Bg-hf regulatory element. A dose increase from two to three failed to cause an essential increase in the number of revertants. The regulatory elements Bg-lf andBg-hfdiffered in their ability to induce excision of the receptor element at the same dose. The frequency of reversion of the receptive alleles was shown to be regulated by epigenetic mechanisms so that high frequency of reversion of receptive alleles requires preliminary premeiotic association between the regulatory and receptor elements. The inheritance of the maize alleles o2-hfand o2-lf proved to be similar to that of an3 mutable alleles in petunia.

Trans-activation of the maize transposable element, Ds, in Brassica napus.

A two-component transposable element system consisting of a stabilized Activator ( Ac(st)) and a chimeric Dissociation ( Ds) element has been introduced into the genome of Brassica napus. Analyses performed on F2 progeny derived from crosses between Ac(st)- and Ds-bearing parents confirm that Ac transposase catalyzes the somatic excision of the Ds element in both embryonic and non-embryonic tissues of this important crop species. The data further reveal that the vast majority of plants containing both Ac(st) and Ds exhibit Ds excision. However, the level of excision is low and germinal Ds excision events are not observed. We estimate that germinal excision of Ds occurs at a frequency of < 0.2%. RT-PCR analysis of the Ac(st) transcript in somatically active seedlings reveals that introns III and IV are highly misprocessed. The pattern of transcript processing is very similar to that observed in germinally inactive but somatically active Arabidopsis seedlings. We suggest that Ds excision activity in B. napus is highly dependent on the efficiency of Ac(st) transcript processing.

Systems for the removal of a selection marker and their combination with a positive marker

Many systems have been developed for the removal of a selection marker in order to generate marker-free transgenic plants. These systems consist of (1) a site-specific recombination system (Cre/lox) or a phage-attachment region (attP) to remove the selectable marker gene and (2) a transposable element system (Ac) or a co-transformation system to segregate the gene of interest from the selectable marker gene. Overall, the process is more time-consuming than conventional transformation methods because two rounds of transformation - two steps of regeneration or sexual crossings - are required to obtain the desired transgenic plants. Recently, removal systems combined with a positive marker, denoted as MAT vectors, have been developed to save time and effort by generating marker-free transgenic plants through a single-step transformation. We summarize here the transformation procedures using these systems and discuss their feasibility for practical use.

The Arabidopsis floral homeotic gene APETALA3 differentially regulates intercellular signaling required for petal and stamen development.

Cell-cell signaling is crucial for the coordination of cell division and differentiation during plant organogenesis. We have developed a novel mosaic analysis method for Arabidopsis, based on the maize Ac/Ds transposable element system, to assess the requirements of individual genes in intercellular signaling. Using this strategy, we have shown that the floral homeotic APETALA3 (AP3) gene has distinct roles in regulating intercellular signaling in different tissues. In petals, AP3 acts primarily in a cell-autonomous fashion to regulate cell type differentiation, but its function is also required in a non-cell-autonomous fashion to regulate organ shape. In contrast, AP3-regulated intercellular interactions are required for conferring both cell type identity and organ shape and size in the stamens. Using antibodies raised against AP3, we have shown that the AP3 protein does not traffic between cells. These observations imply that AP3 acts by differentially regulating the production of intercellular signals in a whorl-specific manner.

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.

Genetic analyses of two independent chlorophyll-deficient mutants identified among the progeny of a single chimeric foliage soybean plant

Chimeric (variegated) foliage plants are frequently observed in many species. In soybean [Glycine max(L.) Merr.], progeny of chimeric plants are a source of nuclear and cytoplasmically inherited mutants. Self-pollinated progeny of a single chimeric plant derived from tissue culture of PI 427099 (Jilin 3) included plants with green foliage, chimeric foliage, yellow foliage (viable), and yellow foliage (lethal). Our objectives were to determine (1) inheritance, linkage, and allelism of the lethal-yellow mutant with known chlorophyll-deficient mutants; (2) inheritance, linkage, and allelism of the viable-yellow mutant with known chlorophyll-deficient mutants; (3) allelism of the lethal-yellow mutant with the viable-yellow mutant; and (4) male and female gamete transmission of the viable-yellow mutant trait. The viable-yellow mutant was allelic to T323, y20 y20 (Ames 2) Mdh1-n Mdh1-n (Ames 2) and was assigned genetic type collection number T361 and gene symbol y20 y20 (Ames 24) Mdh1-n Mdh1-n (Ames 22). The lethal-yellow mutant was allelic to T225H (Y18 y18) and was assigned genetic type collection number T362H and gene symbol Y18 y18 (Ames 2). T225H became Y18 y18 (Ames 1). The two chlorophyll-deficient mutants were not linked to each other. There was no significant difference in F(1) male or female gamete transmission of the viable-yellow mutant. However, many cross-combinations gave significant deviations from the expected 3 green plants:1 viable-yellow plant in the F(2) generation. The allelism of these two chlorophyll-deficient mutants with mutants T225H and T323, derived from putative transposable element systems, is intriguing. An explanation of this phenomenon awaits molecular experimentation.

Regulation of cell proliferation patterns by homeotic genes during Arabidopsis floral development.

The shoot apical meristem of Arabidopsis thaliana consists of three cell layers that proliferate to give rise to the aerial organs of the plant. By labeling cells in each layer using an Ac-based transposable element system, we mapped their contributions to the floral organs, as well as determined the degree of plasticity in this developmental process. We found that each cell layer proliferates to give rise to predictable derivatives: the L1 contributes to the epidermis, the stigma, part of the transmitting tract and the integument of the ovules, while the L2 and L3 contribute, to different degrees, to the mesophyll and other internal tissues. In order to test the roles of the floral homeotic genes in regulating these patterns of cell proliferation, we carried out similar clonal analyses in apetala3-3 and agamous-1 mutant plants. Our results suggest that cell division patterns are regulated differently at different stages of floral development. In early floral stages, the pattern of cell divisions is dependent on position in the floral meristem, and not on future organ identity. Later, during organogenesis, the layer contributions to the organs are controlled by the homeotic genes. We also show that AGAMOUS is required to maintain the layered structure of the meristem prior to organ initiation, as well as having a non-autonomous role in the regulation of the layer contributions to the petals.

Target selected insertional mutagenesis on chromosome IV of Arabidopsis using the En–I transposon system

Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The maize Enhancer–Inhibitor ( En–I) transposable element system was used to develop an effective reverse genetics strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing programme. These genes were of particular interest since homologies to other genes and gene families were identified, but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants. Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify germinal insertions in the progeny.

Genetic and Agronomic Evaluation of wp‐m in Soybean

Transposable element systems have been proposed to explain instability in floral pigmentation of several plant species, including soybean [Glycine max (L.) Merr.]. Soybean lines with chimeric (purple and pink sectored) flowers are hypothesized to contain wp‐m, an active transposable element that is able to excise from the wp locus during morphological development. The objectives of this research were (i) to determine the inheritance of the chimeric flower phenotype when crossed to stable pink or purple flowered revertant lines and (ii) to determine the effect of wp on agronomic traits in stable flowered lines derived from wp‐m Chimeric flowers crossed to pink flowered revertant (wp*) lines produced four F2 populations with unusual segregation ratios of 52 pink, three purple, and two chimeric flowered plants. Crossing chimeric flowers to revertant purple flowered (Wp*) lines resulted in F2 populations that did not have the chimeric flower phenotype evident. In the agronomic evaluations, stable wp* lines were later in maturity and averaged 4 g kg−1 higher in protein content and 3 g kg−1 lower in oil content than Wp* lines. The data suggest wp acts in a pleiotropic manner to influence protein synthesis, as purple flowered revertant lines from a pink flower source had lower protein content than sister lines with wp*. Pink flowered lines derived from a purple flower source had higher levels of protein than sister lines with Wp*. The influence of wp on the anthocyanin pathway, plant morphology, and protein accumulation is a unique phenomenon that has not been reported in other plant species.

Functional analysis of deletion derivatives of the maize transposon MuDR delineates roles for the MURA and MURB proteins.

The regulatory transposon of the Mutator system of transposable elements in maize is MuDR. MuDR elements produce two transcripts, from genes mudrA and mudrB, encoding proteins MURA and MURB, respectively. Like many other transposons, MuDR elements often undergo deletions, usually of internal sequences. Analysis of a deletion that is restricted to the region encoding MURB demonstrates that this gene is not required to cause excisions of a reporter element, although it may be required for transposition or suppression of suppressible alleles. Conversely, a derivative that lacks the region encoding MURA but that produces MURB is nonfunctional for all aspects of Mutator activity. Northern analysis of these derivatives reveals that each of the two transcripts can be independently transcribed, and analysis using an antibody specific for MURB reveals that mudrB transcript can also be successfully translated and its product appropriately localized in the absence of mudrA. A third deletion derivative provides evidence for a source of previously reported antisense transcript.

ALBINO3, an Arabidopsis nuclear gene essential for chloroplast differentiation, encodes a chloroplast protein that shows homology to proteins present in bacterial membranes and yeast mitochondria.

The albino3 (alb3) mutant of Arabidopsis forms white or light yellow cotyledons and leaves and when germinated on soil does not survive beyond the seedling stage. The chloroplasts of the mutant are abnormal, as determined by electron microscopy, and contain reduced levels of chlorophyll. However, the chloroplasts of alb3 mutants are sufficiently differentiated to enable the expression of two nuclear genes whose transcription requires the presence of chloroplasts. The ALB3 gene was isolated by transposon tagging with the Activator/Dissociation transposable element system. ALB3 is a novel plant gene whose product shows homology to a bacterial membrane protein previously identified in five bacterial species and to a yeast protein, OXA1, and its human homolog. OXA1 is required in the mitochondria for proper assembly of the cytochrome oxidase complex. ALB3 does not have a function identical to OXA1 because mitochondrial cytochrome oxidase activity is not affected in the mutant, and immunogold labeling as well as chloroplast import experiments performed in vitro demonstrated that the ALB3 protein is present in chloroplast membranes. ALB3 might have a function related to that of OXA1 and be involved in the assembly of a chloroplast enzyme complex.

A Novel Suppressor of Cell Death in Plants Encoded by the Lls1 Gene of Maize

The lls1 (lethal leaf spot1) locus of maize is defined by a recessive mutation characterized by the initiation, in a developmentally programmed manner, of necrotic lesions that expand to kill leaves cell autonomously. The loss-of-function nature of all lls1 mutants implies that the Lls1 gene is required to limit the spread of cell death in mature leaves. We have cloned the Lls1 gene by tagging with Mutator, a transposable element system in maize, and we show that it encodes a novel protein highly conserved in plants. Two consensus binding motifs of aromatic ring-hydroxylating dioxygenases are present in the predicted LLS1 protein, suggesting that it may function to degrade a phenolic mediator of cell death.

Tissue-specific accumulation of MURB, a protein encoded by MuDR, the autonomous regulator of the Mutator transposable element family.

The Mutator (Mu) system of transposable elements is highly mutagenic and can maintain high levels of activity through multiple generations due to frequent transpositions of both its autonomous and nonautonomous components. This family also shows pronounced developmental regulation. Most notable is the very low frequency of germinal reversions, despite the high levels of somatic transpositions and excisions, and the high frequency of germinally transmitted duplication events. Here, we report the production of antibodies raised against MURB, one of two proteins encoded by MuDR, the autonomous regulator of the Mu family. Immunolocalizations performed using anti-MURB antibodies reveal that this protein is present in specific tissues during male inflorescence development. Throughout much of development, MURB is detected at the highest levels in cell lineages that may find themselves in the germ line, but no MURB is detected in microspore mother cells. These cells are the direct precursors to pollen. Based on these observations as well as previous data, we discuss the relationship between the expression of MURB and developmental regulation of Mu activity.