<Emphasis Type="Italic">Mutator</Emphasis>-like elements identified in melon,Arabidopsis and rice contain ULP1 protease domains

The transposon Mutator was first identified in maize, and is one of the most active mobile elements in plants. The Arabidopsis thaliana genome contains at least 200 Mutator-like elements (MULEs), which contain the Mutator-like transposase gene, and often additional genes. We have detected a novel type of MULEs in melon (CUMULE), which, besides the transposase, contains two ubiquitin-like specific protease-like sequences (ULP1). This element is not present in the observed location in some melon cultivars. Multiple copies of this element exist in the Cucumis melo genome, and it has been detected in other Cucurbitaceae species. Analysis of the A. thaliana genome revealed more than 90 CUMULE-like elements, containing one or two Ulp1-like sequences, although no evidence of mobility exists for these elements. We detected various putative transposable elements containing ULP1-like sequences in rice. The discovery of these MULEs in melon and Arabidopsis, and the existence of similar elements in rice and maize, suggest that a proteolytic function may be important for this subset of the MULE transposable elements. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Nucleotide sequence data reported are available in the GenBank database under the accession number AY524004.     

Tag1 is an autonomous transposable element that shows somatic excision in both Arabidopsis and tobacco.

Tag1 is a transposable element first identified as an insertion in the CHL1 gene of Arabidopsis. The chl1::Tag1 mutant originated from a plant (ecotype Landsberg erecta) that had been transformed with the maize transposon Activator (Ac), which is distantly related to Tag1. Genomic analysis of untransformed Landsberg erecta plants demonstrated that two identical Tag1 elements are present in the Landsberg erecta genome. To determine what provides transposase function for Tag1 transposition, we examined Tag1 excision in different genetic backgrounds. First, the chl1::Tag1 mutant was backcrossed to untransformed wild-type Arabidopsis plants to remove the Ac element(s) from the genome. F2 progeny that had no Ac elements but still retained Tag1 in the CHL1 gene were identified. Tag1 still excised in these Ac-minus progeny producing CHL1 revertants; therefore, Ac is not required for Tag1 excision. Next, Tag1 was inserted between a cauliflower mosaic virus 35S promoter and a beta-glucuronidase (GUS) marker gene and transformed into tobacco. Transformants showed blue-staining sectors indicative of Tag1 excision. Transgenic tobacco containing a defective Tag1 element, which was constructed in vitro by deleting an internal 1.4-kb EcoRI fragment, did not show blue-staining sectors. We conclude that Tag1 is an autonomous element capable of independent excision. The 35S-GUS::Tag1 construct was then introduced into Arabidopsis. Blue-staining sectors were found in cotyledons, leaves, and roots, showing that Tag1 undergoes somatic excision during vegetative development in its native host.     

A transposable element insertion disturbed starch synthase gene SSIIb in maize

Transposable elements, as the most active genetic factors, have driven genome evolution in maize and reshaped certain key loci responsible for maize domestication, exemplified by an inserted transposon in teosinte branched1 (tb1), which controls plant architecture. In this study, we detected an insertion of a transposable element in the second exon of the coding sequence of the maize starch synthase gene SSIIb, leading to a splicing modification and gene frameshift. This insertion provided a means of determining the function of SSIIb for starch synthesis during maize domestication. Association and quantitative trait locus (QTL) mappings showed that SSIIb was not associated with starch eating quality and total starch content of kernel, and two maize near-isogenic-line-like lines with and without the insertion of the transposable element further exhibited the same starch content of kernel and leaf; in addition, nucleotide diversity analysis revealed that maize SSIIb was not under selection during domestication. All these results demonstrated that maize SSIIb might serve as a very minor genetic factor or a functional redundancy gene in starch synthesis. Global BLAST showed that the maize genome harbored 1,387 copies of this transposable element, of which 135 copies were located in genic regions. At least three genes beside maize SSIIb were disturbed by this transposable element. Five patterns of transposition, according to the insertion sites close to or within genes such as maize SSIIb in this study, are under discussion and a large quantity of present/absent variations due to the insertion of varieties of transposable elements, discovered by revolutionary next-generation sequencing, would rapidly accelerate QTL and association mappings for maize domestication through candidate gene tactics in the near future.     

Intervening sequences in paralogous genes: a comparative genomic approach to study the evolution of X chromosome introns

The enlargement of the genome size and the decrease in genome compactness with increase in the number and size of introns is a general pattern during the evolution of eukaryotes. Among the possible mechanisms for modifying intron size, it has been suggested that the insertion of transposable elements might have an important role in driving intron evolution. The analysis of large portions of the human genome demonstrated that a relatively recent (50 to 100 MYA) accumulation of transposable elements appears to be biased, favoring a preferential insertion of LINE1 transposons into sex chromosomes rather than into autosomes. In the present work, the effect of chromosomal location on the increase in size of introns was evaluated with a comparative analysis performed on pairs of human paralogous genes, one located on the X chromosome and the second on an autosome. A phylogenetic analysis was also performed on the X-encoded proteins and their paralogs to confirm orthology-paralogy and to approximately estimate the time of gene duplication. Statistical analysis of total intron length for each pair of paralogous genes provided no evidence for a larger size of introns in the gene copies located on the X chromosome. On the opposite, introns of autosomal genes were found to be significantly longer than introns of their X-linked paralogs. Likewise, LINE1 elements were not significantly more frequent in X-chromosome introns, whereas the frequency of SINE elements showed a marginally significant bias toward autosomal introns.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

Identification of active transposon dTok, a member of the hAT family, in rice

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.     

hAT element population genetics in Anopheles gambiae s.l. in Mozambique

Herves is a functional Class II transposable element in Anopheles gambiae belonging to the hAT superfamily of elements. Class II transposable elements are used as gene vectors in this species and are also being considered as genetic drive agents for spreading desirable genes through natural populations as part of an effort to control malaria transmission. In this study, Herves was investigated in populations of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles merus in Mozambique over a period of 2 years. The copy number of Herves within these three species was approximately 5 copies per diploid genome and did not differ among species or between years. Based on the insertion-site occupancy-frequency distribution and existing models of transposable element dynamics, Herves appears to be transpositionally active currently or, at least recently, in all species tested. Ninety-five percent of the individuals within the populations of the three species tested contained intact elements with complete Herves transposase genes and this is consistent with the idea that these elements are currently active.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842?bp long with perfect inverted terminal repeats (ITRs) of 48?bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.     

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

Sequence and Analysis of the Tomato JOINTLESS Locus

A 119-kb bacterial artificial chromosome from the JOINTLESS locus on the tomato (Lycopersicon esculentum) chromosome 11 contained 15 putative genes. Repetitive sequences in this region include one copia-like LTR retrotransposon, 13 simple sequence repeats, three copies of a novel type III foldback transposon, and four putative short DNA repeats. Database searches showed that the foldback transposon and the short DNA repeats seemed to be associated preferably with genes. The predicted tomato genes were compared with the complete Arabidopsis genome. Eleven out of 15 tomato open reading frames were found to be colinear with segments on five Arabidopsis bacterial artificial chromosome/P1-derived artificial chromosome clones. The synteny patterns, however, did not reveal duplicated segments in Arabidopsis, where over half of the genome is duplicated. Our analysis indicated that the microsynteny between the tomato and Arabidopsis genomes was still conserved at a very small scale but was complicated by the large number of gene families in the Arabidopsis genome.     

Use of a mammalian interspersed repetitive (MIR) element in the coding and processing sequences of mammalian genes.

The mammalian interspersed repetitive (MIR) element was amplified in mammals 130 million years ago. The MIR element is at least 260 bp in length and is found in approximately 105 copies in the mammalian genome. We analyzed copies of the MIR element in the DNA of various mammals to determine its relationship to the structure and function of genes, in an attempt to identify specific uses of the MIR element within the mammalian genome. We found that alternative splicing within the acetylcholine receptor gene in humans takes place within the MIR element and results in the incorporation of part of the MIR element into the coding sequence of this gene. Furthermore, the polyadenylation signal (AATAAA) at the 3' end of four different mammalian genes is derived from the MIR element. These uses of the MIR element suggest that other regulatory sequences found within the mammalian genome originated from ancient transposable elements, many of which may no longer be recognizable.     

Identification of transposon-like elements in non-coding regions of tomato ACC oxidase genes

1-aminocyclopropane-1-carboxylate (ACC) oxidase, which catalyses the terminal step in ethylene biosynthesis, is encoded by a small multigene family in tomato that is differentially expressed in response to developmental and environmental cues. In this study we report the isolation and sequencing of approximately 2 kb of 5′-flanking sequence of three tomato ACC oxidase genes (LEACO1, LEACO2, LEACO3) and the occurrence of class I and class II mobile element-like insertions in promoter and intron regions of two of them. The LEACO1 upstream region contains a 420-bp direct repeat which is present in multiple copies in the tomato genome and is very similar to sequences in the promoters of the tomato E4 and 2A11 genes. The region covering the repeats resembles the remnant of a retrotransposon. Two copies of a small transposable element, belonging to the Stowaway inverted repeat element family, have been found in the 5′-flanking sequence and the third intron of LEACO3. Received: 8 August 1996 / Accepted: 4 November 1996     

The origin of interspersed repeats in the human genome

Over a third of the human genome consists of interspersed repetitive sequences which are primarily degenerate copies of transposable elements. In the past year, the identities of many of these transposable elements were revealed. The emerging concept is that only three mechanisms of amplification are responsible for the vast majority of interspersed repeats and that with each autonomous element a number of dependent non-autonomous sequences have co-amplified.     

The behaviour of the autonomous maize transposable element En/Spm in Arabidopsis thaliana allows efficient mutagenesis

The behavior of the autonomous maize transposable element En/Spm of maize was studied in Arabidopsis. Transgenic Arabidopsis plants carrying En-1 elements were propagated for 12 generations using a single seed descent procedure. The distribution and activity of the En-1 element was monitored using Southern DNA hybridisations in generations 1, 6 and 12. In the first generation the highest number of En-1 insertions per line was 7, which increased to 20 in generation 12. The average number of En-1 insertions increased only slightly in the population, due to a gradual accumulation of segregants that lost the transposable element. During the development of the En-1 mutagenised population the element remained active even in the high-copy lines. In situ hybridisation demonstrated that multiple En-1 insertions were distributed over all Arabidopsis chromosomes. From the initial En-1 mutagenised populations many unstable gene mutations were recovered, indicating that En-1 can be used as a efficient tool for gene tagging in Arabidopsis.     

The Transposable Element Mariner Mediates Germline Transformation in Drosophila Melanogaster

A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the β-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the β-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide hostrange transformation vector for insects.     

Definition and interactions of a positive regulatory element of the Arabidopsis INNER NO OUTER promoter

INNER NO OUTER (INO) expression is limited to the abaxial cell layer of the incipient and developing outer integument in Arabidopsis ovules. Using deletion analysis of the previously defined INO promoter (P-INO), at least three distinct regions that contribute to the endogenous INO expression pattern were identified. One such positive element, designated POS9, which comprises at least three distinct subelements, was found to include sufficient information to duplicate the INO expression pattern when four or more copies were used in conjunction with a heterologous minimal promoter. While known regulators of INO, including INO, SUPERMAN, BELL1, and AINTEGUMENTA, did not detectably interact with POS9 in yeast one-hybrid assays, two groups of proteins that interact specifically with POS9 were identified in one-hybrid library screens. Members of one group include C2H2 zinc finger motifs. Members of the second group contain a novel, conserved DNA-binding region and were designated the BASIC PENTACYSTEINE (BPC) proteins on the basis of conserved features of this region. The BPC proteins are nuclear localized and specifically bind in vitro to GA dinucleotide repeats located within POS9. The widespread expression patterns of the BPCs and the large number of GA repeat potential target sequences in the Arabidopsis genome indicate that BPC proteins may affect expression of genes involved in a variety of plant processes.