New transposable elements identified as insertions in rice transposon Tnr1

Tnr1 (235 bp long) is a transposable element in rice. Polymerase chain reactions (PCRs) done with a primer(s) that hybridizes to terminal inverted repeat sequences (TIRs) of Tnr1 detected new Tnr1 members with one or two insertions in rice genomes. Six identified insertion sequences (Tnr4, Tnr5, Tnr11, Tnr12, Tnr13 and RIRE9) did not have extensive homology to known transposable elements, rather they had structural features characteristic of transposable elements. Tnr4 (1767 bp long) had imperfect 64-bp TIRs and appeared to generate duplication of a 9-bp sequence at the target site. However, the TIR sequences were not homologous to those of known transposable elements, indicative that Tnr4 is a new transposable element. Tnr5 (209 bp long) had imperfect 46-bp TIRs and appeared to generate duplication of sequence TTA like that of some elements of the Tourist family. Tnr11 (811 bp long) had 73-bp TIRs with significant homology to those of Tnr1 and Stowaway and appeared to generate duplication of sequence TA, indicative that Tnr11 is a transposable element of the Tnr1/Stowaway family. Tnr12 (2426 bp long) carried perfect 9-bp TIRs, which began with 5'-CACTA- -3' from both ends and appeared to generate duplication of a 3-bp target sequence, indicative that Tnr12 is a transposable element of the En/Spm family. Tnr13 (347 bp long) had 31-bp TIRs and appeared to generate duplication of an 8-bp target sequence. Two sequences, one the transposon-like element Crackle, had partial homology in the Tnr13 ends. All five insertions appear to be defective elements derived from autonomous ones encoding the transposase gene. All had characteristic tandem repeat sequences which may be recognized by transposase. The sixth insertion sequence, named RIRE9 (3852 bp long), which begins with 5'-TG- -3' and ends with 5'- -CA-3', appeared to generate duplication of a 5-bp target sequence. These and other structural features indicate that this insertion is a solo LTR (long terminal repeat) of a retrotransposon. The transposable elements described above could be identified as insertions into Tnr1, which do not deleteriously affect the growth of rice cells.     

Jittery, a Mutator distant relative with a paradoxical mobile behavior: excision without reinsertion

The unstable mutation bz-m039 arose in a maize (Zea mays) stock that originated from a plant infected with barley stripe mosaic virus. The instability of the mutation is caused by a 3.9-kb mobile element that has been named Jittery (Jit). Jit has terminal inverted repeats (TIRs) of 181 bp, causes a 9-bp direct duplication of the target site, and appears to excise autonomously. It is predicted to encode a single 709-amino acid protein, JITA, which is distantly related to the MURA transposase protein of the Mutator system but is more closely related to the MURA protein of Mutator-like elements (MULEs) from Arabidopsis thaliana and rice (Oryza sativa). Like MULEs, Jit resembles Mutator in the length of the element's TIRs, the size of the target site duplication, and in the makeup of its transposase but differs from the autonomous element Mutator-Don Robertson in that it encodes a single protein. Jit also differs from Mutator elements in the high frequency with which it excises to produce germinal revertants and in its copy number in the maize genome: Jit-like TIRs are present at low copy number in all maize lines and teosinte accessions examined, and JITA sequences occur in only a few maize inbreds. However, Jit cannot be considered a bona fide transposon in its present host line because it does not leave footprints upon excision and does not reinsert in the genome. These unusual mobile element properties are discussed in light of the structure and gene organization of Jit and related elements.     

The primary structure and characteristics of the ISAfe600, an insertion sequence from Acidithiobacillus ferrooxidans strains

A new IS-like element (604 bp) was revealed in the genome of several Acidithiobacillus ferrooxidans strains isolated from diverse biotopes. It includes 26-bp imperfectly matched terminal inverted repeats (TIRs), similar in structure to the TIRs of ISAfel insertion element. The 60-bp DNA fragment adjacent to the right TIR (TIRR) exhibits pronounced homology with the similarly located DNA fragments in ISAfel and IST445, as well as with the internal fragment of ISAfel encoding the transposase gene (nucleotides from 254 to 311 bp). The central section of ISAfe600 is unique and exhibits no homology with any prokaryotic DNA. A duplication of 8 bp of the target DNA was found in the ISAfe600 insertion site. One to four copies of ISAfe600 were revealed by Southern hybridization in the genome of A. ferrooxidans strains studied. The number of ISAfe600 copies varies depending on the growth conditions (energy substrate). Since the open reading frames big enough to encode transposase are not presert in the structure of ISAfe600, it may be a deficient IS element; its translocation is possibly achieved under control of the ISAfel transposase.     

Mx-rMx, a family of interacting transposons in the growing hAT superfamily of maize

More than half a century after the discovery of transposable elements, the number of genetically defined autonomous elements that have been isolated and characterized molecularly in any one species remains surprisingly small. Because of its rich genetic history, maize (Zea mays) is, by far, the plant with the largest number of such elements. Yet, even in maize, a maximum of only two autonomous elements have been characterized in any transposon superfamily. This article describes the isolation and molecular and genetic characterization of Mx (for mobile element induced by x-rays), a third autonomous member of the hAT transposon superfamily in maize. Mx is 3731 bp long, ends in 13-bp terminal inverted repeats (TIRs), and causes an 8-bp duplication of the target site. Mx and rMx (for responder to Mx), its 571-bp nonautonomous partner, define a classical family of interacting transposable elements. Surprisingly, the TIRs of Mx and rMx are only 73% identical, and the subterminal sequences are even less so, suggesting that Mx and rMx may represent diverging transposable elements still capable of mobilization by the same transposase. Sequences that are closer to the ends of either Mx or rMx are present in the maize genome. Mx is predicted to encode a 674-amino acid protein that is homologous to the Ac transposase. Although Mx and Ac are closely related, they do not interact. Other data suggest that maize may possess at least five families of hAT transposons that do not interact with each other. The possible origin of noninteracting transposon families within the same superfamily is discussed.     

The primary structure and characteristics of ISAfe600, an insertion sequence from <Emphasis Type="Italic">Acidithiobacillus ferrooxidans</Emphasis> strains

A new IS-like element (604 bp) was revealed in the genome of several Acidithiobacillus ferrooxidans strains isolated from diverse biotopes. It includes 26-bp imperfectly matched terminal inverted repeats (TIRs), similar in structure to the TIRs of the ISAfel insertion element. The 60-bp DNA fragment adjacent to the right TIR (TIR_R) exhibits pronounced homology with the similarly located DNA fragments in ISAfel and IST445, as well as with the internal fragment of ISAfel encoding the transposase gene (nucleotides from 254 to 311 bp). The central section of ISAfe600 is unique and exhibits no homology with any prokaryotic DNA. A duplication of 8 bp of the target DNA was found in the ISAfe600 insertion site. One to four copies of ISAfe600 were revealed by Southern hybridization in the genome of A. ferrooxidans strains studied. The number of ISAfe600 copies varies depending on the growth conditions (energy substrate). Since open reading frames big enough to encode transposase are not presert in the structure of ISAfe600, it may be a deficient IS element; its translocation is possibly achieved under control of the ISAfel transposase.     

Isolation of a new insertion sequence, IS13655, and its application to Corynebacterium glutamicum genome mutagenesis

A new functional Corynebacterium glutamicum insertion sequence (IS) element, IS13655, was isolated using a suicide vector. The IS element was 1,293 bp in size and contained 26-bp imperfect inverted repeats (IRs) and 3-bp target site duplication as direct repeats (DRs). IS13655 harbored two ORFs with high similarity to the transposase of IS1206, an IS3 family element. IS13655 revealed relatively high transposition efficiency, with low target site selectivity along the Corynebacterium glutamicum R genome, making it a potentially useful genetic engineering tool.     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.     

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.     

Structure and evolution of the hAT transposon superfamily.

The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new tools for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 hAT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.     

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.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae.

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.     

Spring: a novel family of miniature inverted-repeat transposable elements is associated with genes in apple

The apple, Malusxdomestica Borkh., belongs to the family Rosaceae and subfamily Maloideae and has a genome size of approximately 750 Mb. In this study, a novel family of transposable elements, designated Spring, has been identified in the apple genome. The four Spring elements, Spring-1 to Spring-4, share all the classic features of miniature inverted-repeat transposable elements (MITEs), including small size (approximately 148 bp), no coding potential, A/T richness, insertion bias toward noncoding regions, terminal inverted repeats (TIRs), target site duplications, and potential for forming secondary structures. Evidence of previous mobility of Spring-4 is demonstrated by sequence alignment of genes encoding 1-aminocyclopropane-1-carboxylic acid synthase from both apple and a related member of the Maloideae subfamily, pear. The Spring elements are flanked by either 8- or 9-bp direct repeats, and they differ significantly in size compared to other previously reported MITEs in plants. The TIRs of these Spring elements are not found in any other previously reported plant genes or transposons, except for apple. The possible role of Spring elements in the apple genome is discussed.     

Pot2, an inverted repeat transposon from the rice blast fungus Magnaporthe grisea

We report the cloning and characterisation of Pot2, a putative transposable element from Magnaporthe grisea. The element is 1857 by in size, has 43-bp perfect terminal inverted repeats (TIRs) and 16-bp direct repeats within the TIRs. A large open reading frame, potentially coding for a transposase-like protein, was identified. This putative protein coding region showed extensive identity to that of Fott, a transposable element from another phytopathogenic fungus, Fusarium oxysporum. Pot2, like the transposable elements Tc1 and Mariner of Caenorhabditis elegans and Drosophila, respectively, duplicates the dinucleotide TA at the target insertion site. Sequence analysis of DNA flanking 12 Pot2 elements revealed similarity to the consensus insertion sequence of Tct. Pot2 is present at a copy number of approximately 100 per haploid genome and represents one of the major repetitive DNAs shared by both rice and non-rice pathogens of M. grisea.     

Characterization of Stowaway MITEs in pea (Pisum sativum L.) and identification of their potential master elements.

We have investigated miniature inverted-repeat transposable elements (MITEs) of the Stowaway family and corresponding Mariner-like master elements that could potentially facilitate their mobilization in the genome of the garden pea (Pisum sativum L.). The population of pea Stowaway MITEs consists of 103-104 copies dispersed in the genome. Judging from a sequence analysis of 17 isolated Stowaway elements and their flanking genomic regions, the elements are relatively uniform in size and sequence and occur in the vicinity of genes as well as within repetitive sequences. Insertional polymorphism of several elements was detected among various Pisum accessions, suggesting they were still transpositionally active during diversification of these taxa. The identification of several Mariner-like elements (MLEs) harboring intact open reading frames, capable of encoding a transposase, further supports a recent mobilization of the Stowaway elements. Using transposase-coding sequences as a hybridization probe, we estimated that there are about 50 MLE sequences in the pea genome. Among the 5 elements sequenced, 3 distinct subfamilies showing mutual similarities within their transposase-coding regions, but otherwise diverged in sequence, were distinguished and designated as Psmar-1 to Psmar-3. The terminal inverted repeats (TIRs) of these MLE subfamilies differed in their homology to the TIRs of Stowaway MITEs. The homlogy ranged from 9 bp in Psmar-3 to 30 bp in Psmar-1, which corresponds to the complete Stowaway TIR sequence. Based on this feature, the Psmar-1 elements are believed to be the most likely candidates for the master elements of the Stowaway MITEs in pea.     

Tnr8, a foldback transposable element from rice

An insertion sequence 418 bp in length was found in one member of rice retroposon p-SINE1 in Oryza glaberrima. This sequence had long terminal inverted repeats (TIRs) and is flanked by direct repeats of a 9-bp sequence at the target site, indicative that the insertion sequence is a rice transposable element, which we named Tnr8. Interestingly, each TIR sequence consisted of a unique 9-bp terminal sequence and six tandem repeats of a sequence about 30 bp in length, like the foldback transposable element first identified in Drosophila. A homology search of databases and analysis by PCR revealed that a large number of Tnr8 members with sequence variations were present in the rice genome. Some of these members were not present at given loci in several rice species with the AA genome. These findings suggest that the Tnr8 family members transposed long ago, but some appear to have mobilized after rice strains with the AA genome diverged. The Tnr8 members are thought to be involved in rearrangements of the rice genome.     

Mobilization of a Minos Transposon in Drosophila Melanogaster Chromosomes and Chromatid Repair by Heteroduplex Formation

Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in ~75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.