Microuli, a Family of Miniature Subterminal Inverted-Repeat Transposable Elements (MSITEs): Transposition Without Terminal Inverted Repeats

Zhijian TuGo,* and Stephanos P. Orphanidis{dagger}

*Department of Biochemistry, Virginia Polytechnic Institute and State University; and
{dagger}Department of Biochemistry, University of Arizona

The first Microuli element, Microuli-Aa1, was discovered as a 209-bp insertion in the 5' long terminal repeat of a retrotransposon named Mosqcopia-Aa2 (unpublished data, GenBank AY009101) in the yellow fever mosquito, Aedes aegypti. As shown in figure 1A, the Microuli insertion resulted in a TTAA target duplication, indicating past mobility of the element. To further characterize this mobile element, we screened an A. aegypti genomic library, using a digoxigenin-labeled single-stranded DNA probe generated by asymmetric PCR using the entire Microuli-Aa1 as the template. The library construction, probe labeling, and library screening procedures were as previously described (Tu 2000Citation ). The final washing stringency was 0.5 x SSC with 0.1% SDS at 55°C, which allows approximately 15%–25% mismatch (Meinkoth and Wahl 1984Citation ). Because 462 positive plaques were identified among 64,000 total plaques screened, it was estimated using a previously described calculation method (Tu 2000Citation ) that there were approximately 3,000 copies of Microuli per haploid genome. The entire inserts of three positive genomic clones isolated during the above screening experiment were sequenced and deposited in GenBank (AY009102AY009104). The boundaries of Microuli elements were deduced based on sequence comparisons between the four genomic clones. As shown in figure 1B, three of the four elements have the same termini and are flanked by putative TTAA target duplications. There were no sequence similarities between these Microuli clones outside the TTAA target duplications. Although we cannot determine the extent of the diversity of Microuli elements based on the analysis of four elements, the evidence of insertion, sequence and size homogeneity between three out of the four copies, and the conservation of putative target duplications suggest that Microuli has been an intact unit of transposition which has generated a large number of copies.



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Fig. 1.—A, Evidence of past mobility of Microuli-Aa1. Mosqcopia-Aa1 and Mosqcopia-Aa2 (GenBank accession numbers AF134899 and AY009101) are two copies of a retrotransposon in Aedes aegypti. The TTAA target site duplication is underlined. Among four sequenced Mosqcopia elements (data not shown), only Mosqcopia-Aa2 contains a Microuli insertion. B, Multiple-sequence alignment of the Microuli elements. The sequences were aligned using Pileup of GCG (gap weight = 3 and gap length weight = 0). The consensus was created using Pretty of GCG by simple majority rule. The TTAA target site is underlined. Dots indicate sequences that are identical to the consensus. Lowercase letters indicate sequence variation. The thick and thin filled arrows indicate the subterminal inverted repeats (SIRs). The three open arrows indicate the imperfect subterminal direct repeats. C, Comparison between the consensus sequences of Microuli and an A. aegypti MITE Wuneng (Tu 1997Citation ). The arrows indicate inverted repeats. Note that the inverted repeats marked by open arrows and arrows with dashed lines are between Microuli and Wuneng.

 
Analysis of the Microuli elements and their consensus showed that they are small and highly AT-rich (68.8%–72.6%) and have no coding capacity. As shown in figure 1B, there is a 61–62-bp internal subterminal inverted repeat (SIR), as well as a 7-bp SIR 11 bp from the two termini. These inverted repeats suggest a strong potential for Microuli to form stable secondary structures, as indicated by the low predicted {Delta}G value (-66.4 kcal/mol) of the consensus, calculated using Mfold of the GCG package, version 10 (Genetics Computer Group, Madison, Wis.). In addition, there are three imperfect subterminal direct repeats near the 5' end. All of the above characteristics clearly resemble the structural features of miniature inverted-repeat transposable elements (MITEs), which have recently been discovered in plants, vertebrates, nematodes, and insects (e.g., Morgan and Middleton 1990Citation ; Bureau and Wessler 1992Citation ; Morgan 1995Citation ; Oosumi, Garlick, and Belknap 1995, 1996Citation ; Wessler, Bureau, and White 1995Citation ; Smit and Riggs 1996Citation ; Tu 1997, 2000Citation ; Izsvák et al. 1999Citation ; Feschotte and Mouches 2000aCitation ). The only feature that separates Microuli from MITEs is that Microuli elements lack TIRs. Therefore, we use the phrase "miniature subterminal inverted-repeat transposable elements" (MSITEs) to refer to the structural characteristics of the Microuli elements. Short insertion sequences that contain SIRs but lack TIRs have been identified in the genomes of rice and a Culex mosquito (Song et al. 1998Citation ; Feschotte and Mouches 2000aCitation ). However, it is not yet clear how repetitive these elements are in their respective genomes and whether each forms a relatively homogeneous and conserved family. To our knowledge, data described here on the Microuli elements represent the first attempt to characterize these unique MSITEs.

There is no structural or sequence similarity between Microuli and SINEs or any other RNA-mediated retrotransposons. However, as mentioned above, Microuli resembles MITEs in its structure. Moreover, as shown in figure 1C, 14 of the 19 nucleotides at the 5' terminus of Microuli are identical to the TIR of Wuneng, a previously characterized MITE which may also insert specifically into the TTAA target (Tu 1997Citation ). It has been suggested that MITEs and the autonomous DNA transposons share the same transposition machinery based on common TIRs (Morgan 1995Citation ; Oosumi, Garlick, and Belknap 1996Citation ). Indeed, many MITEs have been found to have TIRs that are highly similar to DNA transposons which encode transposases (Morgan 1995Citation ; Oosumi, Garlick, and Belknap 1996Citation ; Tu 2000Citation ; Feschotte and Mouches 2000bCitation ). It is not yet clear whether the TIRs alone are sufficient to support MITE transposition. Nevertheless, TIRs are very important for the transposition of DNA transposons, either as binding sites for transposases (Lampe, Churchill, and Robertson 1996Citation ) or as recombination signal sequences indicating the location of strand cleavage (Beall and Rio 1997Citation ). How did Microuli transpose without the TIRs? As shown in figure 1B and C, the 7-bp SIR, which is conserved between Microuli and Wuneng, and the three subterminal direct repeats could potentially be the binding sites for transposases because SIRs and subterminal direct repeats have been shown to bind transposases in several autonomous DNA transposons (e.g., Groenen, Timmers, and van de Putte 1985Citation ; Morgan and Middleton 1990Citation ; Beall and Rio 1997Citation ; Becker and Kunze 1997Citation ). It remains unclear how the termini of Microuli are determined at the strand cleavage step without a TIR. In this regard, it is interesting to note that the TIR of the Mu transposon is only 2 bp long (Groenen, Timmers, and van de Putte 1985Citation ). In addition, the TTAA target duplication and a 3-bp TIR are essential for the excision of an autonomous transposon, piggyBac (Bauser, Elick, and Fraser 1999Citation ). Therefore, it is possible that Microuli may also be able to use the TTAA target sequence as part of the recombination signal. The significance of the long internal SIR is not yet clear, apart from its contribution to the potential for Microuli to form stable secondary structures, like most MITEs. It should be noted that there is an alternative hypothesis that MITEs can transpose by a DNA intermediate resulting from the folding back of a single-strand DNA generated during replication (Izsvák et al. 1999Citation ). However, it is difficult to use the folding-back mechanism to explain the lack of TIRs in Microuli.

The sequence similarities between Microuli and Wuneng are limited to a terminal 19-bp region and two additional short stretches (fig. 1C ). Therefore, Microuli probably did not originate directly from Wuneng, or vice versa. It has been shown that some MITEs may be derived from deleted DNA transposons that code for transposases (Feschotte and Mouches 2000bCitation ). However, in other cases, the similarities between MITEs and their corresponding DNA transposons are limited to the TIRs (MacRae and Clegg 1992Citation ; Morgan 1995Citation ; Oosumi, Garlick, and Belknap 1996Citation ; Tu 2000Citation ). Chance mutational events that resulted in 11-bp TIRs were suggested to be responsible for the generation of Ds1 elements, which are similar to Ac only in the 11-bp TIRs (MacRae and Clegg 1992Citation ). In light of the discovery of successful transposition of the MSITE family Microuli, it is tempting to hypothesize that some MITEs could evolve from MSITEs through mutation and/or recombination events at the termini which would result in TIRs. Therefore, we suggest that in some cases MSITEs could be the intermediates for the genesis of novel MITEs. If the mutated MSITE, or the primordial MITE, has an evolutionary advantage because it may be more successful in transposition due to the newly acquired TIR, it may ultimately outcompete and replace the old MSITEs. On the other hand, a MSITE could evolve from a MITE as a result of the degeneration of the TIRs. However, such a MSITE may not be able to compete against a preexisting MITE if the TIR confers any evolutionary advantage. MSITEs such as Microuli are not "dead-end" derivatives of DNA transposons, because Microuli is, or has been, a unit of highly successful transposition. Transposition of some Microuli elements may be relatively recent, as the four elements showed 81%–92% sequence identities. Furthermore, the target of Microuli-Aa1 insertion, Mosqcopia-Aa2, seems to be a relatively recent copy itself, because it is a full-length copy with an intact open reading frame and it is 98.7% identical to Mosqcopia-Aa1 (GenBank accession number AF134899). The transition from MSITEs to MITEs would be more likely to occur when one end of the MSITE is derived from the TIRs of a DNA transposon or a preexisting MITE through recombination. Indeed, as described above, one end of Microuli is similar to the TIR of a previously characterized MITE named Wuneng (Tu 1997Citation ). Moreover, the fragmented similarity between the two elements (fig. 1C ) could be evidence of recombination events. However, so far we have not found a MITE which is directly derived from Microuli in A. aegypti. Our hypothesis does not predict that every MSITE would ultimately evolve to a MITE. Such evolutionary events would require the occurrence of some chance events and that the newly derived MITEs have an evolutionary advantage. Direct evidence to support the above hypothesis may be difficult to come by, because one has to find the right moment, at which the new MITE has evolved while the old MSITE has not been totally lost. However, such evidence may someday be within reach, considering the scale and speed of the ongoing genome projects.

Acknowledgements

We thank Henry Hagedorn for support and advice, James Biedler for comments on the manuscript, and the sequencing facilities at the University of Arizona and Virginia Tech for their services. This work was supported by NIH grant AI42121 to Z.T. and by the Agricultural Experimental Station at Virginia Tech.

Footnotes

Pierre Capy, Reviewing Editor

1 Abbreviations: MITE, miniature inverted-repeat transposable element; MSITE, miniature subterminal inverted-repeat transposable element; SIR, subterminal inverted repeat; TIR, terminal inverted repeat. Back

2 Keywords: MITE MSITE subterminal inverted repeats genome mosquito Aedes aegypti. Back

3 Address for correspondence and reprints: Zhijian Tu, Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. jaketu{at}vt.edu Back

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Accepted for publication January 4, 2001.