Department of Biochemistry, Virginia Polytechnic Institute and State University
Abstract
A novel family of miniature inverted repeat transposable elements (MITEs) named Pony was discovered in the yellow fever mosquito, Aedes aegypti. It has all the characteristics of MITEs, including terminal inverted repeats, no coding potential, A+T richness, small size, and the potential to form stable secondary structures. Past mobility of Pony was indicated by the identification of two Pony insertions which resulted in the duplication of the TA dinucleotide targets. Two highly divergent subfamilies, A and B, were identified in A. aegypti based on sequence comparison and phylogenetic analysis of 38 elements. These subfamilies showed less than 62% sequence similarity. However, within each subfamily, most elements were highly conserved, and multiple subgroups could be identified, indicating recent amplifications from different source genes. Different scenarios are presented to explain the evolutionary history of these subfamilies. Both subfamilies share conserved terminal inverted repeats similar to those of the Tc2 DNA transposons in Caenorhabditis elegans, indicating that Pony may have been borrowing the transposition machinery from a Tc2-like transposon in mosquitoes. In addition to the terminal inverted repeats, full-length and partial subterminal repeats of a sequence motif TTGATTCAWATTCCGRACA represent the majority of the conservation between the two subfamilies, indicating that they may be important structural and/or functional components of the Pony elements. In contrast to known autonomous DNA transposons, both subfamilies of Pony are highly reiterated in the A. aegypti genome (8,400 and 9,900 copies, respectively). Together, they constitute approximately 1.1% of the entire genome. Pony elements were frequently found near other transposable elements or in the noncoding regions of genes. The relative abundance of MITEs varies in eukaryotic genomes, which may have in part contributed to the different organizations of the genomes and reflect different types of interactions between the hosts and these widespread transposable elements.
Introduction
Transposable elements can be classified on the basis of the mechanisms of their transposition as DNA-mediated or RNA-mediated elements (Finnegan 1992
). RNA-mediated transposable elements include long terminal repeat (LTR) retrotransposons, non-LTR retrotransposons, and short interspersed repetitive elements (SINEs). Their transposition involves a reverse transcription step which generates cDNA from RNA molecules. DNA-mediated elements such as P, hobo, and mariner use a cut-and-paste mechanism directly from DNA to DNA, and they are characterized by terminal inverted repeats flanking a gene encoding a transposase.
Recently, several families of short interspersed elements with terminal inverted repeats have been found in plants (e.g., Bureau and Wessler 1992
; Bureau, Ronald, and Wessler 1996
; Charrier et al. 1999
; Surzycki and Belknap 1999
), vertebrates (Morgan and Middleton 1990
; Morgan 1995
; Ünsal and Morgan 1995
; Smit and Riggs 1996
; Izsvák et al. 1999
), a nematode (Oosumi, Garlick, and Belknap 1995, 1996
), and two species of insects (Tu 1997
; Braquart, Royer, and Bouhin 1999
). These elements can be grouped as miniature inverted repeat transposable elements (MITEs) based on their common structural characteristics, as proposed by Wessler, Bureau, and White (1995)
. These features include small size, no coding potential, conserved terminal inverted repeats, A+T richness, and, in many cases, the potential to form stable secondary structures. The distribution of many families of MITEs in the genome appears to be biased. Many plant MITEs are associated with genes, where more than 90% of them are found in the noncoding regions, mostly in the 5' and 3' flanking sequences (Bureau and Wessler 1992, 1994a, 1994b
; Bureau, Ronald, and Wessler 1996
). The three families of MITEs previously reported in the yellow fever mosquito, Aedes aegypti, are also associated with genes (Tu 1997
). A few MITEs have been found to have terminal inverted repeats that are highly similar to some autonomous DNA-mediated elements which encode transposases (Morgan 1995
; Oosumi, Garlick, and Belknap 1996
). However, the sequence similarity between these MITEs and their corresponding DNA-mediated elements is confined to the terminal inverted repeats. In this regard, MITEs are similar to the Ds1 transposable elements in maize (Federoff 1989
; MacRae and Clegg 1992
), and they are not simply nonautonomous deletion derivatives of the DNA-mediated elements. MITEs are generally homogeneous in size and are units of highly successful transposition. It has been suggested that some MITEs and the DNA-mediated elements share the same transposition machinery based on common terminal inverted repeats (Morgan 1995
; Oosumi, Garlick, and Belknap 1996
). On the other hand, Izsvák et al. (1999)
propose that MITEs transpose by a DNA intermediate resulting from the folding back of a single strand of DNA during replication. They suggest that the DNA intermediate is reintegrated into the genome using host factors which are involved in cellular replication.
Here, I report the discovery and characterization of a novel family of highly divergent and highly reiterated MITEs named Pony in A. aegypti. The phylogenetic relationships between a large number of Pony elements are analyzed. Different scenarios are presented to explain the evolution of these elements. The transposition mechanism employed by Pony and other MITEs is assessed. The genomic distribution of Pony elements and evolutionary implications of the relative abundance of MITEs in different eukaryotic genomes are discussed.
Materials and Methods
Mosquitoes
Mosquitoes used in this study were from the Rock strain of A. aegypti.
Construction of a Genomic Library
To facilitate the characterization of the short Pony elements in A. aegypti, a genomic library that contains inserts from 1.3 to 2.5 kb was prepared using a ZapExpress vector kit from Stratagene Cloning Systems (La Jolla, Calif.). Genomic DNA was prepared from the Rock strain of A. aegypti. The vector was predigested with BamHI and treated with calf intestine alkaline phosphatase. The genomic DNA was partially digested with Sau3AI, which produces ends that are compatible to BamHI cuts. The digestion conditions were optimized to produce mostly 13-kb fragments. The digested fragments were separated on an agarose gel. Fragments between 1.3 and 2.5 kb were cut out and purified using the Sephaglas Bandprep Kit from Amersham Pharmacia Biotech (Arlington Heights, Ill.). These fragments were ligated to the vector of approximately the same molarity to minimize tandem inserts in one clone. The primary library had 2.8 x 106 original plaque-forming units, with a 1.7% background. A total of 2.0 x 106 original plaque-forming units were amplified and stored. Aliquots of the remaining unamplified library were used in the screening experiments described below.
Screening of the ZapExpress Genomic Library
The unamplified library was screened using two digoxigenin-labeled ssDNA probes, obtained using either Pony-Aa-A1 or Pony-Aa-B1 as template. For the Pony-Aa-A1 probe, the template for the labeling reaction was a gel-purified polymerase chain reaction (PCR) product obtained using a plasmid that contains Pony-Aa-A1 and a primer matching the terminal inverted repeat TACCGTTTTGNYTCANATTNCGNACA. For the Pony-Aa-B1 probe, the template for the labeling reaction was a gel-purified PCR product obtained using a plasmid that contains Pony-Aa-B1 and the primers AACACTTAACTTTCGAATGGT and TATTCCGGACACTCTACTTTG. The above PCR products were labeled in two separate asymmetric PCR reactions using GTATGGTACAGCATTTTGATT and AACACTTAACTTTCGAATGGT as respective primers. The labeling conditions were the same as those described in Tu and Hagedorn (1997)
, using a digoxigenin-dUTP labeling mixture. MagnaGraph Nylon membranes (Micron Separation Inc., Westborough, Mass.) were used to lift the plaques. The prehybridization solution was 5 x SSC with 2% nonfat milk, 0.1% N-lauroylsarcosine, and 0.02% SDS. Approximately 20 ng of probe per milliliter of prehybridization solution was used for hybridization. Hybridization was carried out at 55°C in a Gene Roller from Savant Instruments, Inc. (Holbrook, N.Y.). The final set of washes were at 55°C with 0.5 x SSC containing 0.1% SDS. After washing, the membranes were incubated in a solution of an alkaline phosphataselinked anti-digoxigenin antibody and two phosphatase substrates X-phosphate and nitroblue tetrazolium salt, following the protocol of Boehringer Mannheim Biochemicals (Indianapolis, Ind.). Secondary screening was performed to confirm and purify the positive clones isolated during the primary screening. Inserts in
ZapExpress clones were excised in vivo and inserted into the pBK-CMV phagemid vector using the ExAssist helper phage from Stratagene Cloning Systems. There should be no cross-hybridization between the two Pony probes which are 59.5% identical, because the screening stringency described above allows slightly more than 20% mismatch. This was later confirmed by sequence comparisons between the positive clones isolated using the two different probes.
Estimation of the Copy Number of Pony Elements
A total of four 150-mm plates that contained 31,500 plaques from the unamplified A. aegypti genomic library were screened under the conditions described above. The copy numbers of the two subfamilies were calculated based on the ratio of positive plaques to the total number of plaques screened, taking into account the known size of the haploid genome of the A. aegypti Rock strain (800 Mb; Rao and Rai 1987
), the 1.9-kb average insert size of the genomic library, and the level of background. This method was previously described in Tu, Isoe, and Guzova (1998)
.
PCR and TA Cloning
Pony elements were amplified by PCR using approximately 3 ng of genomic DNA isolated from the Rock strain of A. aegypti. A single primer, TACCGTTTTGNYTCANATTNCGNACA, which corresponds to the TA insertion site plus the consensus sequence of the terminal inverted repeats of known Pony elements, was used in each reaction. The calculated melting temperature of the primer was 68°C. Three different annealing temperatures were used: 60°C, 56°C, and 51°C, respectively. Samples were denatured at 94°C for 35 s and extended at 72°C for 140 s. Approximately 1 U of TakaRa Taq polymerase (Takara, Shuzo Co., Otsu, Shiga, Japan), 1.5 mM MgCl2, 0.2 mM dNTP, and 2 µM primer were used per 20-µl reaction. PCR products were run on a 0.9% agarose gel and purified using a Sephaglas Bandprep Kit from Amersham Pharmacia Biotech. Purified PCR products were cloned in a pCR 2.1 vector using an Original TA Cloning Kit from Invitrogen (Carlsbad, Calif.).
DNA Sequencing
Sequencing of the genomic clones was done with the sequencing facilities at the University of Arizona and Virginia Tech with either T3/T7 primers or custom synthetic primers using an automated sequencer (Model 377, Applied Biosystem International, Foster City, Calif.). Cloned PCR products were sequenced with an IRD800 dye-labeled T7 primer using a 4200S Gene ReadIR sequencing instrument from Li-Cor (Lincoln, Nebr.).
Sequence Analysis and Phylogenetic Inference
Searches for matches of either nucleotide or amino acid sequences in the database (Non-redundant GenBank + EMBL + DDBJ + PDB) were done using Fasta of GCG (Genetics Computer Group, Madison, Wis., version 9.0, 1996) and BLAST (Altschul et al. 1997
). Pairwise comparisons were done by Gap and Bestfit of GCG. Multiple sequences were aligned by Pileup, which is a progressive, pairwise method from GCG. Specific parameters such as gap weight and gap length weight are described in the figure legend of the alignment. Consensus of the multiple-sequence alignment was obtained using Pretty of GCG. Phylogenetic trees were constructed using the minimum-evolution, neighbor-joining, and maximum-parsimony methods of PAUP*, version 4.0 b2 (Swofford 1999
). Specific parameters used in the phylogenetic analyses are described in the corresponding figure legend. One thousand bootstrap replicates were used to assess the confidence in the grouping (Felsenstein and Kishino 1993
). Direct and inverted repeats within MITE sequences were analyzed using GeneQuest of LaserGene (DNASTAR, Inc., Madison, Wis.). DNA secondary structure was predicted using the RNA folding program of GeneQuest, which uses the Vienna modifications (Hofacker et al. 1994
) of the Zuker (1989)
algorithm.
Results
Discovery of a Novel Family of MITEs Named Pony in A. aegypti
The first copy of Pony, Pony-Aa-A1, was discovered in A. aegypti inside a previously reported Wujin element (Tu 1997
). This 514-bp Pony insertion was not detected in the previous paper because a more stringent multiple-sequence alignment method was used to compare different copies of Wujin elements. However, as shown in figure 1A,
a pairwise comparison between two Wujin elements using a condition that imposes less penalty on gap creation indicates that Pony-Aa-A1 was inserted in Wujin-Aa4, resulting in a TA target site duplication. Subsequent database searches identified three additional copies of Pony near other transposable elements and eight copies in the noncoding regions of genes in A. aegypti (table 1
). As described below, analysis of these 12 Pony elements and 26 additional copies suggests that Pony is a novel family of MITEs.
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Additional Pony Sequences Obtained by Library Screening and PCR
Fifteen additional Pony elements isolated from the genomic library were sequenced (table 2
). Analysis of all available sequences indicated that the 24-bp terminal inverted repeats were highly conserved. To survey the diversity of Pony elements and to obtain additional sequences, the consensus of the 24-bp terminal inverted repeats of all available Pony sequences plus the specific TA target sequence was used as the primer to amplify Pony elements from genomic DNA of the Rock strain of A. aegypti in a PCR experiment, as shown in figure 2
. A predominant 500-bp product and a small amount of a 1,000-bp product were obtained in three different reactions at different annealing temperatures (60°C, 56°C, and 51°C, respectively). The 500-bp product obtained at the lowest annealing temperature was cloned. Eleven clones, representing different copies of Pony elements, were sequenced (GenBank accession numbers AF259802AF259812). The 1,000-bp product is likely a dimer of the Pony elements, because when it was purified and reamplified using the same primer, a predominant 500-bp product was obtained (data not shown).
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Despite a Relatively Low Sequence Similarity, the Two Subfamilies Share Several Structural Characteristics, Including a Tc2-like Terminal Inverted Repeat and Conserved Subterminal Motifs
The consensus sequences of the two subfamilies are less than 62% similar, as shown in a pairwise comparison (fig. 5A
). Like all characterized MITEs, the two subfamilies of Pony elements are bordered by imperfect terminal inverted repeats. As shown in figure 5B,
14 of the 18 terminal nucleotides of the two subfamilies of Pony are similar to the terminal sequences of the Tc2 DNA transposon (Ruvolo, Hill, and Levitt 1992
) and the three MITE-like elements in C. elegans (Oosumi, Garlick, and Belknap 1995, 1996
). In addition, as discussed below, Pony tends to specifically insert in a TA dinucleotide target, which is common among Tc2-like elements. A 20-bp sequence motif named
(TTGATTCAWATTCCGRACAS) was identified in both consensus sequences (fig. 5C
). It overlaps the terminal inverted repeats but starts 5 bp inside the termini. Two additional copies of this
sequence were found in the internal regions, one of which had multiple substitutions in the consensus of the B subfamily. Two copies of a segment of
, the 15-bp ß sequence TCAWATTCCGRACAS and the reverse sequence of another short fragment of
, the 8 bp
sequence WATTCCGG, were also highly conserved between the two subfamilies. The majority of the conserved sequences were related to these subterminal repeat motifs (
, ß, and
). The consensus sequences of both subfamilies contain more than 62% A+T. They all have the potential to form stable secondary structures, with
G values lower than -78 kcal/mol (fig. 5D
).
|
Pony Elements Were Often Found near Genes and Other Transposable Elements
As shown in table 1
, five Pony-Aa-A elements and three Pony-Aa-B elements were found in the introns and flanking regions of six genes in A. aegypti. The gene sequences used in the above analysis include 21 genes retrieved from the nonredundant GenBank database (September 1999) and four additional genes, including VgB, VgC (Isoe and Hagedorn, personal communication), AaE74 (unpublished data), and a ferritin gene (Pham and Law, personal communication). It is not yet clear whether this association between Pony elements and the noncoding regions of genes in A. aegypti is random. A large number of randomly selected genes need to be analyzed to address this question. Pony elements were also frequently found near other transposable elements. As shown in table 1
, four Pony elements were found near known transposable elements. In addition, all of the Pony elements found in the noncoding regions of genes had at least one other transposable element nearby (data not shown). Furthermore, as shown in table 2
, 11 of the 14 Pony clones isolated from the genomic library contained at least one other transposable element. Most of the transposable elements found near Pony were either elements of the Feilai family (Tu 1999
), fragments of non-LTR retrotransposons, or MITEs themselves.
Discussion
Diversity and Evolutionary History of Pony Elements
Sequence comparison and phylogenetic analysis indicate that there are two highly divergent subfamilies of Pony elements in A. aegypti (figs. 3 and 4
). The consensus sequences of these subfamilies share less than 62% similarity. Because all 11 elements sequenced from cloned PCR products belong to either the A or the B subfamily and because a degenerate primer that matched the conserved terminal inverted repeats of all available Pony sequences was used in the above PCR experiment at low annealing temperatures (fig. 2
), it is likely that there are only two major subfamilies of Pony elements in A. aegypti. However, we cannot rule out the presence of other subfamilies that are much less abundant. Although the two subfamilies are highly divergent, all but 2 of the 28 full-length Pony elements are more than 85% similar to the consensus sequences of their respective subfamilies. Moreover, most of them are more than 90% similar to their consensus, and phylogenetic analysis showed the presence of multiple subgroups within each of the subfamilies. A small but significant fraction of both subfamilies are truncated copies. Truncations were occurring at both 5' and 3' termini, and the truncated copies were not flanked by TA target duplications. Thus, these truncations could have resulted from recombination, insertion of other transposable elements, and deletions of large terminal segments. Like the full-length copies, a small fraction of the truncated copies showed less than 75% similarity to the consensus of their subfamily, while the majority of them showed 79%95% similarity to their consensus. Although it is not clear how Pony, or any other MITE, might have originated in the genome, different hypotheses can be proposed to explain the evolutionary history of the Pony elements. One hypothesis is that the two subfamilies could have coexisted for a long time in the ancestral species and they were only extensively amplified relatively recently, which could explain the presence of a small number of divergent copies of full-length and truncated Pony elements amid a large number of highly conserved copies within each of these subfamilies. In addition, more than one source gene was amplified which resulted in multiple subgroups within each subfamily. If we assume similar rates of evolution for most Pony elements, amplification events at different times could account for the varied levels of divergence in different subgroups. Alternatively, the two subfamilies could be relatively new in the mosquito genome. Under this hypothesis, to account for the presence of a small number of highly divergent copies within each subfamily, it is necessary to assume that they have been evolving at a much faster rate than the rest of the Pony sequences. To distinguish between these hypotheses, it is necessary to determine the distribution and diversity of Pony in different mosquitoes and perhaps in other insects. Such analysis would also help to address issues such as vertical transmission versus horizontal transfer of MITE families.
The Importance of Subterminal Repeats in Pony and Other MITEs
Sequence comparisons between the two highly divergent subfamilies of Pony elements present a unique opportunity to analyze what features may be important for this and other families of MITEs. In addition to the terminal inverted repeats, the , ß, and
subterminal repeats represent the majority of the conservation between the two subfamilies (fig. 5
), indicating that these repeats may be important structural and/or functional components of the Pony elements. One of the
repeats is less conserved between the two subfamilies because of three substitutions in the consensus of the B subfamily, which could indicate that not all of the repeats are equally important. The occurrence of subterminal repeats has been previously documented in a few MITEs (Morgan and Middleton 1990
; Bureau and Wessler 1992
; Charrier et al. 1999
). New subterminal repeats have been identified in a number of MITE families, as shown in table 3
, although not all of the MITEs surveyed contained significant subterminal repeats. Therefore, subterminal repeats could be an important feature for a subgroup of MITEs. It is possible that some of the conserved repeat motifs in the Pony sequences and other MITEs may serve as multiple binding sites for transposases or other proteins that are involved in the transposition process. The ATTTGCAT octamer repeats in a MITE-like element from Xenopus have been shown to bind to an oocyte nuclear protein (Morgan and Middleton 1990
). A segment of the
motif of the Pony elements, TTGATTCAW, is similar to one of the two repeat motifs (TTGATTCAY) that constitute the strong binding site of the bacterial transposon Mu (Groenen, Timmers, and van de Putte 1985
), although there is no evidence yet for the function of this sequence in Pony elements.
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Genomic Distribution of MITEs and the Evolution of Eukaryotic Genomes
Both of the two subfamilies of Pony elements are highly reiterated in A. aegypti. They constitute approximately 1.1% of the entire genome. There was no close physical linkage between the two subfamilies. However, as shown in tables 1 and 2,
Pony elements are frequently found near other transposable elements. Most of the transposable elements found near Pony are either elements of the Feilai family, fragments of non-LTR retrotransposons, or MITEs themselves, indicating a possible nonrandom distribution of Pony elements in the genome. On the other hand, Pony elements are also frequently found in the noncoding regions of genes in A. aegypti. It seems that Pony elements are not biased against genic regions. This is consistent with our preliminary analysis showing concentrations of a number of repetitive elements in the noncoding regions of a number of genes in A. aegypti (unpublished data). Nonrandom distribution of MITEs and other transposable elements have previously been indicated in A. aegypti (Tu 1997, 1999
; Tu, Isoe, and Guzova 1998
; Tu and Hill 1999
). Together with the other MITEs (Tu 1997
), the highly reiterated Pony elements may have contributed to the highly repetitive nature and the pattern of "short period interspersion" of the A. aegypti genome (Warren and Crampton 1991
). In addition, many families of highly repetitive MITEs have been discovered in genomes that have high levels of repetitive sequences, such as several cereal grasses, a frog, and humans (e.g., Morgan 1995
; Ünsal and Morgan 1995
; Bureau, Ronald, and Wessler 1996
; Smit and Riggs 1996
; Izsvák et al. 1999
). In contrast, no highly repetitive MITEs have been reported in the genome of Drosophila melanogaster, which has a low level of repetitive sequences and a pattern of "long period interspersion" (Crain et al. 1976
). Although a number of families of MITEs have been found in C. elegans, which has a small and compact genome (Oosumi, Garlick, and Belknap 1996
), the copy numbers of these MITEs are generally low. Similarly, MITEs are much less abundant in the genome of Arabidopsis thaliana, which has only approximately 4% repetitive sequences (Casacuberta et al. 1998
; Surzycki and Belknap 1999
). The distribution of MITE-like elements in these various genomes suggests that the massive proliferation of MITEs may be associated with more repetitive genomes in both the plant and the animal kingdoms. The difference in the relative abundance of MITEs may have in part contributed to the different organizations of the eukaryotic genomes and may reflect different types of interactions between the hosts and these widespread transposable elements.
In summary, this study describes a novel family of highly divergent and highly reiterated MITEs in the yellow fever mosquito, A. aegypti. Evolutionary insights were brought to view regarding the expansion of the Pony family and its impact on the organization of the genome. Pony may have been borrowing the transposition machinery from an autonomous DNA transposon, although the detailed mechanisms remain to be determined. Specifically, it remains to be seen how Pony might have attained a very high copy number through a DNA-mediated transposition mechanism. The rapid accumulation of genomic sequences from a wide range of organisms will almost certainly provide a better understanding of the diversity and common characteristics of this large group of transposable elements. Such information, as well as the knowledge of the mechanism of their transposition and expansion, will undoubtedly shed light on the evolution of eukaryotic genomes.
Supplementary Material
The sequences reported in this manuscript have been deposited in GenBank with accession numbers AF208664AF208681 and AF259802AF259812.
Acknowledgements
I thank Shirley Luckhart, Jiann-Shin Chen, Andrea Crampton, and Chunhong Mao for critical comments on the manuscript. I thank Jennifer Hill and Yumin Qi for valuable technical assistance. I also thank the sequencing facilities at the University of Arizona and Virginia Tech for their service. I am indebted to Jun Isoe, Henry Hagedorn, Daphne Pham, and John Law for sharing unpublished data. This work was supported by NIH grant AI42421 to Z.T. and by the Agricultural Experimental Station at Virginia Tech.
Footnotes
1 Abbreviations: MITE, miniature inverted repeat transposable element; SINE, short interspersed repetitive element.
2 Keywords: MITES
SINES
interspersed repeats
genome
evolution
phylogenetics
3 Address for correspondence and reprints: Zhijian Tu, Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. E-mail: jaketu{at}vt.edu
.
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