Insertion Preference of Maize and Rice Miniature Inverted Repeat Transposable Elements as Revealed by the Analysis of Nested Elements

Tourist Multimers in Maize

The first reported MITE was the B2 element, found as a 128-bp insertion into the maize waxy (wx) gene in the mutant wxB2 allele (Wessler and Varagona, 1985; Bureau and Wessler, 1992). Subsequent database searches revealed that this element belongs to a large family of related elements, called Tourist, whose members are associated with the noncoding regions of genes from maize, sorghum, barley, and rice (Bureau and Wessler, 1992, 1994a). Tourist multimers were discovered initially in a polymerase chain reaction (PCR) assay that was intended to identify additional B2-like (Tourist) elements in maize. Genomic DNA from maize inbred B79 was amplified with primers derived from the B2 TIR (14 bp) and 11 bp of internal element sequence. In addition to a product corresponding to the size of the B2 element (128 bp), we observed larger fragments that varied in size depending on the annealing temperature (Figure 1) . PCR products from all size classes were cloned, and several were sequenced, revealing monomers, dimers, and a trimer (Figure 1). Four different multimers were found among the six dimer-sized clones that were sequenced. In contrast, the largest PCR fragment corresponded to a single trimer. All multimers contained a variety of elements that, like B2, are members of Tourist subfamily A (Bureau and Wessler, 1994a).

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Figure 1.

The Identity of Multimers Resulting from PCR Amplification of B2-Tourist Elements in Maize Line B79.

PCR products resolved by agarose gel electrophoresis and visualized by ethidium bromide staining were purified, cloned, and sequenced (see Methods). The annealing temperature for PCR was 60°C for the two samples at left and 55°C for the three samples at right. The identities of PCR bands are diagrammed at right. The positions of the insertion sites of the various Tourist subfamily members (Zm3, Zm11, Zm22, and Zm29) (Bureau and Wessler, 1992, 1994a; N. Jiang and S. Wessler, unpublished data) into B2 elements are shown along with the TSD. M, DNA molecular weight standard (bp).

To exclude the possibility that the Tourist multimers were artifacts of PCR amplification, we used dimer and trimer products to probe a small insert library derived from B79 genomic DNA. Three of 11 sequenced clones contained Tourist multimers, thus confirming the presence of Tourist multimers in the genome.

Insertion into Preexisting MITEs

Among maize inbred lines, the insertion sites of MITEs frequently were polymorphic with respect to the presence or absence of an element at a particular locus (Casa et al., 2000; Zhang et al., 2000). Polymorphism of this type usually is associated with the recent spread of transposon families through the genome. In light of these findings, we designed a PCR assay to detect insertion site polymorphism within Tourist multimers. In this way, evidence might be obtained for the sequential insertion of one element into another.

The locus harboring the Tourist trimer (Figure 1) was investigated for possible insertion polymorphism among different maize lines. Following the methodology described in Methods, we obtained B79 genomic sequence adjacent to one end of the trimer, revealing that another Tourist element (Tourist-Zm22) had inserted adjacent to the trimer with only an intervening target site duplication (TSD) (Figure 2) . A locus-specific primer was designed from the sequence flanking Tourist-Zm22 and used to amplify B37 genomic DNA together with a B2 terminal primer (PB2r). The resulting PCR product, which harbored an additional Tourist element (Tourist-Zm3) (Figure 2), provided evidence for the progressive formation of multimers (tetramers from trimers).

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Figure 2.

Diagram of a MITE Trimer and Tetramer Found at the Same Locus in Different Maize Lines.

As in Figure 1, the positions of the insertion sites of the various Tourist subfamily members into Tourist-B2 and other Tourist-Zm elements are shown along with the TSD. The sequence flanking the trimer in B79 was amplified with primer Pb and an adapter primer (see Methods for details). For B37, the tetramer was amplified using a locus-specific primer, Pf, and the B2 primer PB2r. The number above each element in B79 indicates the similarity between the element in B79 and its counterpart in B37. The asterisk indicates that the primer was labeled with ³³P.

Nonrandom Insertion Sites

Insertion sites within the sequenced multimers clearly were nonrandom. For ease of comparison, insertion sites have been calculated as the number of base pairs from the closest end of the target element to the first nucleotide of the TIR of the insertion element. For all insertions examined, this value corresponded to 27, 37, or 47 bp (Figure 1). To determine whether this periodicity was representative of the multimers in the maize genome, a two-step PCR assay was used to isolate additional multimers. In this assay (see Methods), the length of the PCR products reflects the position of the insertion sites within the multimers. That is, if the insertion sites are 10 bp apart, the PCR products will appear, more or less, as a 10-bp “ladder” on the gel. Such a ladder was observed (Figure 3) . Furthermore, sequencing of selected PCR products revealed that all contained a Tourist-Zm3 element inserted into another Tourist element at ∼10-bp intervals. The composition of some of the multimers is diagrammed in Figure 3. In addition, these data and the data from all previous multimer sequences are summarized in Table 1.

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Figure 3.

Autoradiograph of PCR Products Resolved on an Acrylamide Gel Showing an ∼10-bp Ladder.

The positions of primers used to obtain the PCR products from maize lines or plasmid DNA are indicated by horizontal arrows; the composition of multimers represented by the indicated PCR products also is diagrammed. Vertical arrows over some multimers represent Zm3 insertions. Lane 1, Zm3-B2 dimer containing plasmid; lane 2, B79 genomic DNA; lane 3, B73 genomic DNA; lane 4, recombinant inbred DNA from a B73 × Mo17 mapping population. The asterisk indicates that the primer was labeled with ³³P.

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Table 1.

Insertion Sites within Tourist Dimers and Trimers

MITE Multimers in Rice

In the absence of a significant amount of maize genomic sequence, analysis of maize multimers is restricted to a description of the phenomenon and the characterization of a small fraction of the existing elements. A more thorough survey, including an estimate of the proportion of the multimers present, is possible for rice because a large amount of rice genomic sequence is available publicly (Yuan et al., 2001) and the rice genome contains thousands of MITEs (Mao et al., 2000; Tarchini et al., 2000).

Self-Insertions

The data presented in Table 2 reveal a slight preference for insertion of MITEs into MITEs. However, analysis of these data for individual MITE families indicates that this preference is not displayed by all families and is attributable largely to self-insertions. Four MITE families were analyzed in detail (Table 3). These families were chosen because they are abundant and they represent different groups of MITEs. Among the four families analyzed, Castaway, Gaijin, and Ditto are related to Tourist elements in maize, whereas Stowaway elements belong to another superfamily (see Discussion). As shown in Table 3, all of the Tourist-related elements have sustained more insertions per kb of DNA than has the genome as a whole (insertion frequencies of 0.38, 0.32, and 0.60, respectively, versus 0.22 for all DNA; Tables 2 and 3). In contrast, Stowaway has sustained insertions at approximately the same frequency as the rest of the genome. Although the increased insertions into Castaway and Gaijin elements can be accounted for completely by self-insertions, Ditto elements appear to attract a variety of MITEs (see Discussion). The cluster of six elements from chromosome 1 (Figure 4) illustrates the propensity for self-insertion among Castaway family members.

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Table 3.

Self-Insertion Preference of Some MITEs in Rice

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Figure 4.

A Cluster of Six Castaway Elements on a Rice P1-derived Artificial Chromosomes (PACs) Clone (GenBank Accession Number ap002844) from Chromosome 1.

The distance from the end of the element to the insertion site is shown. Castaway-2 is a member of a subfamily of Castaway. The +3 indicates the inclusion of the 3-bp TSD that was generated upon insertion.

One could argue that the observed higher self-insertion frequency of MITEs reflects a preference of MITEs for particular regions of the genome rather than a preference for other members of the same family. If this is the case, for a certain family of MITEs there would be a comparable number of insertions into sequences flanking MITEs as there are into MITEs. Fortunately, the availability of 30.2 Mb of rice contigs permits an analysis of the insertions into MITEs and their flanking sequences. On the basis of the data presented in Figure 5 , it is evident that for all families examined, except Stowaway, the self-insertion frequencies of MITEs are significantly higher than the insertion frequencies into their flanking sequences (P < 0.01 by χ² test).

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Figure 5.

The Frequencies of Self-Insertions and Insertions into Flanking Sequences for Four MITE Families in Rice.

Frequencies were calculated in the same way as in Table 2 (see Methods for details).

Self-Insertion Preference for Some MITE Families

As calculated in Table 2, the insertion frequency of all MITEs into other MITEs is slightly higher than the average value into the whole genome. However, there is a threefold variation in the frequency of MITE insertions into MITEs when individual families are examined (Table 3). More significantly, self-insertions constituted a major part of the multimers for several families. For Castaway, Gaijin, and Stowaway, self-insertions account for two-thirds of all insertions. These data indicate that the preferential insertion of MITEs into MITEs that is displayed by some families can be attributed, to a great extent, to self-insertions. One exception is the Ditto element. Among the rice MITEs, Ditto elements are targeted frequently by various types of elements, including other Ditto elements. In addition to being targeted 53 times by 12 families of MITEs, we detected five cases of insertions by four different LTR retrotransposons and 22 examples of MITEs inserted in adjacent (with an intervening TSD) sequences.

Composite elements, arising from self-insertion, have been reported previously in maize, in which double Ds and Ac elements were shown to be responsible for chromosome breakage and more complex rearrangements (McClintock, 1949; Courage-Tebbe et al., 1983; Döring and Starlinger, 1984; Weck et al., 1984; Döring et al., 1989; Michel et al., 1994). It was later hypothesized that chromosome breakage resulted from aberrant transposition of composite or adjacent Ds elements (English et al., 1993; Weil and Wessler, 1993). In contrast to the composite Ds elements that are still capable of transposition, the uniqueness of each MITE multimer suggests that self-insertion creates an inactive composite element. Inactivating self-insertions of the Tp1 element of Physarum polycephalum have been observed previously (Rothnie et al., 1991). It has been proposed that a preference for inactivating self-insertions minimizes deleterious effects on the host by providing a safe haven for insertion while simultaneously limiting the overall transposition frequency (Rothnie et al., 1991).

Regional versus Self-Insertion Preference

Previous studies indicate that some MITE families insert preferentially into genic regions (Mao et al., 2000; Zhang et al., 2000). A preference for genic regions also has been observed for the maize class 2 families Ac/Ds and Mutator (Chen et al., 1992; Cresse et al., 1995). Regional preferences have been demonstrated for many elements in a wide variety of species. For example, yeast Ty5 elements integrate preferentially into regions of silent chromatin at the telomeres and the mating loci (Zou et al., 1996), and for P elements, euchromatic sites, especially 5′ regions of genes, are targeted more often than heterochromatin (Berg and Spradling, 1991; Liao et al., 2000).

Regardless of the mechanism responsible, an element with a regional preference is more likely to have a higher frequency of self-insertion than an element with no such preference. If the regional preference is the major factor leading to a high self-insertion frequency, comparable insertion frequencies are expected into elements and into their flanking genomic sequences. The availability of 30.2 Mb of rice sequence allowed us to test this assumption (Figure 5). For Castaway, Gaijin, and Ditto, the self-insertion preference is more likely to be caused by the targeting of preexisting elements than by a regional preference. In contrast, Stowaway elements show no significant difference between insertion into preexisting elements and insertion into flanking DNA, thus suggesting that the high ratio of self-insertions results from a regional preference. Alternatively, the presence of one Stowaway element may alter the flanking DNA in some manner, thereby creating a better target for future insertions. A similar effect was observed for the in vitro transposition of the C. elegans Tc1 element (Ketting et al., 1997). Interestingly, Stowaway elements, like Tc1, use TA dinucleotide targets.

The difference between Stowaway and the three other MITE families may indicate distinct integration mechanisms for different MITE families. Like the Tourist elements in maize, Castaway, Ditto, and Gaijin all create a 3-bp TSD upon insertion (Bureau et al., 1996). More importantly, the TIRs of Castaway, Ditto, and Gaijin are related to the TIR of Tourist elements in maize, suggesting that they may belong to the same superfamily. In contrast, Stowaway elements appear to belong to another superfamily based on their TIR and TSD (Bureau and Wessler, 1994b). Therefore, it is likely that these two superfamilies rely on distinct sources of transposases.

Target Site Preference in Maize Multimers

The potential to form secondary structures has been noted for several MITE families since the discovery of the Tourist family in maize (Bureau and Wessler, 1992; Izsvák et al., 1999). Given the occurrence of multimers among maize Tourist-B2 elements, we hypothesized that secondary structures might play a role in targeting. Consistent with this notion is the inability to detect MITE multimers involving two other maize MITE families (Hbr and mPIF) lacking significant secondary structures (N. Jiang, Q. Zhang, X. Zhang, and S.R. Wessler, unpublished data). However, in the rice genome, multimer formation does not correlate with the potential to form significant secondary structures. In rice, the MITEs that sustained the most insertions, Castaway and Ditto, are those without significant secondary structures (Bureau et al., 1996). In contrast, Stowaway elements usually have significant secondary structures but do not show a targeting bias. However, these data cannot rule out the possibility that small, local stem loops, such as the 14-bp palindrome targeted by P elements (Liao et al., 2000), might influence targeting of MITEs.

The analysis of MITE multimers in rice also was prompted by the discovery of nonrandom insertion sites among Tourist multimers in maize (Figures 1 and 2, Table 1). The 10-bp periodicity observed for Tourist multimers is reminiscent of the integration of human immunodeficiency virus. Integration of human immunodeficiency virus in vitro occurs preferentially into bent DNA in which the major groove is on the exposed face of the nucleosome (Pryciak and Varmus, 1992; Pruss et al., 1994). The 10-bp periodicity for Tourist multimers could be produced in a similar pattern (i.e., the transposition machinery attacks only major or minor grooves of the DNA double helix).

In rice, some “hot” spots for insertion were observed inside the sequence of some MITEs, and some of the insertion sites are ∼10 bp apart. However, insertions that are not 10 bp apart also were observed. Because of the fact that the rice MITEs that sustained most insertions are much larger than maize Tourist elements (maize Tourist, 130 bp; Ditto, 244 bp; Castaway, 364 bp), the distribution of insertion sites appears to be sporadic within rice MITEs. Thus, more rice multimers need to be examined to determine whether or not the 10-bp pattern is statistically significant. Alternatively, this feature may belong only to Tourist elements in maize.

To date, no autonomous element responsible for the transposition of MITEs has been available. The isolation of such elements and their associated protein(s) will ultimately facilitate the biochemical analysis of the various levels of targeting exhibited by MITE families.

Deficiency of MITE Insertions into Non-MITE Elements: Targeting Preference or Temporal Differences in Amplification?

A surprising and dramatic conclusion of the data presented in Table 2 is that MITEs have inserted into MITEs 80 times more often than they have inserted into LTR retrotransposons and 32 times more often than they have inserted into other DNA elements (one MITE insertion versus 4, 330, and 127 kb, respectively). In contrast, the frequency of insertion of LTR retrotransposons and other DNA elements into MITEs is only slightly lower than the overall frequency of insertion of these elements into rice genomic DNA (one insertion per 17 kb of MITEs versus one insertion per 14 kb of genomic DNA).

Previous studies have noted a genic preference for maize class 2 elements, including members of the Ac/Ds and Mutator families (Chen et al., 1992; Cresse et al., 1995). Differences in chromatin density and/or the extent of DNA methylation between gene-rich and other regions of the genome have been proposed as possible target recognition mechanisms (Chen et al., 1992). A similar preference for genic regions has been demonstrated for members of the MITE families Hbr and mPIF (Casa et al., 2000; Zhang et al., 2000; X. Zhang, N. Jiang, and S.R. Wessler, unpublished data). In contrast, MITEs appear to be underrepresented in regions of the maize and barley genomes containing nested or clustered LTR retrotransposons (Tikhonov et al., 1999; Dubcovsky et al., 2001). Although MITEs may target gene-rich regions by the same or similar mechanisms as other class 2 elements, the analysis of MITE multimers in rice provides at least two alternate explanations for the observed (skewed) distribution. Enrichment for MITEs in genic regions and their apparent absence from retrotransposon clusters or domains could reflect a self-insertion preference coupled with avoidance of retrotransposon targets. Alternatively, a dearth of MITE insertions into non-MITE transposons also would result if the bulk of MITE amplification occurred before the amplification of LTR retrotransposons and other class 2 elements. To unambiguously distinguish between these seemingly mutually exclusive hypotheses, it will be necessary to identify an active MITE system that can be exploited to experimentally determine MITE target preference(s). In the mean time, we must rely on the comparative analysis of related genomes to provide clues to the mechanisms underlying the observed distributions of transposable elements.

Plant Material, DNA Extraction, and Library Construction

Maize (Zea mays) lines B79 and B37 were obtained from the U.S. Department of Agriculture, Agricultural Research Service Plant Introduction Station at Ames, Iowa. Maize line B73 and recombinant inbred lines from a cross between B73 and Mo17 were provided by Michael Lee (Iowa State University, Ames). Maize line Spanco was provided by Andy Tull (University of Georgia, Athens). Plant DNA was extracted as described (McCouch et al., 1988). The small insert genomic library from B79 genomic DNA was constructed as described (Zhang et al., 2000).

Polymerase Chain Reaction and Gel Electrophoresis

Polymerase chain reaction (PCR) was performed as described (Bureau and Wessler, 1992) with annealing temperature ranging from 55 to 60°C, depending on the primers. Sequences of primers are available on request.

To clone the flanking sequence of the Tourist trimer in Figure 1, B79 genomic DNA was digested with MseI and ligated with adapters. The DNA then was amplified with a primer complementary to the adapter and primer Pb, which contains the sequence at the junction of (Tourist) Zm3 and the B2-like element (Figure 2). To separate PCR products that resulted only from adapters and PCR products from the two primers, primer Pb was labeled with ³³P, and the PCR products were loaded on 6% denaturing acrylamide-bisacrylamide gels and electrophoresed as described previously (Casa et al., 2000).

The two-step PCR assay described in Figure 3 involved amplification of genomic DNA with primers P1 and P2, followed by amplification of the PCR products with primers P2 and P3 (P3 was labeled with ³³P). PCR products were resolved by PAGE, as described above.

Recovery of Gel Bands

DNA fragments were excised from radioactive gels by scratching the dried gel with yellow tips (Stumm et al., 1997; Elsevier Trends Journals Technical Tips online, http://tto.biomednet.com/cgi-bin/tto/pr), placing the tip in 20 μL of PCR reaction mix with relevant primers for 1 min before discarding, and reamplifying with the same cycling parameters as that of the original reaction. PCR products were resolved on 0.8% agarose gels, and fragments were excised, purified (QIAquick; Qiagen, Chatsworth, CA), and cloned (TA cloning kit; Invitrogen, Carlsbad, CA). DNA templates were sequenced at the Molecular Genetics Instrumentation Facility (University of Georgia).

DNA Sequence Analysis

DNA sequence analysis (pairwise comparisons, multiple sequence alignments, and sequence assembling and formatting) was performed with programs in the University of Wisconsin Genetics Computer Group program suite (GCG, version 10.1) accessed through Research Computing Resources (University of Georgia).