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Genetics, Vol. 176, 2477-2487, August 2007, Copyright © 2007
doi:10.1534/genetics.107.071811
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* Center for Biosystems Research, University of Maryland Biotechnology Institute, Rockville, Maryland 20850 and Department of Entomology, University of California, Riverside, California 92521-0314
1 Corresponding author: Center for Biosystems Research, University of Maryland Biotechnology Institute, 9600 Gudelsky Drive, Rockville, MD 20850.
E-mail: obrochta{at}umbi.umd.edu
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ABSTRACT |
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= 0.0079 in noncoding sequences and 0.0046 in coding sequences) relative to that observed in some of the more well-studied elements in Drosophila melanogaster. In total, 33 distinct forms of Herves were found on the basis of the sequence of the first 528 bp of the transposase open reading frame. Only two forms were found in all six study populations. Although Herves elements in An. gambiae are quite diverse, 85% of the individuals examined had evidence of complete forms of the element. Evidence was found for the lateral transfer of Herves from an unknown source into the An. gambiae lineage prior to the diversification of the An. gambiae species complex. The characteristics of Herves in An. gambiae are somewhat unlike those of P elements in D. melanogaster.
Designing gene vectors and effector transgenes for refractoriness such that they will increase in natural populations and eventually reach fixation is a considerable challenge, and transposable elements may provide a means by which this can be accomplished (BRAIG and YAN 2001). The replicative nature of transposable element movement (even by elements that move by a cut-and-past fashion i.e., Class II elements) results in elements acquiring a transmission advantage, resulting in their gradual increase in frequency in populations (RIBEIRO and KIDWELL 1994; KISZEWSKI and SPIELMAN 1998). The magnitude of that transmission advantage is determined by the rate of transposition; the degree to which transposition is conservative or replicative; the spatial patterns of element transposition within a genome; the biology of the transposable element and its interactions with the host insect; and the size, structure, and characteristics of the target population (RASGON and GOULD 2005).
While intraspecies spreading of transposable elements through transposition has been observed in nature following recent horizontal transfer events involving transposable elements (e.g., P- and hobo elements), population modification has never been attempted by the deliberate and intentional release of an active autonomous transposable element into natural populations of insects (ROBERTSON 2002). Predicting the outcome of such an intentional release of transgenic insects containing active autonomous transposable element gene vectors is an enormous challenge but one that must be successfully met if population-replacement biological control using transposable elements is to be successful (ALPHEY et al. 2002). Data that might inform those predictions include an understanding of the dynamics of endogenous Class II transposable elements within the host insect. Endogenous elements are likely to reveal temporal and spatial patterns of spread as well as how population structure has influenced those patterns. Currently our understanding of the population dynamics of Class II transposable elements in insects is based almost entirely on studies of P- and hobo elements in Drosophila melanogaster and closely related species (ANXOLABEHERE et al. 1988, 1990; BUCHETON et al. 1992; SIMMONS 1992; SILVA and KIDWELL 2004). These studies have documented the ability of these elements to spread rapidly through populations and for the elements to become structurally modified over time, most often by internal deletion. The propensity of these elements to accumulate internal deletions rapidly has raised a serious concern about using transposable elements as transgene spreading agents, namely, the frequent loss of transgenes. Maintaining tight linkage between the antiparasite effector gene and the associated gene drive system has been repeatedly stated as an essential characteristic of this biological control strategy (CURTIS 2003; JAMES 2005). To what extent these characteristics of P, hobo, and mariner elements are general characteristics of Class II elements remains to be fully explored. Because a proposed target species for this novel population-replacement–based biological control strategy is the human malaria vector Anopheles gambiae, the study of Class II transposable element dynamics in this species is particularly relevant.
Recently, a functional hAT element Herves was discovered in An. gambiae, providing an opportunity to examine the dynamics of an active Class II transposable element in this insect (ARENSBURGER et al. 2005). Herves is notably different at the sequence level from the well-studied hobo element from D. melanogaster and Hermes from Musca domestica, sharing only 
20% amino acid identity with these elements (ARENSBURGER et al. 2005). A Herves element isolated from the reduced susceptibility to permethrin (RSP) strain of An. gambiae that was established as a laboratory colony in the early 1990s (VULULE et al. 1994) was shown to be transpositionally active in laboratory-based mobility assays in D. melanogaster (ARENSBURGER et al. 2005) and Aedes aegypti (P. ARENSBURGER and P. ATKINSON, unpublished data). A recent study of the element's abundance and site-occupancy frequency in natural populations of An. gambiae s.s., An. merus, and An. arabiensis in Mozambique revealed that it was present in all three species at approximately five copies per diploid genome, and site-occupancy frequency distributions suggested that Herves had been recently active in the three species examined (O'BROCHTA et al. 2006). In the population of An. gambiae examined in Mozambique, 95% of the individuals tested contained intact (nondeleted) forms of the element, which is quite unlike P elements in D. melanogaster in which most elements are internally deleted derivatives of the canonical element (O'HARE et al. 1992). Here Herves has been investigated in six populations of An. gambiae using a variety of methods to see if the characteristics of the element observed in Mozambique were general features of the element and how it compares to other well-studied Class II elements.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Species identification:
Species identification was performed using the method of SCOTT et al. (1993) as described (O'BROCHTA et al. 2006) using 1/100th of the total genomic DNA from a single mosquito in a volume of 1 µl (SCOTT et al. 1993). This method permits the identification of species-specific polymorphisms in the intergenic spacer region of ribosomal RNA genes using PCR. Only An. gambiae s.s. samples yielding unambiguous species identification results were used in subsequent analyses.
Transposable element display:
Transposable element display is a PCR-based DNA fingerprinting method derived from the amplified fragment length polymorphism (AFLP) method (VOS et al. 1995). It was performed here as described previously with only minor modifications (O'BROCHTA et al. 2006). Transposable element display was performed in triplicate using 2–5 µl (200 ng) of genomic DNA for each replicate. Genomic DNA was digested for 4 hr in a volume of 40 µl at 37° with 4 units of the restriction endonuclease MseI using conditions recommended by the manufacturer (New England Biolabs, Ipswich, MA). Sixty picomoles of adapters were ligated to the MseI digestion products by adding 10 µl of 1x restriction enzyme buffer containing 5 mM ATP, 50 mM DTT (dithiothreitol), 10 µg BSA (bovine serum albumin), 4 units of MseI, and 1 Weiss unit of T4 DNA ligase and incubated at 37° overnight. The adapters were prepared by mixing equimolar amounts of oligonucleotides HhaIa (5'-GAT GAG TCC TGA GTA CG-3') and MseIb2 (5'-TAC GTA CTC AGG ACT CAT CAA G-3'), heating them to 100° for 10 min, and then allowing the mixture to very slowly cool to room temperature. The design of the adapters and the digestion/ligation reaction conditions result in the efficient creation of only monomeric MseI-cut genomic DNA fragments with terminal adapters.
Five microliters of the restriction/ligation reaction were used as the template in a polymerase chain reaction ("preselective PCR") performed in a 50-µl reaction volume containing 1x PCR Buffer II (Applied Biosystems, Foster City, CA), 0.2 mM dNTPs (an equimolar mixture of dATP, dTTP, dCTP, and dGTP), 2.5 mM MgCl2, 1 unit AmpliTaq DNA polymerase (Applied Biosystems), and 24 pmol of primer HhaIa and primer HervTEDAL1a (5'-ATT TCG ACG GGT TCC TAC C-3'). HervTEDAL1a is a Herves-specific primer that anneals to sequences 150 bp from the 5' end of the element. The DNA polymerase was added as a complex with TaqStart Antibody (ClonTech, Mountain View, CA) as described by the manufacturer for the purpose of "hot-starting" the reaction. The reaction conditions were 95°/3 min followed by 25 cycles of 95°/15 sec, 63°/30 sec, and 72°/1.0 min and a final cycle of 72°/5 min. A second PCR was performed ("selective PCR") using 5 µl of the preselective PCR products as template in a 20-µl reaction containing 1x PCR Buffer II, 0.2 mM dNTPs, 2.5 mM MgCl2, 1 unit AmpliTaq DNA polymerase (bound to TaqStart Antibody as above), and 9 pmol each of primers HhaIa and Cy5-labeled HervTEDAL2 (5'-GTT GAT TAG ATG AAC GTA GG-3'). The Cy5-labeled primers were purified by HPLC prior to their use. HervTEDAL2 anneals to sequences 80 bp from the 5' end of the element. Following a denaturation step at 95° for 3 min, "touchdown" PCR conditions were created in which during the first 5 cycles the annealing temperature was decreased 1° after each cycle with the first of these cycles being 95°/15 sec, 64°/30 sec, and 72°/1.0 min. Following these 5 cycles an additional 25 cycles were performed at 95°/15 sec, 60° /30 sec, and 72° /1.0 min with a final cycle of 72°/5 min.
To visualize products of transposable element display, 5 µl of selective PCR products were mixed with 5 µl of loading buffer (95% deionized formamide and 10 mM EDTA) heated to 95° for 3 min and cooled quickly on ice, and 6 µl were loaded on a 6% polyacrylamide gel (19:1 acrylamide:bisacrylamide) containing 6.7 M urea in 1x TBE buffer (90 mM Tris-borate and 2 mM EDTA). ALFExpress Sizer 50-500 (GE Healthcare/Amersham, Piscataway, NJ) was used as a size standard. Electrophoresis was performed at 70 W (constant) for 2.5 hr at which time the gel was transferred to 3 mm filter paper and dried. The dried gel was scanned on a STORM 860 phosphoimager (GE Healthcare/Molecular Dynamics, Piscataway, NJ). The products obtained from the three independent replicate reactions of the same sample were run on the same gel to assist with determining the presence of bands. On the basis of the combined results of three transposable element display experiments, a band was called as present or absent if it was unambiguously present in at least two of the three replicates. Determining the presence of bands in this way resulted in a single scoring matrix that was then used in subsequent analyses.
Site-occupancy frequency distributions were estimated using transposable element display data. Using the frequency distributions and assuming the model of CHARLESWORTH and CHARLESWORTH (1983), the model parameter ß, which measured in part the forces removing insertions from natural populations, was estimated. The model parameter ß is equal to the product of four times the effective population size and the rate of element loss. Estimation of ß and the copy number of Herves per diploid genome were performed as described by WRIGHT et al. (2001) who considered the dominant nature of transposable element display signals and the application of the parameter estimation methods of CHARLESWORTH and CHARLESWORTH (1983) to diploid organisms. Note that although each sample was analyzed three times for transposable element display, these replicates were used to produce a single scoring matrix. The advantage of this procedure is that it increased the accuracy of determining the presence of bands and minimized errors that tended to result in overestimations of ß.
Transposase open reading frame detection:
To assess Herves open reading frames for the presence of deletions and insertions, PCR primers were designed that were complementary to sequences flanking the transposase ORF: 1372f (5'-CCA CAA ATT GAT CTA CGC TCC-3') and 3469r (5'-GAT GCA TCT ATT ATG ATT AAG GC-3'). One-fiftieth of the genomic DNA from one mosquito (2 µl) was used as template in a 50-µl reaction containing 1x ThermalAce (Invitrogen, Carlsbad, CA), 0.2 mM dNTPs (an equimolar mixture of dATP, dTTP, dCTP, and dGTP), 2.5 mM MgCl2, 2 units ThermalAce DNA polymerase (Invitrogen), and 24 pmol of primers 1372f and 3469r. Amplification reactions were performed under the following conditions: 95°/3 min followed by 30 cycles of 95°/30 sec, 48°/30 sec, and 72°/3.0 min and a final cycle of 72°/10 min. Reaction products were fractionated on a 1% agarose gel. PCR products of the samples that failed to produce a detectable product following one round of PCR were used as templates (5 µl) in a second PCR under the same conditions described above but with primers 1407f (5'-GAT CAA AGG TAA CAT TAG TCT TG-3') and 3294r (5'-CCA TGT TAC AAA TTT TGC AAC G-3') and rechecked on a 1% agarose gel. Open reading frames free of deletions and insertions yielded PCR products 2100 bp after the first PCR and 1900 bp after the second PCR. We estimate that elements with deletions as small as 100 bp would be detectable using this strategy.
Sampling and PCR for population analysis:
Transposable element display permitted occupied sites to be identified, and these data were used in determining the composition of the subset of individual mosquito genomic DNAs that would be used in the analysis of sequence diversity of 1474 bp of the noncoding region and the first 528 bp of the transposase open reading frame. This selected subset of individual mosquito genomic DNAs was such that Herves elements at most occupied sites, as determined by transposable element display, were included in the PCR template pool. So, a total of 49 individuals containing elements at the 130 different sites identified by transposable element display were included in the PCR template pool to give us an opportunity to amplify Herves elements inserted at different genomic sites within the populations. Using this subset of genomic DNAs, a portion of the left end of the element was amplified using a nested PCR strategy. Five microliters of genomic DNA from each of the 49 individuals were used as template in separate 20-µl reactions containing 5x Phusion HF Buffer (New England Biolabs), 0.2 µM dNTPs (an equimolar mixture of dATP, dTTP, dCTP, and dGTP), 0.4 units Finnzymes Phusion DNA polymerase (New England Biolabs; error rate = 4.4 x 10–7), and 24 pmol of primer 24F (5'-TAG AGT TGT GCC TCA AGA ACC AGA-3') and primer 2035R (5'-TGG TTC AGG TTT GTC CAT CC-3'). Amplification reactions were performed under the following conditions: 98°/1 min followed by 25 cycles of 98°/10 sec, 65°/30 sec, and 72°/1 min 30 sec and a final cycle of 72°/10 min. Reaction products were fractionated in a 1% agarose gel. PCR products from samples that failed to produce detectable products on an agarose gel following one round of PCR were used as templates (5 µl) in a second PCR under the same conditions described above using primers 24F (5'-TAG AGT TGT GCC TCA AGA ACC AGA-3') and 2002r (5'-GCT ATA GCT TTG GCG GTC G-3') and rechecked on a 1% agarose gel. The 2-kb amplification product was eluted from the gel, precipitated, resuspended in 20 µl dH2O, and cloned into the pCR-Blunt II TOPO vector (Invitrogen). Up to five clones per individual were sequenced, and these sequences were used in subsequent analyses. From samples Zenet, Asembo, Bakin-Kogi, Kisian, Furvela, and Malindi a total of 57, 51, 40, 29, 33, and 28 sequences, respectively, were obtained. Note, the methods used to obtain the sequences for this analysis did not permit these elements to be assigned to specific sites identified in the site-occupancy (transposable element display) analysis.
Sequence analysis:
Sequences were aligned using AlignX, a ClustalW-base alignment program in VectorNTI Advance 10.0.1 (Invitrogen). Nucleotide diversity was estimated from average pair-wise number of differences between elements, (NEI and LI 1979), and from the number of polymorphic sites, 
(WATTERSON 1975). and were estimated using DnaSP 3 (ROZAS and ROZAS 1995; ROZAS et al. 2003). Estimates of the observed silent site diversity in the first 528 bp of the 5' end of the transposase coding region was computed using the Kumar method (NEI and KUMAR 2000) as implemented in MEGA 3.1 (KUMAR et al. 2004). Expected values of silent site diversity were calculated following SANCHEZ-GRACIA et al. (2005) and were the product of the haploid copy number and the average synonymous diversity (0.0209) from a sample of 35 nuclear genes (MORLAIS et al. 2004). Tajima's D was calculated using DnaSP 3. Further analysis was performed on the first 528 bp of the 5' end of the transposase open reading frame. Unique variants of elements were identified (referred to as forms), their frequencies determined, and the relationship of the forms determined using TCS1.21 (CLEMENT et al. 2000). Alignment gaps were treated as missing data in this analysis. Estimates of the number of synonymous substitutions per synonymous site (dS) and of nonsynonymous substitutions per nonsynonymous site (dN) and their ratio, 
= dN/dS, were obtained using maximum likelihood (ML) methods employed by CODEML in PAML 3.13 (YANG 1997) using the alignment of the 33 different forms for the analysis (supplemental Figure 1 at http://www.genetics.org/supplmental). PAML permits an assessment of the observed substitution data after assuming different codon substitution models that differ in the way selection pressure is distributed within the gene. Here we have examined our data in light of three simple models: a single ratio model (M0) that assumes one for all sites; a neutral model (M1) that assumes that there are two classes of sites within the gene, those that are conserved (p0) with 0 = 0 and those that are neutral (p1 = 1 – p0) with 1 = 1; and finally, a discrete model (M3) that assumes three classes of sites each having a unique value of that is estimated from the data (YANG 1997). A likelihood ratio test (LRT), which is twice the log-likelihood difference between two models being compared, was used to determine which model best reflected the observed data. The LRT statistic has a 
2 distribution with degrees of freedom equal to the difference in the number of parameters between the two models (YANG et al. 2000).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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= 0.3244. Therefore, 0.415 of all fragments were calculated to be >80 bp and 0.0017 of all fragments were >1 kb.) Consequently, few elements will be undetected because they are on excessively long templates. Restriction site polymorphism can result in increased estimates of site-occupancy diversity since an element at one site would be displayed as two bands of different lengths resulting in those bands being scored as two elements occupying two sites. While restriction site polymorphism will have this effect on the analysis, the frequency of such polymorphisms is expected to be very low on the basis of the known level of nucleotide polymorphism in An. gambiae s.s. (MORLAIS et al. 2004) and our failure to detect the same element in two different positions in transposable element displays following band isolation, reamplification, and DNA sequencing (GUIMOND et al. 2003; R. A. SUBRAMANIAN and D. O'BROCHTA, unpublished data). Confounding effects of restriction site polymorphism will be small and are not a significant source of variation in transposable element display. All individuals in this study that were analyzed by transposable element display (N = 218) contained at least one Herves element (Table 1). Element copy numbers within the six populations analyzed ranged from 2.9 to 4.4 elements per diploid genome as calculated using the method of WRIGHT et al. (2001). No individuals were found in any population that contained >7.0 elements. In all populations there was an abundance of occupied sites that were observed in only small numbers of individuals (Figure 2). In Zenet, Malindi and Furvela elements with high site-occupancy frequencies were observed although none of these elements were shared among these populations (Figure 2).
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Nucleotide polymorphism:
Approximately 2 kb of sequence beginning at the left (5') inverted terminal repeat and through the first 528 bp of the transposase open reading frame was amplified, cloned, and sequenced (Figure 3). A total of 238 sequences containing the first 528 bp of the transposase open reading frame were analyzed from six different locations. The average nucleotide polymorphism in the 1474 bp of noncoding sequence ( = 0.0079) was significantly different from the polymorphism observed in the coding region ( = 0.0046; P < 0.001) (Table 2). Within the noncoding region the observed polymorphisms were nonuniformly distributed in a 666-bp region beginning at nucleotide 568 having a highly reduced level of polymorphism (Figure 3). This region corresponds to a large stretch of DNA with unknown function 5' of the transposase-coding region and just 3' of a pair of 100 bp subterminal tandem repeats (ARENSBURGER et al. 2005).
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1.8 kb in length, and the structural integrity of Herves elements was assessed by amplifying this region using primers flanking it. Herves elements without any deletions resulted in PCR products of 2 kb in length, and elements with deletions 100 bp or more produced distinct products <2 kb. Of the 218 individuals tested from six locations, 85% showed evidence of the presence of complete open reading frames (Table 4). Individuals with complete elements were least abundant in Nigeria (Bakin Kogi) where only 44% showed evidence of complete open reading frames (N = 32). In western Kenya intact forms of the element were found in 100% of the individuals from Asembo (N = 24) and 90% of the individuals from Kisian (N = 15). In eastern coastal Kenya (Malindi, N = 25) and northeastern coastal Tanzania (Zenet, N = 73), 85% of the individuals tested contained intact forms of the element. In southern Mozambique (Furvela, N = 49) 95% of the individuals sampled contained intact elements.
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(the ratio dN/dS) using ML. The ratios ranged from 0.41 to 0.71 under all models (M0, M1, and M3; see MATERIALS and METHODS) revealing evidence of purifying selection (YANG 1997). The neutral model (M1) was rejected when compared to the discrete model (M3) that allows for three classes of sites with different values of . The LRT statistic, 2
l (2l = 2[–1037.77 – (–1028.00)], for this comparison was 19.54, which was greater than the critical value of 
= 13.816. |
DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Here we examined the dynamics of Herves by measuring the site-occupancy frequency, nucleotide-sequence diversity, and by performing a genealogical analysis of the element. The rare occurrence of locally fixed, Herves-occupied sites and the widespread abundance of sites that are occupied in only a few individuals are consistent with the recent activity of Herves within An. gambiae. The site-occupancy levels observed in this study (ßHerves = 1.9 – 11.0) were similar or somewhat lower than those reported for putatively active transposable elements in D. melanogaster: ßP element = 16.6 (AJIOKA and EANES 1989), ßP element = 5.85 (BIEMONT et al. 1994), ßcopia = 9.79 (BIEMONT et al. 1994), ßcopia = 16.9 (LEIGH-BROWN and MOSS 1987), and ßcopia = 48.3 (KAPLAN and BROOKFIELD 1983).
An. gambiae is distributed almost continuously throughout its range in Africa, and demes are likely to be large and diffuse (LEHMANN et al. 1998). Little population differentiation between populations separated by up to 50 km has been reported (LEHMANN et al. 1997), and this has also been found over distances of 6000 km (LEHMANN et al. 1996). LEHMANN et al. (1998) suggest that Wright's isolation by distance model may best describe the relationships among populations (WRIGHT 1951). Population admixture might be contributing to the pattern of site occupancy observed in this study. However, consistent with the idea that Herves is currently capable of transposing in natural populations of An. gambiae is the finding that Herves elements isolated from An. gambiae collected from the field within the last 20 years are active when introduced into other insects in the laboratory (ARENSBURGER et al. 2005).
A number of pieces of data indicate that Herves entered the An. gambiae lineage via a horizontal gene transfer. A comparison of the silent site diversity among Herves elements and 35 nuclear genes (MORLAIS et al. 2004) revealed less diversity within Herves transposable elements than expected assuming similar mutation rates apply to Class II transposable elements and nuclear genes (SANCHEZ-GRACIA et al. 2005). Others have used intra- and interspecific diversity comparisons to infer the introduction of transposable elements into host genomes (SILVA and KIDWELL 2000; SANCHEZ-GRACIA et al. 2005), and the diversity data for Herves are qualitatively similar to those data. Second, when elements are horizontally transferred to a new host species there is a period of time when natural selection will favor active autonomous elements and this will leave a distinct molecular signature within the elements in the form of a skewed ratio of synonymous and nonsynonymous substitution rates (ROBERTSON and LAMPE 1995). In this study a comparison of the synonymous and nonsynonymous substitution rates within the Herves transposase-coding region detected evidence of purifying selection and is consistent with the hypothesis that Herves was laterally introduced into this lineage from an unknown source.
Although Herves displays evidence of being horizontally introduced into the An. gambiae lineage, the timing of this event remains uncertain. The intensity of the molecular signals indicating horizontal transfer suggests that this event was not in the very recent past. SANCHEZ-GRACIA et al. (2005) recently examined 14 transposable elements in D. melanogaster and, on the basis of silent site diversity, concluded that 13 were products of horizontal transfer that probably occurred 5–12 million years ago. SANCHEZ-GRACIA et al. (2005) observed levels of silent diversity within the transposable elements studied 100-fold less than that observed in 21 nuclear genes, while in this study silent site diversity was only 6-fold less than expected when the data were pooled and ranged from 3-fold to 125-fold less than expected depending on the location from which the samples were collected. These data appear consistent with an historical lateral transfer event, although not one that has occurred recently.
The form diversity observed in this study is also consistent with Herves having an extended residence time within the An. gambiae lineage. Interestingly however, while the number of forms of Herves as determined by the sequence of the 5' end of the transposase gene totaled 33, the frequency of individuals with at least one copy of an element that had either no internal deletions or deletions <100 bp (the limits of the detection method) was >90%. Internally deleted elements can arise quickly following the introduction of a transposable element as has been displayed by the well-studied P element in Drosophila species (O'HARE et al. 1992). This is distinctly not the case for Herves and may be due to a number of factors. First, if deleted elements are preferentially removed from the genome then one would see a relative abundance of intact forms as observed here. Currently there are no data for the differential removal of smaller, internally deleted forms of an element, and indeed, smaller nonautonomous elements can have an activity advantage in the presence of functional transposase (SPRADLING 1986; LAMPE et al. 1998). An alternative possibility is that Herves elements may have reduced opportunities to form internally deleted elements. Internal deletions of Class II transposable elements arise in some cases during the double-stranded DNA gap repair process following element excision. For example, following P element excision in D. melanogaster the resulting double-stranded gap is filled during a homology-dependent recombination process in which homologous or ectopic copies of a P element are copied into the gap (ENGELS et al. 1990). Premature resolution of these recombination products before this templated gap repair process is complete results in the creation of incomplete elements. The extent to which postexcision repair involves homology-dependent recombination or nonhomologous end joining will determine, to some extent, how often internally deleted elements are created within a genome (RIO 2002). A preference for Herves excision products to be repaired using nonhomologous end-joining mechanisms could explain two aspects of Herves observed in An. gambiae—the relative abundance of intact elements and their low copy number.
hAT element excision results in double-stranded breaks in the chromosome in which the ends of each chromosome are sealed by hairpin structures (ZHOU et al. 2004). These hairpin structures are resolved by a nicking event followed by end joining. The hairpin structures that arise on the empty donor site following hAT element excision are not seen following P element excision. We speculate that this predisposes Herves postexcision repair to occur via nonhomologous end joining and thereby reduces the frequency with which internally deleted elements are created.
Herves is present at low copy numbers within An. gambiae, and the data suggest that copy-number equilibrium has not been reached (Tajima's D statistic for pooled data = –1.91). The low copy number of Herves, while not unique among Class II transposable elements, tends to be somewhat unexpected if the element was introduced into this lineage in the distant past. Class II transposable elements tend to increase in copy number when they are active within a genome. This increase in copy number occurs despite the conservative cut-and-paste nature of Class II element movement because the double-stranded breaks that arise following element excision can be repaired using homology-dependent repair processes that result in a copy of the element being inserted into the gap (RIO 2002). Alternatively, an increase in copy number can occur as a result of Class II transposable elements moving from replicated regions of the genome to unreplicated regions of the genome during S-phase (WILSON et al. 2003). Although the mechanisms of copy number increase may vary, it seems well established that element copy number is expected to increase during periods of element activity. The low number of Herves elements in all individuals sampled therefore seems at odds with the diversity data that point to an extended residence time in the An. gambiae lineage. The tendency of different Class II transposable elements to increase in copy number has never been systematically compared although it is reasonable to think that some elements might be more "replicative" than others. hAT elements, and Herves in particular, may have a relatively low replication potential because of the presence of hairpin-containing intermediates following excision.
The structure of the population of An. gambiae in Africa has been studied, and it has been proposed that there are two main divisions of the gene pool: a northwestern division including Senegal, Ghana, Nigeria, Cameroon, Gabon, Democratic Republic of Congo, and western Kenya and a southeastern division including Kenya, Tanzania, Malawi, and Zambia (LEHMANN et al. 2003). It has been proposed that there has been a recent bottleneck in the southeast division resulting in reduced genetic diversity followed by colonization from the northwest division. (LEHMANN et al. 2003). The data presented here show little evidence of geographical variation and are inconsistent with the above model. Samples from Mozambique showed the highest levels of silent site diversity and no reduction in the diversity of forms as might be expected following a bottleneck. In fact, samples from Nigeria not only showed the least silent site diversity but also had the least amount of form diversity. Further sampling of Herves from populations in western Africa is needed to confirm the modest trends revealed in this study.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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