*Laboratoire Dynamique du Génome et Evolution, Institut J. Monod, Paris Cedex, France;
Laboratoire Population, Génétique et Evolution, Gif sur Yvette, France
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Abstract |
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Introduction |
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Using TPE repeats as molecular markers revealed a characteristic geographical distribution, which seems to have been stable since the early 1960s (Bonnivard et al. 2000
). Most populations are monomorphic and contain only hobo elements with three TPE repeats (3TPE elements). These populations belonging to the [3] class (with solely 3TPE elements) are found worldwide. In contrast, polymorphic populations (with several types of element) are restricted to three regions: western Europe, South America, and Equatorial Africa. These polymorphic populations display marked geographical structuring, with adjacent populations showing similar polymorphism. In the light of this stable distribution, we proposed a new scenario for the dynamics of hobo elements based on there having been two distinct invasion stages: a successful and total invasion by 3TPE elements followed by the start of new invasions involving other types of hobo elements, especially 5TPE elements (Bonnivard et al. 2000
).
The existence of polymorphic populations raised questions about the origin of this polymorphism. Does it result from existence of polymorphic flies (i.e., different types of hobo sequences within a fly) or from variability between flies within the same population (i.e., flies belong to different classes with regard to TPE repeats), or from both? To describe the components of the polymorphism that was observed previously and to test our model, we investigated 25 current, natural populations through an analysis of the isofemale lines. In current populations, this analysis revealed the existence of seven new types of hobo elements with regard to the TPE repeats. Moreover, we observed interfly variability within the populations that made it possible to distinguish between populations that belong to the same polymorphic class. In particular, we describe in western Europe a centrifugal decrease in the frequency of 5TPE hobo elements that start from western France.
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Materials and Methods |
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For the 50 representative lines identified, PCR products were also separated on polyacrylamide gel to confirm the different numbers of TPE repeats observed. For this purpose, PCR amplification was performed using a 33P-labeled h6 primer. After amplification, samples were diluted with one volume of loading dye (95% formamide, 0.005% xylene cyanol FF, and 0.005% bromophenol blue), heat-denatured at 94°C for 5 min, and then directly cooled on ice. For each sample, a 5- to 7-µl aliquot was loaded onto a 6% denaturing polyacrylamide gel. After migration, gels were transferred to Whatman 3 MM paper and vacuum dried at 80°C for 1 h, and the dried gels were exposed to X-ray film.
More than 35 TPE repeat bands were chosen on the basis of their size and the population from which they had come, such that at least an example of each type of TPE repeat was included from each of the geographical regions. PCR amplifications were performed on these selected TPE repeat bands excised from the dried gels, according to Melayah et al. (2001)
, and sequenced using the specific amplifying primer h11.
Southern Blot
The Southern blot method was used to estimate the number of full-size hobo elements (2.6 kb XhoI fragments) using the method described by Bonnivard and Higuet (1999)
. For this purpose we used a hobo probe corresponding to the 17562168 internal sequence obtained by PCR amplification using the h6 and h11 primers. The full-size elements were quantified using the CyHBL1 strain as a reference (one Hfl copy per diploid genome, Calvi and Gelbart 1994
). This enabled us to estimate the number of these elements present in the different strains.
Sequence Polymorphism Analysis
To research polymorphism beside the S region, we first used the sequences of h6-h11 PCR amplification products. Second, we investigated part of the no-coding region in 3' of the ORF1 (Streck, Mac Gaffey, and Beckendorf 1986
) using two primers, h21 and h4, which have sequences corresponding to bases 22672286 (5'-ACAAAAACCTAAACAACTCG-3') and 28792859 (5'-ACCCTACTTGCGGCAACACA-3'), respectively, in the Hfl1 element (Calvi et al. 1991
). PCR amplifications were performed on DNA extracted from single fly with only one type of element with regard to the TPE repeats and were sequenced using the specific amplifying primer h21.
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Results |
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Existence of Polymorphism Within Flies
This study of isofemale lines enables us to define a new class of monomorphic flies, the [5] class, represented by two French lines (fig. 2
). Hence, it appears that some flies do not contain the 3TPE hobo element. However, this element type is still found to be predominant, as it is present in all the other 244 lines tested (being sequenced in 14 flies representing 11 populations, table 1
). The 137 other monomorphic flies all belonged to the [3] class. In addition, several different polymorphic classes of flies can be defined (fig. 2
). Two of them seem to be preponderant: 5TPE elements were found in 75 flies from 14 different populations (sequenced in eight of them, table 1
), and 4TPE elements were found in 18 flies from six different populations (sequenced in all of them, table 1
). All the other types of elements seemed to occur at low frequencies, as they occurred in less than two lines (six lines for the 8TPE element). Moreover, the flies that harbor these types of elements present a higher signal for 3TPE, 4TPE, or 5TPE elements in agarose gel. Such differences in the intensity of the PCR product can be interpreted as differences in the ratio of the number of elements of each type, according to Brunet et al. (1996)
. To confirm this hypothesis, we used two transformed lines, which have a single 3TPE or 5TPE hobo element, respectively. DNA extractions were performed on different mixes of these flies (ratio 1:9, 2:8, 5:5, 8:2, and 9:1). PCR amplification results show a difference in the intensity of the PCR products that correlates with the ratio of the transformed flies. Hence, according to the intensity of the PCR product, these particular types of element also seem to be in the minority within the genome.
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Polymorphic populations show different levels of interfly variability, depending on the frequency of polymorphic flies that they display. Some populations have only a few fractions of polymorphic flies, about 1 or 2 out of 10. They are mainly located around the edge of the region where TPE-repeat polymorphism is observed (e.g., Poznan [j] or Costa [m]). For the sake of clarity, the uncommon classes of flies are not shown in figure 2 . They harbor: the 2TPE element in one fly in Nimba (v); the 9TPE element in one fly in Madagascar (y); the 3 + 1SLE element in two independent flies in Poznan (j) and Nimba (v); and -1TPR, 2TPE, or 6TPE elements in few flies from Kourou (p), some of which harbored up to four different types of hobo elements, belonging to the [-1TPR 2, 3, 5, 6] class. In contrast, several populations contain a majority of polymorphic flies. This polymorphism mainly results from 4TPE or 5TPE elements in addition to 3TPE elements. For example, elements with four and five TPE repeats are accountable for the high polymorphism level observed in eastern Africa and western Europe, respectively.
A Gradient of 5TPE hobo Elements in Western European Populations
In western Europe, populations contain a majority of flies of the [3,5] class (fig. 2B
), especially in France, where only two monomorphic [5] flies were observed. The other 48 French flies were polymorphic, and all but four belonged to the [3,5] class. The remaining four flies also contained 8TPE elements. In the three German populations, monomorphic flies of the [3] class were observed but were still a minority. They make up a majority only in the Italian population Firenze (i), and, to the east, in Poland, Poznan (j). This implies that the western European populations display marked geographically determined structuring, as geographically adjacent populations show similar kinds of interfly variability.
To study the marked geographical structuring, 3TPE or 5TPE hobo elements were considered separately, and their frequency in each of the European populations was determined (fig. 3 ). 3TPE hobo elements were present in all flies, except one in Brest (a) and one in Naussac (d). On the other hand, there was a marked decrease in the number of isofemale lines harboring 5TPE elements that correlated with the distance from Brest. 5TPE hobo elements were present in all French flies but were present in progressively fewer and fewer flies in the German and Italian populations and were completely absent from the Polish flies. The data therefore revealed a relationship between the number of flies presenting 5TPE elements and the location of each population. This conclusion is reinforced when we consider the intensity of the different PCR products. Indeed, the eight [3,5] flies in which the 3TPE repeats product could not be detected easily were all French. This fits in with the fact that two French flies belonged to the [5] class. In contrast, the 11 [3,5] flies in which the 5TPE repeat product could not be detected easily were all German. In conclusion, the frequency of 5TPE hobo elements seems to decline steadily and centrifugally with distance, irrespective of the direction from Brest.
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Discussion |
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Characterization of Populations with Regard to TPE Repeats
The polymorphism observed in some populations using mass culture can be redefined on the basis of the interfly variability. Of the seven populations of the [3] class, chosen to confirm that they really are monomorphic, only populations from Costa Rica and Poznan contained one polymorphic fly. Hence, if we assume that mass cultures had been established at least two generations before the molecular analysis, it is quite conceivable that they had escaped detection in the previous study. It also appears that these two populations were chosen for their particular location, on the borders of regions where polymorphic populations were found (see Materials and Methods). On the other hand, all the populations located in regions where only monomorphic populations were found displayed only 3TPE hobo elements, as expected.
Considering polymorphic populations, isofemale lines analyses not only make it possible to achieve better distinction between populations in different classes but also are able to discriminate between populations belonging to the same polymorphic class. Some differences appear between mass culture and isofemale lines data (table 1 ). Such differences have two causes. The first cause is that the isofemale line approach is able to detect types of elements that are only present at a low frequency within a population, such as 2TPE, 8TPE, or 9TPE elements, and may not be detected using mass culture analysis. The second cause is that polyacrylamide gel analysis can be used for directly sequencing elements of interest. This can account for the detection of 4TPE elements that were probably mistaken for 5TPE elements in earlier studies using only agarose gel.
Molecular Origin of the Different Types of Elements
According to the length of the repeats unit, TPE repeats can be consider as microsatellites or minisatellites, depending on the authors (Tautz 1993
; Debrauwere, Gendrel, and Dutreix 1997
), even though their size corresponds more closely to microsatellites. Two mechanisms have been proposed to account for the instability of such repetitive sequences: DNA polymerase slippage, which could account for the low modification of the number of repeats, and unequal recombination, which reshuffles repeat variants (for review see Debrauwere, Gendrel, and Dutreix 1997
). The existence of 3TPE, 4TPE, and 5TPE elements in the same region (e.g., Bolivia or Kenya), as well as the deletion of the degenerate TPR motif in 5' or the duplication of the degenerate SLE motif in 3', can be used to argue for a mechanism linked to polymerase slippage. On the other hand, the absence of elements with an intermediate number of TPE repeats, between five and eight in Europe or between four and nine in Madagascar, conflicts with such an evolutionary model and could argue for recombination events. The instability of microsatellites depends on numerous parameters such as the size and sequence of the repeated motif, the number of repeats, the monotony of the series, the locus, and positive mutational bias (Estoup and Cornuet 1999
). All these parameters can also interact with the mutation rate; hence, the evolution processes of microsatellites depend on the locus considered. In this special case of a microsatellite within the coding region of a transposable element, more information is needed to infer the evolution of the number of TPE repeats. However, some particularities of this locus can already be pointed out. First, TPE repeats present unexpectedly great diversity, given that their worldwide invasion of D. melanogaster is supposed to be a recent event, occurring within the past 70 years. This diversity reflects a high mutation rate that contrasts with the conservation of other parts of the element and with the average mutation rates of microsatellite loci in D. melanogaster (6.5 x 10-6 per locus per generation; Schug, Mackay, and Aquadro 1997
). This high mutation rate could be related to the number of copies and to replication resulting from the transposition of the elements. Second, variations always involve sequences of nine nucleotides, usually TPE motifs but also motifs that are different at the protein level. Third, it is necessary to consider that the S region may not be neutral because it is located in the coding sequence. Thus, it is also possible than some types of elements were undetected because they were eliminated by selection pressure.
The Two-Step Invasion Model
The results obtained in this study are still consistent with the scenario of a two-stage invasion, consisting of a total invasion by 3TPE elements followed by new invasions involving other types of hobo elements (Bonnivard et al. 2000
). Each of the less common types of hobo element is found only in restricted geographical regions; as a result, at present, no data can argue for their invasion capacity. Indeed, elements may be considered as invasive only if they are found in several populations on a large geographical area, for example, 5TPE elements in Europe.
The centrifugal gradient of 5TPE hobo elements described in western Europe can reflect a new invasion of the European populations by these elements, corresponding to the second invasion stage. An alternative hypothesis is that 5TPE elements occurred earlier in Europe, but if this were so, there is no way of explaining the origin of these elements or how they have faded out in many European flies. Fly migrations are preponderant factors in the transposable-element invasion of D. melanogaster (Quesneville 1996
; Bonnivard 1999
). Step-by-step recurrent migrations play a major role in establishing centrifugal gradients such as the one described in this study or the one described in the investigation of natural populations with regard to the number of full-size P elements (Bonnivard and Higuet 1999
). Hence, if we compare the two systems, the stable distributions in western Europe present a remarkable spatial likeness with the centrifugal gradient centered in the west of France that decreases sharply in central Europe. This similarity is difficult to interpret because it cannot result from fly migration alone. Indeed, in the P-M system, the distribution also results directly from the activity and repression properties of the elements (Bonnivard and Higuet 1999
). Indeed, mobility of the element within the genome is a second factor that may be implicated in their invasion capacity because elements that present activity can be amplified within the genome. Considering 5TPE elements, this mobility could reflect the activity of the elements themselves or their mobilization in trans because we are still not sure that a hobo element with more than three TPE repeats corresponds to complete elements. However, some data argued for the likely activity properties of 5TPE elements. First, most of the hobo elements carrying TPE repeats are full-size elements, as deleted elements that display the S region remain a minority, especially in the European populations (Bonnivard et al. 2000
). Second, according to the preponderance of these elements in France, it seems that these elements not only invade French populations but also replace 3TPE elements.
Considering 4TPE elements, their geographical distribution, especially their high frequencies in eastern Africa, suggests that these elements are also potentially invasive and could participated in the second invasion stage. However, at present, the analysis of natural populations suffers from the lack of samples from South America and South Africa. Studying natural populations will allow us to determine if there is a continuum between these two regions and a gradient such as the one described for 5TPE elements.
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Acknowledgements |
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Footnotes |
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Keywords: hobo element
TPE repeats
microsatellites
Drosophila melanogaster
isofemale lines
structuring populations
Address for correspondence and reprints: Eric Bonnivard, Laboratoire Dynamique du Génome et Evolution, Institut J. Monod, 2 place Jussieu, 75251 Paris Cedex 05, France. E-mail: bonni{at}ccr.jussieu.fr
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