*Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS 5558, Université Lyon 1, Villeurbanne Cedex, France;
INSERM-U503, CERVI, Lyon Cedex 07, France
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Abstract |
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Introduction |
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Genome size variability is usually attributed to differences in the amount of repetitive DNA, which corresponds to a large fraction of the genome (Pagel and Johnston 1992
). For example, the human genome consists of only 5% of coding sequences but more than 40% of transposable elements (TEs) (International Human Genome Sequencing Consortium 2001
). A link between genome size and the amount of TEs is therefore to be expected. Recent data provide evidence that the TE copy number is related to environmental conditions and to the colonization process (Arnault and Dufournel 1994
; Labrador and Fontdevila 1994
; Wisotzkey, Felger, and Hunt 1997
; Labrador et al. 1999
). Kalendar et al. (2000)
have shown that the Bare-1 element in barley, is responsible for a large genome size variation, and its copy number increases in relation to the height and dryness of the environment. An association between genome size variation and TE content has also been found in rice, which contains 14% of LTR retrotransposons (Tarchini et al. 2000
). An increase in the genome size of maize is the result of recent TE amplifications, which have occurred within the last 2 to 6 Myr (SanMiguel and Bennetzen 1998
). TEs may also contribute to an increase in genome size after crosses between different species. This was observed when two wallaby species, Macropus eugenii and Wallabia bicolor, were crossed (O'Neill, O'Neill, and Graves 1998
). The centromeric regions of the hybrids were amplified, and the amplified sequences presented homologies to human endogenous retroviral elements. In Drosophila, crosses between Drosophila buzzatti and D. koepferae mobilize the osvaldo element in hybrids (Labrador and Fontdevila 1994
; Labrador et al. 1999
).
Most of the work done on genome size is based on comparisons between different species. However, the few studies which have been done at the population level reveal differences in genome size between populations in mammals, such as the pocket Gophers (Sherwod and Patton 1982
), fishes (Gold and Amemiya 1987
; Gold, Ragland, and Schliesling 1990
; Lockwood and Derr 1990
), and insects, such as mosquitoes (Rao and Rai 1987
; Black and Rai 1988
; Warren and Crampton 1991
) and Drosophila (Dawley 1997
, pp. 143184). One way to explain these intraspecific differences is to view genome size as a selective trait (Gregory 2001
). An alternative hypothesis, however, is that TEs may be mobilized under stressful environmental conditions (Arnault and Dufournel 1994
) or genetic conditions (Wisotzkey, Felger, and Hunt 1997
; Biémont et al. 1999
; Labrador et al. 1999
), in some cases after horizontal transfer (Kidwell and Lish 1997
). As a consequence, the colonization of new habitats could lead to the mobilization of certain TEs in the genome of invasive species, leading to an increase in genome size. In the present study we used flow cytometry to estimate genome size of individuals from various geographically distinct populations of D. melanogaster and D. simulans, and we correlated genome size with the total amount of TEs previously estimated by in situ hybridization (Biémont et al. 1999
; Vieira et al. 1999
). We show that differences in TE amount in the euchromatic part of the chromosomes do indeed account for some of the differences in genome size between species and between populations within a given species. The amount of TEs in centromeric heterochromatin does not seem to be involved in this correlation in a simple way.
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Materials and Methods |
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Estimation of the Euchromatic TE Copy Number
The TE insertion site number of polytene chromosomes had previously been determined for 31 elements in samples of the populations described above (Biémont et al. 1999
; Vieira et al. 1999
).
Estimation of the Total Amount of TEs
We estimated the relative total amount of each TE individually by an inverse dot blot technique, which was developed by D. Lepetit. One microgram of each plasmidic DNA containing a TE sequence and 1 µg of actin DNA used as control were blotted on a nylon N+ membrane (Amersham). The membrane was hybridized with 100 ng of DNA extracted by classical technique from 20 adult female flies from a given population. This DNA was sonicated, and the resulting fragments were labeled with 32P dCTP by random priming. The same blot was thus hybridized successively with the DNA from four different populations, Canton and Senegal for D. melanogaster and Canberra and Madagascar for D. simulans. The list of the TEs used in the dot blot experiment and their euchromatic copy number determined by in situ hybridization are given in table 1
. Autoradiographic signal intensity was acquired with the Molecular Imager System, analyzed with Biorad Molecular Analyst R System and Biorad software, and adjusted as a function of the actin activity. Because the labeling of the probe by random priming is not independent of the base composition, we considered the ratio between populations for each TE. This hybridization with whole fly DNA made it possible to estimate the amount of heterochromatic and euchromatic copies for each TE. In comparison, in situ hybridization gave only the number of TE insertion sites distributed along the euchromatic part of the chromosomes; therefore, it was an underestimation of the real TE copy number because each site labeled could in fact bear more than one TE copy.
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Results |
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Contribution of TEs to Genome Size Variation
To determine the contribution of the TEs to genome size variation, we estimated the amount of TEs in picograms for the populations of extreme genome size for both species (Canton and Senegal for D. melanogaster; Canberra and Madagascar for D. simulans). The number of copies of each TE, estimated by in situ hybridization (table 1
), was multiplied by the TE length in pg (1 pg equals 0.9869 x 109 bp). The difference in TE amount between the two extreme genome size populations within each species was then expressed as a percent of the genome size difference. It appears that variations in TE copy number in euchromatin account for only 5% to 9% of the observed difference in genome size. Other factors, such as the proportion of repetitive sequences in heterochromatin or the interspersion of highly repetitive DNA in the genome, must be involved to account for the difference in genome size, for which the TE copy number seems to be a distinctive label. We thus determined by dot blot the total amount of TEs in the heterochromatic and euchromatic parts of the genome of whole flies from the two populations with extreme size in each species (see an example of dot blot in fig. 3
). In D. simulans, the ratio of the blot intensities from whole DNA between Canberra and Madagascar was equal to 1.10, whereas the ratio of the copy number estimated by the in situ technique was equal to 1.72 (the TEs with no insertion sites were excluded from the calculus). If we removed the 412 element from the calculation (the element that is overrepresented in the Canberra population), the dot blot and the in situ techniques gave copy number ratios between Canberra and Madagascar equal to 1.03 and 1.34, respectively. This shows that the Canberra and Madagascar populations did not differ significantly when the total genomic amount of TEs was considered. The number of insertion sites on the chromosome arms was thus not associated with the amount of TEs within the heterochromatin. It is thus the variation in the number of insertion sites along the chromosomes which was responsible for the main variation in total amount of TEs in D. simulans. This conclusion is sustained by the strong correlation between the ratios of the dot intensity and the in situ insertion site number of Canberra over Madagascar (r = 0.895, P < 0.0001). Again, when we excluded the 412 element from the analysis, the correlation dropped to r = 0.693 but was still significant (P < 0.01) (fig. 4
). Drosophila melanogaster presented a different picture. The ratios of the most extreme populations for genome size, Canton and Senegal, were equal to 1.24 and 1.36 for the dot blot and the in situ hybridization techniques, respectively. This suggests that (1) the TE insertions on the euchromatin, to a great extent, determine the total amount of TEs, (2) the amount of TEs in heterochromatin parallels the amount on euchromatin, or (3) in Canton, the TE amount on heterochromatin increased, whereas it varied independently of the insertion site number in euchromatin. The first hypothesis can be discounted because the ratio of the total genomic amount of eight TEs with very low insertion sites determined by in situ was equal to 1.47 and thus did not differ from the value obtained when all TEs were considered. The second hypothesis can be eliminated because correlation between the ratios of dot blot intensity and the in situ insertion site number of Canton and Senegal (r = -0.13) (fig. 4
) was low and nonsignificant, in contrast with that in D. simulans. This suggests that the TE amount within heterochromatin was higher in Canton than in Senegal and that this TE amount in heterochromatin increased from Senegal to Canton independent of the increase in insertion sites on euchromatin, as proposed by the third hypothesis.
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Discussion |
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Historical processes have led to the invasion of the D. melanogaster genome by many TEs a long time ago, apart from the P and I elements, which only invaded this species at the beginning of the 1900s. In contrast, populations of the D. simulans species still seem to be in the process of being invaded by TEs (Vieira et al. 1999
). Increases in genome size could therefore result from an increase in TE copy number, which could result from mobilization of TEs induced by environmental stresses, as has been reported in bacteria, yeast, and plants (Newton, Wakamiya, and Price 1993
, pp. 321345; Arnault and Dufournel 1994
; Kidwell and Lish 1997
). When populations are subjected to new environmental conditions, as may happen in populations that have invaded novel habitats, some of their TEs may be mobilized (Capy et al. 2000
; Kalendar et al. 2000
) and their genome size increased, leading to the observation that genome size increases during geographical colonization (Cros et al. 1995
). Drosophila melanogaster and D. simulans both originated in East Africa (Lachaise et al. 1988
), and the relationship between genome size and geography in D. melanogaster could therefore mainly reflect the geographic distance from the African birthplace, rather than any adaptive process. The high copy number of the uhu and LOA elements in Hawaiian Drosophila suggests that the colonization of new islands from older islands may have been associated with a significant increase in TE copy number (Wisotzkey, Felger, and Hunt 1997
). The unusual distribution of the osvaldo element in populations from the Iberian Peninsula compared with the original populations in Argentina (Labrador et al. 1999
) also implies an association between an increase in TE copies and colonization processes. TE transposition has also been seen to increase in both Drosophila hybrids (Evgen'ev et al. 1982
; Labrador et al. 1999
) and in the hybrid Australian wallaby (O'Neill, O'Neill, and Graves 1998
), resulting from confronting populations or species that did not previously overlap.
We still have much to learn about the genome. This paper and other recent studies on Drosophila, plants, fishes, and yeast, should trigger a fresh debate on genome changes associated with species invasion of new habitats, geographical locations, and evolution. Progress in this area of research can benefit from the comparative analysis of genomes of native and newly derived populations of the numerous pest species that are continually invading new areas of our world. The Zebra mussel, cheatgrass, European house sparrow, Argentine ant (Orr and Smith 1998
; Duke and Mooney 1999
; Duvernell and Turner 1999
), and many other invaders, in addition to the more common Drosophila species, should help us to understand the link between chromatin structure, TE mobilization, genome size, success of colonization, and various adaptive characteristics.
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Acknowledgements |
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Footnotes |
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Keywords: genome size
Drosophila
transposable elements
Address for correspondence and reprints: Cristina Vieira, Laboratoire de Biométrie et Biologie Evolutive, UMR CNRS 5558, 43 bd 11 novembre, Université Lyon 1, 69622 Villeurbanne Cedex, France. vieira{at}biomserv.univ-lyon1.fr
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