The evolutionary history of Drosophila buzzatii. XXXIV. The distribution of the retrotransposon Osvaldo in original and colonizing populations

The frequency distribution of the retrotransposon Osvaldo in the haploid genome of Drosophila buzzatii has been studied in five natural populations from the Iberian Peninsula and six natural populations from Argentina. In Iberian populations, Osvaldo insertion sites do not follow a Poisson distribution, most probably due to eight euchromatic sites with high occupancy, found in all populations. The estimated alpha and beta parameters, which measure the relative importance of drift and negative selection in shaping frequency distributions, indicate that drift is the main force acting upon the distribution of Osvaldo in natural populations of D. buzzatii in the Iberian Peninsula. On the other hand, Osvaldo distribution in populations from Argentina is similar to the distribution of elements with low copy numbers, such as those described for Drosophila melanogaster and Drosophila simulans: there are no indications for deviation from a Poisson distribution, there is a low occupancy per insertion site, and genetic drift has no apparent effect on the frequency distribution. We propose that the unusual distribution found in the populations from the Iberian Peninsula is a consequence of the colonization process. Iberian Peninsula populations suffered a genomic redistribution of Osvaldo, most probably after a founder effect. Consequently, certain copies that arrived at high frequencies are showing a high occupancies today, and the mean copy number of Osvaldo is higher in Iberian Peninsula populations than in populations from Argentina. All other copies are the result of recent (after colonization) transposition events.     

Interspecific hybridization increases transposition rates of Osvaldo.

Several authors have postulated that genetic divergence between populations could result in genomic incompatibilities that would cause an increase in transposition in their hybrids, producing secondary effects such as sterility and therefore starting a speciation process. It has been demonstrated that transposition largely depends on intraspecific hybridization for P, hobo, and I elements in Drosophila melanogaster and for several elements, including long terminal repeat (LTR) and non-LTR retrotransposons, in D. virilis. However, in order to demonstrate the putative effect of transposable elements on speciation, high levels of transposition should also be induced in hybrids between species that could have been originated by this process and that are still able to interbreed. To test this hypothesis, we studied the transposition of the LTR retrotransposon Osvaldo in Drosophila buzzatii-Drosophila koepferae hybrids. We used a simple and robust experimental design, analyzing large samples of single-pair mate offspring, which allowed us to detect new insertions by in situ hybridization to polytene chromosomes. In order to compare transposition rates, we also used a stock recently obtained from the field and a highly inbred D. buzzatii strain. Our results show that the transposition rate of Osvaldo is 10(-3) transpositions per element per generation in all nonhybrid samples, very high when compared with those of other transposable elements. In hybrids, the transposition rate was always 10(-2), significantly higher than in nonhybrids. We show that inbreeding has no effect on transposition in the strains used, concluding that hybridization significantly increases the Osvaldo transposition rate.     

Chromosomal distribution of the transposable elements Osvaldo and blanco in original and colonizer populations of Drosophila buzzatii

Chromosomal distribution of transposable elements (TEs) Osvaldo and blanco in D. buzzatii was studied in three original natural populations from Argentina (Berna, Puerto Tirol and La Nostalgia) and a colonizer population from the Iberian Peninsula (Carboneras). The Spanish population showed significant differences for Osvaldo and blanco copy numbers when we compared the X chromosome and the autosomes; but it is mainly the accumulation of copies in chromosome 2, where most sites with high insertion frequency were located, that causes the discrepancy with the negative selection model. We found no significant differences in TE frequency between chromosomal regions with different exchange rates, and no evident accumulation of TE was detected within chromosomal inversions where recombination rate is reduced. The Carboneras population shows euchromatic sites of Osvaldo and blanco with high occupancy and others with low copy number. On the contrary, Argentinian populations show only a generalized low occupancy per insertion site. Moreover, the mean copy number of both elements is higher in Spain than in Argentina. All these results suggest an important role of the colonization process in the distribution of TEs. The increase in the copy number of the TEs analysed and their elevated frequency in some chromosomal sites in Carboneras is, most probably, a sequel of the founder event and drift that took place at the time of the colonization of the Old World by D. buzzatii from the New World some 300 years ago.     

Mechanisms regulating the copy numbers of six LTR retrotransposons in the genome of Drosophila melanogaster

There has been debate over the mechanisms that control the copy number of transposable elements in the genome of Drosophila melanogaster. Target sites in D. melanogaster populations are occupied at low frequencies, suggesting that there is some form of selection acting against transposable elements. Three main theories have been proposed to explain how selection acts against transposable elements: insertions of a copy of a transposable element are selected against; chromosomal rearrangements caused by ectopic exchange between element copies are selected against; or the process of transposition itself is selected against. The three theories give different predictions for the pattern of transposable element insertions in the chromosomes of D. melanogaster. We analysed the abundance of six LTR (long terminal repeat) retrotransposons on the X and fourth chromosomes of multiple strains of D. melanogaster, which we compare with the predictions of each theory. The data suggest that no one theory can account for the insertion patterns of all six retrotransposons. Comparing our results with earlier work using these transposable element families, we find a significant correlation between studies in the particular model of copy number regulation supported by the proportion of elements on the X for the different transposable element families. This suggests that different retrotransposon families are regulated by different mechanisms.     

Maintenance of transposable element copy number in natural populations of Drosophila melanogaster and D. simulans

To investigate the main forces controlling the containment of transposable elements (TE) in natural populations, we analyzed the copia, mdg1, and 412 elements in various populations of Drosophila melanogaster and D. simulans. A lower proportion of insertion sites on the X chromosome in comparison with the autosomes suggests that selection against the detrimental effects of TE insertions is the major force containing TE copies in populations of Drosophila. This selection effect hypothesis is strengthened by the absence of the negative correlation between recombination rate and TE copy number along the chromosomes, which was expected under the alternative ectopic exchange model (selection against the deleterious rearrangements promoted by recombination between TE insertions). A cline in 412 copy number in relation to latitude was observed among the natural populations of D. simulans, with very high numbers existing in some local populations (around 60 copies in a sample from Canberra, Australia). An apparent absence of selection effects in this Canberra sample and a value of transposition rate equal to 1–2 × 10^-3 whatever the population and its copy number agree with the idea of recent but temporarily drastic TE movements in local populations. The high values of transposition rate in D. simulans clearly disfavor the hypothesis that the low amount of transposable elements in this species could result from a low transposition rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

High transposition rates of Osvaldo, a new Drosophila buzzatii retrotransposon

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.This paper is dedicated posthumously to Osvaldo A. Reig in recognition of his contributions to evolutionary biology and his early appreciation of the role of transposable elements in evolution     

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK- cells.

L929TK- cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK+ transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK+ transformation. Southern blot analysis of chromosomal DNA of TK+ transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK- cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occurred in chromosomal DNA of TK+ clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK+ transformant clones was noticed.     

Identification of a sporulation-specific promoter regulating divergent transcription of two novel sporulation genes in Saccharomyces cerevisiae

Transposition of a new Drosophila retrotransposon was investigated. Total genomic Southern analysis and polytene in situ hybridizations in D. buzzatii strains and other related species using a 6 kb D. buzzatii clone (cDb314) showed a dispersed, repetitive DNA pattern, suggesting that this clone contains a transposable element (TE). We have sequenced the cDb314 clone and demonstrated that it contains all the conserved protein sequences and motifs typical of retrovirus-related sequences. Although cDb314 does not include the complete TE, the protein sequence alignment demonstrates that it includes a defective copy of a new long terminal repeat (LTR) retrotransposon, related to the gypsy family, which we have named Osvaldo. Using a D. buzzatii inbred line in which all insertion sites are known, we have measured Osvaldo transposition rates in hybrids between this D. buzzatii line and its sibling species D. koepferae. The results show that Osvaldo transposes in bursts at high rate, both in the D. buzzatii inbred line and in species hybrids.     

Nucleotide sequence and phylogenetic analysis of long terminal repeats of human endogenous retrovirus K family (HERV-K) on human chromosomes

It has been suggested that human endogenous retroviruses K family (HERV-K) has a role in disease, and solitary long terminal repeats (LTRs) of HERV-K have been potentially capable of affecting the expression of closely located genes. Using the human monochromosomes 8, 9, 17, and 18, with specific PCR primers, we identified thirty-four sequences of new HERV-K LTRs. Those LTR elements were analyzed phylogenetically with the human-specific HERV-K LTRs using neighbor-joining and maximum parsimony methods. Clones HKL8-5, HKL9-5, and HKL9-8 are related by more than 99% homology with the human-specific HERV-K LTRs. The HKL9-5 clone on chromosome 9 was 100% identical with the sequences of human-specific LTR, AC002400, on chromosome 16. The findings suggest that there has been recent proliferation, transposition, or chromosomal translocation of HERV-K LTR elements on human chromosomes.     

Identity by descent and DNA sequence variation of human SINE and LINE elements

To test the hypothesis that Alu and L1 elements are genetic characters that are essentially homoplasy-free, we sequenced a total of five human L1 elements and eleven recently integrated Alu elements from 160 chromosomes (80 individuals representing four diverse human populations). Analysis of worldwide samples at L1 loci revealed 292 segregating sites and a nucleotide diversity of 0.0050. For Ya5 Alu loci, there were 129 segregating sites and nucleotide diversity was estimated at 0.0045. The Alu and L1 sequence diversity varied element to element. No completely or partially deleted Alu or L1 alleles were identified during the analysis. These data suggest that mobile element insertions are identical by descent characters for the study of human population genetics.     

The origins of premating reproductive isolation: testing hypotheses in the grasshopper Chorthippus parallelus

There are many proposed routes for the origin of premating reproductive isolation, but few systematic studies aimed at testing their relative importance. Accumulated information about the biogeographical history of the European meadow grasshopper, Chorthippus parallelus, has allowed us to make a planned series of comparisons among populations aimed at distinguishing the contributions of some of these hypotheses. We have compared the effects on assortative mating of long-term isolation in glacial refugia, founder events during postglacial colonization, and sympatry with a closely related species. A likelihood-based analysis allowed us to separate effects of variation in male and female mating propensity among populations from variation in mate choice leading to assortative mating. All three effects contributed significantly to the overall variation in mating pattern in a set of 21 pairwise comparisons among seven populations. Male cuticular composition, but not other candidate signals, was significantly associated with the level of assortative mating. Of the hypotheses for the origin of reproductive isolation, only the predictions of the founder hypothesis explained a significant amount of the variation in assortative mating. This does not rule out the possiblity that there may be some other explanation. Having established the pattern of divergence, it is possible to generate hypotheses that explain our results at least as well as the founder hypothesis. However, because many such post hoc hypotheses are possible, they cannot be tested with this dataset. On this basis, our results favor the hypothesis that some aspect of the colonization process tends to accelerate divergence in mating signals leading to premating reproductive isolation. This could be accomplished through any one of several mechanisms. Colonization involves many bottlenecks as new populations are established at the edge of the range by long-distance migrants. Genetic effects may be important, but these bottlenecks may also alter the conditions under which mates are found and chosen, as suggested by Kaneshiro. At the same time, the colonizing populations may encounter novel environmental challenges.     

Population dynamics of an Ac-like transposable element in self- and cross-pollinating arabidopsis.

Theoretical models predict that the mating system should be an important factor driving the dynamics of transposable elements in natural populations due to differences in selective pressure on both element and host. We used a PCR-based approach to examine the abundance and levels of insertion polymorphism of Ac-III, a recently identified Ac-like transposon family, in natural populations of the selfing plant Arabidopsis thaliana and its close outcrossing relative, Arabidopsis lyrata. Although several insertions appeared to be ancient and shared between species, there is strong evidence for recent activity of this element family in both species. Sequences of the regions flanking insertions indicate that all Ac-III transposons segregating in natural populations are in noncoding regions and provide no evidence for local transposition events. Transposon display analysis suggests the presence of slightly higher numbers of insertion sites per individual but fewer total polymorphic insertions in the self-pollinating A. thaliana than A. lyrata. Element insertions appear to be segregating at significantly lower frequencies in A. lyrata than A. thaliana, which is consistent with a reduction in transposition rate, reduction in effective population size, or reduced efficacy of natural selection against element insertions in selfing populations.     

Alterations in the U3 region of the long terminal repeat of an infectious thymotropic type B retrovirus.

We isolated and characterized a type B thymotropic retrovirus (DMBA-LV) which is highly related to mouse mammary tumor virus (MMTV) isolates and which induces T-cell thymomas with a high incidence and a very short latent period. Regions of nonhomology between the DMBA-LV genome and the MMTV genome were identified by heteroduplex mapping and nucleotide sequence studies. In the electron microscope heteroduplex mapping studies the EcoRI-generated 5' and 3' fragments of the DMBA-LV genome were compared with the corresponding fragments of the MMTV (C3H and GR) genome isolated from mammary tumors. The results indicated that DMBA-LV contained a region of nonhomologous nucleotide sequences in the 3' half of the U3 region of the long terminal repeat (LTR). Nucleotide sequence studies confirmed these results and showed that in this region 440 nucleotides of the MMTV (C3H) sequences were deleted and substituted with a segment of 122 nucleotides. This substituted segment in the form of a tandem repeat structure contained nucleotide sequences derived exclusively from sequences which flanked the substitution loop. The distal glucocorticoid regulatory element was unaltered, and two additional copies of the distal glucocorticoid regulatory element-binding site were present in the substituted region. The restriction endonuclease map of the reconstructed molecular clone of DMBA-LV was identical to that corresponding to unintegrated linear DMBA-LV DNA present in DMBA-LV-induced tumor cell lines. Since the nucleotide sequences of the LTRs present in four different DMBA-LV proviral copies isolated from a single thymoma were identical, we concluded that they were derived from the same parental virus and that this type B retrovirus containing an alteration in the U3 region of its LTR could induce thymic lymphomas. Thus, DMBA-LV represents the first example of a productively replicating type B retrovirus that contains an LTR modified in the U3 region and that has target cell and disease specificity for T cells.     

Mechanism of Is1 Transposition in E. COLI: Choice between Simple Insertion and Cointegration

Insertion element IS1 and IS1-based transposon Tn9 generate cointegrates (containing vector and target DNAs joined by duplicate copies of IS1 or Tn9) and simple insertions (containing IS1 or Tn9 detached from vector sequences). Based on studies of transposon Tn5 we had proposed a conservative (non-replicative) model for simple insertion. Others had proposed that all transposition is replicative, occurring in a rolling circle structure, and that the way DNA strands are joined when replication terminates determines whether a simple insertion or a cointegrate is formed.--We selected for the transposition of amp and cam resistance markers from pBR322::Tn9 plasmids to an F factor in recA-E. coli and identified products containing three and four copies of IS1, corresponding to true cointegrates (from monomeric plasmids), and simple insertions (from dimeric plasmids). The simple insertions with four copies of IS1 outnumbered those with three by a ratio of about 3:1, whereas true cointegrates containing three copies of IS1 were more numerous than those with four.--A straightforward rolling circle model had predicted that the simple insertions containing three copies of IS1 should be more frequent than those with four. Because we obtained the opposite result we propose that simple insertions only arise when the element fails to replicate or if replication starts but then terminates prematurely. The two classes of products, simple insertions and cointegrates, reflect alternative conservative and replicative fates, respectively, of an early intermediate in transposition.     

Physical Analysis of Tn10- and Is10-Promoted Transpositions and Rearrangements

We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10(-4) per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.     

Spontaneous transposition in the bacteriophage lambda cro gene residing on a plasmid.

A new mutagenesis assay system based on the phage lambda cro repressor gene residing on a plasmid was developed. The assay detects mutations in cro that decrease the binding of the repressor to the OR operator in an OR PR-lacZ fusion present in a lambda prophage. Mutations arose spontaneously during growth of E. coli cells harboring cro plasmids at a frequency of 3-6 x 10(-6). Analysis of some 200 cro mutants from several 'wild-type' strains revealed a substantial fraction of 25-70% insertion events caused by transposition of IS elements. Most of the insertions were caused by IS1, but IS5 insertions were observed too. In strains harboring Tn10, IS10 was responsible for most insertions. Restriction nuclease digestion analysis revealed a preference for insertion of IS10 into the C-terminal half of cro, despite the absence of sequences which are known hot spots for Tn10 insertions. The frequency of IS1 insertions into cro decreased 25-60-fold and that of IS10 insertions decreased 200-fold in cells carrying the recA56 mutation, suggesting that RecA is involved in transposition of these elements. During the logarithmic phase of growth, the mutation frequency was constant for at least 22 generations; however, upon continuous incubation at the stationary phase, the mutation frequency gradually increased, yielding a 3-fold increase in the frequency of insertion and a 4-5-fold increase in point mutation. Genomic Southern analysis of chromosomal IS elements in cells which underwent a transposition from the chromosome into the cro plasmid revealed that the number and distribution of IS1 and IS5 were usually unaltered compared to cells which did not undergo a transposition event. In contrast, essentially each IS10 transposition was accompanied by multiple events which led to changes in the number and distribution of chromosomal IS10 elements.