Hobo transposons causing chromosomal breakpoints.

Several laboratory surveys have shown that transposable elements (TEs) can cause chromosomal breaks and lead to inversions, as in dysgenic crosses involving P-elements. However, it is not presently clear what causes inversions in natural populations of Drosophila. The only direct molecular studies must be taken as evidence against the involvement of mobile elements. Here, in Drosophila lines transformed with the hobo transposable element, and followed for 100 generations, we show the appearance of five different inversions with hobo inserts at breakpoints. Almost all breakpoints occurred in hobo insertion sites detected in previous generations. Therefore, it can be assumed that such elements are responsible for restructuring genomes in natural populations.     

The Distribution of Mutation Effects on Viability in Drosophila Melanogaster

Parameters of continuous distributions of effects and rates of spontaneous mutation for relative viability in Drosophila are estimated by maximum likelihood from data of two published experiments on accumulation of mutations on protected second chromosomes. A model of equal mutant effects gives a poor fit to the data of the two experiments; higher likelihoods are obtained with leptokurtic distributions or for models in which there is more than one class of mutation effect. Minimum estimates of mutation rates (events per generation) at polygenes affecting viability on chromosome 2 are 0.14 and 0.068, but estimates are strongly confounded with other parameters in the model. Separate information on rates of molecular divergence between Drosophila species and from rates of movement of transposable elements is used to infer the overall genomic mutation rate in Drosophila, and the viability data are analyzed with mutation rate as a known parameter. If, for example, a mutation rate for chromosome 2 of 0.4 is assumed, maximum likelihood estimates of mean mutant effect on relative viability are 0.4% and 1%, but the majority of mutations have very much smaller effects than these values as distributions are highly leptokurtic. The methodology is applied to estimate viability effects of single P element insertional mutations. The mean effect per insertion is found to be higher, and their distribution is found to be less leptokurtic than for spontaneous mutations. The equilibrium genetic variance of viability predicted by a mutation-selection balance model with parameters estimated from the mutation accumulation experiments is similar to laboratory estimates of genetic variance of viability from natural populations of Drosophila.     

Active miniature transposons from a plant genome and its nonrecombining Y chromosome

Mechanisms involved in eroding fitness of evolving Y chromosomes have been the focus of much theoretical and empirical work. Evolving Y chromosomes are expected to accumulate transposable elements (TEs), but it is not known whether such accumulation contributes to their genetic degeneration. Among TEs, miniature inverted-repeat transposable elements are nonautonomous DNA transposons, often inserted in introns and untranslated regions of genes. Thus, if they invade Y-linked genes and selection against their insertion is ineffective, they could contribute to genetic degeneration of evolving Y chromosomes. Here, we examine the population dynamics of active MITEs in the young Y chromosomes of the plant Silene latifolia and compare their distribution with those in recombining genomic regions. To isolate active MITEs, we developed a straightforward approach on the basis of the assumption that recent transposon insertions or excisions create singleton or low-frequency size polymorphisms that can be detected in alleles from natural populations. Transposon display was then used to infer the distribution of MITE insertion frequencies. The overall frequency spectrum showed an excess of singleton and low-frequency insertions, which suggests that these elements are readily removed from recombining chromosomes. In contrast, insertions on the Y chromosomes were present at high frequencies. Their potential contribution to Y degeneration is discussed.     

Comprehensive analysis of Alu-associated diversity on the human sex chromosomes

A comprehensive analysis of the human sex chromosomes was undertaken to assess Alu-associated human genomic diversity and to identify novel Alu insertion polymorphisms for the study of human evolution. Three hundred forty-five recently integrated Alu elements from eight different Alu subfamilies were identified on the X and Y chromosomes, 225 of which were selected and analyzed by polymerase chain reaction (PCR). From a total of 225 elements analyzed, 16 were found to be polymorphic on the X chromosome and one on the Y chromosome. In line with previous research using other classes of genetic markers, our results indicate reduced Alu-associated insertion polymorphism on the human sex chromosomes, presumably reflective of the reduced recombination rates and lower effective population sizes on the sex chromosomes. The Alu insertion polymorphisms identified in this study should prove useful for the study of human population genetics.     

Cytogeography and the evolutionary significance of B chromosomes in relation to inverted rearrangements in a grasshopper species

The analysis of geographic distribution of polymorphic cytological markers, briefly termed as cytogeography, can be considered an important tool to be applied when studying the evolutionary significance of chromosome variability within a species, either to unravel the adaptive significance of chromosome polymorphisms or to investigate the parasitic nature of some genomic elements. In this article we review cytogeographical studies in Trimerotropis pallidipennis, a grasshopper species whose South American populations display geographical patterns of distribution of inversion and B chromosome polymorphisms. Several lines of evidence that issue from the analysis of the geographic distribution of polymorphic markers suggest that inverted chromosomes are special sequences that are maintained by deterministic forces. On the other hand, the pattern of distribution of B chromosome polymorphism clearly demonstrates its selfish nature, being more frequent in those populations in central environments. We also present the analysis of 272 individuals of T. pallidipennis from Uspallata, and demonstrate that Bs in this population have some influence on body size, enlarging many of the morphometric characters of individuals and we propose it could be the consequence of its genotypic disequilibrium with one inversion. These investigations are finally discussed with regard to the models proposed for the maintenance of B chromosomes in natural populations and in relation to the possible interactions with chromosome inversions.     

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.     

Interaction among different heterocromatic variants in the grasshopper Dichroplus elongatus

Simultaneous chromosome polymorphisms for supernumerary elements allow us to analyse the relationships among different forms of heterochromatic variation in nature. We report simultaneous variation patterns for supernumerary segments in chromosomes S10 (SS10), S9 (SS9) and S6 (SS6) and B chromosomes in nine populations of the grasshopper Dichroplus elongatus from two biogeographic provinces from east Argentina. Our results show spatial chromosome differentiation for three out of four supernumerary heterochromatic variants (B chromosomes, SS6 and SS10). The incidence of B chromosomes was negatively correlated with the SS10 frequency. The distribution pattern analysis shows different degree of differentiation among populations for each supernumerary heterochromatic variant suggesting that the detected chromosome variation cannot be explained by interaction between migration and genetic drift. Moreover, the observed population chromosome differentiation was not in agreement with the hierarchical analysis of molecular of heterogeneity at mitochondrial DNA level (mtDNA). The present results point out the importance of the interaction among heterochromatic variants in the chromosome intraspecific variation in east Argentina natural populations of the grasshopper D. elongatus.     

A new resource of locally transposed Dissociation elements for screening gene-knockout lines in silico on the Arabidopsis genome

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).     

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.     

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.     

B chromosome ancestry revealed by histone genes in the migratory locust

In addition to the standard set of chromosomes (A), about 15% of eukaryote genomes carry B chromosomes. In most cases, B chromosomes behave as genomic parasites being detrimental for the individuals carrying them and prospering in natural populations because of transmission advantages (drive). B chromosomes are mostly made up of repetitive DNA sequences, especially ribosomal DNA (rDNA), satellite DNA and mobile elements. In only two cases have B chromosomes been shown to carry protein-coding genes. Although some B chromosomes seem to have derived from interspecific hybridisation, the most likely source of B chromosomes is the host genome itself, but the specific A chromosome being the B ancestor has not been identified in any B-containing species. Here, we provide strong evidence for B chromosome ancestry in the migratory locust, based on the location of genes for the H3 and H4 histones in the B chromosome and a single A chromosome pair (i.e. the eighth in order of decreasing size). The high DNA sequence similarity of A and B chromosome H3–H4 genes supports B-origin from chromosome 8. The higher variation shown by B sequences, compared to A sequences, suggests that B chromosome sequences are most likely inactive and thus less subjected to purifying selection. Estimates of time of divergence for histone genes from A and B chromosomes suggest that B chromosomes are quite old (>750,000 years), showing the B-chromosome ability to persist in natural populations for long periods of time.     

Chromosome interactions in P-M dysgenic male recombination of Drosophila melanogaster.

The frequency, distribution and structure of P elements on the second and third chromosomes of Texas 1, a wild-type inbred strain of Drosophila melanogaster, were investigated by in situ hybridization. These autosomes were isolated individually and used as P-element donors to study the frequency and distribution of male recombination events generated on recipient chromosomes which were originally devoid of P sequences. The P-element array of chromosome 2 was shown to generate higher male recombination frequencies on chromosome 3 than vice versa, despite having fewer P factors and fewer P elements in general. This is likely to be due to the presence and distribution of specific P-deletion derivatives, which vary in their ability to repress P mobility. The male recombination generated on recipient chromosomes is associated with the insertion of donated P sequences, but only in a small minority of cases could a novel P-element site be detected at, or near, the recombination breakpoint. The majority of such breakpoints appear to be associated either with unsuccessful P insertion, or with the action of P transposase attracted by P elements newly inserted elsewhere on the recipient chromosome. Recent evidence also suggests that a small proportion of the breakpoints may be associated with the action of P transposase alone. Male recombination breakpoints appear to be distributed effectively at random along the recipient autosomes, and their frequency of occurrence was shown to correlate with the physical length of DNA available between markers, as revealed by the polytene map distance.     

Transposable Elements in Mendelian Populations. II. Distribution of Three COPIA-like Elements in a Natural Population of DROSOPHILA MELANOGASTER

Twenty X chromosomes isolated from a natural population of Drosophila melanogaster were surveyed using in situ hybridization to determine the number and cytogenetic location of three families of transposable elements: copia, 412 and 297. We found no sites of insertions in high frequency; in fact, frequencies of specific sites for all three elements were so low that each insertion could be interpreted as being unique. This suggests that rates of transposition and deletion for these elements are very high. Our data also show a higher than expected rate of the co-occurrence of different elements at the same site on the same chromosome.     

P-elements are old components of the Scaptomyza pallida genome

We report the cloning and analysis of a sample representative of all P-elements from Scaptomyza pallida. We have compared four independent stocks of this species, using Southern blot and in situ hybridization experiments to examine the number, structure, and distribution of P-elements. All four stocks give similar results: they contain about 15 P-elements including three to four full-length elements as well as shorter, deleted elements. All elements are divergent from one another and most of them appear to be immobile since they are located at identical positions in the genomes of independent stocks. These data indicate that P-elements are old components of the S. pallida genome. Moreover, the presence of P-sequences in species closely related to S. pallida suggests that they have had a long evolutionary history in the Scaptomyza genus. We have also found that most P-elements of S. pallida are located in the pericentromeric heterochromatin. This corroborates other studies which show that in the course of their evolution, transposable elements tend to accumulate into pericentromeric heterochromatin, where they become immobile and noncoding. Correspondence to: M. Simonelig     

P-related sequences inDrosophila bifasciata: A molecular clue to the understanding of P-element evolution in the genusDrosophila

Summary Two P-elements (bif1 and bif2) were isolated from a genomic library ofDrosophila bifasciata. Both elements are internally deleted and have lost the coding capacity for a functional transposase. One of the elements (bif2) contains an insert consisting of a repetitive sequence. The terminal inverted repeats and the segments necessary for passive mobility are well conserved. Element bif2 has retained rudiments of the coding sequence of exon 0 and exon 3, but the reading frame is destroyed by insertions and deletions. The comparison of theD. bifasciata P-elements with P-elements ofDrosophila melanogaster andDrosophila nebulosa reveals that the two latter sequences are more similar to each other than either of them is to theD. bifasciata elements. This finding contradicts the phylogenetic relationship of the species and can be taken as an indirect but unequivocal evidence for recent horizontal gene transfer from a relative ofD. nebulosa to the gene pool ofD. melanogaster. The P-elements ofD. bifasciata are phylogenetically ancient and have evolved independently for about 50 million years. A higher substitution rate at the third codon position as well as a predominance of conservative replacements at the amino acid level indicates that the P-elements ofD. bifasciata have been under selective constraint over a long period and that immobilization has occurred only recently.     

Mitotically unstable B chromosome polymorphism in the grasshopper Dichroplus elongatus

Dichroplus elongatus, a widespread South American phytophagous grasshopper, exhibits polymorphisms for supernumerary chromosomes and segments (SS) in natural populations in Argentina. In this paper we review the available information on B chromosome polymorphism in D. elongatus related to geographic distribution, patterns of chromosome variation and influence on sperm formation. In D. elongatus the different forms of supernumerary variants are not independent. The proportion of B-carrying individuals (B prevalence) is negatively correlated with SS10 and positively with SS6 frequencies. The analysis of population structure considering the different supernumerary variants would suggest that the patterns of chromosome variation can not be explained only by random factors. Geographic distribution was analyzed scoring the prevalence of B chromosomes in 13 natural populations collected in three different biogeographical provinces from Northwest (Las Yungas province) and East (Espinal and Pampeana provinces) of Argentina. The detected heterogeneity may be explained by significant differentiation between Northwest and East regions and among populations within Las Yungas and Pampeana provinces. Correlation analysis suggested that B chromosome prevalence is associated with maximum temperature and with latitude. Additional information about the nature of the patterns of B chromosome variation was obtained comparing them with those obtained at the mitochondrial DNA level. The hierarchical analysis of molecular differentiation revealed discrepancy with respect to chromosome differentiation and also suggested that the pattern of B chromosomes may not be explained by historical factors. We also discussed the probable influence on fertility of carriers considering the production of abnormal sperm formation (macro and microspermatids) in relation to the number of Bs per follicle.     

Evidence for chromosome number reduction and chromosomal homosequentiality in the 24-chromosome Korean frog Rana dybowskii and related species

The karyotype of the Korean frog Rana dybowskii with its pattern of C-band heterochromatin distribution was numerically analyzed. There are 2n = 24 chromosomes in the karyotype representing a reduction in number from the typical 2n = 26 chromosome karyotype of Rana. The karyotype shows other evidence of reorganization relative to 26-chromosome species. The chromosomes grade smoothly in size from largest to smallest without the two size classes that are characteristic for 26-chromosome species. In contrast to many 26-chromosome species, there are few centromeric C-bands but many interstitial ones. C-bands for each homologous chromosome pair are distinctive. A prominent secondary constriction is located on one of the smallest chromosomes, chromosome 11, in a position similar to that seen in most 26-chromosome species. The karyotype of R. dybowskii is compared to those of other species of Rana known to have 2n = 24 chromosomes; it is most similar to that of R. chensinensis, less so that of R. ornativentris and less still to that of R. arvalis in terms of the positions of centromeres and secondary constrictions. C-bands as well as secondary constrictions in the karyotypes of these frogs show evidence of chromosomal homosequentiality. The process and possible consequences of chromosome number reduction from an ancestral 26-chromosome karyotype is also evident in the karyotypes of these closely allied palearctic frogs. Pericentric inversions followed by fusion of two small elements apparently produced a new chromosome, chromosome 6, occurring originally among northeast Asian populations.     

Mapping of Drosophila mutations using site-specific male recombination.

Although recombination does not usually occur in the male Drosophila germline, site-specific recombination can be induced at the ends of P elements. This finding suggested that male recombination could be used to map Drosophila mutations. In this article, we describe the general method and its application to the mapping of two EMS-induced female-sterile mutations, grauzone and cortex. Within two months, the grauzone gene was mapped relative to seven different P-element insertion sites, and cortex was mapped relative to 23 different P-elements. The results allowed us to map grauzone to a region of about 50 kb, and cortex distal to the chromosomal region 33E. These experiments demonstrate that P-element-induced site-specific male recombination is an efficient and general method to map Drosophila autosomal mutations.     

The mobile nature of acrocentric elements illustrated by three unusual chromosome variants

Variant chromosomes are polymorphic in areas that are rich in repeat sequences such as the pericentromeric regions or in the acrocentric short arm regions. The dynamic nature of these regions is evident in the polymorphisms they exhibit. In this paper three unusual variants are described: a chromosome 21 with additional material on its short arm, a chromosome 7 with an insertion in the short arm and a chromosome 2 with satellites at the end of the long arm. All three variants were shown to involve acrocentric elements using special banding techniques and fluorescence in situ hybridization. The 21 variant was found to be a tricentric with a 21 and two 15 alpha-, two classical and three acrocentric beta-satellite signals interspersed by AgNOR-positive regions. The telomeres were present at the two terminal ends. The insertion on chromosome 7 was found to be C-band positive and to contain acrocentric beta-satellite DNA. However, acrocentric alpha-satellite, classical satellite, whole-chromosome-painting or all-telomeres sequence probes did not hybridize to the insertion. The satellited region of chromosome 2 had two C-bands, a small positive all-centromeres probe signal, and two signals for the beta-satellite probe. Sandwiched between the beta-satellite sequences was an AgNOR-positive region. The telomeres were present at the two ends of the satellited chromosome 2. Chromosome 2 subtelomeric probes hybridized to the terminal ends of the short and long arm of chromosome 2. The common thread in these three variants is the involvement of acrocentric short arm elements. The acrocentric short arm elements are shown to move to other acrocentric or nonacrocentric chromosomes and relocate to both terminal and interstitial positions. The integrations are stable and heritable. Received: 23 September 1997 / Accepted: 23 February 1998     

Strong linkage disequilibrium between the XY274 polymorphism and the pseudoautosomal boundary.

The pseudoautosomal boundary is defined by an Alu repeat element on the Y chromosome. The Alu element is found on all Y chromosomes and on no X chromosomes, establishing it as part of Y-specific sequences. Distal to the Alu element, sequences from the X and Y are strictly homologous, suggesting that the boundary is formed by an abrupt break in sequence homology. Further investigation of the function of the boundary has been undertaken by examining the population structure of an MspI restriction-site polymorphism (XY274), which is located 274 bp distal to the Alu insertion site. Southern blot and polymerase chain reaction analysis demonstrate fixation of the high allele (noncutting or AT base pair) of XY274 on the Y chromosome in most populations, while a full range of high allele frequencies is found on the X chromosomes of different populations. Two exceptions to fixation on the Y chromosome were found in African populations. The level of linkage disequilibrium suggests that the first few hundred base pairs of the pseudoautosomal region on the Y chromosome share a single common origin more recent than the origin of the species.