Polymorphisms in the chromosomal locations of elements of the 412, copia and 297 dispersed repeated gene families in Drosophila.

The number and chromosomal locations of elements of the 412, copia and 297 dispersed repeated gene families differ extensively when the genomes of four D. melanogaster strains are compared. Differences among individuals from the same laboratory stock in the arrangement of these elements are also observed. In contrast to these polymorphisms, the structures of the elements themselves are closely conserved. Our results indicate that 412, copia and 297 are capable of evolutionarily rapid transpositions to new chromosomal sites.     

Mobile dispersed repeated DNA elements in the Drosophila genome

The molecular and cytogenetic organizations of 19 nonhomologous dispersed repeated sequence families were studied in 15 different laboratory strains of Drosophila melanogaster. Elements from each of the families appear to undergo transposition within the Drosophila genome, because there were striking differences in both the number and chromosomal locations of these elements between strains. A significant fraction (greater than 1%) of Drosophila DNA therefore has an unstable genomic organization. Each middle repetitive family exhibited similar variations in the chromosomal distribution of elements between the strains. Although the movements of these elements are not limited to a small number of genomic sites, there are chromosomal regions where elements from the different dispersed repeated DNA families appear to be clustered. The locations of such preferred integration sites are different in each of the D. melanogaster strains examined.     

A complex repeated DNA sequence within the Drosophila transposable element copia.

A 320 nucleotide repeated DNA sequence within the copia coding element of Drosophila melanogaster has been identified and characterized. This sequence has been localized by DNA-DNA hybridization and electron microscopic analysis of heteroduplexes to the approximate middle of the 5 kb copia coding region. The primary sequence of this repeated DNA has been determined. The sequence is composed of three related subunits, 35-37 nucleotides in length (A, B and C). This 105 nucleotide higher order repeat has apparently been duplicated twice to yield a complex repeated sequence, ABCA'B'C'A"B"C", which exhibits divergence among the individual subunits. This sequence is AT rich, as are the direct terminal repeats which flank the copia coding region, but does not contain any apparent homology with the terminal repeats. This repeated sequence contains three presumptive polyadenylation signals and two 25 nucleotide, imperfectly matched, inverted repeat sequences adjacent to two of the polyadenylation sequences.     

Regulatory elements of copia retrotransposons, controlling the level of its expression in Drosophila melanogaster testes

Expression of the lacZ reporter gene under the control of five deletion derivatives of the copia regulatory region including the 5' long terminal repeat (LTR) and the 5' untranslated region (UTR) was assayed in the testes of transgenic Drosophila melanogaster males (larvae and imago). The full-length copia regulatory region (LTR + UTR) ensured expression of the reporter gene in testes of both larvae and adult males. Deletion of UTR or 3' end of LTR increased lacZ expression in the testes, whereas deletion of the 5' end of LTR increased it. This indicated that a positive regulator of copia expression is at the 5' end of LTR and that negative regulators are at the 3' end of LTR and in UTR. The effects of the fragments of the copia regulatory region on reporter gene expression in the testes in vivo did not completely coincide with the effects observed earlier in cultured cells. We suggest that this difference is due to different regulation of expression of the fusion constructs integrated into chromatin as compared to their transient expression.     

Transposition of mobile genetic elements in interspecific hybrids of Drosophila

In situ hybridization of labeled DNA of four mobile dispersed genetic elements (mdg), isolated from D. melanogaster and C. virilis genomes, with polytene chromosomes of the larvae of several Drosophila species has been carried out. The data show that the mdg elements exhibit a high degree of species specificity. The same conclusions are derived from filter hybridization using ³²P-labeled D. melanogaster and D. virilis DNA and cloned mdg sequences immobilized on nitrocellulose filters. We attempted to induce transpositions (jumping) of mdg elements specific for D. virilis chromosomes to the chromosomes of related species (e.g. D. littoralis Meigen) originally lacking the representatives of this family of repeats. For this purpose we produced hybrid stocks with synthetic karyotoypes characterized by different combinations of D. virilis homologous chromosomes and hybrid chromosomes. In one of such stocks we did find by in situ hybridization the insertion of a D. virilis mdg element into the fifth chromosome of D. littoralis Meigen. The transposition (jumping) took place in the only region where somatic pairing between the fifth chromosomes of D. virilis and D. littoralis occurs more or less regularly in the hybrids. Since crossing-over in hybrid chromosomes of males is excluded in such synthetic stocks, gene conversion may be responsible for this transposition. The possible bearing of the phenomenon observed on the problem of hybrid dysgenesis is discussed.     

Properties of dispersed, highly repeated DNA within and near the hamster CAD gene.

Dispersed, highly repeated DNA sequences were found within and near the Syrian hamster gene coding for the multifunctional protein CAD. Most of the repeated sequences were homologous to each other and had similar properties. They hybridized to many cytoplasmic polyadenylated RNAs and to 7S and 4.5S cytoplasmic non-polyadenylated RNAs. Cloned DNA fragments containing repeated sequences were transcribed in vitro by RNA polymerase III. The repeated sequences from Syrian hamsters share many properties with the Alu family of repetitive DNA from humans. The hamster sequences were homologous to total repetitive human DNA but only very weakly homologous to two cloned members of the human Alu family.     

Transposition induction of the mobile genetic element Dm412 in the Drosophila genome using heat shock]

Males of Drosophila melanogaster isogenic line with oligogene mutation radius incompletus (ri) were exposed to standard heat-shock (SHS: t = 37 degrees C, 90 min) and heavy heat-shock (SHS: three-fold transfer of males from t = 37 degrees C, 2h, t0t = 4 degrees C 1 h, and back). At F1 of the treated males with untreated females of the same isogenic line mass transpositions of MGE Dm412 were found. The new positions of MGE seem to be not random, and 5 "hot sites" of transpositions were detected. The probabilities of transpositions were estimated after SHS and HHS and in control sample. They were, correspondingly, 3.4 x 10(-2), 8.7 x 10(-2) and less than 4.1 x 10(-4) transpositions per genome, per site occupied, per generation. Therefore, as a result of HS treatment, the probabilities of transpositions were two orders of magnitude increased as compared to control, directly at next generation after induction. Comparison of these results with those obtained after step-wise temperature treatment shows that induction is dependent rather of "stressor effect" of temperature treatment than of treatment way used.     

Expression of the quantitative trait radius incompletus, temperature effects and localization of mobile genetic elements in Drosophila. II. Mobile genetic elements Dm-412

Two "selection" sub-populations (ris- and ris+), as well as two "temperature" ones (ric113 and ric149) were earlier developed from the control ric sub-population with interrupted vein of the fly wing. All five sub-populations were investigated for hybridization of MGE Dm-412 with drosophila polytene chromosomes in situ. The tree of similarity of MGE Dm-412 hybridization patterns was built by the methods of matrix clusterization. The sub-populations with the most resembling expressions of characters (ris- and ric113, ris+ and ric149) were found to be also most similar in patterns of MGE localization and their changes. Nonrandomness of these changes was shown, the similarity of patterns being demonstrated to be mainly the result of the changes. There is evidence that such effects cannot be accounted for by genetic drift and independent stochastic changes in MGE localization.     

Transposition bursts in analysis of repeated mutations in unstable strains of Drosophila melanogaster

The phenomenon of transposition memory was earlier demonstrated for the cut locus and mdg4. This work has been aimed at finding out, in what way the transposition memory can be realized. An unstable stock cmMR17ctMRpN17 was analysed which had high frequency of double cm+ct+ reversions and cmMRctMRpN repeated mutations. A series of five such transpositions could be followed. The ctMRpN17 mutation is a result of insertion at the cut locus mdg4 with the jockey element inserted within it. As seen from in situ hybridization analysis, transitions to the normal phenotype correlate, as a rule, with the excision of mdg4 and the jockey from the cut locus. Analysis of distribution of mdg1, mdg2, mdg3 and jockey on the X-chromosome of unstable revertants and repeated mutants indicated that not only transpositions of mdg4 and jockey, but also those of all mobile elements tested occur. So, we propose that the transposition memory in our genetic system is manifested in the process of transposition bursts.     

Expression of the quantitative trait radius incompletus in Drosophila and localization of mobile elements MDG1 and copia

A series of subpopulations earlier obtained were studied for hybridization of mobile genetic elements (MGE). The subpopulations examined were two "selected" (ris- and ris+), two "temperature" (ri(c113) and ri(c149)) and the control (ric). The method of in situ hybridization with polytene chromosomes of larval salivary glands was used to determine the patterns of MGE localization for all subpopulations. The patterns obtained appeared to be quite different from that of mdg-2. The trees of similarity for subpopulations according to the patterns of every MGE localization were built by conventional clustering methods. These trees were topologically similar to each other and to mdg-2. Distinction spectra of patterns of four daughter subpopulations, in comparison with the control one, were shown for each of these MGE to be independent and individual. However, there are some common regularities among copia-like MGE-mdg-1, copia, mdg-2 and, probably, mdg-3, namely: non-random property of the majority of changes, the similarity of patterns for subpopulations with similar phenotypes etc. So, Drosophila genome can be conceived as a complex system of patterns of different MGE localization, capable of common or independent mass transpositions after external stress action.     

Transposition rate of the 412 retrotransposable element is independent of copy number in natural populations of Drosophila simulans

The transposition and excision rates of the 412 retrotransposable element were estimated in five populations of Drosophila simulans differing in their average 412 copy numbers, which ranged from 2 to 54. The transposition rate was found to equal 1 x 10(-3) to 2 x 10(-3), independently of copy number. No excision was detected. These values eliminate autoregulation as a force maintaining copy number of the 412 element in natural populations of D. simulans.      

Induction of MGE 412 Transposition in an Isogenic Strain of Drosophila melanogaster by Different Doses of Ethanol Fumes

The effect of treatment of males from an isogenic Drosophila melanogaster strain by limiting doses of ethanol fumes on transpositions of MGE412 was examined. Validity of the phenomenon of transposition induction was demonstrated. We estimated rates of induced transposition (10^–2 events per site, per sperm, per generation versus <10^–3 in control) and showed dose dependence of the rate on the exposure time of the males to ethanol fumes. Experiments with alcohol treatment at limiting doses must end either in death of the individuals or bursts of genetic variability in their progeny. In terms of genetics of an individual, this may mean loss of vital hereditary basis followed by mass degradation of the progeny of the hard drinkers. In terms of populations genetics, this mode of MGE transposition induction can rapidly create a burst of novel genetic variation, which, apart of great losses, may generate a number of advantageous individuals, i.e., be significant for population survival in new, stressful environments.     

Mutation accumulation and the effect of copia insertions in Drosophila melanogaster

Repeated efforts to estimate the genomic deleterious mutation rate per generation (U) in Drosophila melanogaster have yielded inconsistent estimates ranging from 0.01 to nearly 1. We carried out a mutation-accumulation experiment with a cryopreserved control population in hopes of resolving some of the uncertainties raised by these estimates. Mutation accumulation (MA) was carried out by brother sister mating of 150 sublines derived from two inbred lines. Fitness was measured under conditions chosen to mimic the ancestral laboratory environment of these genotypes. We monitored the insertions of a transposable element, copia, that proved to accumulate at the unusually high rate of 0.24 per genome per generation in one of our MA lines. Mutational variance in fitness increased at a rate consistent with previous studies, yielding a mutational coefficient of variation greater than 3%. The performance of the cryopreserved control relative to the MA lines was inconsistent, so estimates of mutation rate by the Bateman-Mukai method are suspect. Taken at face value, these data suggest a modest decline in fitness of about 0.3% per generation. The element number of copia was a significant predictor of fitness within generations; on average, insertions caused a 0.76% loss in fitness, although the confidence limits on this estimate are wide.