Single-cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. Upon electrophoresis, the DNA of lysed, agarose-embedded cells known as nucleoids, extends towards the anode in a structure resembling a comet, the relative intensity of the tail reflecting the frequency of DNA breaks. The structural organization of the DNA within comet preparations is not fully understood. We have used fluorescent in situ hybridization with large-insert genomic probes and human Cot-I DNA to investigate whether the production of the comet tail is simply explained by the relaxation of supercoiled DNA loops. We find that, under neutral electrophoresis conditions, when the tail and head DNA are double-stranded, the probed sequence of DNA is seen as a linear array, consistent with extension from a fixed point on the nuclear core or matrix. After alkaline electrophoresis, the appearance of the fluorescent probes suggests that linear DNA has coalesced into a granular form.

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Retroelements with long-terminal repeats (LTRs) inhabit nearly all eukaryotic genomes. During the time of their rich evolutionary history they have developed highly diverse forms, ranging from ordinary retrotransposons to complex pathogenic retroviruses such as HIV-I. Errantiviruses are a group of insect endogenous LTR elements that share structural and functional features with vertebrate endogenous retroviruses. The errantiviruses illustrate one of the evolutionary strategies of retrotransposons to become infective, which together with their similarities to vertebrate retroviruses make them an attractive object of research promising to shed more light on the evolution of retroviruses.

Mobile genetic elements constitute a substantial part of eukaryotic genome and play an important role in its organization and functioning. Co-evolution of retrotransposons and their hosts resulted in the establishment of control systems employing mechanisms of RNA interference that seem to be impossible to evade. However, "active" copies of endogenous retrovirus gypsy escape cellular control in some cases, while its evolutionary elder "inactive" variants do not. To clarify the evolutionary relationship between "active" and "inactive" gypsy we combined two approaches: the analysis of gypsy sequences, isolated from G32 Drosophila melanogaster strain and from different Drosophila species of the melanogaster subgroup, as well as the study of databases, available on the Internet. No signs of "intermediate" (between "active" and "inactive") gypsy form were found in GenBank, and four full-size G32 gypsy copies demonstrated a convergence that presumably involves gene conversion. No "active" gypsy were revealed among PCR generated gypsy ORF3 sequences from the various Drosophila species indicating that "active" gypsy appeared in some population of D. melanogaster and then started to spread out. Analysis of sequences flanking gypsy variants in G32 revealed their predominantly heterochromatic location. Discrepancy between the structure of actual gypsy sites in G32 and corresponding sequences in database might indicate significant inter-strain heterochromatin diversity.

Transposable elements (TE) are found in all eukaryotic genomes and play a significant role in their structure and functioning. The majority of mobile elements are silent in the genomes indicating the existence of cell control mechanisms of their activity. Establishment of immunity to TE is of great interest, but it cannot be studied directly and there are only few examples of present or recent active transpositions of mobile elements. G32, a Drosophila melanogaster strain, is characterized by the presence of large complex chromosomal aberration in the 3rd chromosome, active transpositions of gtwin in the past, and its stability at present. To address the question as to what had happened to the element while the cell took it under the control, we performed the detailed cytological and molecular analyses of gtwin's structure and its distribution in G32. Two variants of gtwin were found, one of which is amplified in G32 despite the alteration of tRNA-primer binding site. This element is accumulated in the aberrant chromosome and associated with the inversions breakpoints. Gtwin copies are predominantly localized in euchromatic regions and at least three of them are situated in heterochromatin. One copy was found in the piRNA cluster that might have caused silencing of the element.

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Transposable genetic elements constitute an essential component of eukaryotic genome. LTR-retrotransposons of the group Gypsy , many of which are considered as endogenous retroviruses of invertebrates, are of special interest [1,2]. The transposable genetic element MDG4 ( gypsy ) is the best studied retrotransposon of Drosophila melanogaster. Earlier, we demonstrated the existence of MDG4 subfamilies, differing in their ability for transpositions. Both these subfamilies, which were named "active" and "inactive," with different efficiency form new copies in transformed cultured cells of D. hydei. However, only the active MDG4 can undergo transpositions in D. melanogaster lines, whereas no transposition of the inactive MDG4 was detected in any line studied [3,4]. Structural differences between the two MDG4 subfamilies can be detected even at the level of restriction maps (Fig. 1). For example, all MDG4 copies cloned from the insertion mutagenesis systems contain the sites for the restriction endonucleases Hind III (4483), Mlu I (5335), and Cla I (6939) in the coding region and the sites for Xba I (430, 7417) in the long terminal repeats (LTRs) [3]. Because, unlike the active MDG4 variant, the inactive MDG4 is present in the genome of all Drosophila lines studied, it is probably the most ancient variant. To approach the question on MDG4 evolution, we decided to study all sequences of D. melanogaster genome that shared a significant extent of homology with this retrotransposon. Experiments were performed with the D. melanogaster line G32, the genome of which contains unusual MDG4 variants [6].As a result of screening of the genomic library of the D. melanogaster line G32, 28 clones homologous to the MDG4 sequence were selected. Only four out of the 28 clones contained the full-length variants of this retrotransposon: two active copies, one inactive copy, and one unusual copy that contained facultative restriction sites for Hind III (4483) and Xba I (430 and 7417) but had no sites for Mlu I (5335) and Cla I (6939). Probably, this copy occurred as a result of recombination processes. Another four sequences cloned from the genome of line G32 contained copies of a poorly studied retrotransposon gtwin , which is classified in the group of LTR-retrotransposons Gypsy and shares a significant degree of homology with MDG4. The other clones contained sequences whose structure also considerably differed from MDG4 structure.To reveal sequences homologous to MDG4, we created a genomic library of line G32 on the basis of bacteriophage λ . Genomic DNA of λ GEM11 was treated with the specific restriction endonuclease BamH I,