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.

No abstract

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.