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The SF1 insulator was found to contain a polyadenylation signal, which corresponded to the functional polyadenylation signal in embryonic S2 cells and the transgenic lines of Drosophila and bi-directional promoter that functioned in S2 cells. The studies performed did not confirm the ability of the SF1 insulator to protect expression of reporter gene white from the chromosome position effect in transgenic lines.

A neDNA fragment that protects a transgene from position effect variegation when flanking it was tested for insulator properties. The fragment did not act as an insulator. A similarity of neDNA and various barrier elements was examined, and Drosophila melanogaster and Mus musculus chromosome DNA regions homologous to neDNA were analyzed. Additional conserved sites and DNA duplex destabilization sites were found in the neDNA sequence, and DNA conformational specifics were assumed for the chromosomal neighborhood of neDNA sites of the D. melanogaster and M. musculus genomes.

The bithorax complex (BX-C) genes have a regulatory region containing nine independent parasegment-specific enhancers. Inactivation of the enhancers is a result of Pc-mediated repression, which leads to the appearance of active and repressed domains. Active domains are supposed to be separated from the repressed domains by insulators on the domain boundaries. Three insulators were found in the BX-C by the ability to block enhancers intransgenic model systems. It is shown that one of these three well studied insulators, Fab-7, is able to protect the yellow gene transcription against the most powerful Polycomb-dependent silencer from the bxd regulatory domain of the Ultra-bithorax gene (Ubx). The barrier activity requires a region containing GAF-binding sites. Interestingly, the silencer used in the model system (1.5 kb PRE from the regulatory region of Ubx) recruits a transgenic construct with a frequency of up to 47% into genomic regions that completely repress the yellow gene expression.

Insulators are regulatory DNA elements restricting gene activation by enhancers. Interactions between insulators can lead to both insulation and activation of promoters by enhancers. In this work, we analyzed the effects of interaction of two Drosophila insulators, Wari and Su(Hw). The functional interaction between these insulators was found to enhance the activity of the Su(Hw) insulator only, but not of the Wari insulator. This suggests that the formation of a chromatin loop between interacting insulators is not a key factor for enhancement of insulation, which is in disagreement with the main idea of structural models. In addition, the effect of interaction between Wari and Su(Hw) depends on a distance between them and on the position in the system relative to other regulatory elements.

Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated promoter. The best studied insulator of Drosophila melanogaster was found in the 5' regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression, induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines, two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter and functionally opposite elements, responsible for either gene activation, or repression.