[Analysis of the genetic constructs expressing antiviral siRNAs in the non-viral test system].

The efficiency of RNA interference triggered by the genetic constructs encoding siRNAs and directed against human immunodeficiency virus type 1 (HIV-1) transcripts was investigated in the non-viral test system. The investigation showed 6 biologically active siRNAs attacking 6 conservative targets in the HIV-1 genome. Expression of these genetic constructs failed to induce a nonspecific interferon response.

[RNA interference is developmentally regulated: analysis of expression of suffix, a short Drosophila retroelement]. / RNK-interferentsiia reguliruetsia v razvitii: analiz ékspressii suffiksa--korotkogo retroélementa Drozofily.

Sense and antisense transcripts of suffix and F element were detected at different stages of Drosophila development. A short RNA, similar in size to the full-length suffix transcript, was also found. It was suggested that it could originate from the master copy of the element. Though transcription pattern of these elements changed during the development, transcription remained symmetrical. These data indicate that formation of dsRNA in the cells and triggering of the RNA interference mechanisms are possible at all stages of Drosphila development. Analysis of total RNA samples from all stages of Drosophila development showed that 21- to 25-nt long suffix--specific small interfering RNAs (siRNA), the obligatory products of the RNA interference, were detected only in pupae. Thereafter, RNA interference mechanisms are developmentally regulated and the formation of dsRNA is necessary but not sufficient for launching this potent and specific machinery of post-transcription silencing.

[Analysis of the suffix short retroelement copies and the encoded protein domain of Drosophila]. / Analiz kopii suffiksa--korotkogo retroélementa Drozofily--i kodiruemogo im belkovogo domena.

To analyze the copies of the suffix short retro-element, its homologs were sought in nucleic acid sequence databases of the Drosophila melanogaster genome. The search yielded several conserved (near identical in sequence) copies, which are indicative of recent suffix transposition, and numerous divergent copies, which suggest ancient suffix transposition. Analysis of the short suffix ORF revealed a conserved protein domain, which was also found as the eighth C-terminal domain in reverse transcriptases of certain long interspersed elements (LINEs). The suffix-encoded polypeptide proved to be homologous to DNA- and RNA-recognizing domains.

[Generation of Kruppel phenocopies by injecting into Drosophila embryos RNA complementary to mRNA in parallel orientation]. / Poluchenie fenokopii Kruppel pri in''ektsiiakh v émbriony drozofily RNK, komplementarnoi mRNK v parallel'nom napravlenii.

RNA preparations synthesized in vitro were used to study the influence of RNA interference on the Kruppel gene activity in Drosophila embryos. RNA complementary in parallel orientation to the mRNA fragment proved to induce the development of Kruppel phenocopies. The data obtained indicate that mechanisms of specific regulation of gene activity exist in Drosophila cells, which are sensitive to the formation of both parallel and antiparallel RNA-RNA duplexes that include mRNA of the corresponding gene.

[RNA interference in Escherichia coli cells: the expression of molecules that are complementary to the lon gene mRNA in parallel orientation]. / RNK-interferentsiia v kletkakh Escherichia coli pri ékspressii molekul, komplementarnykh v parallel'nom napravlenii mRNK gena lon.

To study the effect of RNA interference (RNAi) on the activity of gene lon in Escherichia coli, genetic constructs were used that could express RNA molecules complementary to the 5' region of lon mRNA in the same direction. These RNAs were termed parallel RNAs (pRNAs). Two approaches were used to control expression. In one approach, lon gene activity was estimated genetically, based on the effect of the Lon protease on bioluminescence determined by the Vibrio fischeri lux regulon. The other approach was direct testing of ATP-dependent proteolysis in vitro. It was found that pRNA considerably suppressed lon expression. The antiparallel RNA (apRNA) was a less effective suppressor of this gene. The specific RNAi was found to decay gradually by the 40th generation. The data obtained indicate that Eubacterium cells have mechanisms for specific regulation of gene activity that are sensitive to the formation of both parallel and antiparallel RNA duplexes involving mRNA of the given gene.

[Localization of transcripts corresponding to different regions of the "cut" locus in Drosophila embryos]. / Lokalizatsiia transkriptov, sootvetstvuiushchikh raznym oblastiam lokusa "cut", v émbryonakh drozofily.

Transcripts of the cut locus were localized in Drosophila embryos with use of nonradioactive in situ hybridization. DIG-labeled probes were generated from two locus regions located at position -130. Distal and proximal regions were found to be transcribed in the same tissues throughout embryogenesis. As only the proximal region transcript was detected, the conclusion was made that two or more various transcripts derived from different regions of the locus and under common control, are present simultaneously in the embryonic cells.

[Molecular analysis of integration sites of the drosophila retrotransposon burdock]. / Molekuliarnyi analiz mest integratsii retrotranspozona drozofily repeinik (burdock).

Data on molecular analysis of the insertion sites of nine random copies of burdock retrotransposon are presented. The 12-bp consensus sequence of the insertion sites, YNNUTUTUYAYA (Y-pyrimidine; U-purine), was determined. Homology between the burdock sequence and ribosomal genes was revealed. Three copies of this element were located within the region of ribosomal repeats: one copy in the 18S RNA gene, and two copies in the same intergenic spacer region, in the so-called Alu-repeats of Drosophila, in different copies of ribosomal genes.

[Molecular analysis of a copy of the novel mobile element Burdock and the region of its insertion into the cut locus of Drosophila melanogaster]. / Molekuliarnyi analiz kopii novogo mobil'nogo élementa--repeinik (Burdock)--i raiona ego insertsii v lokuse cut Drosophila melanogaster.

Molecular analysis of a copy of the novel mobile element burdock and its insertion region into the cut locus of Drosophila was performed. The burdock was shown to be a retrotransposon containing a single open reading frame (ORF). It does not contain domens coding for protease, RNAse H, reverse transcriptase, and integrase, which are required for transposition. However, multiple insertions of this copy of the mobile element into a definite region of the cut locus (hot site) were observed earlier. The polypeptide encoded by the burdock ORF contains two successive regions homologous to the proteins encoded by the ORF1 and ORF2 of the gypsy retrotransposon in N and C regions, respectively. The burdock insertion into this region of the cut locus interrupts its ORF, since the mobile element is transcribed in the opposite direction compared with the transcription in the locus. This is presumed to account for the arising of a lethal mutation. The hot site of this element integration into the locus corresponds to the recognition site of Drosophila topoisomerase II.

[The role of retroelements in evolution: an analysis of the suffix element from the Drosophila genome]. / Rol' retroélementov v évoliutsii: analiz suffiks-élementa iz genoma drozofily.

The investigation of sequences homologous to the suffix chains from Drosophila genome revealed the central domain of the element homologous to 16S ribosomal sequence from endosymbiotic organisms. The opposite strand of the element encodes C-domains of reverse transcriptase enzyme in F- and Doc-elements. 19 DNA clones possessing PCR-amplified stretches of genomic DNA were sequenced. It was found that micro-satellite sequences (CAACA)n/(TGTTG)n and (TTTGT)n/(CACAAA)n are the target sites for the insertions of suffix copies in heterochromatic regions of Drosophila genome in both orientations. It was found that the presence of decanucleotide GCGGCCCGGG (GC-box) followed by the alternating stretch (A)5(T)4(A)3(T)2(A)1(T)n (AT-box) led to the fixed polarity of the suffix insertions in the same micro-satellite sequences.

[Analysis of copies of the suffix-retroposon of Drosophila, produced using PCR on genomic DNA]. / Analiz kopii suffiksa-retropozona drozofily, narobotannykh s pomoshch'iu PTsR na genomnoi DNK.

Nineteen DNA clones, containing copies of the suffix element retroposon of the Drosophila genome and produced by a polymerase chain reaction (PCR), were sequenced. Insertions of the copies in both orientations into microsatellite sequences (CAACA)n/(TGTTG)n and (TTTGT)n/(CACAAA)n were revealed. It was found that, if the microsatellite sequence has both a decanucleotide GCGGCCCGGG (GC-box) and a colinear alternating sequence (A)5(T)4(A)3(T)2(A)1(t)n (AT-box), the insertion of the suffix occurs in only one orientation. It is suggested, that in such apparently heterochromatic sequences, suffix element copies and probably some other retroposons can be inserted a particular orientation by means of site-specific recombination. An approach to analysis of the molecular organization of such sequences is proposed.