RESUMEN
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.
Asunto(s)
Interferones/metabolismo , ARN Interferente Pequeño , Antivirales , Bioensayo , Genoma Viral , Células HEK293 , VIH-1/genética , VIH-1/metabolismo , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/inmunología , ARN Interferente Pequeño/metabolismo , ARN Viral/genéticaRESUMEN
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.
Asunto(s)
Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Interferencia de ARN , Retroelementos , Animales , Secuencia de Bases , Northern Blotting , Drosophila/crecimiento & desarrollo , Silenciador del Gen , Larva , Datos de Secuencia Molecular , Poliadenilación , ARN Interferente Pequeño/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Retroelementos , Secuencia de Aminoácidos , Animales , Elementos de Nucleótido Esparcido Largo , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , ADN Polimerasa Dirigida por ARN/genética , Homología de Secuencia de Aminoácido , Programas InformáticosRESUMEN
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.
Asunto(s)
Drosophila/genética , Genes Homeobox , Fenotipo , ARN Complementario/genética , ARN Mensajero/genética , Animales , Drosophila/embriología , Embrión no Mamífero , MicroinyeccionesRESUMEN
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.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Proteasa La , ARN Mensajero/genética , Serina Endopeptidasas/genética , Proteasas ATP-Dependientes , Regulación Bacteriana de la Expresión GénicaRESUMEN
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.
Asunto(s)
Mapeo Cromosómico , Drosophila/genética , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , ARN Mensajero/análisis , Animales , Drosophila/embriología , Hibridación in SituRESUMEN
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.
Asunto(s)
Drosophila/genética , Retroelementos , Animales , Secuencia de Bases , ADN , Datos de Secuencia Molecular , ARN Ribosómico 18S/genética , Secuencias Repetitivas de Ácidos Nucleicos , Homología de Secuencia de Ácido NucleicoAsunto(s)
Drosophila/genética , Elementos de Facilitación Genéticos , Proteínas de Insectos/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Animales , Secuencia de Bases , ADN , Proteínas de Drosophila , Proteínas de Homeodominio , Proteínas de Insectos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Mapeo Físico de Cromosoma , Unión Proteica , Factores de TranscripciónRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Mapeo Restrictivo , Retroelementos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Hibridación in Situ , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
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.
Asunto(s)
Drosophila melanogaster/genética , Evolución Molecular , Genoma , Retroelementos/genética , Animales , Secuencia de Bases , Clonación Molecular , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , ARN Ribosómico 16S/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
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.