RESUMEN
A full-length copy of the retrotransposon GATE was identified as an insertion in the tandemly repeated, heterochromatic, Stellate genes, which are expressed in the testis of Drosophila melanogaster. Sequencing of this heterochromatic GATE copy revealed that it is closely related to the BEL retrotransposon, a representative of the recently defined BEL-like group of LTR retrotransposons. This copy contains identical LTRs, indicating that the insertion is a recent event. By contrast, the euchromatic part of the D. melanogaster genome contains only profoundly damaged GATE copies or fragments of the transposon. The preferential localization of GATE sequences in heterochromatin was confirmed for the other species in the melanogaster subgroup. The level of GATE expression is dramatically increased in ovaries, but not in testes, of spn-E(1) homozygous flies. We speculate that spn-E is involved in the silencing of GATE via an RNA interference mechanism.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Secuencia de Aminoácidos , Animales , ADN/metabolismo , Femenino , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Mutación , Ovario/metabolismo , Filogenia , Estructura Terciaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Testículo/metabolismoRESUMEN
BACKGROUND: The injection of double-stranded RNA (dsRNA) has been shown to induce a potent sequence-specific inhibition of gene function in diverse invertebrate and vertebrate species. The homology-dependent posttranscriptional gene silencing (PTGS) caused by the introduction of transgenes in plants may be mediated by dsRNA. The analysis of Caenorhabditis elegans mutants impaired with dsRNA-mediated silencing and studies in plants implicate a biological role of dsRNA-mediated silencing as a transposon-repression and antiviral mechanism. RESULTS: We investigated the silencing of testis-expressed Stellate genes by paralogous Su(Ste) tandem repeats, which are known to be involved in the maintenance of male fertility in Drosophila melanogaster. We found that both strands of repressor Su(Ste) repeats are transcribed, producing sense and antisense RNA. The Stellate silencing is associated with the presence of short Su(Ste) RNAs. Cotransfection experiments revealed that Su(Ste) dsRNA can target and eliminate Stellate transcripts in Drosophila cell culture. The short fragment of Stellate gene that is homologous to Su(Ste) was shown to be sufficient to confer Su(Ste)-dependent silencing of a reporter construct in testes. We demonstrated that Su(Ste) dsRNA-mediated silencing affects not only Stellate expression but also the level of sense Su(Ste) RNA providing a negative autogenous regulation of Su(Ste) expression. Mutation in the spindle-E gene relieving Stellate silencing also leads to a derepression of the other genomic tandem repeats and retrotransposons in the germline. CONCLUSIONS: Homology-dependent gene silencing was shown to be used to inhibit Stellate gene expression in the D. melanogaster germline, ensuring male fertility. dsRNA-mediated silencing may provide a basis for negative autogenous control of gene expression. The related surveillance system is implicated to control expression of retrotransposons in the germline.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster/genética , Silenciador del Gen , Genes de Insecto , Proteínas de Insectos/genética , Proteínas Quinasas , ARN Bicatenario/fisiología , Proteínas Represoras/genética , Adenosina Trifosfatasas/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Femenino , Genes Reporteros , Proteínas de Insectos/biosíntesis , Masculino , Mutación , ARN sin Sentido/biosíntesis , ARN Mensajero/biosíntesis , Proteínas Represoras/biosíntesis , Retroelementos , Homología de Secuencia de Ácido Nucleico , Secuencias Repetidas en Tándem , Testículo/metabolismoRESUMEN
Testis-specific expression of tandemly repeated Stellate genes, located in eu- and heterochromatin regions of the X chromosome of Drosophila melanogaster, is suppressed by homologous Suppressor of Stellate repeats located on the Y chromosome. Using transgenic lines, we have demonstrated that three Su(Ste) copies failed to change the expression of the reporter construction carrying the bacterial beta-galactosidase gene under control of the Stellate gene regulatory sequence. Possible mechanisms of the Su(Ste) repeat suppressor activity are discussed.
Asunto(s)
Drosophila melanogaster/genética , Genes de Insecto , Genoma , Secuencias Repetidas en Tándem , AnimalesRESUMEN
Euchromatic genes are often silenced by rearrangements that place them within or near heterochromatin, a phenomenon known as position effect variegation (PEV). However, little is known about molecular structure of cis-acting heterochromatic fragments responsible for PEV. Here we report that heterochromatic cluster containing Stellate repeats, that encode putative regulatory subunit of protein kinase CK2 cause PEV of a reporter white 'mini-gene'. It is the first example of an euchromatic gene being silenced because of the proximity to the natural, well-defined heterochromatic repeat cluster.
Asunto(s)
Cromatina/genética , Drosophila/genética , Heterocromatina/genética , Proteínas de Insectos/genética , Proteínas Quinasas , Animales , Quinasa de la Caseína II , Proteínas de Drosophila , Eucromatina , Ojo/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/genética , Genes de Insecto/genética , Genes Reguladores/genética , Pigmentación/genética , Proteínas Serina-Treonina Quinasas/genética , Secuencias Repetitivas de Ácidos Nucleicos/genética , Transformación GenéticaRESUMEN
The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction.
Asunto(s)
ADN Ribosómico/genética , Drosophila melanogaster/genética , Genes de Insecto , Seudogenes , ARN Ribosómico 18S/genética , Animales , Secuencia de Bases , Datos de Secuencia Molecular , Familia de Multigenes , Análisis de Secuencia de ADNRESUMEN
The 30-kb cluster comprising close to 20 copies of tandemly repeated Stellate genes was localized in the distal heterochromatin of the X chromosome. Of 10 sequenced genes, nine contain undamaged open reading frames with extensive similarity to protein kinase CK2 beta-subunit; one gene is interrupted by an insertion. The heterochromatic array of Stellate repeats is divided into three regions by a 4.5-kb DNA segment of unknown origin and a retrotransposon insertion: the A region (approximately 14 Stellate genes), the adjacent B region (approximately three Stellate genes), and the C region (about four Stellate genes). The sequencing of Stellate copies located along the discontinuous cluster revealed a complex pattern of diversification. The lowest level of divergence was detected in nearby Stellate repeats. The marginal copies of the A region, truncated or interrupted by an insertion, escaped homogenization and demonstrated high levels of divergence. Comparison of copies in the B and C regions, which are separated by a retrotransposon insertion, revealed a high level of diversification. These observations suggest that homogenization takes place in the Stellate cluster, but that inserted sequences may impede this process.