RESUMEN
The homologue of the Drosophila melanogaster mobile element gypsy was cloned from the distantly related species D. virilis. It has three ORFs highly homologous to those of the element from D. melanogaster. gypsy from D. virilis appears to be actively transcribed and is capable of transposition. Comparison of the untranslated regions of both elements revealed conserved sequences including those which had previously been demonstrated to be important in transcription regulation. Distribution of gypsy among the different strains of D. virilis and different species within the D. virilis group was analyzed. Possible involvement of horizontal transmission in the process of spreading and evolution of gypsy is discussed.
Asunto(s)
Elementos Transponibles de ADN/genética , Drosophila/genética , Animales , Northern Blotting , Clonación Molecular , Drosophila melanogaster/genética , Electroforesis en Gel de Agar , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , ARN/análisis , Homología de Secuencia de Ácido Nucleico , Especificidad de la Especie , Transcripción GenéticaRESUMEN
We addressed the possibility of the horizontal transfer of long interspersed element (LINE)-like mobile elements by studying the distribution of the Drosophila melanogaster LINE-like element jockey in different Drosophila species. Outside the D. melanogaster group jockey was detected only in the distantly related species Drosophila funebris. Cloning and sequencing of this element from D. funebris revealed the existence of the two open reading frames highly similar to those of jockey from D. melanogaster. Elements from both species are transcriptionally active and contain in their promoter regions a conserved sequence important for its activity. The high degree of similarity between the D. melanogaster and the D. funebris jockey and the absence of jockey from other sibling species of the D. funebris group provide evidence for the horizontal transmission of jockey into D. funebris.
Asunto(s)
Drosophila/genética , Secuencia de Aminoácidos , Animales , Bacteriófago lambda/genética , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , ADN/genética , Drosophila melanogaster/genética , Biblioteca de Genes , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Sistemas de Lectura Abierta , Poli A/genética , ARN/genética , ARN Mensajero , Mapeo Restrictivo , Especificidad de la Especie , Transfección , Dedos de ZincRESUMEN
The gypsy (mdg4) mobile element of Drosophila contains two closely spaced regions which bind proteins from nuclear extracts. One of these is an imperfect palindrome having homology with the lac-operator of Escherichia coli; the other contains a reiterated sequence (5'PyPuT/C TGCATAC/TPyPy) homologous to the octamer that is the core of many enhancers and upstream promoter elements. Transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators of transcription. As was demonstrated earlier by other authors, mutations induced by the presence of gypsy in different loci are suppressed owing to either repression or activation of gypsy transcription in Drosophila strains carrying unlinked mutations in su(Hw) or su(f) genes. We have shown that binding to a negative regulator (silencer) is weakened in nuclear extracts isolated from fly stocks carrying su(f) mutations which activate gypsy transcription; therefore the su(f) gene seems to code for a protein capable of gypsy repression. Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from fly stocks carrying su(Hw) gene mutations which decrease the level of gypsy transcription; therefore, the su(Hw) gene most likely encodes a protein which activates gypsy transcription.
Asunto(s)
Drosophila melanogaster/genética , Supresión Genética , Transcripción Genética , Animales , Secuencia de Bases , Deleción Cromosómica , ADN/genética , Drosophila melanogaster/metabolismo , Genes Reguladores , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Recent data obtained in the authors' laboratories concerning the behaviour of mobile genetic elements of Drosophila melanogaster are reviewed. It was found that the mobile element jockey represents the typical LINE element. It is efficiently transcribed in D. melanogaster cells in flies and in culture. Transcription is initiated from the +1 nucleotide of jockey and depends on an internal promoter. This is the first case of an internal promoter being used by RNA polymerase II. Several events which take place during the transposition bursts in ctMR2 family of strains were described. Among them are the removal of mobile dispersed genetics (mdg) elements (with solo long terminal repeat (LTR) remaining at the site of excision), complete removal of an mdg element, and reinsertion of the same mdg to the same place either in the presence or in absence of solo LTR sequence. Finally, the formation of deletions was observed. A 462-bp deletion destroying the white locus can be further repaired (w+ reversion). Thus, transposition bursts include many different genetic events. A novel system of prolonged genome destabilization was described. It depends on mobilization of a new mobile element called Stalker. After certain crosses Stalker actively moves for dozens of generations giving rise to large numbers of insertion mutations. Several novel genes were detected using mobilized Stalker. They include a modifier of mdg4 and six enhancers of yellow mutations.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Animales , Secuencia de Bases , Deleción Cromosómica , Genes Reguladores , Datos de Secuencia Molecular , Mutación , Transcripción GenéticaRESUMEN
A detailed investigation of the Drosophila melanogaster mobile dispersed repetitive element jockey was performed. This is similar in its structural organization and coding potential to the long interspersed elements (LINEs) of various organisms. A complete copy of jockey (approx. 5 kb) is terminated with an oligodeoxynucleotide (dA) sequence preceded by two long open reading frames (ORFs) overlapping with a frameshift-1. Judging by the sequence homologies, ORF1 codes for a nucleic-acid-binding protein, and ORF2 for a reverse transcriptase which is most similar in its sequence to putative reverse transcriptase of other LINEs. As demonstrated by sequencing two deleted jockey copies, they contain only a small part of ORF2; however, other regions, including the terminal sequences, are highly conservative. The existence of a large number of jockey copies with a deletion in the second frame may indicate that they can use reverse transcriptase in trans.
Asunto(s)
Elementos Transponibles de ADN , Drosophila melanogaster/genética , Retroviridae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , ADN , Datos de Secuencia Molecular , ARN Polimerasa II/genética , Proteínas de Unión al ARN , ADN Polimerasa Dirigida por ARN/genética , Mapeo RestrictivoRESUMEN
The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. As demonstrated here, two polyadenylated jockey transcripts detected at different stages of Drosophila ontogenesis and in cell cultures have the same length as genomic copies of jockey and correspond to the strand containing ORFs. alpha-amanitin experiments indicate that jockey is transcribed by RNA polymerase II. Analysis of both expression of CAT constructions and initiation of transcription in jockey genomic and transfected copies has shown that jockey transcription is controlled by an internal promoter. Inward location of the promoter allows it to be preserved in the course of replication via reverse transcription and accounts for the distribution of jockey and probably other LINEs throughout the genome. This is the first case of an internal promoter described for RNA polymerase II. The comparison of sequences at the beginning of LINE elements in Drosophila allows one to detect possible core sequences.
Asunto(s)
Elementos Transponibles de ADN , Drosophila/genética , Mamíferos/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Especificidad de la EspecieRESUMEN
Transposition outbursts occur in the destabilized Drosophila melanogaster strain ct carrying a mutation in the locus cut induced by an insertion of mdg4. While the distribution of mobile genetic elements remained unchanged in the great majority of germ cells, in a few cells numerous transpositions had occurred involving mdg (copia-like), fold-back and P-elements. We used in situ hybridization to analyze the distribution of five families of mdg elements in the X-chromosome during several consequent mutational changes in D. melanogaster. Each of them was accompanied by many changes in mdg localization, all of which occurred in one and the same cell. Thus, we could observe the series consisting of up to five successive transposition explosions leading to an almost complete change in the distribution of the mdg elements tested. We also found that in the course of successive transposition explosions, mdg elements often inserted into those sub-sections of the X-chromosome where they had previously been located. This phenomenon, designated as reverse directed transposition, was studied in more detail on insertion into the locus yellow. The rate of reverse transposition of the same mdg element to the corresponding locus was 10-100 times as high as that of primary insertion. In some cases, ;the transposon shuttle' into and out of the locus was observed. The existence of ;transposition memory' partially explains the specificity of mdg localization in closely related strains as well as the co-ordinated behaviour of different mdg elements in independent transposition explosions. The evolutionary significance of transposition explosions and directed reverse transposition (transposon shuttle) is discussed.
RESUMEN
The segment of the locus cut containing the mobile genetic element mdg4 (gypsy) insertions which induce unstable ct and ct mutations has been cloned. Both mutations depend on the insertion of mdg4 into the same sequence, which coincides with that in ct allele. The ct mutation differs from ct by additional insertion of a novel mobile element jockey into mdg4. Jockey is 2.8 kb long, represented by 2-100 copies per genome, very homogeneous and lacks long terminal repeats (LTRs). The excision of mdg4 takes place in stable ct reversions. On the other hand, a complete single LTR is retained in the case of unstable ct reversions characterized by a high level of reverse directed transpositions of mdg4 into the locus cut. The LTR serves as a guide for reinsertion of mdg4 itself or mdg4 with jockey into the same site of the genome. A possible mechanism of transposition memory (homologous recombination with extrachromosomal circular DNA) is discussed.