RESUMO
The I factor is a Drosophila melanogaster LINE-like element that efficiently transposes in the genetic system of I-R hybrid dysgenesis. It has been suggested that some of the I-related sequences located in the heterochromatin of D. melanogaster are involved in the regulation of I factor activity. In this work we have performed fluorescent in situ hybridization (FISH) mapping of I element sequences in mitotic heterochromatin of nine differentially reactive D. melanogaster strains. The results of our analysis showed that a single hybridization site mapping to region h28 of the distal heterochromatin of the X chromosome is present in three strains with low or intermediate levels of reactivity, while it is undetectable in six highly reactive strains. Together, these observations suggest a negative correlation between I sequences located at h28 and the level of reactivity. To this regard, it is intriguing that flamenco and COM, two loci that regulate the activity of D. melanogaster endogenous retroviruses also map to the distal heterochromatin of the X chromosome. Our data represent the first experimental evidence in favour of a silencing effect exerted by naturally occurring I element sequences located in pericentromeric heterochromatin.
Assuntos
Elementos de DNA Transponíveis/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Animais , Drosophila melanogaster/classificação , Drosophila melanogaster/crescimento & desenvolvimento , Heterocromatina/ultraestrutura , Hibridização in Situ Fluorescente , Larva , Mitose , Especificidade da EspécieAssuntos
Baculoviridae/genética , Regulação da Expressão Gênica/fisiologia , Produtos do Gene gag/metabolismo , Transporte Proteico/fisiologia , Proteínas Recombinantes/metabolismo , Retroviridae/metabolismo , Spodoptera/metabolismo , Spodoptera/virologia , Fatores de Transcrição/metabolismo , Animais , Baculoviridae/metabolismo , Células Cultivadas , Produtos do Gene gag/genética , Proteínas Recombinantes/genética , Retroviridae/genética , Spodoptera/genética , Distribuição Tecidual , Fatores de Transcrição/genética , Transfecção/métodosRESUMO
The endogenous retrovirus gypsy is expressed at high levels in mutant flamenco female flies. Gypsy viral particles extracted from such flies can infect naive flamenco individuals raised in the presence of these extracts mixed into their food. This results in the integration of new proviruses into the germline genome. These proviruses can then increase their copy number by (1) expression in the flamenco female somatic cells, (2) transfer into the oocyte and (3) integration into the genome of the progeny. Surprisingly, unlike the infection observed in the feeding experiments, this strategy of endogenous proviral multiplication does not seem to involve the expression of the viral env gene.
Assuntos
Drosophila melanogaster/genética , Retrovirus Endógenos/genética , Genes env , Animais , Animais Geneticamente Modificados , Retrovirus Endógenos/fisiologia , Evolução Molecular , Células Germinativas , Humanos , Provírus/genética , Provírus/fisiologia , Replicação ViralRESUMO
I factors are non-LTR retrotransposons of Drosophila melanogaster that transpose at high frequency in the germline of females resulting from appropriate crosses, allowing in vivo studies of the retrotransposition process. Reverse transcription of a full-length RNA intermediate is thought to occur at the site of integration, using a 3' hydroxyl group generated by endonucleolytic cleavage of the genomic DNA to prime synthesis of the first cDNA strand. This target-primed reverse transcription (TPRT) process is mediated by endonuclease and reverse transcriptase activities encoded by the element. We have designed a molecularly tagged, endonuclease-defective I element that can be mobilised with high efficiency by constructs that express the product of the I factor ORF2 in trans. This indicates that the endonuclease activity required for retrotransposition of the I factor can be provided in trans. Using this system, we show that the endonuclease domain of the R1Bm retrotransposon from Bombyx mori cannot functionally replace that of the I factor.
Assuntos
Drosophila melanogaster/genética , Teste de Complementação Genética , Retroelementos , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Endonucleases/metabolismo , Feminino , Masculino , Análise de Sequência de DNARESUMO
BACKGROUND: The genome of invertebrates is rich in retroelements which are structurally reminiscent of the retroviruses of vertebrates. Those containing three open reading frames (ORFs), including an env-like gene, may well be considered as endogenous retroviruses. Further support to this similarity has been provided by the ability of the env-like gene of DmeGypV (the Gypsy endogenous retrovirus of Drosophila melanogaster) to promote infection of Drosophila cells by a pseudotyped vertebrate retrovirus vector. RESULTS: To gain insights into their evolutionary story, a sample of thirteen insect endogenous retroviruses, which represents the largest sample analysed until now, was studied by computer-assisted comparison of the translated products of their gag, pol and env genes, as well as their LTR structural features. We found that the three phylogenetic trees based respectively on Gag, Pol and Env common motifs are congruent, which suggest a monophyletic origin for these elements. CONCLUSIONS: We showed that most of the insect endogenous retroviruses belong to a major clade group which can be further divided into two main subgroups which also differ by the sequence of their primer binding sites (PBS). We propose to name IERV-K and IERV-S these two major subgroups of Insect Endogenous Retro Viruses (or Insect ERrantiVirus, according to the ICTV nomenclature) which respectively use Lys and Ser tRNAs to prime reverse transcription.
Assuntos
Ceratitis capitata/virologia , Drosophila melanogaster/virologia , Drosophila/virologia , Retrovirus Endógenos/genética , Evolução Molecular , Vírus de Insetos/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Animais , Ceratitis capitata/enzimologia , Bases de Dados Genéticas , Drosophila/enzimologia , Drosophila melanogaster/enzimologia , Retrovirus Endógenos/enzimologia , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene gag/química , Produtos do Gene gag/genética , Produtos do Gene pol/química , Produtos do Gene pol/genética , Vírus de Insetos/enzimologia , Dados de Sequência Molecular , Família Multigênica/genética , Filogenia , DNA Polimerase Dirigida por RNA/genética , Retroelementos/genética , Ribonuclease H/genética , Homologia de Sequência de Aminoácidos , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
The flamenco gene, located at 20A1-3 in the beta-heterochromatin of the Drosophila X chromosome, is a major regulator of the gypsy/mdg4 endogenous retrovirus. As a first step to characterize this gene, approximately 100 kb of genomic DNA flanking a P-element-induced mutation of flamenco was isolated. This DNA is located in a sequencing gap of the Celera Genomics project, i.e., one of those parts of the genome in which the "shotgun" sequence could not be assembled, probably because it contains long stretches of repetitive DNA, especially on the proximal side of the P insertion point. Deficiency mapping indicated that sequences required for the normal flamenco function are located >130 kb proximal to the insertion site. The distal part of the cloned DNA does, nevertheless, contain several unique sequences, including at least four different transcription units. Dip1, the closest one to the P-element insertion point, might be a good candidate for a gypsy regulator, since it putatively encodes a nuclear protein containing two double-stranded RNA-binding domains. However, transgenes containing dip1 genomic DNA were not able to rescue flamenco mutant flies. The possible nature of the missing flamenco sequences is discussed.
Assuntos
Mapeamento Cromossômico , Proteínas de Drosophila , Drosophila melanogaster/genética , Genoma , Animais , Sequência de Bases , Northern Blotting , Núcleo Celular/metabolismo , Clonagem Molecular , Cosmídeos , Cruzamentos Genéticos , DNA/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Ágar , Biblioteca Gênica , Teste de Complementação Genética , Marcadores Genéticos , Heterocromatina/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , RNA/metabolismo , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Retroviridae/genética , Fatores de Transcrição , TransgenesRESUMO
We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos/genética , Análise de Sequência de DNA/métodos , Cromossomo X/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Biologia Computacional , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Feminino , Ordem dos Genes/genética , Masculino , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo/métodos , Fatores de Transcrição/genéticaRESUMO
We have identified two novel, closely related subfamilies of non-long-terminal-repeat (non-LTR) retrotransposons in Drosophila melanogaster, the Waldo-A and Waldo-B subfamilies, that are in the same lineage as site-specific LTR retrotransposons of the R1 clade. Both contain potentially active copies with two large open reading frames, having coding capacities for a nucleoprotein as well as endonuclease and reverse transcriptase activities. Many copies are truncated at the 5' end, and most are surrounded by target site duplications of variable lengths. Elements of both subfamilies have a nonrandom distribution in the genome, often being inserted within or very close to (CA)(n) arrays. At the DNA level, the longest elements of Waldo-A and Waldo-B are 69% identical on their entire length, except for the 5' untranslated regions, which have a mosaic organization, suggesting that one arose from the other following new promoter acquisition. This event occurred before the speciation of the D. melanogaster subgroup of species, since both Waldo-A and Waldo-B coexist in other species of this subgroup.
Assuntos
Drosophila/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Proteínas/genética , Retroelementos/genética , Regiões 5' não Traduzidas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Primers do DNA/química , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
I factors in Drosophila melanogaster are non-LTR retrotransposons that transpose at very high frequencies in the germ line of females resulting from crosses between reactive females (devoid of active I factors) and inducer males (containing active I factors). Constructs containing I factor ORF1 under the control of the hsp70 promoter repress I factor activity. This repressor effect is maternally transmitted and increases with the transgene copy number. It is irrespective of either frame integrity or transcriptional orientation of ORF1, suggesting the involvement of a homology-dependent trans-silencing mechanism. A promoterless transgene displays no repression. The effect of constructs in which ORF1 is controlled by the hsp70 promoter does not depend upon heat-shock treatments. No effect of ORF1 is detected when it is controlled by the I factor promoter. We discuss the relevance of the described regulation to the repression of I factors in I strains.
Assuntos
Drosophila melanogaster/genética , Inativação Gênica , Proteínas/genética , Retroelementos/genética , Animais , Animais Geneticamente Modificados , Cruzamentos Genéticos , Feminino , Fertilidade/genética , Masculino , Regiões Promotoras Genéticas , Recombinação Genética , Sequências Reguladoras de Ácido Nucleico , Transcrição GênicaRESUMO
We conducted a phylogenetic survey of the endogenous retrovirus Gypsy in the eight species of the Drosophila melanogaster subgroup. A 362-bp fragment from the integrase gene (int) was amplified, cloned, and sequenced. Phylogenetic relationships of the elements isolated from independent clones were compared with the host phylogeny. Our results indicate that two main lineages of Gypsy exist in the melanogaster subgroup and that vertical and horizontal transmission have played a crucial role in the evolution of this insect endogenous retrovirus.
Assuntos
Drosophila melanogaster/virologia , Drosophila/virologia , Retrovirus Endógenos/classificação , Retrovirus Endógenos/genética , Evolução Molecular , Filogenia , Sequência de Aminoácidos , Animais , Drosophila/genética , Drosophila melanogaster/genética , Integrases/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Non-long terminal repeat (LTR) retrotransposons or LINEs transpose by reverse transcription of an RNA intermediate and are thought to use the 3' hydroxyl of a chromosomal cleavage to initiate synthesis of the first strand of the cDNA. Many of them terminate in a poly(dA) sequence at the 3' end of the coding strand although some, like the I factor of Drosophila melanogaster, have 3' ends formed by repeats of the trinucleotide TAA. We report results showing that I factor transcripts end a few nucleotides downstream of the TAA repeats and that these extra nucleotides are not integrated into chromosomal DNA during retrotransposition. We also show that the TAA repeats are not required for transposition and that I elements containing mutations affecting the TAA sequences generate transposed copies ending with tandem repeats of various types. Our results suggest that during integration the 3' end of the I factor RNA template can pair with nucleotides at the target site and that tandem duplications are generated by the reverse transcriptase of the I factor in a manner that is reminiscent of the activity of the reverse transcriptases of telomerases. Reverse transcriptases of other non-LTR retrotransposons may function in a similar way.
Assuntos
Drosophila melanogaster/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Mutagênese Insercional/genética , Retroelementos/genética , Sequências de Repetição em Tandem/genética , Animais , Sequência de Bases , Modelos Genéticos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Sequências Repetidas Terminais/genéticaRESUMO
One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.
Assuntos
Drosophila melanogaster/genética , Cromossomo X/genética , Animais , Bandeamento Cromossômico , Biologia Computacional , Cosmídeos , Elementos de DNA Transponíveis , DNA Satélite , Genes de Insetos , Hibridização In Situ , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Cromossomo X/ultraestruturaRESUMO
BACKGROUND: Non-long terminal repeat (non-LTR) retrotransposons are eukaryotic mobile genetic elements that transpose by reverse transcription of an RNA intermediate. We have performed a systematic search for sequences matching the characteristic reverse transcriptase domain of non-LTR retrotransposons in the sequenced regions of the Drosophila melanogaster genome. RESULTS: In addition to previously characterized BS, Doc, F, G, I and Jockey elements, we have identified new non-LTR retrotransposons: Waldo, You and JuanDm. Waldo elements are related to mosquito RTI elements. You to the Drosophila I factor, and JuanDm to mosquito Juan-A and Juan-C. Interestingly, all JuanDm elements are highly homogeneous in sequence, suggesting that they are recent components of the Drosophila genome. CONCLUSIONS: The genome of D. melanogaster contains at least ten families of non-site-specific non-LTR retrotransposons representing three distinct clades. Many of these families contain potentially active members. Fine evolutionary analyses must await the more accurate sequences that are expected in the next future.
Assuntos
Drosophila melanogaster/genética , Genoma , DNA Polimerase Dirigida por RNA/genética , Retroelementos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , DNA/genética , Bases de Dados Factuais , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Gypsy is an infectious endogenous retrovirus of Drosophila melanogaster. The gypsy proviruses replicate very efficiently in the genome of the progeny of females homozygous for permissive alleles of the flamenco gene. This replicative transposition is correlated with derepression of gypsy expression, specifically in the somatic cells of the ovaries of the permissive mothers. The determinism of this amplification was studied further by making chimeric mothers containing different permissive/restrictive and somatic/germinal lineages. We show here that the derepression of active proviruses in the permissive soma is necessary and sufficient to induce proviral insertions in the progeny, even if the F1 flies derive from restrictive germ cells devoid of active proviruses. Therefore, gypsy endogenous multiplication results from the transfer of some gypsy-encoded genetic material from the soma towards the germen of the mother and its subsequent insertion into the chromosomes of the progeny. This transfer, however, is not likely to result from retroviral infection of the germline. Indeed, we also show here that the insertion of a tagged gypsy element, mutant for the env gene, occurs at high frequency, independently of the production of gypsy Env proteins by any transcomplementing helper. The possible role of the env gene for horizontal transfer to new hosts is discussed.
Assuntos
Drosophila melanogaster/genética , Retrovirus Endógenos/genética , Amplificação de Genes , Provírus/genética , Retroelementos , Animais , Linhagem da Célula , Cruzamentos Genéticos , Drosophila melanogaster/virologia , Feminino , Genes de Insetos , Genes env , Óvulo , Fatores Sexuais , Replicação ViralRESUMO
To study the expression of the I factor, a non-long-terminal-repeat retrotransposon responsible for I-R hybrid dysgenesis in Drosophila melanogaster, we have tagged the ORF1 protein (ORF1p) by inserting the HA epitope in its N-terminal region. In transgenic flies, this modification is compatible with a high rate of autonomous transposition and allows direct estimation of the transposition frequency. I factor transposes in the germline of females (SF) that are daughters from crosses between I strain males (which contain active copies of the I factor) and R strain females (which do not). We analyzed the expression pattern of ORF1p by indirect immunofluorescence. Its expression correlates with retrotransposition. During oogenesis ORF1p appears unexpectedly as a cytoplasmic product, which accumulates with a specific pattern into the oocyte. A comparison of the expression patterns under conditions that modify the transposing activity of the element clarifies some aspects of I-factor functioning in the transposition process.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Insetos/genética , Retroelementos/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Sequência de Bases , Drosophila melanogaster/metabolismo , Feminino , Dados de Sequência Molecular , Oócitos/metabolismoRESUMO
The gypsy element of Drosophila melanogaster is the first retrovirus identified in invertebrates. Its transposition is controlled by a host gene called flamenco (flam): restrictive alleles of this gene maintain the retrovirus in a repressed state while permissive alleles allow high levels of transposition. To develop a cell system to study the gypsy element, we established four independent cell lines derived from the Drosophila strain SS, which contains a permissive allele of flamenco, and which is devoid of transposing copies of gypsy. The ultrastructural analysis of three SS cell lines revealed some remarkable characteristics, such as many nuclear virus-like particles, cytoplasmic dense particles, and massive cisternae filled with a fibrous material of unknown origin. Gypsy intragenomic distribution has been compared between the three cell lines and the original SS fly strain, and revealed in two of the cell lines an increase in copy number of a restriction fragment usually present in active gypsy elements. This multiplication seems to have occurred during the passage to the cell culture. Availability of SS cell lines should assist studies of gypsy transposition and infectivity and might be useful to produce high amounts of gypsy viral particles. These new lines already allowed us to show that the Envelope-like products of gypsy can be expressed as membrane proteins.
Assuntos
Técnicas de Cultura de Células/métodos , Linhagem Celular , Drosophila melanogaster/virologia , Genes de Insetos/genética , Retroviridae/genética , Animais , Glicogênio/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Mutação , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/ultraestruturaRESUMO
The I factor is a LINE-like transposable element in Drosophila. Most strains of Drosophila melanogaster, inducer strains, contain 10-15 copies of the I factor per haploid genome located in the euchromatic regions of the chromosome arms. These are not present in a few strains known as reactive strains. I factors transpose at low frequency in inducer strains but at high frequency in the female progeny of crosses between reactive and inducer flies. We have found that the activity of the I factor promoter is sensitive to the number of copies of the first 186 nucleotides of the I factor sequence, which constitutes the 5'-untranslated region. The activity of the I factor decreases as the copy number of this sequence increases.
Assuntos
Elementos de DNA Transponíveis , Drosophila/genética , Animais , Animais Geneticamente Modificados , Cloranfenicol O-Acetiltransferase/genética , Cruzamentos Genéticos , Drosophila melanogaster/genética , Feminino , Fertilidade/genética , Expressão Gênica , Genes de Insetos , Genes Reporter , Genoma , Masculino , Família Multigênica , Ovário/enzimologia , Regiões Promotoras GenéticasRESUMO
Gypsy is an endogenous retrovirus present in the genome of Drosophila melanogaster. This element is mobilized only in the progeny of females which contain active gypsy elements and which are homozygous for permissive alleles of a host gene called flamenco (flam). Some data strongly suggest that gypsy elements bearing a diagnostic HindIII site in the central region of the retrovirus body represent a subfamily that appears to be much more active than elements devoid of this site. We have taken advantage of this structural difference to assess by the Southern blotting technique the genomic distribution of active gypsy elements. In some of the laboratory Drosophila stocks tested, active gypsy elements were found to be restricted to the Y chromosome. Further analyses of 14 strains tested for the permissive vs. restrictive status of their flamenco alleles suggest that the presence of permissive alleles of flam in a stock tends to be associated with the confinement of active gypsy elements to the Y chromosome. This might be the result of the female-specific effect of flamenco on gypsy activity.
Assuntos
Drosophila melanogaster/genética , Genes de Insetos , Retroelementos , Cromossomo Y/genética , Animais , Drosophila melanogaster/virologia , Feminino , Genótipo , Masculino , Mapeamento por Restrição , Retroviridae/genéticaRESUMO
I factors in Drosophila melanogaster are non-LTR retrotransposons similar to mammalian LINEs. They transpose at very high frequencies in the germ line of SF females resulting from crosses between reactive females, devoid of active I factors, and inducer males, containing active I factors. The vermilion marked IviP2 element was designed to allow easy phenotypical screening for retrotransposition events. It is deleted in ORF2 and therefore cannot produce reverse transcriptase. IviP2 can be mobilized at very low frequencies by actively transposing I factors in the germ line of SF females. This paper shows that IviP2 can be mobilized more efficiently in the germ line of strongly reactive females in the absence of active I factors, when it is trans-complemented by the product of ORF2 synthesized from the hsp70 heat-shock promoter. This represents a promising step toward the use of marked I elements to study retrotransposition and as tools for mutagenesis.