Trans-complementation of an endonuclease-defective tagged I element as a tool for the study of retrotransposition in Drosophila melanogaster.

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.

Identification of Waldo-A and Waldo-B, two closely related non-LTR retrotransposons in Drosophila.

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.

New insights on homology-dependent silencing of I factor activity by transgenes containing ORF1 in Drosophila melanogaster.

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.

A search for reverse transcriptase-coding sequences reveals new non-LTR retrotransposons in the genome of Drosophila melanogaster.

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.

High-frequency retrotransposition of a marked I factor in Drosophila melanogaster correlates with a dynamic expression pattern of the ORF1 protein in the cytoplasm of oocytes.

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.

A genetically marked I element in Drosophila melanogaster can be mobilized when ORF2 is provided in trans.

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.

A genetically tagged, defective I element can be complemented by actively transposing I factors in the germline of I-R dysgenic females in Drosophila melanogaster.

Non-LTR retrotransposons, also known as LINEs, transpose by reverse transcription of an RNA intermediate. Their mechanism of transposition is apparently different from that of retrotransposons and similar to that of proviruses of retroviruses. The I factor is responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. Inducer strains contain several functional I factors whereas reactive strains do not. Transposition of I factors can be experimentally induced: they are stable in inducer strains, but transpose at high frequency in the germline of females, known as SF females, produced by crossing reactive females and inducer males. We have constructed an I element, called IviP2, marked with the vermilion gene, the coding sequence of which was interrupted by an intron. Splicing of the intron can only occur in the transcript initiated from the I element promoter. Transposed copies expressing a wild-type vermilion phenotype were recovered in the germline of SF females in which I factors were actively transposing. This indicates that trans-complementation of a defective I element, deficient for the second open reading frame, by functional I factors can occur in the germline of dysgenic females.

A member of the Notch group of interacting loci, deltex encodes a cytoplasmic basic protein.

Prior genetic studies have suggested a functional relationship between the product of the deltex gene and those of three of the so-called "neurogenic" loci, Notch, Delta and mastermind. To gain further insight into this relationship, we have proceeded with a molecular characterization of deltex. We report that deltex encodes a maternally and zygotically expressed transcript that conceptually translates to a basic protein of novel sequence. Immunolocalization of the protein reveals an apparently ubiquitous distribution in embryonic and imaginal tissues. Because our detection methods also reveal a very low level of protein accumulation within the cytoplasm of cells, we have used transgenic flies to confirm this observation by ectopically expressing deltex under the control of a heat shock gene promoter. The resulting overexpression rescues deltex mutant defects but does not produce any obvious phenotypic abnormalities in otherwise wild-type flies. Finally, we examine genetically several Supressor of deltex mutations for evidence of functional integration with deltex and other neurogenic genes. We demonstrate that in addition to suppressing all adult morphological defects of deltex alleles, these suppressors also are capable of suppressing most synergistic effects involving deltex and Notch, Delta and mastermind.

I factors in Drosophila melanogaster: transposition under control.

I factors are responsible for the I-R system of hybrid dysgenesis in Drosophila melanogaster. They belong to the LINE class of mobile elements, which transpose via reverse transcription of a full-length RNA intermediate. I factors are active members of the I element family, which also contains defective I elements that are immobilized within peri-centromeric heterochromatin and represent very old components of the genome. Active I factors have recently invaded natural populations of Drosophila melanogaster, giving rise to inducer strains. Reactive strains, devoid of active I factors, derive from old laboratory stocks established before the invasion. Transposition of I factors is activated at very high frequencies in the germline of hybrid females issued from crosses between females from reactive strains and males from inducer strains. It results in the production of high rates of mutations and chromosomal rearrangements as well as in a particular syndrome of sterility. The frequency of transposition of I factors is dependent on the amount of full-length RNA that is synthesized from an internal promoter. This full-length RNA serves both as an intermediate of transposition and presumably as a messenger for protein synthesis. Regulators of transposition apparently affect transcription initiation from the internal promoter. The data presented here lead to the proposal of a tentative model for transposition.

Identification of a potential RNA intermediate for transposition of the LINE-like element I factor in Drosophila melanogaster.

The I factor, a transposable element related to mammalian LINEs, controls the I-R system of hybrid dysgenesis in Drosophila melanogaster. It transposes at high frequency in the germ-line of the female progeny of crosses between females of the reactive class of strains and males of the inducer class. The structure and DNA sequence of the I factor suggest that it transposes by reverse transcription of an RNA intermediate. Northern blot and S1 mapping experiments show that a full-length RNA of the I factor is synthesized specifically in the conditions of which I factors transpose. This RNA has all characteristics of a transposition intermediate. It is only found in the ovaries of dysgenic females suggesting that I factor activity is restricted to this tissue because of regulation at the level of the initiation of transcription or RNA stability.

A long interspersed repetitive element--the I factor of Drosophila teissieri--is able to transpose in different Drosophila species.

Long interspersed repetitive elements (LINEs) are transposable elements present in many species. In mammals they are difficult to study because most of them are defective and their transposition frequency is low. The I factor of Drosophila melanogaster is a LINE element that is particularly interesting because its transposition occurs at high frequency during I-R hybrid dysgenesis. This phenomenon occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several complete 5.4-kilobase I factors at various sites on the chromosomal arms. Reactive strains are devoid of complete I factors. Many results indicate that active I factors have invaded the D. melanogaster genome recently. To study the evolutionary history of I elements, we have cloned and sequenced a potentially active I factor from Drosophila teissieri. It is flanked by a target-site duplication and terminates at the 3' end by tandem repeats of the sequence TAA. When introduced into the germ line of a reactive strain of D. melanogaster by P element-mediated transformation, it is able to transpose and induces hybrid dysgenesis. This strengthens the hypothesis of a recent reinvasion of the D. melanogaster genome by active I factors giving rise to the inducer strains. They could have originated by horizontal transfer from another species. Such events also could occur for other LINE elements and might explain the spread of new variants in mammalian genomes. Moreover, the results give a further insight into I factor functional organization.

Characterization of 5' truncated transposed copies of the I factor in Drosophila melanogaster.

I factors in Drosophila melanogaster are transposable elements structurally related to Mammalian LINEs. Their transposition is activated at high frequencies during I-R hybrid dysgenesis and is associated with the production of mutations of various sorts. Very few of these mutations have been studied at the molecular level; those reported so far result either from chromosomal rearrangements or from insertions of complete I factors. We have analysed three I-R induced yellow mutations and have found that one of them is due to the insertion of an I element very similar to the complete I factor, whereas the other two are due to insertions of I elements that are truncated at their 5' ends; one of them exhibits an unusual 3' end. We discuss possible mechanisms of production of such modified I elements.

I elements of Drosophila melanogaster generate specific chromosomal rearrangements during transposition.

We report a detailed molecular analysis of three chromosomal rearrangements, which have been produced during I-R hybrid dysgenesis in Drosophila melanogaster. They all disrupt the yellow gene. One of them is a deletion; the other two are inversions, which may be interpreted as the results of recombination events between I elements inserted at their break points. These events appear to occur at the time of transposition and involve integrating rather than resident I elements. They are produced by a mechanism very similar to homologous ectopic recombination.

Structure and genomic organization of I elements involved in I-R hybrid dysgenesis in Drosophila melanogaster.

I-R hybrid dysgenesis in D. melanogaster is controlled by transposable elements known as I factors which terminate at their 3' ends by an A-rich sequence. Inducer strains contain active I factors. Both reactive and inducer stocks possess defective I elements. We have cloned various I elements from both categories of strains. The I elements having recently transposed in inducer strains have a structure closely related to that of active I factors. However we have isolated one such I element that is truncated at its 5' end. The I elements common to reactive and inducer strains are affected by various rearrangements and many point mutations. They do not appear to be simple derivatives of complete I factors.