Biomedical subjects

Y V Ilyin

Publications and source records attributed to Y V Ilyin.

At least 19 recordsLinked to original sources

PubMed2002

Evidence for horizontal transfer of the LTR retrotransposon mdg3, which lacks an env gene.

Horizontal (interspecific) transfer is regarded as a possible strategy for the propagation of transposable elements through evolutionary time. To date, however, conclusive evidence that transposable elements are capable of horizontal transfer from one species to another has been limited to class II or DNA-type elements. We tested the possibility of such transfer for several Drosophila melanogaster LTR retrotransposons of the gypsy group in an experiment in which D. melanogaster and D. virilis somatic cell lines were used as donor and recipient cells, respectively. This approach was chosen in light of the high levels of LTR retrotransposon amplification and expression observed in cultured D. melanogaster cells. In the course of the experiment, parallel analysis for mdg1, mdg3, 17.6, 297, 412 and B104/roo retrotransposons was performed to detect their presence in the genome of recipient cells. Only the mdg3 retrotransposon, which lacks an env gene, was found to be transmitted into recipient cells. This model, based on the use of cultured cells, is a promising system for further investigating the mechanisms of LTR retrotransposon transfer.

Animals↗

PubMed2001

The endogenous Drosophila melanogaster retrovirus gypsy can propagate in Drosophila hydei cells.

The endogenous Drosophila melanogaster retrovirus gypsy (mdg4) forms virus-like particles (VLPs) which are found as extracellular particles in the medium used to culture D. melanogaster cells. The D. hydei somatic cell line DH14, which does not harbour gypsy sequences, was exposed to D. melanogaster VLPs. Subsequent PCR and Southern analysis revealed that gypsy elements had penetrated into the D. hydei cells, suggesting interspecific transmission of the retrovirus. A D. hydei cell line containing gypsy sequences was established and grown in a mixed culture together with the G418-resistant D. hydei cell line DH33, and gypsy was shown to be transmitted from cell to cell. The proportion of cells carrying gypsy increased with time. The rate of gypsy invasion of the lines DH14 and DH33 was 10(-3) and 10(-2) per cell per generation, respectively. The results demonstrate the possibility of interspecific horizontal transfer of gypsy in the form of its VLPs.

Animals↗

PubMed2001

Two variants of the Drosophila melanogaster retrotransposon gypsy (mdg4): structural and functional differences, and distribution in fly stocks.

Two variants of the Drosophila melanogaster retrotransposon gypsy were subjected to detailed structural and functional analysis. A series of hybrid constructs containing various combinations of "active" and "inactive" gypsy copies were tested for their ability to produce new DNA copies in cultured cells by means of reverse transcription. It was shown that the previously demonstrated variations in retrotranspositional activity are associated with either one or both of two amino acid substitutions at the beginning of ORF2. The first substitution is located at the boundary between the putative protease and reverse transcriptase domains and, hence, may influence the processing of the polyprotein. The other substitution may alter reverse transcriptase activity since it is located in the second of the seven conserved domains of the RT gene. To address the question of the evolutionary relationship between the two gypsy variants, their distribution was analyzed in among various fly stocks. Southern analysis revealed that all D. melanogaster strains studied so far contain the "inactive" gypsy variant, while the "active" copies are present only in some strains; most of the latter were established from flies recently isolated from natural populations. Finally, in stocks carrying the flamenco mutation the "active" gypsy variant is much more abundant than the "inactive" form. Possible scenarios for the orgin of the "active" form of gypsy are discussed.

Animals↗

PubMed1994

Precise excision of the retrotransposon gypsy from the forked and cut loci in a genetically unstable D. melanogaster strain.

The genetically unstable Mutator Strain of D. melanogaster is characterised by a high frequency of spontaneous mutations and their reversions. Three forked mutants were obtained independently and several reversions arose spontaneously with frequency of 10(-3)-10(-4). The sites of integration and excision of the gypsy retrotransposon were analysed by Southern blot analysis and sequencing of PCR fragments. In all cases gypsy had inserted at the end of the third exon of the major transcript of the forked gene, causing the duplication of TCCA target sequence. All the reversions resulted from precise excision of the gypsy. A double mutant containing ct6 and f1, caused by gypsy insertions into untranslated regions of the corresponding genes, was constructed. Two spontaneous ct6f+ revertants as well as one ct+f1 revertant were obtained from this line. Sequence analysis of gypsy integration and excision sites revealed that in all cases gypsy excision was also precise. These experiments constitute the first demonstration of precise excision of LTR-containing elements from their host genomes.

Animals↗

PubMed1994

Identification of spliced RNA species of Drosophila melanogaster gypsy retrotransposon. New evidence for retroviral nature of the gypsy element.

We have identified a novel RNA species of Drosophila melanogaster gypsy retrotransposon that is ca. 2 kb in length and corresponds to the third open reading frame (ORF3) of the gypsy element. This RNA is generated by splicing of the primary gypsy transcript, as is the case for retroviral env gene expression. Therefore, the striking resemblance between gypsy and retroviruses has now been extended by this study to the expression strategies of these retroelements. The primary structure of spliced RNA was determined, and its analysis shows that both gypsy subfamilies (6K and 7K) apparently are able to encode functionally active ORF3 translation products.

Animals↗

PubMed1994

The introduction of a transpositionally active copy of retrotransposon GYPSY into the Stable Strain of Drosophila melanogaster causes genetic instability.

A previously described genetic system comprising a Mutator Strain (MS) and the Stable Strain (SS) from which it originated is characterized by genetic instability caused by transpositions of the retrotransposon gypsy. A series of genetic crosses was used to obtain three MS derivatives, each containing one MS chromosome (X, 2 or 3) in the environment of SS chromosomes. All derivatives are characterized by elevated frequencies of spontaneous mutations in both sexes. Mutations appear at the premeiotic stage and are unstable. Transformed derivatives of SS and another stable strain 208 were obtained by microinjection of plasmid DNA containing transpositionally active gypsy inserted into the Casper vector. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives, while the integration of a single copy of gypsy into the genome of 208 does not change the genetic properties of this strain. We propose that genetic instability in the MS system is caused by the combination of two factors: mutation(s) in gene(s) regulating gypsy transposition in SS and its MS derivatives, and the presence of transpositionally active gypsy copies in MS but not SS.

Animals↗

PubMed1993

Two Drosophila retrotransposon gypsy subfamilies differ in ability to produce new DNA copies via reverse transcription in Drosophila cultured cells.

Plasmid DNA constructs containing 5' end truncated retrotransposon gypsy were introduced into Drosophila cultured cells. Appearance of new complete DNA copies with reconstructed via reverse transcription 5'LTR were detected by PCR after transient expression and by Southern blot analysis of genome DNA of stably transformed cells. Two gypsy subfamilies supposed to be different in transpositional activity were analyzed in terms of their ability to produce new DNA copies via reverse transcription in D. hydei cultured cells. It was demonstrated that both gypsy variants undergo retrotransposition but with different efficiency.

Animals↗

PubMed1992

Control of transcription of Drosophila retrotransposons.

Studies of transcriptional control sequences responsible for regulated and basal-level RNA synthesis from promoters of Drosophila melanogaster retrotransposons reveal novel aspects of gene regulation and lead to identification of trans-acting factors that can be involved in RNA polymerase II transcription not only of retrotransposons, but of many other cellular genes. Comparisons between promoters of retrotransposons and some other Drosophila genes demonstrate that there is a greater variety in basal promoter structure than previously thought and that many promoters may contain essential sequences downstream from the RNA start site.

Animals↗

PubMed1991

Leader region of mdg1 Drosophila retrotransposon RNA contains 3'-end processing sites.

Transient expression of the mdg1 deletion mutants revealed sites of 3'-end processing in the leader region of the transcribed RNA. The efficiency of the processing is regulated in different types of cells. The sequences within the mdg1 body and the 3'-LTR are involved in its regulation. We have also shown, that one of the small open reading frames in the mdg1 leader region in principle might be translated.

Animals↗

PubMed1991

Authentic reverse transcriptase is coded by jockey, a mobile Drosophila element related to mammalian LINEs.

The mobile element jockey is similar in structural organization and coding potential to the LINEs of various organisms. It is transcribed at different stages of Drosophila ontogenesis. The Drosophila LINE family includes active transposable elements. Current models for the mechanism of transposition involve reverse transcription of an RNA intermediate and utilization of element-encoded proteins. As demonstrated here, a 2.23 kb DNA fragment from the region of jockey encoding the putative reverse transcriptase was stably introduced into an expression system under inducible control of the Escherichia coli lac regulatory elements. We describe the expression of the 92 kDa protein and identify this polypeptide alone as the authentic jockey reverse transcriptase based on some of its physical and enzymic properties. The jockey polymerase demonstrates RNA and DNA-directed DNA polymerase activities but lacks detectable RNase H, has a temperature optimum at 26 degrees C, requires Mg2+ or Mn2+ as a cofactor and is inactivated by sulphydryl reagent. The enzyme prefers poly(rC) and poly(rA) as template and 'activated' DNA is not effective.

Animals↗

PubMed1991

Properties of promoter regions of mdg1 Drosophila retrotransposon indicate that it belongs to a specific class of promoters.

A sequence 30 bp downstream from the start site of the Drosophila melanogaster retrotransposon mdg1 is shown to be responsible for correct and precise initiation of mdg1 RNA synthesis in combination with the RNA start-site sequence TCAGTT. A sequence-specific DNA binding protein is demonstrated to interact with the +30 sequence, and the efficient binding of this factor is necessary for in vivo transcriptional activity of the plasmid constructs containing mdg1 promoter fragments. The nucleotides -8/+34 of mdg1 represent a minimal promoter which is able to provide correct initiation of transcription by RNA polymerase II at basal levels. A comparison with properties of some other retrotransposable elements and several developmentally regulated cellular genes allows us to conclude that together they form a specific class of RNA polymerase II promoter. This promoter class characteristically lacks upstream sequences necessary for transcription initiation, such as TATA boxes, but requires a specific downstream promoter element within 40 bp downstream of the RNA start site. The level of transcription can, however, be modulated by upstream regulatory elements. The identified sequence-specific downstream initiation factor may be responsible for transcription initiation on promoters of some genes which belong to this class.

Animals↗

PubMed1991

Retrotransposon Gypsy and genetic instability in Drosophila (review).

The laboratory mutator strain (MS) has properties which can be characterized as genetic instability. It exhibits the high level of gypsy autonomous transposition in somatic and germ cells. This paper summarizes all the data concerning this system and gypsy itself that has been obtained in our works during the last years.

Animals↗

PubMed1990

Molecular analysis of the gypsy (mdg4) retrotransposon in two Drosophila melanogaster strains differing by genetic instability.

The structural organization of the retrotransposon gypsy (mdg4) is investigated in two Drosophila melanogaster strains. One of them, the stable w strain (SS), is characterized by a small copy number and stable localization of gypsy. In the other, unstable mutator strain (MS) which is derived from SS, the gypsy copy number and the frequency of its transposition are greatly increased. Genomic gypsy copies cloned from both strains display structural differences allowing them to be divided into two subfamilies. At the nucleotide level, these differences involve single substitutions, deletions and insertions. Southern blot analysis revealed that SS possesses only gypsy elements that belong to one subfamily, while in MS only gypsy copies from the other subfamily were amplified and transposed. The transcriptional activity of gypsy was also studied. Despite the structural differences, plasmid-borne copies of each type of gypsy exhibit equal transcriptional activity in transfected tissue culture cells. Nevertheless, although a high level of gypsy transcription is observed in MS, gypsy poly(A)+RNA is not detected in SS.

Animals↗

PubMed1989

Suppression in Drosophila: su(Hw) and su(f) gene products interact with a region of gypsy (mdg4) regulating its transcriptional activity.

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.

Animals↗

PubMed1989

Mobile genetic elements in Drosophila melanogaster (recent experiments).

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.

Animals↗

PubMed1988

The Drosophila mobile element jockey belongs to LINEs and contains coding sequences homologous to some retroviral proteins.

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.

Amino Acid Sequence↗

PubMed1988

jockey, a mobile Drosophila element similar to mammalian LINEs, is transcribed from the internal promoter by RNA polymerase II.

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.

Animals↗

PubMed1985

The nature of unstable insertion mutations and reversions in the locus cut of Drosophila melanogaster: molecular mechanism of transposition memory.

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.

Journal Article↗