Conservation of structure and expression of the trithorax gene between Drosophila virilis and Drosophila melanogaster.

The Drosophila melanogaster trithorax gene encodes several large RNAs which are expressed in complex patterns in the embryo. The D. virilis trithorax gene was isolated and sequenced. It produces a similar to D. melanogaster set of transcripts, and it encodes a protein that shows sequence similarity in several domains which are also conserved in human homologue, ALL-1/HRX. Previous experiments have suggested that a distinct expression domain of trithorax in the posterior region of the embryo is required to maintain expression of the BX-C genes (Sedkov et al., 1994, Development 120, 1907-1917). At cellular blastoderm, trithorax RNA expression in D. virilis embryos is also confined to the posterior portion of the presumptive mesoderm. This finding supports the idea that the specific BX-C-related expression domain is an essential feature of the trithorax gene.

The Drosophila trithorax gene encodes a chromosomal protein and directly regulates the region-specific homeotic gene fork head.

The activity of the Drosophila gene trithorax is required to maintain the proper spatial pattern of expression of multiple homeotic genes of the Bithorax and Antennapedia complexes, trithorax encodes two large protein isoforms of > 400 kD. We have detected its products at 16 discrete sites on larval salivary gland polytene chromosomes, 12 of which colocalize with binding sites of several Polycomb group proteins. The intensity of trithorax protein binding is strongly decreased in larvae carrying mutations in another trithorax group gene ash-1, and in the Polycomb group gene pco/E(z). A strong trithorax binding site was found at the cytological location of the fork head gene, a region-specific homeotic gene not located within a homeotic complex. Further analysis showed that trithorax protein binds at ectopic sites carrying fork head sequences in transformed lines. Trithorax binding occurs within an 8.4-kb regulatory region that directs fork head expression in several embryonic tissues including salivary glands. Consistently, expression of endogenous fork head RNA is greatly reduced in trithorax mutant embryos and in larval tissues. These results show that trithorax maintains expression of target genes by interaction with their regulatory regions and that this interaction depends on the presence of at least some of the other trithorax and Polycomb group proteins.

The bithorax complex is regulated by trithorax earlier during Drosophila embryogenesis than is the Antennapedia complex, correlating with a bithorax-like expression pattern of distinct early trithorax transcripts.

The trithorax gene is required throughout development to maintain expression of homeotic genes of the bithorax and Antennapedia complexes. We determined complete structures of maternal and zygotic alternatively spliced trithorax transcripts, and found that two RNA isoforms are expressed in a surprising manner in the early embryo. At syncytial blastoderm their expression is confined to the ventral region fated to become mesoderm. An additional broad domain of trithorax expression arises during cellularization and is quickly resolved into four pair-rule-like stripes in the posterior half of the embryo. This early expression pattern suggested the possibility that trithorax is involved in the very early regulation of homeotic genes expressed only in the posterior region of the embryo. Indeed, transcription of bithorax complex genes in the mesoderm and ectoderm is altered in strong trithorax mutants during germ band elongation, while the anteriorly expressed Antennapedia complex genes are affected only at late stages of embryonic development. In addition, in another mutant allele (trxE3), expression of bithorax complex genes is normal, while expression of Antennapedia complex genes is reduced. These results suggest that proper expression of genes in the two homeotic complexes is maintained by products of different trithorax RNAs at different times of embryogenesis.

Cloning and analysis of the mobile element gypsy from D. virilis.

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.

Evidence for horizontal transmission of the mobile element jockey between distant Drosophila species.

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.

[Interaction of products of su(Hw) and su(f) genes with MDG4 region regulating its transcription suppresses mutations in Drosophila caused by insertion of this gene]. / Vzaimodeistvie produktov genov su(Hw) i su(f) s oblast'iu MDG4, reguliruiushchei ego transkriptsiiu, supressiruet mutatsii u drozofily, vyzvannye vstraivaniem étogo gena.

The mdg4 (gypsy) mobile element of Drosophila contains two closely situated regions binding to 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'PyPuTCTGCATACTPyPy) homologous to the octamer which is the core of many enhancers and upstream promoter elements. The transient expression of deletion mutants has shown that these DNA regions are negative and positive regulators, respectively, of mdg4 transcription. As was demonstrated earlier, mutations induced by the presence of mdg4 at different loci are suppressed, owing to either repression or activation of mdg4 transcription in Drosophila lines 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 cell lines carrying su(f) mutations which activate mdg4 transcription; therefore, the su(f) gene codes for a protein capable of mdg4 repression. Furthermore, binding to a positive regulator is weakened in nuclear extracts isolated from cell lines carrying su(Hw) gene mutations which decrease the level of mdg4 transcription; hence, the su(Hw) gene encodes a protein which activates mdg4 transcription.

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.

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.

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.

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.

[The Drosophila mobile element jockey, being a typical LINE, is transcribed from the internal promoter by RNA polymerase II]. / Mobil'nyi élement drozofily zhokei, iavliaiushchiisia tipichnym LINE, transkribiruetsia s vnutrennego promotora RNK-polimerazoi II.

Two polyadenylated transcripts of the jockey are detected at different stages of Drosophila melanogaster ontogenesis and in the cell culture. They have the same length as complete and deleted copies of jockey and correspond to the DNA strand containing open reading frames coding for polypeptides which are homologous to retroviral RNA-(DNA)-binding proteins and to their reverse transcriptases. The results of the experiments, where transcription was inhibited with alpha-amanitin in vivo, indicate that jockey is transcribed by RNA polymerase II. The analysis of expression of CAT constructions made on the basis of jockey, and the detection of a fixed site for transcription initiation in jockey genomic and transfected copies have shown that jockey transcription is controlled by an internal promoter located not farther than 12 nucleotides from the beginning of the element. Such an inward location of the promoter allows it to be preserved in replication via reverse transcription and accounts for the distribution of jockey and probably other LINEs throughout the genome. This is the case of the first internal promoter described for RNA polymerase II. The comparison of starting sequences of LINEs in Drosophila makes it possible to detect core sequences of such a promoter.

[The Drosophila mobile element jockey is a LINE element and contains coding sequences homologous to retroviral proteins]. / Mobil'nyi élement drozofily--zhokei iavliaetsia LINE-élementom i soderzhit kodiruiushchie posledovatel'nosti, gomologichnye nekotorym belkam retrovirusov.

A detailed investigation of Drosophila melanogaster mobile dispersed repetitive element jockey is performed. Its structural features resemble those of LINE elements. Sequencing of the complete jockey 5020 bp in length revealed two long open reading frames ORF1 and ORF2 overlapping with a frameshift-1. Judging by amino acid homologies, ORF1 encodes a nucleic acid binding protein, characteristic of replication competent retroviruses; the 3' part of ORF2 encodes an RNA-dependent DNA polymerase which has an amino acid sequence, similar to recently published sequences of LINE elements of Drosophila, Trypanosoma and mammals. This fact demonstrates their evolutionary relationship. Sequencing of several deleted copies of jockey revealed the absence of the major part of ORF2, though the rest of the element, including the ends, is highly conservative.

Successive transposition explosions in Drosophila melanogaster and reverse transpositions of mobile dispersed genetic elements.

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.

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.