Rapid evolution of a cyclin A inhibitor gene, roughex, in Drosophila.

The recent sequencing of the complete genome of the fruit fly Drosophila melanogaster has yielded about 30% of the predicted genes with no obvious counterparts in other organisms. These rapidly evolving genes remain largely unexplored. Here, we present evidence for a striking variability in an important Drosophila cell cycle regulator encoded by the gene roughex (rux) in closely related fly species. The unusual level of Rux protein variability indicates that there are very low overall constraints on amino acid substitutions. Despite the lack of sequence similarity, certain common features, including the presence of a C-terminal nuclear localization signal and a functionally important N-terminal RXL cyclin-binding motif, exist between Rux and cyclin-dependent kinase inhibitors of the Cip/Kip family. These results indicate that even some genes involved in key regulatory processes in eukaryotes evolve at extremely high rates.

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.

Roughex mediates G(1) arrest through a physical association with cyclin A.

Differentiation in the developing Drosophila eye requires synchronization of cells in the G(1) phase of the cell cycle. The roughex gene product plays a key role in this synchronization by negatively regulating cyclin A protein levels in G(1). We show here that coexpressed Roughex and cyclin A physically interact in vivo. Roughex is a nuclear protein, while cyclin A was previously shown to be exclusively cytoplasmic during interphase in the embryo. In contrast, we demonstrate that in interphase cells in the eye imaginal disk cyclin A is present in both the nucleus and the cytoplasm. In the presence of ectopic Roughex, cyclin A becomes strictly nuclear and is later degraded. Nuclear targeting of both Roughex and cyclin A under these conditions is dependent on a C-terminal nuclear localization signal in Roughex. Disruption of this signal results in cytoplasmic localization of both Roughex and cyclin A, confirming a physical interaction between these molecules. Cyclin A interacts with both Cdc2 and Cdc2c, the Drosophila Cdk2 homolog, and Roughex inhibits the histone H1 kinase activities of both cyclin A-Cdc2 and cyclin A-Cdc2c complexes in whole-cell extracts. Two-hybrid experiments suggested that the inhibition of kinase activity by Roughex results from competition with the cyclin-dependent kinase subunit for binding to cyclin A. These findings suggest that Roughex can influence the intracellular distribution of cyclin A and define Roughex as a distinct and specialized cell cycle inhibitor for cyclin A-dependent kinase activity.

[Trans-mobilization of deletion copies of the mdg3 retrotransposon in cultured cells of Drosophila]. / Trans-mobilizatsiia deletirovannykh kopii retrotranspozona MDG3 v kul'tiviruemykh kletkakh drozofily.

A model system was studied that was associated with the selective amplification of shortened copies of the mdg3 retrotransposon in cultured cells of Drosophila melanogaster. While full-length mdg3 is present in all species phylogenetically closely related to D. melanogaster, the distribution of its deletion copy mdg3del was shown to be restricted only to D. melanogaster strains. mdg3del appeared to be amplified in two Drosophila cell lines of different origin. A "generalized" (statistically averaged) sequence of the shortened copy from a cell line Schneider2 was determined. The structure of this copy was shown to be the same as in the other tested strains of Drosophila. In addition to the major deletion involving the reverse transcriptase domain and a part of the ribonuclease H domain, several other deletions, insertions, and point substitutions were also revealed in shortened copies. The population of shortened copies was shown to result from the amplification of a single mdg3del copy in the genome of Schneider2 cells. The results obtained suggest that defective copies of the retrotransposon can be trans-mobilized in cultured cells and that the mechanisms of retroamplification in cell cultures and in an organism are different.

[Features of the structure of the 7K-copy of Drosophila MDG4 (gypsy) retrotransposon provides evidence that the 7K-subfamily of MDG4 is potentially capable of transposition]. / Osobennosti struktury 7K-kopii retrotranspozona drozofily MDG4 (gypsy)svidetel'stvuiut o potentsial'noi sposobnosti 7K-podsemeistva MDG4 k transpozitsiiam.

The complete nucleotide sequence of the 7K variant of the gypsy retrotransposon of Drosophila melanogaster was determined. This variant belongs to the 7K subfamily of gypsy, which was previously considered inactive. All differences found in the sequenced 7K copy compared to the transpositionally active 6K variants were point mutations. These nucleotide substitutions account for about 1% of the total base pair number of gypsy. Long terminal repeats (LTR) have the highest rate of nucleotide substitutions However, changes in nontranslated regions did not involve the promoter region and other supposed cis-acting elements. Sixteen amino acid substitutions were found in the coding region of gypsy. These substitutions were mainly located on the boarders of potential functional domains and within the third open reading frame (ORF3). Comparative analysis of structures of these two variants of gypsy suggests the potential ability of 7K copies to transpose.

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.

[Expression of the third open reading frame of the drosophila MDG4 retrotransposon similar to the retroviral env-genes, occurring through splicing]. / Ekspressiia tret'ei otkrytoi ramki schityvaniia retrotranspozona drozofily MDG4, podobno retrovirusnym env-genam, osushchestvliaetsiia cherez splaising.

The presence of a third long open reading frame (ORF3) is the common feature of a number of Drosophila retrotransposons, including MDG4 (gypsy). Thus, these elements have a strong structural resemblance to the integrated forms of vertebrate retroviruses. To elucidate the mode of expression of ORF3, transcription analysis of MDG4 for several D. melanogaster strains and Schneider 2 cultured cells was carried out. In all cases the analysis revealed the presence of 2-kb subgenomic ORF3 transcripts in the polyadenylated RNA fractions. It was shown by using the cDNA-PCR technique with different sets of primers that these subgenomic 2-kb RNAs are generated through splicing of full-length MDG4 transcripts. In all cases, only one DNA fragment is amplified for each suitable pair of primers, indicating that there is probable no alternative splicing during the ORF3 expression. The primary structure of spliced RNA was determined, and its analysis shows that both MDG4 subfamilies (6K and 7K) are apparently able to encode functionally active translation products of ORF3. The regulation at the level of splicing is supposed to be one of the most important factors controlling the transposition frequency of MDG4.

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.

[Features of the structural organization of the MDG1 retrotransposon of Drosophila, revealed during its sequencing]. / Osobennosti strukturnoi organizatsii retrotranspozona Drozofily MDG1, vyavlennye pri ego sekvenirovanii.

The two long open reading frames (ORF1 and ORF2) have been identified in the primary structure of the full-length copy of mdg1 Drosophila retrotransposon by the sequence analysis. These two partially overlapping frames code for the necessary information for transposition through the reverse transcription mechanism. There are unusually long leader and terminal regions in mdg1. The leader area contains two small open reading frames. One of them includes the information of a polypeptide with the typical zinc-binding region. Long oligo (dA) sequences separate the small ORFs from each other and from the long ORFs. Considerable homology between the MDG1 and 412 mobile elements has been detected by comparative analysis of their sequences. The common ancestor of these two elements is postulated.