Heat shock protein 70 from a thermotolerant Diptera species provides higher thermoresistance to Drosophila larvae than correspondent endogenous gene.

Heat shock proteins (Hsp70s) from two Diptera species that drastically differ in their heat shock response and longevity were investigated. Drosophila melanogaster is characterized by the absence of Hsp70 and other hsps under normal conditions and the dramatic induction of hsp synthesis after temperature elevation. The other Diptera species examined belongs to the Stratiomyidae family (Stratiomys singularior) and exhibits high levels of inducible Hsp70 under normal conditions coupled with a thermotolerant phenotype and much longer lifespan. To evaluate the impact of hsp70 genes on thermotolerance and longevity, we made use of a D. melanogaster strain that lacks all hsp70 genes. We introduced single copies of either S. singularior or D. melanogaster hsp70 into this strain and monitored the obtained transgenic flies in terms of thermotolerance and longevity. We developed transgenic strains containing the S. singularior hsp70 gene under control of a D. melanogaster hsp70 promoter. Although these adult flies did synthesize the corresponding mRNA after heat shock, they were not superior to the flies containing a single copy of D. melanogaster hsp70 in thermotolerance and longevity. By contrast, Stratiomyidae Hsp70 provided significantly higher thermotolerance at the larval stage in comparison with endogenous Hsp70.

A Drosophila heat shock response represents an exception rather than a rule amongst Diptera species.

Heat shock protein 70 (Hsp70) is the major player that underlies adaptive response to hyperthermia in all organisms studied to date. We investigated patterns of Hsp70 expression in larvae of dipteran species collected from natural populations of species belonging to four families from different evolutionary lineages of the order Diptera: Stratiomyidae, Tabanidae, Chironomidae and Ceratopogonidae. All investigated species showed a Hsp70 expression pattern that was different from the pattern in Drosophila. In contrast to Drosophila, all of the species in the families studied were characterized by high constitutive levels of Hsp70, which was more stable than that in Drosophila. When Stratiomyidae Hsp70 proteins were expressed in Drosophila cells, they became as short-lived as the endogenous Hsp70. Interestingly, three species of Ceratopogonidae and a cold-water species of Chironomidae exhibited high constitutive levels of Hsp70 mRNA and high basal levels of Hsp70. Furthermore, two species of Tabanidae were characterized by significant constitutive levels of Hsp70 and highly stable Hsp70 mRNA. In most cases, heat-resistant species were characterized by a higher basal level of Hsp70 than more thermosensitive species. These data suggest that different trends were realized during the evolution of the molecular mechanisms underlying the regulation of the responses of Hsp70 genes to temperature fluctuations in the studied families.

The peculiarities of piRNA expression upon heat shock exposure in Drosophila melanogaster.

Different types of stress including heat shock may induce genomic instability, due to the derepression and amplification of mobile elements (MEs). It remains unclear, however, whether piRNA-machinery regulating ME expression functions normally under stressful conditions. The aim of this study was to explore the features of piRNA expression after heat shock (HS) exposure in Drosophila melanogaster. We also evaluated functioning of piRNA-machinery in the absence of major stress protein Hsp70 in this species. We analyzed the deep sequence data of piRNA expression after HS treatment and demonstrated that it modulates the expression of certain double-stranded germinal piRNA-clusters. Notable, we demonstrated significant changes in piRNA levels targeting a group of MEs after HS only in the strain containing normal set of hsp70 genes. Surprisingly, we failed to detect any correlation between the levels of piRNAs and the transcription of complementary MEs in the studied strains. We propose that modulation of certain piRNA-clusters expression upon HS exposure in D. melanogaster occurs due to HS-induced altering of chromatin state at certain chromosome regions.

[Localization of the Dras1 sequence to polytene chromosomes in sibling species of the Drosophila virilis group].

Drasl gene was mapped by in situ hybridization to polytene chromosomes of several sibling species of the Drosophila virilis group and hybrids between them. A 1037 bp fragment of the Drasl gene of the D. virilis genome was used as a probe. The gene sequence is localized to the region of the disk 25 A-B on the chromosome 2 of the polytene chromosome map of D. virilis.

[Notch122, mutation of Notch locus Abruptex type in Drosophila virilis: characteristics of genetic interactions].

A Drosophila virilis Notch122 mutant has been isolated and genetically identified which is similar to Notch Drosophila melanogaster Abruptex type alleles. Some possible peculiarities of genetic interactions of Notch alleles of Abruptex type are discussed.

[Morphologic and molecular manifestations of hybrid dysgenesis in ontogenesis of Drosophila virilis].

Disorders of germ track cell development that take place in crossbreeding of certain lines of Drosophila virilis and lead to hybrid dysgenesis (HD) syndrome have been studied. Polar cells and germ line cells were identified with specific antibodies against Vasa protein. It has been shown that abnormalities of formation of primary gonads takes place already at the 11-12 stage of embryo development and the consequences of germ line cell death lead to disorders of gonad development at the following stages of ontogenesis of dysgenic hybrids. The start point of germ line cell death in embryogenesis as well as initiation of transcription of Penelope retroelement, which is supposed to play a significant role in HD syndrome development in D. virilis, were estimated.

[Structural and functional analysis of the representatives of a new class of retroelements in Drosophila species].

Various copies of mobile element Penelope previously described in Drosophila virilis have been investigated in different strains of D. virilis and transgenic strains of D. melanogaster transformed by P-based constructs containing full-size copy of Penelope. It has been demonstrated that most of Penelope copies in both species carry large terminal inverted repeats (TIR) and contain deletions of various size at their 5'end of ORF. Junctions between TIR and ORF usually carry microhomology of various length enabling to postulate a hypothesis explaining the molecular mechanism underlying the origin of such complex structures. Penelope copies usually carry 34 bp repeat located in direct orientation at both end of ORF and target site duplications of various length.

[Qualitative and quantitative analyses of the transpositions of P element--based genetic construction into the region of Drosophila melanogaster hsp70 genes].

The hsp70 genes is among the main systems underlying the adaptation of organisms to adverse environmental factors. The ever increasing amount of data in literature demonstrates an important adaptive role of mobile genetic elements in microevolution. Drosophila hsp70 genes are potential target for transpositions of various mobile elements in natural populations. We have analyzed the frequency and localization of a P element-based genetic construction, EPgy2, in the region of Drosophila melanogaster hsp70 genes. A hot spot for the transposition was discovered in the promoter regions of genes hsp70Aa and hsp70Ab. No insertions of this construction in the coding or 3'-flanking regions of hsp70 genes have been recorded. It was demonstrated that the region of 161 to 7800 bp adjacent to the original construction is in certain cases also involved in the transposition. No transpositions of any other mobile elements have been observed. The inserts were shown to change the activity of hsp70 genes and the thermotolerance of transgenic strains.

[Determination of the endonuclease activity encoded by retrotransposon]. / Endonukleaznaia aktivnost' retrotransposona Penelope.

Mobile element Penelope is mobilized in the course of hybrid dysgenesis in D. virilis. This element is also responsible for the activation of other unrelated families of TE occurring in the progeny of dysgenic crosses. Penelope elements have extremely variable structure and combine some properties of LINEs and LTR-containing elements. Penelope-like elements (PLEs) have been recently described in various organisms including fish species, rotifers and amoebae. Computer analysis enabled to predict the presence of reverse transcriptase domain in Penelope-encoded polyprotein as well as UvrC type endonuclease at the C-end of the element. It is noteworthy that none of the previously described retroelements was shown to contain such a nuclease. Multiple alignments revealed five conservative catalytic motifs and all conservative residues present in GIY-YIG endonuclease family within Penelope-encoded protein. Herein we have demonstrated that Penelope element isolated from D. virilis encodes functionally active endonuclease exhibiting some sequence-specificity to the sequence previously demonstrated to serve as Penelope genomic insertion site.

[The unusual mobile element Penelope and its behavior in distant Drosophila species]. / Neobychnyi mobil'nyi élement Penelope i ego povedenie v otdalennykh vidakh Drozofily.

The retroelement Penelope isolated from Drosophila virilis has a very unusual structure and codes for reverse transcriptase and an endonuclease belonging to the UvrC type. As shown previously, Penelope is a key element in induction of the hybrid dysgenesis syndrome described in D. virilis, which also involves mobilization of several unrelated mobile element families. Here we report a successful introduction of Penelope into the D. melanogaster genome by P element-mediated transformation. In the new host genome, Penelope is actively transcribed producing major transcript which coincides with that detected in dysgenic hybrids of D. virilis. In situ hybridization on D. melanogaster polytene chromosomes and Southern blotting revealed multiple transpositions of Penelope in the transformed D. melanogaster strains. We determined the structure of six Penelope copies inserted into D. melanogaster chromosomes. Some transformed D. melanogaster strains showed dysgenesis effects similar to those observed in hybrids from D. virilis dysgenic crosses.

[Structure and evolutionary role of the Penelope mobile element in Drosophila species of the virilis group]. / Struktura i évoliutsionnaia rol' mobil'nogo élementa Penelopa u vidov Drosophila gruppy virilis.

The mobile element Penelope is activated and mobilizes several other transposons in dysgenic crosses in Drosophila virilis. Its structure proved to be complex and to vary greatly in all examined species of the virilis group. Phylogenetic analysis of the reverse transcriptase (RT) domain assigned Penelope to a new branch, rather than to any known family, of LTR-lacking retroelements. Amino acid sequence analysis showed that the C-terminal domain of the Penelope polyprotein is an active endonuclease, which is related to intron-encoded endonucleases and to bacterial repair endonuclease UrvC, and may act as an integras. Retroelements coding for a putative endonuclease that differs from typical integrase have thus far not been known. The N-terminal domain of the Penelope polyprotein was shown to contain a protease with significant homology to HIV-1 protease. Phylogenetic analysis divided the Penelope copies from several virilis species into two subfamilies, one including virtually identical full-length copies, and the other comprising highly divergent defective copies. The results suggest both vertical and horizontal transfer of the element. Possibly, Penelope invasion recurred during evolution and contributed to genome rearrangement in the virilis species. Chromosome aberrations detected in D. virilis, which is now being invaded by Penelope, is direct evidence for this assumption.

[Genetic characteristics of the wing mutation Odd(22) and its interaction with alleles of the Delta locus in Drosophila virilis]. / Geneticheskaia kharakteristika krylovoi mutatsii Odd(22) i ee vzaimodeistvii s alleliami lokusa Delta u Drosophila virilis.

The dominant sex-linked semilethal mutation Odd22 was isolated from progeny of a dysgenic cross of Drosophila virilis lines. Flies homozygous, heterozygous, and hemizygous for Odd22 displayed multiple wing defects, including enlargements and gaps on the veins; irregularly thickened, branched, shortened, or completely reduced veins; and cuts on the wing margin. The most remarkable feature of the Odd22 expression was a combination of both an increase and a reduction of the wing vein material simultaneously present in the same wing, which is commonly associated with suppression and hyperfunction, respectively, of genes of the Notch (N) signaling system. Phenotypic analysis revealed the interaction of Odd22 with alleles of the Delta (Dl) locus, which codes for the ligand of the NOTCH receptor. Based on these data, Odd22 was assumed to directly or indirectly affect the activity of the genes involved in Dl-N signaling.

[Genetic structure of mobile elements of the "Penelope" family in closely related Drosophila species]. / Geneticheskaia struktura mobil'nykh élementov semeistva "Penelopa" u blizkikh vidov Drosophila.

Genomic libraries were obtained from species belonging to the "virilis" group of Drosophila. Several copies of Penelope elements were isolated from these libraries by using a D. virilis Penelope clone as a probe. The elements were sequenced, and their structure was determined. The geographical distribution of this family of mobile elements in closely related species of the group was studied in detail. Cytological localization of the elements was also carried out. The high variability observed between different copies of Penelope is probably due to recombination between individual copies. The role of these elements in the evolution of closely related species is discussed.

[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.