[Effect of nucleocapsid on multimerization of gypsy structural protein Gag].

The structural protein (Gag) of the gypsy Drosophila retrovirus lacks matrix, but contains capsid and nucleocapsid domains. The Gag forms virus-like particles in a bacterial cell; besides, its capsid alone is able to form aggregates. However, aggregates assembled from the capsid were variable in size and displayed much less organization than particles formed by the whole Gag. The nucleocapsid exerts influence on the organization and structure of particles, and this function is directed by sequence of amino acid residues at its N-terminus (a nucleocapsid proximal part). The particle assembling occurs in the presence of any RNAs or single stranded DNA oligonucleotides.

[The structural protein Gag of the gypsy retrovirus forms virus-like particles in the bacterial cell].

The amino acid sequence of the drosophila retrovirus MDG4 (gypsy) structural protein Gag does not contain a canonical motif known for the majority of vertebrate retroviruses. Moreover, the protein translation can theoretically begin with two separated initiation codons located within its unique open reading frame. We designed constructs for expression of two theoretically possible variants of Gag polypeptide and investigated an ability of the each product to form virus-like particles in the bacterial cell, i.e. in the absence of eukaryotic cell factors. The results obtained showed that the both variants of the gypsy protein Gag form globular particles in the bacterial cell.

[Precise excision of long terminal repeats of the gypsy (mdg4) retrotransposon of Drosophila melanogaster detected in Escherichia coli cells is explained by its integrase function].

An Escherichia coli model system was developed to estimate the capacity of the integrase of the Drosophila melanogaster retrotransposon gypsy (mdg4) for precise excision of the long terminal repeat (LTR) and, hence, the entire gypsy. The gypsy retrotransposon was cloned in the form of a PCR fragment in the pBlueScript II KS+ (pBSLTR) vector, and the region of the second open reading frame (INT ORF2) of this element encoding integrase was cloned under the lacZ promoter in the pUC19 vector and then recloned in pACYC184 compatible with pBSLTR. The LTR was cloned in such a manner that its precise excision from the recombinant plasmid led to the restoration of the nucleotide sequence and the function of the ORF of the lacZ gene contained in the vector; therefore, it was detected by the appearance of blue colonies on a medium containing X-gal upon IPTG induction. Upon IPTG induction of E. coli XL-1 Blue cells obtained by cotransformation with plasmids pACCint and pBSLTR on an X-gal-containing medium, blue clones appeared with a frequency of 1 x 10(-3) to 1 x 10(-4), the frequency of spontaneously appearing blue colonies not exceeding 10(-9) to 10(-8). The presence of blue colonies indicated that that the integrase encoded by the INT ORF2 (pACYC 184) fragment was active. After the expression of the integrase, it recognized and excised the gypsy LTR from pBSLTR, precisely restoring the nucleotide sequence and the function of the lacZ gene, which led to the expression of the beta-galactosidase enzymatic activity. PCR analysis confirmed that the LTR was excised precisely. Thus, the resultant biplasmid model system allowed precise excisions of the gypsy LTR from the target site to be detected. Apparently, the gypsy integrase affected not only the LTR of this mobile element, but also the host genome nucleotide sequences. The system is likely to have detected only some of the events occurring in E. coli cells. Thus, the integrase of gypsy is actually capable of not only transposing this element by inserting DNA copies of the gypsy retrotransposon to chromosomes of Drosophila, but also excising them, gypsy is excised via a precise mechanism, with the original nucleotide sequence of the target site being completely restored. The obtained data demonstrate the existence of alternative ways of the transposition of retrotransposons and, possibly, retroviruses, including gypsy (mdg4).

[Diversity of LTR retrotransposons and their role in genome reorganization].

Current views of retrotransposons possessing long terminal repeats (LTRs) are described. The existing classification and element types isolated by genome organization are considered. Experimental data are summarized to demonstrate that the replicative cycle of a retrotransposon is not restricted to a single cell and that LTR retrotransposons are transferred between somatic cells with a rate comparable with the element transposition rate within the genome of one cell. The major mechanisms mediating the role of LTR retrotransposons in reorganization of the genome are considered with regard to the strategies of their horizontal and vertical transfer.

[Retrotransposon gtwin: structural analysis and distribution in Drosophila melanogaster strains].

A search for noncanonical variants of the gypsy retrotransposon (MDG4) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF1 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. hydei or D. virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.

[Errantiviruses of Drosophila]. / Erantivirusy Drosophila.

The view on Drosophila long terminal repeat (LTR) retrotransposons, which have three reading frames, as endogenous retroviruses or errantiviruses (ERVs, according to the latest ICTV nomenclature) is discussed. Data on the biology of ERVs and the mechanisms of their involvement in genetic instability of Drosophila are considered.

[Retrotransposon MDG4 and its role in genetic instability of a mutator strain of Drosophila melanogaster]. / Retrotranspozon MDG4 i ego rol' v geneticheskoi nestabil'nosti v mutatornoi linii Drosophila melanogaster.

This article summarizes the results of a ten-year study of genetic instability of a mutator strain of Drosophila melanogaster caused by transposition of the gypsy retrotransposon. The results of other authors working with an analogous system are analyzed. Possible mechanisms are suggested for the interaction of gypsy with the cell gene flamenco that participates in transposition control of this mobile element.

[Retrotransposon mdg3 is transferred between somatic cells of unrelated species in coculture]. / Retrotranspozon MDG3 sposoben peremeshchat'sia mezhdu somaticheskimi kletkami nerodstvennykh vidov pri ikh sovmestnom kul'tivirovanii.

Since retrovirus-like particles of gypsy (mdg4) are capable of interspecific transfer, other Drosophila melanogaster gypsy-related retrotransposons were tested for this property. As a donor and a recipient, D. melanogaster and D. virilis cultured cells were used. Recipient cell DNA was analyzed with probes directed to mdg1, mdg3, 17.6, 297, 412, or B104/roo. Transfer was demonstrated for mdg3, which lacks env. The possible mechanism of transfer is discussed.

[Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon]. / Mezhlineinoe raspredelenie i osobennosti struktury dvukh podsemeistv retrotranspozona Drosophila melanogaster MDG4 (gypsy).

The distribution of two variants of MDG4 (gypsy) was analyzed in several Drosophila melanogaster strains. Southern blot hybridization revealed the inactive variant of MDG4 in all strains examined and active MDG4 only in some of them. Most of the strains harboring the active MDG4 variant were recently isolated from natural populations. It is of interest that the active MDG4 prevailed over the inactive one only in strains carrying the mutant flamenco gene. Several lines were analyzed in more detail. The number of MDG4 sites on salivary-gland polytene chromosomes was established via in situ hybridization, and MDG4 was tested for transposition using the ovoD test.

[Mobile genetic element MDG4 (gypsy) in Drosophila melanogaster. Features of structure and regulation of transposition]. / Mobil'nyi geneticheskii élement MDG4 (gypsy) v liniiakh Drosophila melanogaster. Osobennosti struktury i reguliatsiia transpozitsii.

Distribution of two structural functional variants of the MDG4 (gypsy) mobile genetic element was examined in 44 strains of Drosophila melanogaster. The results obtained suggest that less transpositionally active MDG4 variant is more ancient component of the Drosophila genome. Using Southern blotting, five strains characterized by increased copy number of MDG4 with significant prevalence of the active variant over the less active one were selected for further analysis. Genetic analysis of these strains led to the suggestion that some of them carry factors that mobilize MDG4 independently from the cellular flamenco gene known to be responsible for transposition of this element. Other strains probably contained a suppressor of the flam- mutant allele causing active transpositions of the MDG4. Thus, the material for studying poorly examined relationships between the retrovirus and the host cell genome was obtained.

[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.