Interlineage distribution and characteristics of the structure of two subfamilies of Drosophila melanogaster MDG4 (gypsy) retrotransposon

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]

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.     

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.     

Expression of heat shock proteins in response to high and low temperature extremes in diapausing pharate larvae of the gypsy moth,Lymantria dispar

Diapausing pharate first instars of the gypsy moth, Lymantria dispar, respond to high temperature (37–41°C) by suppressing normal protein synthesis and synthesizing a set of seven heat shock proteins with M_rs of 90,000, 75,000, 73,000, 60,000, 42,000, 29,000, and 22,000 as determined by SDS-PAGE. During recovery at 25°C from heat shock, synthesis of the heat shock proteins gradually decreases over a period of 6 h, while normal protein synthesis is restored. A subset of these same heat shock proteins is also expressed during recovery at 4°C or 25°C from brief exposures to low temperature (-10 to 20°C), and its expression is more intense with increased severity of cold exposure. During recovery at 4°C after 24 h at ?20°C, both 90,000 and 75,000 M_r heat shock proteins are expressed for more than 96 h. While normal protein synthesis is suppressed during heat shock and recovery from heat shock, normal protein synthesis coincides with synthesis of the heat shock proteins during recovery from low temperatures, thus implying that expression of the heat shock proteins is not invariably linked to suppression of normal protein synthesis. Western transfer, using a monoclonal antibody that recognizes the inducible form of the human 70,000 M_r heat shock protein, demonstrates that immunologically related proteins in the gypsy moth are expressed at 4°C and during recovery from cold and heat shock.     

The Distribution in Different Strains and Characteristic Features of Two Subfamilies of Drosophila melanogaster Retrotransposon MDG4 (gypsy)

The distribution of two variants of MDG4 (gypsy) was analyzed in severalDrosophila melanogasterstrains. 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 mutantflamenco 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 ovo^D test.     

Study of male mating behavior in some Drosophila melanogaster strains in experiments with fertilized females

Male courtship ritual is among the main behavioral characteristics of Drosophila. This is a complex, genetically determined process consisting of four general stages: orientation, vibration, licking, and attempts at copulation (or successful copulation). Several genes are known that control some stages of this behavior. Most of them have pleiotropic effects and are involved in other biological processes. Earlier, we have shown that a mutation in locus flamenco (20A1-3), which controls transposition and infectivity of retrotransposon gypsy (MDG4), is involved in the genetic control of behavior. In strains mutant for this locus, the male mating activity is decreased and the structure of courtship ritual is changed. To understand the mechanisms of these changes, it is important to study all behavioral stages in genetically identical strains. For this purpose, the normal allele of gene flamenco from the X chromosome of the wild-type strain (stock) Canton S was introduced into strain SS carrying flamMS. This offers new opportunities in studying the role of gene flamenco in the control of mating behavior in Drosophila.     

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.     

Comparative and functional studies of Drosophila species invasion by the gypsy endogenous retrovirus.

Gypsy is an endogenous retrovirus of Drosophila melanogaster. Phylogenetic studies suggest that occasional horizontal transfer events of gypsy occur between Drosophila species. gypsy possesses infective properties associated with the products of the envelope gene that might be at the origin of these interspecies transfers. We report here the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster, suggesting that potentially infective gypsy copies able to spread between sexually isolated species can occur. The ability of gypsy to invade the genome of a new species is conditioned by its capacity to be expressed in the naive genome. The genetic basis for the regulation of gypsy activity in D. melanogaster is now well known, and it has been assigned to an X-linked gene called flamenco. We established an experimental simulation of the invasion of the D. melanogaster genome by gypsy elements derived from other Drosophila species, which demonstrates that these non- D. melanogaster gypsy elements escape the repression exerted by the D. melanogaster flamenco gene.     

Maintenance of the copy number of retrotransposon MDG3 in the Drosophila melanogaster genome]

The genomes of laboratory stocks and natural population of Drosophila melanogaster contain 8-12 copies of retrotransposon MDG3 detected by in situ hybridization. Construction of genotypes with decreased MDG3 copy number using X-chromosome and chromosome 3 free of MDG3 copies results in appearance of hybrid genomes carrying up to 7-10 copies, instead of 2-4 copies expected. New MDG3 copies are detected in different genome regions, including the 42B hot spot of their location. The chromosomes, where new clusters of MDG3 were observed, carry conserved "parental pattern" of MDG1 arrangement. The data obtained suggest the existence of genomic mechanism for maintenance of retrotransposon copy number on a definite level.     

Comparative analysis of the localization and mobility of retrotransposons in sibling species Drosophila simulans and Drosophila melanogaster]

The distribution of four retrotransposon families (MDG1, MDG3, MDG4 and copia) on polytene chromosomes of different (from 9 to 15) Drosophila simulans strains is studied. The mean number of MDG1 and copia euchromatic hybridization sites (3 sites for each element) is drastically decreased in D. simulans in comparison with D. melanogaster (24 and 18 sites respectively). The mean number of MDG3 sites of hybridization is 5 in D. simulans against 12 in D. melanogaster. As for MDG4 both species have on the average about 2-3 euchromatic sites. The majority of MDG1 and copia and about a half of MDG3 euchromatic copies are localized in restricted number of sites (hot spots) on D. simulans polytene chromosomes. In D. melanogaster these elements are scattered along the chromosomes though there are some hot spots too. It appears that euchromatic copies of MDG1 and copia are considerably less mobile in D. simulans in contrast to D. melanogaster. Some common hot spots of retrotransposon localization in D. simulans and D. melanogaster were earlier described as intercalary heterochromatin regions in D. melanogaster. The level of interstrain variability of MDG4 hybridization sites is comparable in both species. Comparative blot-analysis of adult and larval salivary gland DNA shows that MDG1 and copia are situated mainly in euchromatic regions of D. melanogaster chromosomes. In D. simulans genome they are located mainly in heterochromatic regions underreplicated in salivary gland polytene chromosomes. There are interspecies differences in the distribution of retrotransposons in beta-heterochromatic chromosome regions.     

Evolution of gypsy endogenous retrovirus in the Drosophila obscura species group

The Ty3/gypsy family of retroelements is closely related to retroviruses, and some of their members have an open reading frame resembling the retroviral gene env. Sequences homologous to the gypsy element from Drosophila melanogaster are widely distributed among Drosophila species. In this work, we report a phylogenetic study based mainly on the analysis of the 5' region of the env gene from several species of the obscura group, and also from sequences already reported of D. melanogaster, Drosophila virilis, and Drosophila hydei. Our results indicate that the gypsy elements from species of the obscura group constitute a monophyletic group which has strongly diverged from the prototypic D. melanogaster gypsy element. Phylogenetic relationships between gypsy sequences from the obscura group are consistent with those of their hosts, indicating vertical transmission. However, D. hydei and D. virilis gypsy sequences are closely related to those of the affinis subgroup, which could be indicative of horizontal transmission.     

About the origin of retroviruses and the co-evolution of the gypsy retrovirus with the Drosophila flamenco host gene

The gypsy element of Drosophila melanogaster is the first retrovirus identified so far in invertebrates. According to phylogenetic data, gypsy belongs to the same group as the Ty3 class of LTR-retrotransposons, which suggests that retroviruses evolved from this kind of retroelements before the radiation of vertebrates. There are other invertebrate retroelements that are also likely to be endogenous retroviruses because they share with gypsy some structural and functional retroviral-like characteristics. Gypsy is controlled by a Drosophila gene called flamenco, the restrictive alleles of which maintain the retrovirus in a repressed state. In permissive strains, functional gypsy elements transpose at high frequency and produce infective particles. Defective gypsy proviruses located in pericentromeric heterochromatin of all strains seem to be very old components of the genome of Drosophila melanogaster, which indicates that gypsy invaded this species, or an ancestor, a long time ago. At that time, Drosophila melanogaster presumably contained permissive alleles of the flamenco gene. One can imagine that the species survived to the increase of genetic load caused by the retroviral invasion because restrictive alleles of flamenco were selected. The characterization of a retrovirus in Drosophila, one of the most advanced model organisms for molecular genetics, provides us with an exceptional clue to study how a species can resist a retroviral invasion. This revised version was published online in August 2006 with corrections to the Cover Date.