A. I. Kim

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.

Autonomous transposition of gypsy mobile elements and genetic instability in Drosopbila melanogaster

SummaryThe laboratory imitator strain (MS) of Drosophila melanogaster is characterized by an elevated frequency of spontaneous mutation (10−3–10−4). Mutations occur in both sexes at premeiotic stages of germ cell development. The increased mutability is a characteristic feature of MS itself, since it appears in the absence of outcrossing. Most of the mutations arising in this strain are unstable: reversions to wild type, high frequency mutation to new mutant states and replicating instability were observed. We have investigated the localization of the transposable genetic elements mdg1, 412, mdg3, gypsy (mdg4), copia and P in the X chromosomes of the MS and in the mutant lines y, ct, sbt derived from it by in situ hybridization. The P element was not found in any of these strains. The distributions of mdg1, 412, mdg3 and copia were identical in the X chromosomes of the MS and its derivatives. However, the sites of hybridization with gypsy differ in the various lines tested. In the polytene chromosomes of MS animals significant variation in location and number of copies of the gypsy element was demonstrated between different larvae; copy numbers as high as 30–40 were observed. These results suggest autonomous transposition of gypsy in the MS genome while several other mobile elements remain stable.

The introduction of a transpositionally active copy of retrotransposon GYPSY into the Stable Strain of Drosophila melanogaster causes genetic instability

A previously described genetic system comprising a Mutator Strain (MS) and the Stable Strain (SS) from which it originated is characterized by genetic instability caused by transpositions of the retrotransposon gypsy. A series of genetic crosses was used to obtain three MS derivatives, each containing one MS chromosome (X, 2 or 3) in the environment of SS chromosomes. All derivatives are characterized by elevated frequencies of spontaneous mutations in both sexes. Mutations appear at the premeiotic stage and are unstable. Transformed derivatives of SS and another stable strain 208 were obtained by microinjection of plasmid DNA containing transpositionally active gypsy inserted into the Casper vector. In situ hybridization experiments revealed amplification and active transposition of gypsy in SS derivatives, while the integration of a single copy of gypsy into the genome of 208 does not change the genetic properties of this strain. We propose that genetic instability in the MS system is caused by the combination of two factors: mutation(s) in gene(s) regulating gypsy transposition in SS and its MS derivatives, and the presence of transpositionally active gypsy copies in MS but not SS.

Transposition of mobile elements gypsy (mdg4) and hobo in germ-line and somatic cells of a genetically unstable mutator strain of Drosophila melanogaster

SummaryUsing the in situ hybridization technique, we have analysed the distribution of mobile elements in the X chromosomes of male offspring of individual mutator strain (MS) males crossed to attached-X females. The experiments demonstrate varying cytological localization of the mobile elements gypsy (mdg4) and hobo among different individuals. The other mobile elements investigated (mdgl, mdg3, 412, 297, copia, 17.6, Doc, H.M.S. Beagle, Springer, FB) display no changes in insertion sites. Such an experiment is equivalent to analysis of separate gametes of an MS individual. Thus, the ability of gypsy and hobo to transpose in germ-line cells is demonstrated directly. Transpositions occur at premeiotic stages of germ cell development, since they appear in clusters. Analysis of gypsy and hobo transposition events shows that they occur independently. The same experiment demonstrates that gypsy localization varies significantly between different salivary gland cells of an MS individual. Two types of gypsy hybridization sites can be distinguished: “permanent” sites, common to all cells, and “additional” ones varying between neighbouring salivary gland cells. These additional sites indicate gypsy transposition in somatic cells of the MS. Transposition of the hobo element in somatic cells has also been observed.

Molecular analysis of the gypsy (mdg4) retrotransposon in two Drosophila melanogaster strains differing by genetic instability

SummaryThe structural organization of the retrotransposon gypsy (mdg4) is investigated in two Drosophila melanogaster strains. One of them, the stable w strain (SS), is characterized by a small copy number and stable localization of gypsy. In the other, unstable mutator strain (MS) which is derived from SS, the gypsy copy number and the frequency of its transposition are greatly increased. Genomic gypsy copies cloned from both strains display structural differences allowing them to be divided into two subfamilies. At the nucleotide level, these differences involve single substitutions, deletions and insertions. Southern blot analysis revealed that SS possesses only gypsy elements that belong to one subfamily, while in MS only gypsy copies from the other subfamily were amplified and transposed. The transcriptional activity of gypsy was also studied. Despite the structural differences, plasmid-borne copies of each type of gypsy exhibit equal transcriptional activity in transfected tissue culture cells. Nevertheless, although a high level of gypsy transcription is observed in MS, gypsy poly(A)+RNA is not detected in SS.

Divergence of Chemical Function in the Alkaline Phosphatase Superfamily: Structure and Mechanism of the P-C Bond Cleaving Enzyme Phosphonoacetate Hydrolase

Phosphonates constitute a class of natural products that mimic the properties of the more common organophosphate ester metabolite yet are not readily degraded owing to the direct linkage of the phosphorus atom to the carbon atom. Phosphonate hydrolases have evolved to allow bacteria to utilize environmental phosphonates as a source of carbon and phosphorus. The work reported in this paper examines one such enzyme, phosphonoacetate hydrolase. By using a bioinformatic approach, we circumscribed the biological range of phosphonoacetate hydrolase to a select group of bacterial species from different classes of Proteobacteria. In addition, using gene context, we identified a novel 2-aminoethylphosphonate degradation pathway in which phosphonoacetate hydrolase is a participant. The X-ray structure of phosphonoformate-bound phosphonoacetate hydrolase was determined to reveal that this enzyme is most closely related to nucleotide pyrophosphatase/diesterase, a promiscuous two-zinc ion metalloenzyme of the alkaline phosphatase enzyme superfamily. The X-ray structure and metal ion specificity tests showed that phosphonoacetate hydrolase is also a two-zinc ion metalloenzyme. By using site-directed mutagenesis and (32)P-labeling strategies, the catalytic nucleophile was shown to be Thr64. A structure-guided, site-directed mutation-based inquiry of the catalytic contributions of active site residues identified Lys126 and Lys128 as the most likely candidates for stabilization of the aci-carboxylate dianion leaving group. A catalytic mechanism is proposed which combines Lys12/Lys128 leaving group stabilization with zinc ion activation of the Thr64 nucleophile and the substrate phosphoryl group.

Estrogen receptors, antiestrogens, and non-small cell lung cancer.

This review considers data on expression of different types of estrogen receptors (ERα and ERβ) in in vitro cultured cells of non-small cell lung cancer and also in human and animal lung tumors. Estrogens are shown to play an important role in genesis and development of non-small cell lung cancer because the estrogen-stimulated cell proliferation as well as antiestrogen-caused inhibition of proliferation occurred only in the cells expressing different types of estrogen receptors. In general, the situation is similar to that observed in breast cancer, but in the cells of non-small cell lung cancer not ERα are expressed in more than half of cases but ERβ. Just estrogen receptors β play the crucial role in inducing cell proliferation in response to estrogens, and ERβ is a prognostic marker of a favorable course of non-small cell lung cancer. Data on the interactions between ER and EGFR signaling pathways, as well as on the additive antitumor effect of antiestrogens (tamoxifen and fulvestrant) combined with tyrosine kinase inhibitors (gefitinib, erlotinib, and vandetanib) are considered. The review also includes data on the influence of estrogens on genesis and development of lung cancer in humans and animals and the frequency of ERα and ERβ expression in non-small cell lung cancer in tissues from patients of the two sexes. Problems of quantitative determination of α and β estrogen receptors in the tumor cells are also discussed.

Integration specificity of LTR-retrotransposons and retroviruses in the Drosophila melanogaster genome

Integration of DNA copies in a host genome is a necessary stage in the life cycle of retroviruses and LTR-retrotransposons. There is still no clear understanding of integration specificity of retroelements into a target site. The selection of the target DNA is believed to potentially affect a number of factors such as transcriptional status, association with histones and other DNA-binding proteins, and DNA bending. The authors performed a comprehensive computer analysis of the integration specificity of Drosophilamelanogaster LTR-retrotransposons and retroviruses including an analysis of the nucleotide composition of targets, terminal sequences of LTRs, and integrase sequences. A classification of LTR-retrotransposons based on the integration specificity was developed. All the LTR-retrotransposons of the gypsy group with three open frames (errantiviruses) and their derivatives with two open frames demonstrate strict specificity to a target DNA selection. Such specificity correlates with the structural features of the target DNA: bendability, A-philicity, or protein-induced deformability. The remaining LTR-retrotransposons (copia and BEL groups, blastopia and 412 subgroups of the gypsy group) do not show specificity of integration. Chromodomain is present in the integrase structures of blastopia and 412 subgroup LTR-retrotransposons and may facilitate the process of non-specific integration.

Molecular phylogeny and systematics of drosophila retrotransposons and retroviruses

Full classification of Drosophila melanogaster retrotransposons with long terminal repeats (LTR-retrotransposons) has been recomposed, and their evolutional analysis in sequenced genomes of different species of drosophila and other arthropods has been carried out. D. melanogaster LTR-retrotransposons are divided into three groups: gypsy (one, two, or three open reading frames (ORFs)), copia (one ORF), and BEL (one ORF). The gypsy group is divided into three subgroups. Subgroup I is underrepresented by retrotransposons-retroviruses with three ORFs and their derivatives, which have lost the env gene (ORF3). Subgroup II is underrepresented by retrotransposons with two ORFs, and subgroup III is underrepresented by retrotransposons with one ORF. A comparative analysis of homologs of gypsy group LTR-retrotransposons evidences that subgroups I and II are represented only in the genomes of Lepidoptera and Diptera. The gypsy group of LTR-retrotransposons with one and two ORFs is found in almost all genomes of arthropods. Most of the families of D. melanogaster gypsy group LTR-retrotransposons have close homologs in the genomes of other species of drosophila. A degree of identity of retrotransposons sequences is correlated with a degree of relation between species of drosophila, indicating vertical transmission of retrotransposons. Obvious cases of horizontal transfer of some mobile elements have been detected including retrotransposons without the env gene. Homologs of distinct ORFs of retrotransposons—genes gag and env—have been found. Gene-homolog of the gag gene—Grp (CG5680)—is under purifying selection, so it has an important function in drosophila genome.

Domesticated retroviral GAG gene in Drosophila: New functions for an old gene

The domestication of foreign genes is a powerful mechanism for new gene formation and genome evolution. It is known that domesticated retroviral gag genes in mammals not only take part in protecting against viral infection but also control cell division, apoptosis, function of the placenta, and other biological processes. In this study, we focused on the domesticated retroviral gag gene homolog (Grp) in the Drosophila melanogaster genome. According to the results of a bioinformatic analysis, the Grp gene product is primarily under purifying selection in Drosophilidae family. The Grp protein has been shown to be transmembrane. The Grp gene is expressed at the adult stage of D. melanogaster in gender-specific and tissue-specific manner. Also the Grp gene expression is increased in response to the gypsy retrovirus. A function of the protein as a component of the endosomic membrane is considered.