M. V. Kostyuchenko

New insights into the induction of the heat shock proteins in baculovirus infected insect cells.

Eight members of the HSP/HSC70 family were identified in Spodoptera frugiperda Sf9 cells infected with Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by 2D electrophoresis followed by mass spectrometry (MALDI/TOF) and a Mascot search. The family includes five HSP70s induced by AcMNPV-infection and three constitutive cognate HSC70s that remained abundant in infected cells. Confocal microscopy revealed dynamic changes in subcellular localization of HSP/HSC70s in the course of infection. At the early stages (4 to 10 hpi), a fraction of HSPs is localized in distinct speckles in cytoplasm. The speckles contained ubiquitinylated proteins suggesting that they may be aggresomes where proteins targeted by ubiquitin are sequestered or processed for proteolysis. S. frugiperda HSP90 was identified in the 2D gels by Western blotting. Its amount was unchanged during infection. A selective inhibitor of HSP90, 17-AAG, decreased the rate of viral DNA synthesis in infected cells suggesting a supportive role of HSP90 in virus replication.

Zeste can facilitate long-range enhancer-promoter communication and insulator bypass in Drosophila melanogaster.

The looping model of enhancer–promoter interactions predicts that these specific long-range interactions are supported by a certain class of proteins. In particular, the Drosophila transcription factor Zeste was hypothesized to facilitate long-distance associations between enhancers and promoters. We have re-examined the role of Zeste in supporting long-range interactions between an enhancer and a promoter using the white gene as a model system. The results show that Zeste binds to the upstream white promoter region and the enhancer that is responsible for white activation in the eyes. We have confirmed the previous finding that Zeste is not required for the activity of the eye enhancer and the promoter when they are located in close proximity to each other. However, inactivation of Zeste markedly affects the enhancer–promoter communication in transgenes when the eye enhancer and the white promoter are separated by a 3-kb spacer or the yellow gene. Zeste is also required for insulator bypass by the eye enhancer. Taken together, these results show that Zeste can support specific long-range interactions between enhancers and promoters.

Polymorphism of Bovine Prolactin Gene: Microsatellites, PCR-RFLP

In the samples of Russian Ayrshire and Gorbatov Red cattle breeds, distribution of frequencies of prolactin (PRL) gene alleles generated due to the presence of polymorphic RsaI site in exon 3 were studied. In the breeds, the frequencies of the Ballele of the PRLgene (with RsaI(+) site) detected by the PCR–RFLP method were 14.1 and 8.6%, respectively. In Black Pied, Ayrshire and Gorbatov Red cattle breeds, variation of the microsatellite dinucleotide repeat in the regulatory region of the gene PRLwas also studied. Gorbatov Red breed was monomorphic at the microsatellite locus with the only allele 164 bp in length. Two alleles (164 bp and 162 bp) were detected in the other breeds studied. The frequencies of 164-bp allele of the microsatellite locus were 93.7 and 90.0% in Black Pied and Ayrshire breeds, respectively. In Gorbatov Red breed of dairy type with good beef qualities and low milk-fat yield, lower level of heterozygosity for PRLgene was demonstrated compared to Ayrshire and Black Pied breeds that have high milk-fat yield. In three cattle breeds, higher mean estimate of polymorphism information content of PCR–RFLP in exon 3 (PIC = 0.21) was revealed compared with the same estimate (PIC = 0.09) for the microsatellite locus variability in the regulatory region of the PRLgene. Characteristics of allele Bdistribution of thePRLgene in the representatives of the Bovidae family are considered.

EAST Organizes Drosophila Insulator Proteins in the Interchromosomal Nuclear Compartment and Modulates CP190 Binding to Chromatin

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best studied Drosophila insulator proteins, dCTCF (a homolog of the vertebrate insulator protein CTCF) and Su(Hw), are DNA-binding zinc finger proteins. Different isoforms of the BTB-containing protein Mod(mdg4) interact with Su(Hw) and dCTCF. The CP190 protein is a cofactor for the dCTCF and Su(Hw) insulators. CP190 is required for the functional activity of insulator proteins and is involved in the aggregation of the insulator proteins into specific structures named nuclear speckles. Here, we have shown that the nuclear distribution of CP190 is dependent on the level of EAST protein, an essential component of the interchromatin compartment. EAST interacts with CP190 and Mod(mdg4)-67.2 proteins in vitro and in vivo. Over-expression of EAST in S2 cells leads to an extrusion of the CP190 from the insulator bodies containing Su(Hw), Mod(mdg4)-67.2, and dCTCF. In consistent with the role of the insulator bodies in assembly of protein complexes, EAST over-expression led to a striking decrease of the CP190 binding with the dCTCF and Su(Hw) dependent insulators and promoters. These results suggest that EAST is involved in the regulation of CP190 nuclear localization.

EAST affects the activity of Su(Hw) insulators by two different mechanisms in Drosophila melanogaster

Recent data suggest that insulators organize chromatin architecture in the nucleus. The best characterized Drosophila insulator, found in the gypsy retrotransposon, contains 12 binding sites for the Su(Hw) protein. Enhancer blocking, along with Su(Hw), requires BTB/POZ domain proteins, Mod(mdg4)-67.2 and CP190. Inactivation of Mod(mdg4)-67.2 leads to a direct repression of the yellow gene promoter by the gypsy insulator. Here, we have shown that such repression is regulated by the level of the EAST protein, which is an essential component of the interchromatin compartment. Deletion of the EAST C-terminal domain suppresses Su(Hw)-mediated repression. Partial inactivation of EAST by mutations in the east gene suppresses the enhancer-blocking activity of the gypsy insulator. The binding of insulator proteins to chromatin is highly sensitive to the level of EAST expression. These results suggest that EAST, one of the main components of the interchromatin compartment, can regulate the activity of chromatin insulators.

MOD(MDG4)-64.2 protein, isoform of MOD(MDG4) loci, directly interacts with the Tweedle protein family of Drosophila melanogaster.

225 Transcription regulation in higher eukaryotes is ensured by the interaction between intricate protein complexes that are formed on the sequences of genomic regulatory elements. The promoters that define the transcription initiation and its basic level as well as the cisregulatory elements, which either enhance (enhancers) or attenuate (silencers) the basic level of transcription, have been studied sufficiently well. Unlike them, insulators are a relatively new class of regulatory elements. Initially, insulators described as regulatory elements that (1) are located between the enhancer and promoter and can block the interaction between them, without affecting their functionality and (2) exhibit barrier properties, i.e., prevent the inactivating effect of heterochromatin on the trann scription of the gene surrounded by them. It was then found that the role of insulators in the regulation of transcription is not unambiguous [1]. Currently, the mechanism of action of insulators and their role in the genome are not understood completely. There are sevv eral models of functioning of insulators, each of which has its advantages and disadvantages. Unfortunately, none of them is universal because it is not supported by sufficient experimental data. The best studied insulator is the Su(Hw) insulator of D. melanogaster [2, 3]. One of the main compoo nents of the Su(Hw))dependent insulator complex is the Mod(mdg4) protein. It is required for the insulator function and is connected to it through the interaction with the Su(Hw) protein [4, 5]. The mod(mdg4) gene was identified in many various genetic experiments. It was independently identified in the screening of mutations that affect PEV, alter the properties of insuu lator sequences, and influence the development of nervous cells, conjugation of chromosomes during meiosis, and apoptosis [6]. The molecular analysis of the mod(mdg4) locus showed that it encodes a family consisting of at least of 26 protein isoforms. It was shown that, to produce mature transcripts, this locus uses the mechanism of transssplicing of mRNA. The NNterminus of all splice variants contains the BTB/POZ domain and a glutaminee rich region [7, 8]. The BTB/POZ domain provides the interaction between proteins and the formation of homodimers, heterodimers, and oligomers [9, 10]. However, all splice variants differ in their CCterminal domains [4, 5]. The majority of CCtermini contain the conserved protein motif Cys 2 His 2 , which is called FLYWCH [4–6]. Mutations that affecting the mod(mdg4) gene region that encodes the sequence common for all isoforms of the Mod(mdg4) protein …

Mod(mdg4)-58.8, isoform of mod(mdg4) loci, directly interacts with MTACP1A and MTACP1B proteins of Drosophila melanogaster

As a result of experiments in yeast two-hybrid system, coimmunoprecipitation of proteins from D. melanogaster embryo cell lysate, and immunostaining, it was shown for the first time that Mod(mdg4)-58.8 protein (isoform P), a product of mod(mdg4) locus, directly interacts with mtACP1A and mtACP1B proteins. These proteins are involved in de novo biosynthesis of fatty acids in mitochondria and are required for normal gametogenesis in males and females and, possibly, for the trachea development. This result expands the understanding of the role of mod(mdg4) locus products in the regulation of life activity of the eukaryotic cell.

Interactions between BTB domain of CP190 and two adjacent regions in Su(Hw) are required for the insulator complex formation

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited cooperatively to chromatin through interactions with the DNA-binding architectural protein Su(Hw). While Mod(mdg4)-67.2 interacts only with Su(Hw), CP190 interacts with many other architectural proteins. In spite of the fact that CP190 is critical for the activity of Su(Hw) insulators, interaction between these proteins has not been studied yet. Therefore, we have performed a detailed analysis of domains involved in the interaction between the Su(Hw) and CP190. The results show that the BTB domain of CP190 interacts with two adjacent regions at the N-terminus of Su(Hw). Deletion of either region in Su(Hw) only weakly affected recruiting of CP190 to the Su(Hw) sites in the presence of Mod(mdg4)-67.2. Deletion of both regions in Su(Hw) prevents its interaction with CP190. Using mutations in vivo, we found that interactions with Su(Hw) and Mod(mdg4)-67.2 are essential for recruiting of CP190 to the Su(Hw) genomic sites.

Multiple interactions are involved in a highly specific association of the Mod(mdg4)-67.2 isoform with the Su(Hw) sites in Drosophila

The best-studied Drosophila insulator complex consists of two BTB-containing proteins, the Mod(mdg4)-67.2 isoform and CP190, which are recruited to the chromatin through interactions with the DNA-binding Su(Hw) protein. It was shown previously that Mod(mdg4)-67.2 is critical for the enhancer-blocking activity of the Su(Hw) insulators and it differs from more than 30 other Mod(mdg4) isoforms by the C-terminal domain required for a specific interaction with Su(Hw) only. The mechanism of the highly specific association between Mod(mdg4)-67.2 and Su(Hw) is not well understood. Therefore, we have performed a detailed analysis of domains involved in the interaction of Mod(mdg4)-67.2 with Su(Hw) and CP190. We found that the N-terminal region of Su(Hw) interacts with the glutamine-rich domain common to all the Mod(mdg4) isoforms. The unique C-terminal part of Mod(mdg4)-67.2 contains the Su(Hw)-interacting domain and the FLYWCH domain that facilitates a specific association between Mod(mdg4)-67.2 and the CP190/Su(Hw) complex. Finally, interaction between the BTB domain of Mod(mdg4)-67.2 and the M domain of CP190 has been demonstrated. By using transgenic lines expressing different protein variants, we have shown that all the newly identified interactions are to a greater or lesser extent redundant, which increases the reliability in the formation of the protein complexes.

Functional organization of the white gene enhancer in Drosophila melanogaster

89 Enhancers are DNA elements that promote tran scription by recruiting tissue specific transcription factors (TFs), RNA polymerase II (RNAPII), and other cofactors involved in the activation of transcrip tion [1–5]. In recent years, due to the genome wide mapping of histone modifications, binding sites of var ious TFs, and other chromatin features, significant advances in understanding the structure and functions of enhancers were made [6]. However, the experimen tal data obtained in various model systems and various eukaryotic organisms, do not allow a clear conclusion about the complexity of these regulatory elements. One of the key features of eukaryotic enhancers is that the distance between them and specific promoters can be very large and often reaches hundreds of kb [1, 3, 5, 7]. To date, the question as to how the enhancer can interact with its specific target gene, located at a large distance, remains open.