Transcription factor DREF regulates expression of the microRNA gene bantam in Drosophila melanogaster.

The bantam gene encodes a vital microRNA and has a complex expression pattern in various tissues at different stages of Drosophila development. This microRNA is involved in the control of normal development of the ocular and wing imaginal discs, the central nervous system, and also in maintaining the undifferentiated state of stem cells in the ovaries of adult females. At the cellular level, bantam stimulates cell proliferation and prevents apoptosis. The bantam gene is a target of several conserved signaling cascades, in particular, Hippo. At the moment, at least ten proteins are known to directly regulate the expression of this gene in different tissues of Drosophila. In this study, we found that the bantam regulatory region contains motifs characteristic of binding sites for DREF, a transcription factor that regulates the expression of Hippo cascade genes. Using transgenic lines containing a full-length bantam lethality-rescuing deletion fragment and a fragment with a disrupted DREF binding site, we show that these motifs are functionally significant because their disruption at the bantam locus reduces expression levels in the larvae and ovaries of homozygous flies, which correlates with reduced vitality and fertility. The effect of DREF binding to the promoter region of the bantam gene on its expression level suggests an additional level of complexity in the regulation of expression of this microRNA. A decrease in the number of eggs laid and a shortening of the reproductive period in females when the DREF binding site in the regulatory region of the bantam gene is disrupted suggests that, through bantam, DREF is also involved in the regulation of Drosophila oogenesis.

CHARACTERISTIC OF THE CHROMATIN TYPE CORRESPONDING TO THIN «GREY¼ BANDS IN POLYTHENE CHROMOSOMES OF DROSOPHILA MELANOGASTER.

Recently, we developed a bioinformatic algorithm dividing drosophila genome into 4 types of chromatin which differ in protein composition. This allows us to propose a model of structural and functional organization of interphase chromosomes which postulates an existence of correlation between the chromatin types and morphological structures of polytene chromosomes. So, constantly and everywhere open chromatin type named «aquamarine¼ is characteristic of interbands, while the combinations of the other three types («lazurite¼, «malachite¼ and «ruby¼) form the bands. In this study, we characterized protein composition, genetic organization and morphological features of 39 «lazurite¼-chromatin regions in polytene chromosomes. We found out that «lazurite¼-chromatin usually form thin «grey¼ bands and more rarely ­ boundary portions of large bands. This type of chromatin contains coding parts and 3R-ends of genes and is enriched with proteins and histone modifications associated with active transcription at the stage of elongation. The expression patterns of these genes differ greatly depending on the type of chromatin in their 5R-regions.

A COMBINED METHOD FOR MAPPING POLYTHENE CHROMOSOMES ON THE EXAMPLE OF THE FOURTH MICROCHROMOSOME OF DROSOPHILA MELANOGASTER.

Genetic activity of interphase chromosomes is associated with their structural organization, but the mechanism of these relations is still unclear. Classic polythene chromosomes of dipteran insects are a convenient model for such investigations. Despite intensive study of polythene chromosomes of Drosophila melanogaster is carried out, an exact conformity of bands and interbands to the molecular map of the genome remains unknown in most cases. For addressing this issue, the genetic map and molecular characteristics of chromatin have been compared with the banding pattern of the fourth chromosome, which is the smallest chromosome in the D. melanogaster genome and is different in many ways from other chromosomes. This is a unique chromatin domain of D. melanogaster, which is characterized by specific proteins, including HP1, POF and EGG. Matching of cytological and physical maps of the fourth chromosome has been carried out by FISH. Genomic coordinates of bands and interbands have been determined. This result makes it possible to investigate the regulation of gene activity of the fourth chromosome in the context of molecular characteristics of cytological structures in which these genes are located.

ECTOPIC TETHERING OF THE CHROMATOR PROTEIN IN UASDBD(GAL4) SYSTEM AS APPROACH FOR STUDYING OF THE INSULATOR PROTEINS IN DROSOPHILA MELANOGASTER POLYTENE CHROMOSOMES.

Chromatin insulator proteins are one of the major components that determine the domain organization of the genome. According to the latest data, they can mark the boundaries of topological domains and prevent the spread of silent chromatin to adjacent areas. One approach to the analysis of the actions of these proteins is to use the ectopic involvement in the UAS>DBD(GAL4). The method allows to evaluate the effect of selected protein in chromatin organization, to establish its association with other insulator proteins and influence on the processes of transcription and replication. and influence the processes of transcription and replication. In this study, we have developed and tested the functionality of the system components in ectopic tethering of the Chromator (Chriz) to the region of intercalary heterochromatin 10A1-2. Preliminary data have been obtained showing that ectopically tethered Chromator to the band 10A1-2 can induce a partial decompactization of the band chromatin. Further use of this experimental model provides the opportunity to investigate the effect of insulator proteins on the chromatin structure.

Targeted mutagenesis of Drosophila RNaseZ gene by homologous recombination.

For the first time we used a homologous recombination method to obtain complete and precise deletion of Drosophila dRNaseZ gene. In the founder line of flies in which the RNaseZ sequence was replaced by attP site, the full-length sequence of the gene was reintegrated, and its functionality was shown. This approach will allow us to generate further gene mutations in different domains of dRNaseZ protein and discover a broad spectrum and uncover functions outside of tRNA processing.

[Chromomeric organization of interphase chromosomes in Drosophila melanogaster].

As a result of treatment of bioinformatic data on the genome localization of structural proteins, histone modifications, DNase-hypersensitive regions, replication origins (taken from modENCODE) and their cytological localization to polytene chromosome structures, it is shown here that two types of interphase chromosomes -polytene chromosomes from salivary glands and from mitotically dividing cells cultures - demonstrate identical pictures of interband/band, i. e. the same localization and length on physical map and the same sets of proteins. In the interbands of both chromosome types we find the proteins that control initiation of transcription (RNA-polymerase II, transcription factors), replication (ORC2) as well as proteins modifying nucleosome structure (WDS, NURF) and proteins of insulators (BEAF). The nucleosome density and H1 histone concentration in the interbands are depleted; localization of DNase-hypersensitive regions corresponds strictly to the interbands. So, we conclude that both polytene and cell line interphase chromosomes are arranged according to general principle and polytene chromosomes represent precise model of interphase chromosomes. The interbands play a critical role in the initiation of transcription and replication. The interbands of interphase chromosomes are the sites of 5' parts of genes, while the 3' gene ends are located in the adjacent bands. The constancy of interbands decondensation results in the conclusion that the "interbands" genes are constantly active, i. e. they contain "house-keeping" genes. The large late replicating bands contain genes that do not have direct contact to the adjoining interbands are usually polygenic and contain tissue-specific genes.

[Chromatin decompaction in the interbands of Drosophila polytene chromosomes does not correlate with high transcription level].

The search for correlation between structural organization of particular chromosome regions and their functions is among key directions of molecular cytogenetics. In this study, we used polytene chromosomes of Drosophila melanogaster as a convenient model for examining transcriptional activity of chromomeres (bands) and interchromomeres (interbands) in eukaryotic interphase chromosomes. Using cloning of the interband DNA sequences and determination of the molecular limits for some interbands, we analyzed the transcriptional activity of these regions and compared the band and interband transcriptional activity in polytene chromosomes. Our results showed the absence of correlation between the decompacted state of the interbands examined and the levels of transcription observed in these regions.

[Identification and molecular genetic characterization of the polytene chromosome interbands in Drosophila melanogaster].

Being inserted into the polytene chromosome interbands, P transposable elements integrated in the genome of Drosophila produce new bands, enabling their use as markers of interband positions on the physical map. Molecular genetic analysis of 13 interbands marked as described showed that in most cases these regions were represented by intergenic spacers and by 5' noncoding regions of the genes. The interband regions consist of unique chromatin type whose decondensation is not obviously associated with transcription. In addition, interbands are enriched with the specific CHRIZ protein. Comparison of chromosomal protein sets and histone modifications in the polytene chromosome interband regions and in the corresponding sequences of the diploid cell chromosomes demonstrated their complete similarity relative these characteristics. In both cell types, interband regions contained open chromatin markers, including RNA polymerase II, ORC, GAF, TRX, and acetylated histones. At the same time, these regions appeared to be depleted of the repressed chromatin proteins, PC, E(Z), H3K9Me3, H3K27Me3, and some others. The similarity between interband chromosomal regions from different cell types is also manifested in the sets of DNAse I hypersensitive sites, which proved to be hot spots for transposon insertions. Our results suggest that band-interband structure is a fundamental principle of the interphase chromosome organization.

[Molecular combing method in the research of DNA replication parameters in isolated organs of Drosophyla melanogaster].

Methods of physical DNA mapping and direct visualization of replication and transcription in specific regions of genome play crucial role in the researches of structural and functional organization of eukaryotic genomes. Since DNA strands in the cells are organized into high-fold structure and present as highly compacted chromosomes, the majority of these methods have lower resolution at chromosomal level. One of the approaches to enhance the resolution and mapping accuracy is the method of molecular combing. The method is based on the process of stretching and alignment of DNA molecules that are covalently attached with one of the ends to the cover glass surface. In this article we describe the major methodological steps of molecular combing and their adaptation for researches of DNA replication parameters in polyploidy and diploid tissues of Drosophyla larvae.

[Molecular combing in studies of the genome organization and DNA replication].

Molecular combing (MC) yields preparations where individual DNA molecules are uniformly stretched and are parallel to each other. Fluorescence in situ hybridization on such preparations allows an exact mapping of DNA sequences, and pulsed inclusion of halogenated deoxyuridine analogs and their detection using fluorochrome-conjugated antibodies makes it possible to visualize replication. The MC technique was adapted for studying DNA replication in isolated Drosophila melanogaster organs, and it was checked whether a mutation of the Suppressor of UnderReplication (SuUR) gene directly affected the replication fork rate.

[Intercalary heterochromatin in the genome of Drosophila].

The modern concept of intercalary heterochromatin as polytene chromosome regions exhibiting a number of specific characteristics is formulated. DNA constituting these regions is replicated late in the S period; therefore, some strands of polytene chromosomes are underrepresented; i.e., they are underreplicated. Late-replicating regions account for about 7% of the genome; genes are located there in clusters of as many as 40. In general, the gene density in the clusters is substantially lower than in the main part of the genome. Late-replicating regions have an inactivating capacity: genes incorporated into these regions as parts of transposons are inactivated with a higher probability. These regions contain a specific protein SUUR affecting the rate of replication completion.

[Functional organization of interbands in Drosophila polytene chromosomes].

The functional organization of particular chromosome regions is tightly associated with their function in eukaryotic cells. Details of this association are among the most topical problems of modem genetics. The paper characterizes the results of recent research of the specifics of the genetic organization and chromatin decondensation in interbands of Drosophila polytene chromosomes. Data on functional heterogeneity of interbands are considered. Experimental findings point to a lack of correlation between the decondensed chromatin state and the observed transcription level in particular interbands. The DNA sequences responsible for the interband formation are principally identifiable via site-specific homologous FRT/FLP recombination between two P transposons contained in chromosomes. The results allow a search for particular protein factors that are involved in the decondensed state of interbands and structural and functional differentiation of polytene chromosomes.

Polytene chromosomes: 70 years of genetic research.

Polytene chromosomes were described in 1881 and since 1934 they have served as an outstanding model for a variety of genetic experiments. Using the polytene chromosomes, numerous biological phenomena were discovered. First the polytene chromosomes served as a model of the interphase chromosomes in general. In polytene chromosomes, condensed (bands), decondensed (interbands), genetically active (puffs), and silent (pericentric and intercalary heterochromatin as well as regions subject to position effect variegation) regions were found and their features were described in detail. Analysis of the general organization of replication and transcription at the cytological level has become possible using polytene chromosomes. In studies of sequential puff formation it was found for the first time that the steroid hormone (ecdysone) exerts its action through gene activation, and that the process of gene activation upon ecdysone proceeds as a cascade. Namely on the polytene chromosomes a new phenomenon of cellular stress response (heat shock) was discovered. Subsequently chromatin boundaries (insulators) were discovered to flank the heat shock puffs. Major progress in solving the problems of dosage compensation and position effect variegation phenomena was mainly related to studies on polytene chromosomes. This review summarizes the current status of studies of polytene chromosomes and of various phenomena described using this successful model.

[Creation of a new construct for cloning DNA and modeling the structure of Drosophila polytene chromosomes]. / Sozdanie novoi konstruktsii dlia klonirovaniia DNK i modelirovaniia struktur politennykh khoromsom drozofily.

Modification of P element-based transformation vector pCaSpeR3New yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5' and 3' regulatory regions of hsp70, pUC19, and two tandem FRTs. Owing to the hsp70 promoter, the pICon insertion site may be localized on polytene chromosomes after heat shock by light or electron microscopy. The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3' end of pICon by the rescue of the P-element target. Functional FRTs allow insertion or deletion of various DNA fragments. The construct is large (22046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation. To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.

[Creation of a convenient vector for transformation of Drosophila with functional FRT-pFRT sites]. / Sozdanie udobnogo vektora transformatsii drozofily s funktsional'nymi saitami FRT-pFRT.

Modification of Drosophila transformation vector pCaSper3 with the P element was used to construct a new vector, pFRT. The vector contains two tandem FRT sites flanked with several unique restriction sites and separated by a polylinker of five restriction sites, and allows easy cloning of DNA fragments between or close to the FRT sites. FRT-mediated excision of DNA sequences cloned between the FRT sites was demonstrated in vivo. The vector was proposed for molecular genetic studies of the position effect variegation, structural and molecular organization of Drosophila polytene chromosomes, etc.

[DNA from Drosophila melanogaster region 61C7/C8 has high evolution rate]. / DNK iz mezhdiska 61C7/C8 Drosophila melanogaster évoliutsioniruet s vysokoi skorost'iu.

The results of a comparative study of cloned DNA fragments of Drosophila simulans, D. mauritiana, D. teissieri, and D. erecta are presented. The fragments were amplified in PCR with primers specified to the region of D. melanogaster interband 61C7/C8. The uniqueness of all cloned fragments in the genomes of these species was confirmed. A comparative analysis of nucleotide sequences revealed that the rate of evolution of DNA from D. melanogaster interband 61C7/C8 is close to the rate of neutral evolution in the genus Drosophila.

[Allele distribution at the HVR-IgH hypervariable locus in unrelated individuals living in the Ural, Siberia, and northern Kazakhstan]. / Analiz raspredeleniia allelei gipervariabel'nogo lokusa HVR-IgH sredi nerodstvennykh predstavitelei naseleniia, prozhivaiushchego na territorii Urala, Sibiri i severnogo Kazakhstana.

Allelic polymorphism at a hypervariable locus mapped to the 5'-region of the gene cluster encoding JH segments of human immunoglobulin heavy chain H (IgH-VNTR) was typed in 462 unrelated individuals living in the Urals, Siberia, and Northern Kazakhstan. Molecular characteristics of the three previously unknown IgH-VNTR alleles is presented. The observed genotype frequencies were concordant with those expected under the Hardy-Weinberg distribution. The data obtained can be used in the work of regional forensic laboratories.

Polytene chromosome interband DNA is organized into nucleosomes.

The molecular basis that underlies the maintenance of polytene chromosome banding pattern remains unclear. To test the possibility that the decondensed state of interbands is provoked by the absence of nucleosomes, we have subjected chromatin from the previously defined 61C7/C8 interband to digestion with micrococcal nuclease. We have demonstrated that interband DNA forms nucleosomes both in salivary glands and in the bulk of larval tissues. This finding strongly suggests that the difference in compaction between DNA in polytene chromosome bands and interbands results from differences that appear at the higher levels of chromatin organization.