Developmental and Housekeeping Genes: Two Types of Genetic Organization in the Drosophila Genome.

We developed a procedure for locating genes on Drosophila melanogaster polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which differed markedly in morphological and genetic properties. This was reached through the use of our original methods of molecular and genetic analysis, electron microscopy, and bioinformatics data processing. Analysis of the genome-wide distribution of these properties led us to a bioinformatics model of the Drosophila genome organization, in which the genome was divided into two groups of genes. One was constituted by 65, in which the genome was divided into two groups, 62 genes that are expressed in most cell types during life cycle and perform basic cellular functions (the so-called "housekeeping genes"). The other one was made up of 3162 genes that are expressed only at particular stages of development ("developmental genes"). These two groups of genes are so different that we may state that the genome has two types of genetic organization. Different are the timings of their expression, chromatin packaging levels, the composition of activating and deactivating proteins, the sizes of these genes, the lengths of their introns, the organization of the promoter regions of the genes, the locations of origin recognition complexes (ORCs), and DNA replication timings.

North African Endemism: A New Species of Black Fly (Diptera: Simuliidae) from the Djurdjura Mountains of Algeria.

Discoveries of endemic species highlight areas of biogeographic and conservation interest. Endemic species, however, are often morphologically disguised as more common and widespread species. The larval polytene chromosomes revealed a new species of black fly, Prosimulium fungiforme, from the Djurdjura Mountains of northern Algeria, and its female, male, pupa, and larva are described. The species is chromosomally unique; none of its 11 chromosomal rearrangements are shared with other species. Although the new species structurally resembles Prosimulium rufipes (Meigen) with which it previously has been confused, it can be distinguished from all other known species of Prosimulium in the Western Palearctic based on at least one character in each described life stage. Symbiotic organisms included two species of microsporidia, at least one of which is probably undescribed, one unknown protozoan pathogen novel in simuliids, and the trichomycete fungus Harpella melusinae Léger and Duboscq. Associated simuliid species included at least one new species of the genus Helodon. The new species of Prosimulium is tentatively considered endemic to the mountains of northern Algeria but might be expected in the mountains of eastern Morocco and northern Tunisia and perhaps in Sicily. If its endemic status holds, it would be the only nominal species of black fly unique to Algeria.

Different Protein Groups Involved in Transcription Regulation in Development and Housekeeping Genes in Drosophila.

Antibodies to histone modifications and an insulator protein involved in the processes of transcription initiation and elongation are mapped in Drosophila polytene chromosomes. The CHRIZ protein (chromatin insulator) and H3K36me3 histone modification (RNA elongation) are detected only in the localization of housekeeping genes (interbands and gray bands of polytene chromosomes) and never in the regions of developmental genes (black bands and large puffs arising from them). Antibodies to H3S10P histone modification, which is associated with the initial elongation of the RNA strand during transcription, are found exclusively in small puffs, but not in housekeeping gene localization sites or large ecdysone-induced puffs, where housekeeping genes are localized. Antibodies to H4R3me2 histone modification (a co-repressor of the ecdysone receptor) are detected only in large ecdysone-induced puffs.

A hybrid RNA FISH immunofluorescence protocol on Drosophila polytene chromosomes.

OBJECTIVES: Investigating protein-DNA interactions is imperative to understanding fundamental concepts such as cell growth, differentiation, and cell development in many systems. Sequencing techniques such as ChIP-seq can yield genome-wide DNA binding profiles of transcription factors; however this assay can be expensive, time-consuming, may not be informative for repetitive regions of the genome, and depend heavily upon antibody suitability. Combining DNA fluorescence in situ hybridization (FISH) with immunofluorescence (IF) is a quicker and inexpensive approach which has historically been used to investigate protein-DNA interactions in individual nuclei. However, these assays are sometimes incompatible due to the required denaturation step in DNA FISH that can alter protein epitopes, hindering primary antibody binding. Additionally, combining DNA FISH with IF may be challenging for less experienced trainees. Our goal was to develop an alternative technique to investigate protein-DNA interactions by combining RNA FISH with IF. RESULTS: We developed a hybrid RNA FISH-IF protocol for use on Drosophila melanogaster polytene chromosome spreads in order to visualize colocalization of proteins and DNA loci. We demonstrate that this assay is sensitive enough to determine if our protein of interest, Multi sex combs (Mxc), localizes to single-copy target transgenes carrying histone genes. Overall, this study provides an alternative, accessible method for investigating protein-DNA interactions at the single gene level in Drosophila melanogaster polytene chromosomes.

A comparative analysis of the chromosomes of three FARQ species complex members, Ceratitis rosa, C. quilicii, and C. fasciventris F2 (Diptera: Tephritidae).

The Ceratitis FARQ species complex consists of four highly destructive agricultural pests of Africa, namely C. fasciventris, C. anonae, C. rosa, and C. quilicii. The members of the complex are considered very closely related and the species limits among them are rather obscure. Their economic significance and the need for developing biological methods for their control makes species identification within the complex an important issue, which has become clear that can only be addressed by multidisciplinary approaches. Chromosomes, both mitotic and polytene, can provide a useful tool for species characterization and phylogenetic inference among closely related dipteran species. In the current study, we present the mitotic karyotype and the polytene chromosomes of C. rosa and C. quilicii together with in situ hybridization data. We performed a comparative cytogenetic analysis among the above two species and C. fasciventris, the only other cytogenetically studied member of the FARQ complex, by comparing the mitotic complement and the banding pattern of the polytene chromosomes of each species to the others, as well as by studying the polytene chromosomes of hybrids between them. Our analysis revealed no detectable chromosomal rearrangements discriminating the three FARQ members studied, confirming their close phylogenetic relationships.

Variability of polyteny of giant chromosomes in Drosophila melanogaster salivary glands.

Polyteny is an effective mechanism for accelerating growth and enhancing gene expression in eukaryotes. The purpose of investigation was to study the genetic variability of polyteny degree of giant chromosomes in the salivary glands of Drosophila melanogaster Meig. in relation to the differential fitness of different genotypes. 16 strains, lines and hybrids of fruit flies were studied. This study demonstrates the significant influence of hereditary factors on the level of polytenization of giant chromosomes in Drosophila. This is manifested in the differences between strains and lines, the effect of inbreeding, chromosome isogenization, hybridization, adaptively significant selection, sexual differences, and varying degrees of individual variability of a trait in different strains, lines, and hybrids. The genetic component in the variability of the degree of chromosome polyteny in Drosophila salivary glands was 45.3%, the effect of sex was 9.5%. It has been shown that genetic distances during inbreeding, outbreeding or hybridization, which largely determine the selective value of different genotypes, also affect polyteny patterns. Genetic, humoral, and epigenetic aspects of endocycle regulation, which may underlie the variations in the degree of chromosome polyteny, as well as the biological significance of the phenomenon of endopolyploidy, are discussed.

Cytological Approaches to Visualize Intracellular Dynamics of RNA-Binding Proteins at Active Genes in Drosophila.

Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of RNA-binding proteins that modulate multiple aspects of gene activity and RNA processing, including transcription, splicing, localization, translation, and decay of RNA. Interaction of hnRNPs with RNA is a highly dynamic but regulated process. Poly(ADP-ribose) polymerase (PARP)-dependent PARylation of different hnRNPs is a well-known posttranslational modification that affects their interactions with RNA. Here, we described a protocol for in situ localization of RNA-binding proteins (RBPs) on giant polytene chromosomes in Drosophila larval salivary glands, which have been widely used to visualize the dynamic binding profiles of various RBPs and other transcription-related proteins at specific loci on chromosomes. This chapter also includes a stepwise description of RNA:RNA in situ hybridization, in conjunction with immunostaining, using polytene chromosome squashes or intact tissues. We also highlight advanced live cell imaging methods, including FRAP and FLIP, using transgenic lines that express fluorescent-tagged hnRNPs. These cytological approaches can be used to visualize the localization of RNA-binding proteins and their interacting RNAs under different cellular conditions.

Otu and Rif1 Double Mutant Enables Analysis of Satellite DNA in Polytene Chromosomes of Ovarian Germ Cells in Drosophila melanogaster.

Polytene chromosomes in Drosophila serve as a classical model for cytogenetic studies. However, heterochromatic regions of chromosomes are typically under-replicated, hindering their analysis. Mutations in the Rif1 gene lead to additional replication of heterochromatic sequences, including satellite DNA, in salivary gland cells. Here, we investigated the impact of the Rif1 mutation on heterochromatin in polytene chromosomes formed in ovarian germ cells due to the otu gene mutation. By the analysis of otu11; Rif11 double mutants, we found that, in the presence of the Rif1 mutation, ovarian cells undergo additional polytenization of pericentromeric regions. This includes the formation of large chromatin blocks composed of satellite DNA. Thus, the effects of the Rif1 mutation are similar in salivary gland and germ cells. The otu11; Rif11 system opens new possibilities for studying factors associated with heterochromatin during oogenesis.

Drosophila SUMM4 complex couples insulator function and DNA replication control.

Asynchronous replication of chromosome domains during S phase is essential for eukaryotic genome function, but the mechanisms establishing which domains replicate early versus late in different cell types remain incompletely understood. Intercalary heterochromatin domains replicate very late in both diploid chromosomes of dividing cells and in endoreplicating polytene chromosomes where they are also underreplicated. Drosophila SNF2-related factor SUUR imparts locus-specific underreplication of polytene chromosomes. SUUR negatively regulates DNA replication fork progression; however, its mechanism of action remains obscure. Here, we developed a novel method termed MS-Enabled Rapid protein Complex Identification (MERCI) to isolate a stable stoichiometric native complex SUMM4 that comprises SUUR and a chromatin boundary protein Mod(Mdg4)-67.2. Mod(Mdg4) stimulates SUUR ATPase activity and is required for a normal spatiotemporal distribution of SUUR in vivo. SUUR and Mod(Mdg4)-67.2 together mediate the activities of gypsy insulator that prevent certain enhancer-promoter interactions and establish euchromatin-heterochromatin barriers in the genome. Furthermore, SuUR or mod(mdg4) mutations reverse underreplication of intercalary heterochromatin. Thus, SUMM4 can impart late replication of intercalary heterochromatin by attenuating the progression of replication forks through euchromatin/heterochromatin boundaries. Our findings implicate a SNF2 family ATP-dependent motor protein SUUR in the insulator function, reveal that DNA replication can be delayed by a chromatin barrier, and uncover a critical role for architectural proteins in replication control. They suggest a mechanism for the establishment of late replication that does not depend on an asynchronous firing of late replication origins.

Inside cells, molecules of DNA provide the instructions needed to make proteins. Cells carefully maintain and repair their DNA, and typically make a complete copy of the genome before they divide to ensure that after division, each daughter cell has a full set. Within human, fly and other eukaryotic nuclei, DNA is packaged into structures known as chromosomes. Cells follow precisely controlled programs to replicate distinct regions of chromosomes at different times. To start copying a particular region, the cell machinery that replicates DNA binds to a sequence known as the origin of replication. It is thought that as-yet unknown cues from the cell may lead the replication machinery to bind to different origins of replication at different times. In some circumstances, cells make extra copies of their DNA without dividing. For example, many cells in the larvae of fruit flies contain hundreds of extra DNA copies to sustain their increased sizes. However, the entire genome is not copied during this process, so cells end up with more copies of some regions of the genome than others. A protein called SUUR is required for hindering the replication of the 'underrepresented' regions, but it is not clear how it works. To address this question, Andreyeva, Emelyanov et al. developed a new approach based on liquid chromatography and quantitative proteomics to identify the native form of SUUR in fruit flies. This revealed that SUUR exists as a stable complex with a protein called Mod(Mdg4), which is needed to recruit SUUR to the chromosomes. Further experiments suggested that SUUR and Mod(Mdg4) work together to bind to regions of DNA known as gypsy insulator elements, creating a physical barrier that hinders the replication machinery from accessing some parts of the genome. The findings of Andreyeva, Emelyanov et al. provide an alternative explanation for how individual cells may stagger the process of copying their DNA without relying on the replication machinery binding to various replication origins at different times. Rather, late replication timing may be instructed by an insulator-born delay of the progression of replication over particular genomic regions. This mechanism adds to the list of nuclear processes (chromosome partitioning, transcriptional regulation, etc.) that are known to be directed by insulators and associated architectural proteins.

A Spontaneous Inversion of the X Chromosome Heterochromatin Provides a Tool for Studying the Structure and Activity of the Nucleolus in Drosophila melanogaster.

The pericentromeric heterochromatin is largely composed of repetitive sequences, making it difficult to analyze with standard molecular biological methods. At the same time, it carries many functional elements with poorly understood mechanisms of action. The search for new experimental models for the analysis of heterochromatin is an urgent task. In this work, we used the Rif1 mutation, which suppresses the underreplication of all types of repeated sequences, to analyze heterochromatin regions in polytene chromosomes of Drosophila melanogaster. In the Rif1 background, we discovered and described in detail a new inversion, In(1)19EHet, which arose on a chromosome already carrying the In(1)sc8 inversion and transferred a large part of X chromosome heterochromatin, including the nucleolar organizer to a new euchromatic environment. Using nanopore sequencing and FISH, we have identified the eu- and heterochromatin breakpoints of In(1)19EHet. The combination of the new inversion and the Rif1 mutation provides a promising tool for studies of X chromosome heterochromatin structure, nucleolar organization, and the nucleolar dominance phenomenon. In particular, we found that, with the complete polytenization of rDNA repeats, the nucleolus consists of a cloud-like structure corresponding to the classical nucleolus of polytene chromosomes, as well as an unusual intrachromosomal structure containing alternating transcriptionally active and inactive regions.

Chromosomes as Barcodes: Discovery of a New Species of Black Fly (Diptera: Simuliidae) from California, USA.

One of the most popular tools for species discovery and resolution is the DNA barcode, typically based on the cytochrome c oxidase I (COI) gene. However, other non-genic barcodes are available for Diptera. The banding sequence of polytene chromosomes in some dipteran cells, particularly of the larval silk glands, can provide a unique species barcode. We used the sequence of bands to reveal a new species of black fly in the Simulium (Boreosimulium) annulus species group from California, USA. To further characterize the species and provide more integrated taxonomy, we morphologically described all life stages above the egg, formally named the species Simulium ustulatum n. sp., and provided a conventional COI barcode. The COI barcode confirmed the chromosomal and morphological evidence that the species is a new member of the S. annulus group, and enabled identification of the larva and female, which are structurally similar to those of other species. The chromosomal barcode shows that this species has the most rearranged complement, compared with the eight other North American members of its species group, with up to 12 times the number of fixed rearrangements. Up to six chromosomal rearrangements, including autosomal polymorphisms and sex-linked phenomena, are shared with other members of the group. The most unique and conspicuous chromosomal feature of this new species is a large, pale-staining chromocenter from which the six chromosomal arms radiate. The distribution of this univoltine species in lowland rivers of California's Central Valley could make it vulnerable, given climate change and increasing land development.

Wheat Antipodal Cells with Polytene Chromosomes in the Embryo Sac Are Key to Understanding the Formation of Grain in Cereals.

The ultrastructure of antipodal cells of the Triticum aestivum embryo sac was studied at different stages of differentiation and programmed cell death. The importance of cell function in the antipodal complex is evidenced by the fact that it is fully formed before double fertilization, past the stages of proliferation of three initial cells, and several rounds of genome endoreduplication during differentiation. In this study, we showed that the actively synthesizing organelles, the granular reticulum, and Golgi apparatus, alter their structure during differentiation and death. The polymorphism of the shape of the mitochondria and plastids was demonstrated. For the first time, the actin filaments of the cytoskeleton and numerous multivesicular bodies associated with the plasma membrane were detected in the cytoplasm. The transfer of cytoplasm and organelles between antipodal cells and into the coenocyte of the endosperm was confirmed. DNA breaks and the release of cytochrome c at various stages of death were revealed. To understand the function of the antipodal cells, a quantitative PCR analysis of the expression of wheat genes involved in protective, antistress, and metabolic processes was carried out. We found that gene expression in the antipodal cell fraction was increased compared with that in the whole embryo sac. On the basis of the data, we assume that antipodal cells produce both nutrients and numerous antistress factors that ensure the normal development of the endosperm of the grain, which, in turn, further ensures the development of the embryo.

The Organization of Pericentromeric Heterochromatin in Polytene Chromosome 3 of the Drosophilamelanogaster Line with the Rif11; SuURES Su(var)3-906 Mutations Suppressing Underreplication.

Although heterochromatin makes up 40% of the Drosophila melanogaster genome, its organization remains little explored, especially in polytene chromosomes, as it is virtually not represented in them due to underreplication. Two all-new approaches were used in this work: (i) with the use of a newly synthesized Drosophila line that carries three mutations, Rif11, SuURES and Su(var)3-906, suppressing the underreplication of heterochromatic regions, we obtained their fullest representation in polytene chromosomes and described their structure; (ii) 20 DNA fragments with known positions on the physical map as well as molecular genetic features of the genome (gene density, histone marks, heterochromatin proteins, origin recognition complex proteins, replication timing sites and satellite DNAs) were mapped in the newly polytenized heterochromatin using FISH and bioinformatics data. The borders of the heterochromatic regions and variations in their positions on arm 3L have been determined for the first time. The newly polytenized heterochromatic material exhibits two main types of morphology: a banding pattern (locations of genes and short satellites) and reticular chromatin (locations of large blocks of satellite DNA). The locations of the banding and reticular polytene heterochromatin was determined on the physical map.

Chromosomal and Molecular Diversity in the Simulium ornatum Group (Diptera: Simuliidae) in the Western Tian Shan Range of Central Asia.

By any measure, such as abundance, species diversity or geographic range, the Simulium ornatum species group is one of the most successful Palearctic taxa of black flies. To explore potential diversity in this group in the Tian Shan range of Central Asia, we focused on Kyrgyzstan, in which three nominal morphospecies have been recorded. Among our samples, we morphologically identified S. mesasiaticum Rubtsov and a second possible species tentatively identified as S. ferganicum Rubtsov. By analyzing banding patterns of the larval polytene chromosomes, we discovered two fixed inversions, two sex-linked rearrangements, and 19 autosomal rearrangements, including supernumerary B chromosomes. The chromosomal data indicate minimal diversity of only one or two species across the surveyed area of nearly 50,000 km2. Mitochondrial DNA (CO1) sequences fell into three distinct clusters, possibly representing separate species. The chromosomal, molecular, and morphological data indicate that Kyrgyz populations are unique within the S. ornatum group, but the data sets are not entirely congruent. Thus, reconciling data sets and assigning existing names is tentative. Simulium mesasiaticum is linked with undifferentiated sex chromosomes, one of the three CO1 clades, and higher elevations, whereas S. ferganicum is tenuously associated with differentiated sex chromosomes, a separate CO1 clade, and lower elevations. These associations leave one Kyrgyz larva, which is in a third CO1 clade, unlinked to a formal species name. Our analyses also indicate that S. ornatum Meigen sensu stricto, contrary to previous reports, does not occur in Kyrgyzstan and should be deleted from the country's faunal list.

Chromatin Structure and "DNA Sequence View": The Role of Satellite DNA in Ectopic Pairing of the Drosophila X Polytene Chromosome.

Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with different epigenetic properties, protein compositions and transcription activity. This leaves specific DNA sequences that affect chromosome interactions. The Drosophila melanogaster polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain agnts3, a Drosophila model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain Canton-S (CS). Ectopic pairing can be mediated by some specific DNA sequences. In this study, using our Homology Segment Analysis software, we estimated the correlation between FEC and frequency of short matching DNA fragments (FMF) for all sections of the X chromosome of Drosophila CS and agnts3 strains. With fragment lengths of 50 nucleotides (nt), CS showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in agnts3, which may indicate the alternative epigenetic mechanisms affecting FEC in the mutant strain. Most of the fragments that specifically contributed to FMF were related to 1.688 or 372-bp middle repeats. Thus, middle repetitive DNA may serve as an organizer of ectopic pairing.

Mutations of Phosphorylation Sites in MSL1 Protein Do Not Affect Dosage Compensation in Drosophila melanogaster.

Proteins MSL1 and MSL2 form the core of the Drosophila dosage compensation complex, which specifically binds to the X chromosome of males. Phosphorylation of certain amino acid residues was previously shown to regulate MSL1 activity. In the present work, transgenic lines of Drosophila expressing mutant variants of the MSL1 protein were obtained, in which amino acids undergoing phosphorylation were replaced. As a result, it was shown that inactivation of phosphorylation sites does not affect the efficiency of specific binding of the dosage compensation complex to the X chromosome of males and its functional activity.

Structure of antipodal cells nuclei of wheat embryo sac during programmed cell death.

MAIN CONCLUSION: During antipodal cells PCD, polytene chromosomes rearrangement, segregation of nucleoli components and extrusion of nuclear components occur, cytochrome c is released from the mitochondria and DNA breaks appear. We studied in detail the nuclei of cells of the antipodal complex of wheat embryo sac (Triticum aestivum L.) during programmed cell death (PCD). The antipodal complex has been reported to be formed before double fertilisation of the embryo sac. Polyploidisation leads to the formation of giant polytene chromosomes in the nuclei of antipodal cells. These chromosomes are involved in secretory functions and are important for the development of cellular endosperm. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and immunodetection revealed DNA breaks in the nuclei and release of cytochrome c from mitochondria into the cytoplasm of antipodal cells during PCD. We used transmission electron microscopy, immunodetection and histochemistry to analyse the characteristic structural changes in the nuclei of antipodal cells during PCD. These included sequential structural changes in the nuclei containing polytene chromosomes, segregation of some components of the nucleolus into the bodies of polytene chromosomes, extrusion of nucleolar components and parts of chromosomes into the cytoplasm of antipodal cells and then into the endosperm coenocyte. The obtained results expand the understanding of the structural changes of plant cells with giant polytene chromosomes during PCD.

In vivo analysis reveals that ATP-hydrolysis couples remodeling to SWI/SNF release from chromatin.

ATP-dependent chromatin remodelers control the accessibility of genomic DNA through nucleosome mobilization. However, the dynamics of genome exploration by remodelers, and the role of ATP hydrolysis in this process remain unclear. We used live-cell imaging of Drosophila polytene nuclei to monitor Brahma (BRM) remodeler interactions with its chromosomal targets. In parallel, we measured local chromatin condensation and its effect on BRM association. Surprisingly, only a small portion of BRM is bound to chromatin at any given time. BRM binds decondensed chromatin but is excluded from condensed chromatin, limiting its genomic search space. BRM-chromatin interactions are highly dynamic, whereas histone-exchange is limited and much slower. Intriguingly, loss of ATP hydrolysis enhanced chromatin retention and clustering of BRM, which was associated with reduced histone turnover. Thus, ATP hydrolysis couples nucleosome remodeling to remodeler release, driving a continuous transient probing of the genome.

In Vivo Silencing of Genes Coding for dTip60 Chromatin Remodeling Complex Subunits Affects Polytene Chromosome Organization and Proper Development in Drosophila melanogaster.

Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In Drosophila melanogaster, the Tip60 chromatin remodeling complex (dTip60) play roles in chromatin regulation, which are shared by evolutionarily-related complexes identified in animal and plants. Recently, it was found that most subunits previously assigned to the dTip60 complex are shared by two related complexes, DOM-A.C and DOM-B.C, defined by DOM-A and DOM-B isoforms, respectively. In this work, we combined classical genetics, cell biology, and reverse genetics approaches to further investigate the biological roles played during Drosophila melanogaster development by a number of subunits originally assigned to the dTip60 complex.