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Journal of Visualized Experiments : JoVE Aug 2021Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster....
Visualization of heterochromatin aggregates by immunostaining can be challenging. Many mammalian components of chromatin are conserved in Drosophila melanogaster. Therefore, it is an excellent model to study heterochromatin formation and maintenance. Polytenized cells, such as the ones found in salivary glands of third instar D. melanogaster larvae, provide an excellent tool to observe the chromatin amplified nearly a thousand times and have allowed researchers to study changes in the distribution of heterochromatin in the nucleus. Although the observation of heterochromatin components can be carried out directly in polytene chromosome preparations, the localization of some proteins can be altered by the severity of the treatment. Therefore, the direct visualization of heterochromatin in cells complements this type of study. In this protocol, we describe the immunostaining techniques used for this tissue, the use of secondary fluorescent antibodies, and confocal microscopy to observe these heterochromatin aggregates with greater precision and detail.
Topics: Animals; Chromosomes; Drosophila; Drosophila Proteins; Drosophila melanogaster; Heterochromatin; Salivary Glands; Staining and Labeling
PubMed: 34487109
DOI: 10.3791/62408 -
BMC Biology Mar 2020Aedes aegypti is the principal mosquito vector of Zika, dengue, and yellow fever viruses. Two subspecies of Ae. aegypti exhibit phenotypic divergence with regard to...
BACKGROUND
Aedes aegypti is the principal mosquito vector of Zika, dengue, and yellow fever viruses. Two subspecies of Ae. aegypti exhibit phenotypic divergence with regard to habitat, host preference, and vectorial capacity. Chromosomal inversions have been shown to play a major role in adaptation and speciation in dipteran insects and would be of great utility for studies of Ae. aegypti. However, the large and highly repetitive genome of Ae. aegypti makes it difficult to detect inversions with paired-end short-read sequencing data, and polytene chromosome analysis does not provide sufficient resolution to detect chromosome banding patterns indicative of inversions.
RESULTS
To characterize chromosomal diversity in this species, we have carried out deep Illumina sequencing of linked-read (10X Genomics) libraries in order to discover inversion loci as well as SNPs. We analyzed individuals from colonies representing the geographic limits of each subspecies, one contact zone between subspecies, and a closely related sister species. Despite genome-wide SNP divergence and abundant microinversions, we do not find any inversions occurring as fixed differences between subspecies. Many microinversions are found in regions that have introgressed and have captured genes that could impact behavior, such as a cluster of odorant-binding proteins that may play a role in host feeding preference.
CONCLUSIONS
Our study shows that inversions are abundant and widely shared among subspecies of Aedes aegypti and that introgression has occurred in regions of secondary contact. This library of 32 novel chromosomal inversions demonstrates the capacity for linked-read sequencing to identify previously intractable genomic rearrangements and provides a foundation for future population genetics studies in this species.
Topics: Aedes; Animals; Chromosome Inversion; Chromosomes; Genetic Introgression; Genetic Variation; High-Throughput Nucleotide Sequencing; Mosquito Vectors
PubMed: 32164699
DOI: 10.1186/s12915-020-0757-y -
International Journal of Molecular... Apr 2024We developed a procedure for locating genes on polytene chromosomes and described three types of chromosome structures (gray bands, black bands, and interbands), which... (Review)
Review
We developed a procedure for locating genes on 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 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.
Topics: Animals; Genes, Essential; Drosophila; Drosophila melanogaster; Chromatin; Introns
PubMed: 38612878
DOI: 10.3390/ijms25074068 -
International Journal of Molecular... Apr 2021Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In , the Tip60...
Chromatin organization is developmentally regulated by epigenetic changes mediated by histone-modifying enzymes and chromatin remodeling complexes. In , 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 development by a number of subunits originally assigned to the dTip60 complex.
Topics: Animals; Chromatin; Chromatin Assembly and Disassembly; Drosophila Proteins; Drosophila melanogaster; Epigenesis, Genetic; Histone Acetyltransferases; Histones; Polytene Chromosomes; Transcription Factors
PubMed: 33926075
DOI: 10.3390/ijms22094525 -
Parasites & Vectors Nov 2016Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical... (Comparative Study)
Comparative Study
BACKGROUND
Anopheles lesteri and Anopheles sinensis are two major malaria vectors in China and Southeast Asia. They are dramatically different in terms of geographical distribution, host preference, resting habitats, and other traits associated with ecological adaptation and malaria transmission. Both species belong to the Anopheles hyrcanus group, but the extent of genetic differences between them is not well understood. To provide an effective way to differentiate between species and to find useful markers for population genetics studies, we performed a comparative cytogenetic analysis of these two malaria vectors.
RESULTS
Presented here is a standard cytogenetic map for An. lesteri, and a comparative analysis of chromosome structure and gene order between An. lesteri and An. sinensis. Our results demonstrate that much of the gene order on chromosomes X and 2 was reshuffled between the two species. However, the banding pattern and the gene order on chromosome 3 appeared to be conserved. We also found two new polymorphic inversions, 2Lc and 3Rb, in An. lesteri, and we mapped the breakpoints of these two inversions on polytene chromosomes.
CONCLUSIONS
Our results demonstrate the extent of structural divergence of chromosomes between An. lesteri and An. sinensis, and provide a new taxonomic cytogenetic tool to distinguish between these two species. Polymorphic inversions of An. lesteri could serve as markers for studies of the population structure and ecological adaptations of this major malaria vector.
Topics: Animals; Anopheles; Chromosome Banding; Chromosomes, Insect; Cytogenetic Analysis; DNA Probes; Mosquito Vectors; Polymorphism, Genetic; Species Specificity
PubMed: 27887641
DOI: 10.1186/s13071-016-1855-0 -
Doklady. Biochemistry and Biophysics Dec 2023In Drosophila, a large group of actively transcribed genes is located in pericentromeric heterochromatin. It is assumed that heterochromatic proteins recruit...
In Drosophila, a large group of actively transcribed genes is located in pericentromeric heterochromatin. It is assumed that heterochromatic proteins recruit transcription factors to gene promoters. Two proteins, Ouib and Nom, were previously shown to bind to the promoters of the heterochromatic genes nvd and spok. Interestingly, Ouib and Nom are paralogs of the M1BP protein, which binds to the promoters of euchromatic genes. We have shown that, like M1BP, the Quib and Nom proteins bind to CP190, which is involved in the recruitment of transcription complexes to promoters. Unlike heterochromatic proteins, Ouib and Nom do not interact with the major heterochromatic protein HP1a and bind to euchromatic promoters on polytene chromosomes from the larval salivary glands. The results suggest a new mechanism for the recruitment of transcription factors into the heterochromatic compartment of the nucleus.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Heterochromatin; Microtubule-Associated Proteins; Nuclear Proteins; Transcription Factors
PubMed: 38472665
DOI: 10.1134/S1607672924700741 -
Bio-protocol Jul 2020larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and...
larval salivary gland polytene chromosome squashes have been used for decades to analyze genome-wide protein-binding patterns, transcriptional activation processes, and changes in chromatin structure at specific genetic loci. There have been many evolutions of the squashing protocol over the years, with sub-optimal reproducibility and low sample success rate as accepted caveats. However, low sample success rates are an obvious disadvantage when polytene chromosomes are used for more high-throughput approaches, such as genetic or antibody screens, or for experiments requiring high-quality chromosome structure preservation. Here we present an exceptionally reproducible squashing and fluorescence staining protocol, which generates high-quality fluorescence images on well-spread chromosomes. This is followed by our novel, semi-automated MATLAB analysis program used to determine correlations between fluorescence signals of interest at a single site on polytene chromosomes, in a pixel-by-pixel manner. In our case, we have used this approach to assess chromatin changes at genomic sites, ectopically targeted by nuclear pore proteins. The use of our analysis program increases the ability to make unbiased conclusions on changes in chromatin structure, or in protein recruitment to chromatin, regardless of sample variation in immunofluorescence staining. As it is simply based upon differences in fluorescence intensity at a defined location, the provided analysis program is not limited to analysis of polytene chromosome, and could be applied to many different contexts where correlation between fluorescent signals at any particular location is of interest.
PubMed: 33659343
DOI: 10.21769/BioProtoc.3673 -
Cells Jun 2020In salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations and results in the polytenization of...
In salivary gland polytene chromosomes, a substantial portion of heterochromatin is underreplicated. The combination of mutations and results in the polytenization of a substantial fraction of unique and moderately repeated sequences but has almost no effect on satellite DNA replication. The Rap1 interacting factor 1 (Rif) protein is a conserved regulator of replication timing, and in , it affects underreplication in polytene chromosomes. We compared the morphology of pericentromeric regions and labeling patterns of in situ hybridization of heterochromatin-specific DNA probes between wild-type salivary gland polytene chromosomes and the chromosomes of mutants and double mutants. We show that, despite general similarities, heterochromatin zones exist that are polytenized only in the mutants, and that there are zones that are under specific control of . In the mutants, we found additional polytenization of the largest blocks of satellite DNA (in particular, satellite 1.688 of chromosome X and simple satellites in chromosomes X and 4) as well as partial polytenization of chromosome Y. Data on pulsed incorporation of 5-ethynyl-2'-deoxyuridine (EdU) into polytene chromosomes indicated that in the mutants, just as in the wild type, most of the heterochromatin becomes replicated during the late S phase. Nevertheless, a significantly increased number of heterochromatin replicons was noted. These results suggest that regulates the activation probability of heterochromatic origins in the satellite DNA region.
Topics: Animals; Carrier Proteins; Drosophila Proteins; Drosophila melanogaster; Heterochromatin; Mutation; Polytene Chromosomes; Salivary Glands; Telomere-Binding Proteins
PubMed: 32575592
DOI: 10.3390/cells9061501 -
Genetica Feb 2023Polyteny is an effective mechanism for accelerating growth and enhancing gene expression in eukaryotes. The purpose of investigation was to study the genetic variability...
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.
Topics: Animals; Drosophila melanogaster; Chromosomes; Genotype; Hybridization, Genetic; Salivary Glands
PubMed: 36163579
DOI: 10.1007/s10709-022-00168-4 -
Open Biology Nov 2021Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap...
Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of .
Topics: Animals; Chromosome Mapping; Computational Biology; Connexins; Gene Expression Regulation, Developmental; Insect Proteins; Models, Molecular; Nematocera; Polytene Chromosomes; Protein Conformation; Sequence Analysis, DNA; Tissue Distribution
PubMed: 34753320
DOI: 10.1098/rsob.210224