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Genes Apr 2020The polytene chromosomes are the best model for studying the genome organization during interphase. Despite of the long-term studies available on genetic organization...
The polytene chromosomes are the best model for studying the genome organization during interphase. Despite of the long-term studies available on genetic organization of polytene chromosome bands and interbands, little is known regarding long gene location on chromosomes. To analyze it, we used bioinformatic approaches and characterized genome-wide distribution of introns in gene bodies and in different chromatin states, and using fluorescent in situ hybridization we juxtaposed them with the chromosome structures. Short introns up to 2 kb in length are located in the bodies of housekeeping genes (grey bands or chromatin). In the group of 70 longest genes in the genome, 95% of total gene length accrues to introns. The mapping of the 15 long genes showed that they could occupy extended sections of polytene chromosomes containing band and interband series, with promoters located in the interband fragments ( chromatin). Introns ( and chromatin) in polytene chromosomes form independent bands, which can contain either both introns and exons or intron material only. Thus, a novel type of the gene arrangement in polytene chromosomes was discovered; peculiarities of such genetic organization are discussed.
Topics: Animals; Chromatin; Drosophila Proteins; Drosophila melanogaster; Genome; Introns; Polytene Chromosomes
PubMed: 32290448
DOI: 10.3390/genes11040417 -
PloS One 2014Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years....
Drosophila melanogaster polytene chromosomes display specific banding pattern; the underlying genetic organization of this pattern has remained elusive for many years. In the present paper, we analyze 32 cytology-mapped polytene chromosome interbands. We estimated molecular locations of these interbands, described their molecular and genetic organization and demonstrate that polytene chromosome interbands contain the 5' ends of housekeeping genes. As a rule, interbands display preferential "head-to-head" orientation of genes. They are enriched for "broad" class promoters characteristic of housekeeping genes and associate with open chromatin proteins and Origin Recognition Complex (ORC) components. In two regions, 10A and 100B, coding sequences of genes whose 5'-ends reside in interbands map to constantly loosely compacted, early-replicating, so-called "grey" bands. Comparison of expression patterns of genes mapping to late-replicating dense bands vs genes whose promoter regions map to interbands shows that the former are generally tissue-specific, whereas the latter are represented by ubiquitously active genes. Analysis of RNA-seq data (modENCODE-FlyBase) indicates that transcripts from interband-mapping genes are present in most tissues and cell lines studied, across most developmental stages and upon various treatment conditions. We developed a special algorithm to computationally process protein localization data generated by the modENCODE project and show that Drosophila genome has about 5700 sites that demonstrate all the features shared by the interbands cytologically mapped to date.
Topics: Animals; Chromatin; Chromosome Banding; Chromosomes, Insect; DNA Transposable Elements; DNA-Binding Proteins; Drosophila melanogaster; Genome-Wide Association Study; Genomics; Histones; Interphase; Physical Chromosome Mapping; Polytene Chromosomes
PubMed: 25072930
DOI: 10.1371/journal.pone.0101631 -
PloS One 2017Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown...
Spatial organization of chromosome territories is important for maintenance of genomic stability and regulation of gene expression. Recent studies have shown tissue-specific features of chromosome attachments to the nuclear envelope in various organisms including malaria mosquitoes. However, other spatial characteristics of nucleus organization, like volume and shape of chromosome territories, have not been studied in Anopheles. We conducted a thorough analysis of tissue-specific features of the X chromosome and nucleolus volume and shape in follicular epithelium and nurse cells of the Anopheles atroparvus ovaries using a modern open-source software. DNA of the polytene X chromosome from ovarian nurse cells was obtained by microdissection and was used as a template for amplification with degenerate oligo primers. A fluorescently labeled X chromosome painting probe was hybridized with formaldehyde-fixed ovaries of mosquitoes using a 3D-FISH method. The nucleolus was stained by immunostaining with an anti-fibrillarin antibody. The analysis was conducted with TANGO-a software for a chromosome spatial organization analysis. We show that the volume and position of the X chromosome have tissue-specific characteristics. Unlike nurse cell nuclei, the growth of follicular epithelium nuclei is not accompanied with the proportional growth of the X chromosome. However, the shape of the X chromosome does not differ between the tissues. The dynamics of the X chromosome attachment regions location is tissue-specific and it is correlated with the process of nucleus growth in follicular epithelium and nurse cells.
Topics: Animals; Anopheles; Cell Nucleolus; Female; In Situ Hybridization, Fluorescence; Malaria; Organ Specificity; Polytene Chromosomes; X Chromosome
PubMed: 28158219
DOI: 10.1371/journal.pone.0171290 -
PloS One 2016During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and...
During embryogenesis, primordial germ cells (PGCs) and somatic gonadal precursor cells (SGPs) migrate and coalesce to form the early gonad. A failure of the PGCs and SGPs to form a gonad with the proper architecture not only affects germ cell development, but can also lead to infertility. Therefore, it is critical to identify the molecular mechanisms that function within both the PGCs and SGPs to promote gonad morphogenesis. We have characterized the phenotypes of two genes, longitudinals lacking (lola) and ribbon (rib), that are required for the coalescence and compaction of the embryonic gonad in Drosophila melanogaster. rib and lola are expressed in the SGPs of the developing gonad, and genetic interaction analysis suggests these proteins cooperate to regulate gonad development. Both genes encode proteins with DNA binding motifs and a conserved protein-protein interaction domain, known as the Broad complex, Tramtrack, Bric-à-brac (BTB) domain. Through molecular modeling and yeast-two hybrid studies, we demonstrate that Rib and Lola homo- and heterodimerize via their BTB domains. In addition, analysis of the colocalization of Rib and Lola with marks of transcriptional activation and repression on polytene chromosomes reveals that Rib and Lola colocalize with both repressive and activating marks and with each other. While previous studies have identified Rib and Lola targets in other tissues, we find that Rib and Lola are likely to function via different downstream targets in the gonad. These results suggest that Rib and Lola act as dual-function transcription factors to cooperatively regulate embryonic gonad morphogenesis.
Topics: Animals; Cytoskeletal Proteins; Dimerization; Drosophila Proteins; Drosophila melanogaster; Germ Cells; Gonads; Immunohistochemistry; Membrane Proteins; Mesoderm; Microscopy, Fluorescence; Morphogenesis; Mutation; Protein Isoforms; Salivary Glands; Transcription Factors; Two-Hybrid System Techniques
PubMed: 27898696
DOI: 10.1371/journal.pone.0167283 -
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 -
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 -
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 -
International Journal of Molecular... Aug 2021Chromatin 3D structure plays a crucial role in regulation of gene activity. Previous studies have envisioned spatial contact formations between chromatin domains with...
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 polytene chromosomes are involved in non-allelic ectopic pairing. The mutant strain , a model for Williams-Beuren syndrome, has an increased frequency of ectopic contacts (FEC) compared to the wild-type strain (). 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 and strains. With fragment lengths of 50 nucleotides (nt), showed a specific FEC-FMF correlation for 20% of the sections involved in ectopic contacts. The correlation was unspecific in , 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.
Topics: Animals; Base Pairing; Chromatin; Computational Biology; DNA, Satellite; Disease Models, Animal; Drosophila melanogaster; Humans; Polytene Chromosomes; Software; Williams Syndrome; X Chromosome
PubMed: 34445413
DOI: 10.3390/ijms22168713