Browsing by Author "George, Phillip"

Now showing 1 - 5 of 5
  • Thumbnail Image
    2D and 3D Chromosome Painting in Malaria Mosquitoes
    George, Phillip; Sharma, Atashi; Sharakhov, Igor V. (Journal of Visualized Experiments, 2014-01-01)
    Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
  • Thumbnail Image
    Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies
    Waterhouse, Robert M.; Aganezov, Sergey; Anselmetti, Yoann; Lee, Jiyoung; Ruzzante, Livio; Reijnders, Maarten J. M. F.; Feron, Romain; Bérard, Sèverine; George, Phillip; Hahn, Matthew W.; Howell, Paul I.; Kamali, Maryam; Koren, Sergey; Lawson, Daniel; Maslen, Gareth; Peery, Ashley; Phillippy, Adam M.; Sharakhova, Maria V.; Tannier, Eric; Unger, Maria F.; Zhang, Simo V.; Alekseyev, Max A.; Besansky, Nora J.; Chauve, Cedric; Emrich, Scott J.; Sharakhov, Igor V. (2020-01-02)
    Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from ‘finished’. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies. Results We evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi. Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.
  • Thumbnail Image
    Genome mapping and characterization of the Anopheles gambiae heterochromatin
    Sharakhova, Maria V.; George, Phillip; Brusentsova, Irina V.; Leman, Scotland C.; Bailey, Jeffrey A.; Smith, Christopher D.; Sharakhov, Igor V. (Biomed Central, 2010-08-04)
    Background Heterochromatin plays an important role in chromosome function and gene regulation. Despite the availability of polytene chromosomes and genome sequence, the heterochromatin of the major malaria vector Anopheles gambiae has not been mapped and characterized. Results To determine the extent of heterochromatin within the An. gambiae genome, genes were physically mapped to the euchromatin-heterochromatin transition zone of polytene chromosomes. The study found that a minimum of 232 genes reside in 16.6 Mb of mapped heterochromatin. Gene ontology analysis revealed that heterochromatin is enriched in genes with DNA-binding and regulatory activities. Immunostaining of the An. gambiae chromosomes with antibodies against Drosophila melanogaster heterochromatin protein 1 (HP1) and the nuclear envelope protein lamin Dm0 identified the major invariable sites of the proteins' localization in all regions of pericentric heterochromatin, diffuse intercalary heterochromatin, and euchromatic region 9C of the 2R arm, but not in the compact intercalary heterochromatin. To better understand the molecular differences among chromatin types, novel Bayesian statistical models were developed to analyze genome features. The study found that heterochromatin and euchromatin differ in gene density and the coverage of retroelements and segmental duplications. The pericentric heterochromatin had the highest coverage of retroelements and tandem repeats, while intercalary heterochromatin was enriched with segmental duplications. We also provide evidence that the diffuse intercalary heterochromatin has a higher coverage of DNA transposable elements, minisatellites, and satellites than does the compact intercalary heterochromatin. The investigation of 42-Mb assembly of unmapped genomic scaffolds showed that it has molecular characteristics similar to cytologically mapped heterochromatin. Conclusions Our results demonstrate that Anopheles polytene chromosomes and whole-genome shotgun assembly render the mapping and characterization of a significant part of heterochromatic scaffolds a possibility. These results reveal the strong association between characteristics of the genome features and morphological types of chromatin. Initial analysis of the An. gambiae heterochromatin provides a framework for its functional characterization and comparative genomic analyses with other organisms.
  • Thumbnail Image
    Increased production of piRNAs from euchromatic clusters and genes in Anopheles gambiae compared with Drosophila melanogaster
    George, Phillip; Jensen, Silke; Pogorelcnik, Romain; Lee, Jiyoung; Xing, Yi; Brasset, Emilie; Vaury, Chantal; Sharakhov, Igor V. (Biomed Central, 2015-11-27)
    Background Specific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential. Results To analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway. Conclusions Here, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in the genome of D. melanogaster. Also, protein-coding genes have an increased role in production of piRNAs in the germline of this mosquito. Genes involved in germline and embryonic development of An. gambiae generate a substantial portion of piRNAs, suggesting a role of the piRNA pathway in the epigenetic regulation of the reproductive processes in the African malaria vector.
  • Thumbnail Image
    Three-dimensional Organization of Polytene Chromosomes in Somatic and Germline Tissues of Malaria Mosquitoes
    George, Phillip; Kinney, Nicholas A.; Liang, Jiangtao; Onufriev, Alexey V.; Sharakhov, Igor V. (MDPI, 2020-02-01)
    Spatial organization of chromosome territories and interactions between interphase chromosomes themselves, as well as with the nuclear periphery, play important roles in epigenetic regulation of the genome function. However, the interplay between inter-chromosomal contacts and chromosome-nuclear envelope attachments in an organism’s development is not well-understood. To address this question, we conducted microscopic analyses of the three-dimensional chromosome organization in malaria mosquitoes. We employed multi-colored oligonucleotide painting probes, spaced 1 Mb apart along the euchromatin, to quantitatively study chromosome territories in larval salivary gland cells and adult ovarian nurse cells of Anopheles gambiae, An. coluzzii, and An. merus. We found that the X chromosome territory has a significantly smaller volume and is more compact than the autosomal arm territories. The number of inter-chromosomal, and the percentage of the chromosome–nuclear envelope, contacts were conserved among the species within the same cell type. However, the percentage of chromosome regions located at the nuclear periphery was typically higher, while the number of inter-chromosomal contacts was lower, in salivary gland cells than in ovarian nurse cells. The inverse correlation was considerably stronger for the autosomes. Consistent with previous theoretical arguments, our data indicate that, at the genome-wide level, there is an inverse relationship between chromosome-nuclear envelope attachments and chromosome–chromosome interactions, which is a key feature of the cell type-specific nuclear architecture.