Novel generation of human satellite DNA-based artificial chromosomes in mammalian cells

2000 ◽  
Vol 113 (18) ◽  
pp. 3207-3216 ◽  
Author(s):  
E. Csonka ◽  
I. Cserpan ◽  
K. Fodor ◽  
G. Hollo ◽  
R. Katona ◽  
...  

An in vivo approach has been developed for generation of artificial chromosomes, based on the induction of intrinsic, large-scale amplification mechanisms of mammalian cells. Here, we describe the successful generation of prototype human satellite DNA-based artificial chromosomes via amplification-dependent de novo chromosome formations induced by integration of exogenous DNA sequences into the centromeric/rDNA regions of human acrocentric chromosomes. Subclones with mitotically stable de novo chromosomes were established, which allowed the initial characterization and purification of these artificial chromosomes. Because of the low complexity of their DNA content, they may serve as a useful tool to study the structure and function of higher eukaryotic chromosomes. Human satellite DNA-based artificial chromosomes containing amplified satellite DNA, rDNA, and exogenous DNA sequences were heterochromatic, however, they provided a suitable chromosomal environment for the expression of the integrated exogenous genetic material. We demonstrate that induced de novo chromosome formation is a reproducible and effective methodology in generating artificial chromosomes from predictable sequences of different mammalian species. Satellite DNA-based artificial chromosomes formed by induced large-scale amplifications on the short arm of human acrocentric chromosomes may become safe or low risk vectors in gene therapy.

2020 ◽  
Author(s):  
Agata Motyka-Pomagruk ◽  
Sabina Zoledowska ◽  
Agnieszka Emilia Misztak ◽  
Wojciech Sledz ◽  
Alessio Mengoni ◽  
...  

Abstract Background: Dickeya solani is an important plant pathogenic bacterium causing severe losses in European potato production. This species draws a lot of attention due to its remarkable virulence, great devastating potential and easier spread in contrast to other Dickeya spp. In view of a high need for extensive studies on economically important soft rot Pectobacteriaceae , we performed a comparative genomics analysis on D. solani strains to search for genetic foundations that would explain the differences in the observed virulence levels within the D. solani population. Results: High quality assemblies of 8 de novo sequenced D. solani genomes have been obtained. Whole-sequence comparison, ANIb, ANIm, Tetra and pangenome-oriented analyses performed on these genomes and the sequences of 14 additional strains revealed an exceptionally high level of homogeneity among the studied genetic material of D. solani strains. With the use of 22 genomes, the pangenome of D. solani , comprising 84.7% core, 7.2% accessory and 8.1% unique genes, has been almost completely determined, suggesting the presence of a nearly closed pangenome structure. Attribution of the genes included in the D. solani pangenome fractions to functional COG categories showed that higher percentages of accessory and unique pangenome parts in contrast to the core section are encountered in phage/mobile elements- and transcription- associated groups with the genome of RNS 05.1.2A strain having the most significant impact. Also, the first D. solani large-scale genome-wide phylogeny computed on concatenated core gene alignments is herein reported. Conclusions: The almost closed status of D. solani pangenome achieved in this work points to the fact that the unique gene pool of this species should no longer expand. Such a feature is characteristic of taxa whose representatives either occupy isolated ecological niches or lack efficient mechanisms for gene exchange and recombination, which seems rational concerning a strictly pathogenic species with clonal population structure. Finally, no obvious correlations between the geographical origin of D. solani strains and their phylogeny were found, which might reflect the specificity of the international seed potato market.


2016 ◽  
Author(s):  
Shaun D Jackman ◽  
Benjamin P Vandervalk ◽  
Hamid Mohamadi ◽  
Justin Chu ◽  
Sarah Yeo ◽  
...  

AbstractThe assembly of DNA sequences de novo is fundamental to genomics research. It is the first of many steps towards elucidating and characterizing whole genomes. Downstream applications, including analysis of genomic variation between species, between or within individuals critically depends on robustly assembled sequences. In the span of a single decade, the sequence throughput of leading DNA sequencing instruments has increased drastically, and coupled with established and planned large-scale, personalized medicine initiatives to sequence genomes in the thousands and even millions, the development of efficient, scalable and accurate bioinformatics tools for producing high-quality reference draft genomes is timely.With ABySS 1.0, we originally showed that assembling the human genome using short 50 bp sequencing reads was possible by aggregating the half terabyte of compute memory needed over several computers using a standardized message-passing system (MPI). We present here its re-design, which departs from MPI and instead implements algorithms that employ a Bloom filter, a probabilistic data structure, to represent a de Bruijn graph and reduce memory requirements.We present assembly benchmarks of human Genome in a Bottle 250 bp Illumina paired-end and 6 kbp mate-pair libraries from a single individual, yielding a NG50 (NGA50) scaffold contiguity of 3.5 (3.0) Mbp using less than 35 GB of RAM, a modest memory requirement by today’s standard that is often available on a single computer. We also investigate the use of BioNano Genomics and 10x Genomics’ Chromium data to further improve the scaffold contiguity of this assembly to 42 (15) Mbp.


Science ◽  
2019 ◽  
Vol 364 (6441) ◽  
pp. 658-664 ◽  
Author(s):  
Scott E. Boyken ◽  
Mark A. Benhaim ◽  
Florian Busch ◽  
Mengxuan Jia ◽  
Matthew J. Bick ◽  
...  

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.


Genetics ◽  
2021 ◽  
Author(s):  
Leslie A Mitchell ◽  
Laura H McCulloch ◽  
Sudarshan Pinglay ◽  
Henri Berger ◽  
Nazario Bosco ◽  
...  

Abstract Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.


Author(s):  
Dmitry Schigel ◽  
Thomas Jeppesen ◽  
Robert Finn ◽  
Guy Cochrane ◽  
Urmas Kõljalg ◽  
...  

The Global Biodiversity Information Facility (GBIF) was established by governments in 2001, largely through the initiative and leadership of the natural history collections community, following the 1999 recommendation by a working group under the Megascience Forum (predecessor of the Global Science Forum) of the Organization for Economic Cooperation and Development (OECD). Over 20 years, GBIF has helped develop standards and convened a global community of data-publishing institutions, aggregrating over one billion specimen occurrence records freely and openly available for use in research and policy making. These GBIF mediated data range from vouchered museum specimens to observation records generated by humans and machines. New data are being generated from integrated remote sensing, ecological sampling, and molecular sequencing that have strong geospatial components but lack traditional vouchers. GBIF is working with partners to develop best practices of bringing this data into the GBIF architecture. Following discussions during the second Global Biodiversity Information Conference in 2018, GBIF and the European Bioinformatics Institute (EMBL-EBI), supported by ELIXIR, have extended collaboration to share species occurrence records known only from their genetic material. When these data providers contribute data coordinates along with the sequences to the European Nucleotide Archive (ENA), the records will appear on GBIF maps and in spatial searches. This collaboration enables significant new molecular data streams to become discoverable through GBIF.org: by mid-March 2019, over 7.8m individual occurrence records via the ENA, and over 13.2m records as standardized Darwin Core sampling-event datasets via MGnify, a resource that provides taxonomic and functional annotations on sequences derived from environmental sequencing projects. Sequence-based occurrence records published by ENA and MGnify boost representation of microbial diversity which was underrepresented at GBIF. The ELIXIR-ENA-MGnify-GBIF partnership is working on further refinement of the dynamic data linkages, frequency of updates and other improvements. The API-based tool that connects GBIF data infrastructures is open to new data contributors and for indexes of molecular occurrences. Indexing of these data streams is dependent on the presence of a name (any rank) with the sequence. Under the current Codes of nomenclature, animals, fungi, plants, and algae cannot be described based on exclusively sequence data. Yet, a significant volume of biodiversity data has only been represented by DNA sequences. Barcoding and sequence clustering procedures vary among taxa and research communities, but clusters can be related to a taxon with a Latin name. Many DNA similarity clusters do not contain a sequence from a formally described taxon; however these sequence clusters provide provisional molecular names for nomenclatural communication. In the best cases, curated libraries of reference sequences, their metadata, clusters, alignments, and links to individuals and physical material become de facto naming conventions for certain taxonomic groups, and co-exist with Latin names. Integration of molecular names into the taxonomic backbone of GBIF started with Fungi and UNITE, a data management and identification environment for fungal ITS barcodes with 87,000+ fungal species hypotheses demarcating 800,000+ sequence specimens as of March 2019. Checklist publication of all names in UNITE through GBIF.org including Linnaean names and stable, DOI-trackable molecular sequence based ‘species hypotheses’, enables indexing of fungal metabarcoding data worldwide, such as BIOWIDE. As names are currently essential to indexing the world’s occurrence data, GBIF will develop similar linkages with names in the Barcode of Life data system (BOLD) and in SILVA - a resource for high-quality ribosomal RNA sequence data and taxonomy, and welcomes other reference systems to this development. Expanding the molecular data streams (Fig. 1) allows GBIF to address spatial, temporal and taxonomic gaps and biases, and to support large-scale data-intensive research openly and worldwide.


2003 ◽  
Vol 23 (21) ◽  
pp. 7689-7697 ◽  
Author(s):  
M. Katharine Rudd ◽  
Robert W. Mays ◽  
Stuart Schwartz ◽  
Huntington F. Willard

ABSTRACT Human artificial chromosomes have been used to model requirements for human chromosome segregation and to explore the nature of sequences competent for centromere function. Normal human centromeres require specialized chromatin that consists of alpha satellite DNA complexed with epigenetically modified histones and centromere-specific proteins. While several types of alpha satellite DNA have been used to assemble de novo centromeres in artificial chromosome assays, the extent to which they fully recapitulate normal centromere function has not been explored. Here, we have used two kinds of alpha satellite DNA, DXZ1 (from the X chromosome) and D17Z1 (from chromosome 17), to generate human artificial chromosomes. Although artificial chromosomes are mitotically stable over many months in culture, when we examined their segregation in individual cell divisions using an anaphase assay, artificial chromosomes exhibited more segregation errors than natural human chromosomes (P < 0.001). Naturally occurring, but abnormal small ring chromosomes derived from chromosome 17 and the X chromosome also missegregate more than normal chromosomes, implicating overall chromosome size and/or structure in the fidelity of chromosome segregation. As different artificial chromosomes missegregate over a fivefold range, the data suggest that variable centromeric DNA content and/or epigenetic assembly can influence the mitotic behavior of artificial chromosomes.


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