scholarly journals DNA Sequence Preference for De Novo Centromere Formation on a Caenorhabditis elegans Artificial Chromosome

2020 ◽  
Author(s):  
Zhongyang Lin ◽  
Karen Wing Yee Yuen
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Sequence ◽  
Dna Sequences ◽  
De Novo ◽  
Repetitive Sequences ◽  
Model Organism ◽  
Artificial Chromosome ◽  
Centromeric Dna ◽  
Artificial Chromosomes ◽  
Non Homologous End Joining

ABSTRACTCentromeric DNA sequences vary in different species, but share common characteristics, like high AT-content, repetitiveness, and low, but not no, transcriptional activity. Yet, neocentromeres can be found on non-centromeric, ectopic sequences, suggesting that centromeres can be established and maintained epigenetically. In contrast, canonical centromeric DNA sequences are more competent in de novo centromere formation on artificial chromosomes (ACs). To determine if specific DNA sequence features are preferred for new centromere formation, we injected different DNA sequences into the gonad of a holocentric model organism, Caenorhabditis elegans, to form ACs in embryos, and monitored mitotic AC segregation. We demonstrated that AT-rich sequences, but not repetitive sequences, accelerated de novo centromere formation on ACs. We also injected fragmented Saccharomyces cerevisiae genomic DNA to construct a less repetitive, more complex AC that can propagate through generations. By whole-genome sequencing and de novo assembly of AC sequences, we deduced that this AC was formed through non-homologous end joining. By CENP-AHCP-3 chromatin immunoprecipitation followed by sequencing (ChIP-seq), we found that CENP-AHCP-3 domain width on both the AC and endogenous chromosomes is positively correlated with AT-content. Besides, CENP-AHCP-3 binds to unexpressed gene loci or non-genic regions on the AC, consistent with the organization of endogenous holocentromeres.

scholarly journals CENP-B box is required for de novo centromere chromatin assembly on human alphoid DNA

2002 ◽  
Vol 159 (5) ◽  
pp. 765-775 ◽  
Author(s):  
Jun-ichirou Ohzeki ◽  
Megumi Nakano ◽  
Teruaki Okada ◽  
Hiroshi Masumoto
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
De Novo ◽  
Repetitive Sequences ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Binding Activity ◽  
Chromatin Assembly ◽  
Direct Assembly ◽  
Alphoid Dna

Centromere protein (CENP) B boxes, recognition sequences of CENP-B, appear at regular intervals in human centromeric α-satellite DNA (alphoid DNA). In this study, to determine whether information carried by the primary sequence of alphoid DNA is involved in assembly of functional human centromeres, we created four kinds of synthetic repetitive sequences: modified alphoid DNA with point mutations in all CENP-B boxes, resulting in loss of all CENP-B binding activity; unmodified alphoid DNA containing functional CENP-B boxes; and nonalphoid repetitive DNA sequences with or without functional CENP-B boxes. These four synthetic repetitive DNAs were introduced into cultured human cells (HT1080), and de novo centromere assembly was assessed using the mammalian artificial chromosome (MAC) formation assay. We found that both the CENP-B box and the alphoid DNA sequence are required for de novo MAC formation and assembly of functional centromere components such as CENP-A, CENP-C, and CENP-E. Using the chromatin immunoprecipitation assay, we found that direct assembly of CENP-A and CENP-B in cells with synthetic alphoid DNA required functional CENP-B boxes. To the best of our knowledge, this is the first reported evidence of a functional molecular link between a centromere-specific DNA sequence and centromeric chromatin assembly in humans.

Construction and analysis of artificial chromosomes with de novo holocentromeres in Caenorhabditis elegans

2020 ◽  
Vol 64 (2) ◽  
pp. 233-249
Author(s):  
Zhongyang Lin ◽  
Karen Wing Yee Yuen
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Sequence ◽  
De Novo ◽  
Complex Mixture ◽  
Chromosome Length ◽  
Cis Acting ◽  
Artificial Chromosomes ◽  
C Elegans ◽  
Successive Generations

Abstract Artificial chromosomes (ACs), generated in yeast (YACs) and human cells (HACs), have facilitated our understanding of the trans-acting proteins, cis-acting elements, such as the centromere, and epigenetic environments that are necessary to maintain chromosome stability. The centromere is the unique chromosomal region that assembles the kinetochore and connects to microtubules to orchestrate chromosome movement during cell division. While monocentromeres are the most commonly characterized centromere organization found in studied organisms, diffused holocentromeres along the chromosome length are observed in some plants, insects and nematodes. Based on the well-established DNA microinjection method in holocentric Caenorhabditis elegans, concatemerization of foreign DNA can efficiently generate megabase-sized extrachromosomal arrays (Exs), or worm ACs (WACs), for analyzing the mechanisms of WAC formation, de novo centromere formation, and segregation through mitosis and meiosis. This review summarizes the structural, size and stability characteristics of WACs. Incorporating LacO repeats in WACs and expressing LacI::GFP allows real-time tracking of newly formed WACs in vivo, whereas expressing LacI::GFP-chromatin modifier fusions can specifically adjust the chromatin environment of WACs. The WACs mature from passive transmission to autonomous segregation by establishing a holocentromere efficiently in a few cell cycles. Importantly, WAC formation does not require any C. elegans genomic DNA sequence. Thus, DNA substrates injected can be changed to evaluate the effects of DNA sequence and structure in WAC segregation. By injecting a complex mixture of DNA, a less repetitive WAC can be generated and propagated in successive generations for DNA sequencing and analysis of the established holocentromere on the WAC.

scholarly journals An Artificially Constructed De Novo Human Chromosome Behaves Almost Identically to Its Natural Counterpart during Metaphase and Anaphase in Living Cells

2006 ◽  
Vol 26 (20) ◽  
pp. 7682-7695 ◽  
Author(s):  
Tomohiro Tsuduki ◽  
Megumi Nakano ◽  
Nao Yasuoka ◽  
Saeko Yamazaki ◽  
Teruaki Okada ◽  
...  
Keyword(s):  
Yeast Artificial Chromosome ◽  
Fluorescent Protein ◽  
De Novo ◽  
Artificial Chromosome ◽  
Time Lapse ◽  
Living Cells ◽  
Lac Repressor ◽  
Host Chromosome ◽  
Artificial Chromosomes ◽  
Spindle Midzone

ABSTRACT Human artificial chromosomes (HACs) are promising reagents for the analysis of chromosome function. While HACs are maintained stably, the segregation mechanisms of HACs have not been investigated in detail. To analyze HACs in living cells, we integrated 256 copies of the Lac operator into a precursor yeast artificial chromosome (YAC) containing α-satellite DNA and generated green fluorescent protein (GFP)-tagged HACs in HT1080 cells expressing a GFP-Lac repressor fusion protein. Time-lapse analyses of GFP-HACs and host centromeres in living mitotic cells indicated that the HAC was properly aligned at the spindle midzone and that sister chromatids of the HAC separated with the same timing as host chromosomes and moved to the spindle poles with mobility similar to that of the host centromeres. These results indicate that a HAC composed of a multimer of input α-satellite YACs retains most of the functions of the centromeres on natural chromosomes. The only difference between the HAC and the host chromosome was that the HAC oscillated more frequently, at higher velocity, across the spindle midzone during metaphase. However, this provides important evidence that an individual HAC has the capacity to maintain tensional balance in the pole-to-pole direction, thereby stabilizing its position around the spindle midzone.

A mushroom-inducing DNA sequence isolated from the Basidiomycete, Schizophyllum commune.

Genetics ◽  
1991 ◽  
Vol 129 (3) ◽  
pp. 707-716 ◽  
Author(s):  
J S Horton ◽  
C A Raper
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
De Novo ◽  
Schizophyllum Commune ◽  
Sequence Divergence ◽  
Fruiting Bodies ◽  
Genomic Dnas ◽  
Different Strains ◽  
Mating Interaction ◽  
Normal Requirement

Abstract A DNA sequence capable of inducing the de novo development of fruiting bodies (mushrooms) when integrated into the genome of unmated, nonfruiting strains of the Basidiomycete Schizophyllum commune has been isolated and partially characterized. This sequence, designated FRT1, overrides the normal requirement of a mating interaction for fruiting in this organism. It has been shown to integrate stably in different chromosome locations and appears to be trans-acting. It also enhances the normal process of fruiting that occurs after mating. Additional DNA sequences with similarity to FRT1 were detected within the genome of the strain of origin by hybridization of labeled FRT1 DNA to blots of digested genomic DNAs. FRT1 and the genomic sequences similar to it were shown to be genetically linked. Southern hybridization experiments suggested sequence divergence at the FRT1 locus between different strains of S. commune. A testable model for how FRT1 may act as a key element in the pathway for the differentiation of fruiting bodies is presented as a working hypothesis for further investigation.

scholarly journals Sequential de novo centromere formation and inactivation on a chromosomal fragment in maize

2015 ◽  
Vol 112 (11) ◽  
pp. E1263-E1271 ◽  
Author(s):  
Yalin Liu ◽  
Handong Su ◽  
Junling Pang ◽  
Zhi Gao ◽  
Xiu-Jie Wang ◽  
...  
Keyword(s):  
Dna Sequences ◽  
De Novo ◽  
B Chromosome ◽  
Chromosome 9 ◽  
Centromeric Dna ◽  
Repeat Sequences ◽  
Centromeric Repeat ◽  
And Function ◽  
Centromere Assembly ◽  
Derivatives Of

The ability of centromeres to alternate between active and inactive states indicates significant epigenetic aspects controlling centromere assembly and function. In maize (Zea mays), misdivision of the B chromosome centromere on a translocation with the short arm of chromosome 9 (TB-9Sb) can produce many variants with varying centromere sizes and centromeric DNA sequences. In such derivatives of TB-9Sb, we found a de novo centromere on chromosome derivative 3-3, which has no canonical centromeric repeat sequences. This centromere is derived from a 288-kb region on the short arm of chromosome 9, and is 19 megabases (Mb) removed from the translocation breakpoint of chromosome 9 in TB-9Sb. The functional B centromere in progenitor telo2-2 is deleted from derivative 3-3, but some B-repeat sequences remain. The de novo centromere of derivative 3-3 becomes inactive in three further derivatives with new centromeres being formed elsewhere on each chromosome. Our results suggest that de novo centromere initiation is quite common and can persist on chromosomal fragments without a canonical centromere. However, we hypothesize that when de novo centromeres are initiated in opposition to a larger normal centromere, they are cleared from the chromosome by inactivation, thus maintaining karyotype integrity.

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 ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Satellite Dna ◽  
Mammalian Cells ◽  
Large Scale ◽  
De Novo ◽  
Mammalian Species ◽  
Genetic Material ◽  
Exogenous Dna ◽  
Artificial Chromosomes ◽  
Acrocentric Chromosomes

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.

scholarly journals RbAp46/48LIN-53 and HAT-1 are required for initial CENP-AHCP-3 deposition and de novo centromere formation in Caenorhabditis elegans embryos

2020 ◽  
Author(s):  
Zhongyang Lin ◽  
Karen Wing Yee Yuen
Keyword(s):  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
De Novo ◽  
Dna Arrays ◽  
Replicative Helicase ◽  
Cell Cycles ◽  
Artificial Chromosomes ◽  
Hmw Dna ◽  
Foreign Dna ◽  
Initial Deposition

ABSTRACTForeign DNA microinjected into the Caenorhabditis elegans germline forms episomal extra-chromosomal arrays, or artificial chromosomes (ACs), in embryos. Injected linear, short DNA fragments concatemerize into high molecular weight (HMW)-DNA arrays that are visible as punctate DAPI-stained foci in oocytes, which undergo chromatinization and centromerization in embryos. The inner centromere, inner and outer kinetochore components, including AIR-2, CENP-AHCP-3, Mis18BP1KNL-2 and BUB-1, assemble onto the nascent ACs during the first mitosis. Yet, due to incomplete DNA replication of the nascent ACs, centromeric proteins are not oriented at the poleward faces of the nascent ACs in mitosis, resulting in lagging ACs. The DNA replication efficiency of ACs improves over several cell cycles. We found that a condensin subunit, SMC-4, but not the replicative helicase component, MCM-2, facilitates de novo CENP-AHCP-3 deposition on nascent ACs. Furthermore, H3K9ac, H4K5ac, and H4K12ac are highly enriched on newly chromatinized ACs. HAT-1 and RbAp46/48LIN-53, which are essential for de novo centromere formation and segregation competency of nascent ACs, also hyperacetylate histone H3 and H4. Different from centromere maintenance on endogenous chromosomes, where Mis18BP1KNL-2 functions upstream of RbAp46/48LIN-53, RbAp46/48LIN-53 depletion causes the loss of both CENP-AHCP-3 and Mis18BP1KNL-2 initial deposition at de novo centromeres on ACs.

scholarly journals Human AlphoidtetO Artificial Chromosome as a Gene Therapy Vector for the Developing Hemophilia A Model in Mice

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 879 ◽  
Author(s):  
Sergey V. Ponomartsev ◽  
Sergey A. Sinenko ◽  
Elena V. Skvortsova ◽  
Mikhail A. Liskovykh ◽  
Ivan N. Voropaev ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Hemophilia A ◽  
De Novo ◽  
Ex Vivo ◽  
Embryonic Stem ◽  
Artificial Chromosome ◽  
Monogenic Disease ◽  
Gene Therapy Vector ◽  
Artificial Chromosomes ◽  
Integrative Vector

Human artificial chromosomes (HACs), including the de novo synthesized alphoidtetO-HAC, are a powerful tool for introducing genes of interest into eukaryotic cells. HACs are mitotically stable, non-integrative episomal units that have a large transgene insertion capacity and allow efficient and stable transgene expression. Previously, we have shown that the alphoidtetO-HAC vector does not interfere with the pluripotent state and provides stable transgene expression in human induced pluripotent cells (iPSCs) and mouse embryonic stem cells (ESCs). In this study, we have elaborated on a mouse model of ex vivo iPSC- and HAC-based treatment of hemophilia A monogenic disease. iPSCs were developed from FVIIIY/− mutant mice fibroblasts and FVIII cDNA, driven by a ubiquitous promoter, was introduced into the alphoidtetO-HAC in hamster CHO cells. Subsequently, the therapeutic alphoidtetO-HAC-FVIII was transferred into the FVIIIY/– iPSCs via the retro-microcell-mediated chromosome transfer method. The therapeutic HAC was maintained as an episomal non-integrative vector in the mouse iPSCs, showing a constitutive FVIII expression. This study is the first step towards treatment development for hemophilia A monogenic disease with the use of a new generation of the synthetic chromosome vector—the alphoidtetO-HAC.

scholarly journals An artificial chromosome for data storage

2021 ◽  
Author(s):  
Weigang Chen ◽  
Mingzhe Han ◽  
Jianting Zhou ◽  
Qi Ge ◽  
Panpan Wang ◽  
...  
Keyword(s):  
Data Storage ◽  
De Novo ◽  
Video Clip ◽  
Data Retrieval ◽  
Artificial Chromosome ◽  
Information Storage ◽  
Artificial Chromosomes ◽  
Design And Synthesis ◽  
The Stability

Abstract DNA digital storage provides an alternative for information storage with high density and long-term stability. Here, we report the de novo design and synthesis of an artificial chromosome that encodes two pictures and a video clip. The encoding paradigm utilizing the superposition of sparsified error correction codewords and pseudo-random sequences tolerates base insertions/deletions and is well suited to error-prone nanopore sequencing for data retrieval. The entire 254 kb sequence was 95.27% occupied by encoded data. The Transformation-Associated Recombination method was used in the construction of this chromosome from DNA fragments and necessary autonomous replication sequences. The stability was demonstrated by transmitting the data-carrying chromosome to the 100th generation. This study demonstrates a data storage method using encoded artificial chromosomes via in vivo assembly for write-once and stable replication for multiple retrievals, similar to a compact disc, with potential in economically massive data distribution.

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