scholarly journals De novo formed satellite DNA-based mammalian artificial chromosomes and their possible applications

2015 ◽  
Vol 23 (1) ◽  
pp. 143-157 ◽  
Author(s):  
Robert L. Katona
Keyword(s):  
Satellite Dna ◽  
De Novo ◽  
Artificial Chromosomes

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 Human Artificial Chromosomes with Alpha Satellite-Based De Novo Centromeres Show Increased Frequency of Nondisjunction and Anaphase Lag

2003 ◽  
Vol 23 (21) ◽  
pp. 7689-7697 ◽  
Author(s):  
M. Katharine Rudd ◽  
Robert W. Mays ◽  
Stuart Schwartz ◽  
Huntington F. Willard
Keyword(s):  
Chromosome Segregation ◽  
X Chromosome ◽  
Satellite Dna ◽  
De Novo ◽  
Chromosome 17 ◽  
Small Ring ◽  
Alpha Satellite ◽  
Artificial Chromosomes ◽  
Centromere Function ◽  
Alpha Satellite Dna

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.

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.

Isolation and structural analysis of a 1.2-megabase N-myc amplicon from a human neuroblastoma

1992 ◽  
Vol 12 (12) ◽  
pp. 5563-5570
Author(s):  
S S Schneider ◽  
J L Hiemstra ◽  
B A Zehnbauer ◽  
P Taillon-Miller ◽  
D L Le Paslier ◽  
...  
Keyword(s):  
Cell Line ◽  
Gene Amplification ◽  
De Novo ◽  
Physical Map ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cell Line ◽  
Chromosome 2 ◽  
Human Neuroblastoma ◽  
Artificial Chromosomes ◽  
Yeast Artificial Chromosomes

Oncogene amplification is observed frequently in human cancers, but little is known about the mechanism of gene amplification or the structure of amplified DNA in tumor cells. We have studied the N-myc amplified domain from a representative neuroblastoma cell line, SMS-KAN, and compared the map of the amplicon in this cell line with that seen in normal DNA. The SMS-KAN cell line DNA was cloned into yeast artificial chromosomes (YACs), and clones were identified by screening the YAC library with amplified DNA probes that were obtained previously (B. Zehnbauer, D. Small, G. M. Brodeur, R. Seeger, and B. Vogelstein, Mol. Cell. Biol. 8:522-530, 1988). In addition, YAC clones corresponding to the normal N-myc locus on chromosome 2 were obtained by screening two normal human YAC libraries with these probes, and the restriction maps of the two sets of overlapping YACs were compared. Our results suggest that the amplified domain in this cell line is a approximately 1.2-Mb circular molecule with a head-to-tail configuration, and the physical map of the normal N-myc locus generally is conserved in the amplicon. These results provide a physical map of the amplified domain of a neuroblastoma cell line that has de novo amplification of an oncogene. The head-to-tail organization, the general conservation of the normal physical map in the amplicon, and the extrachromosomal location of the amplified DNA are most consistent with the episome formation-plus-segregation mechanism of gene amplification in these tumors.

scholarly journals Bacterial artificial chromosomes establish replication timing and sub-nuclear compartment de novo as extra-chromosomal vectors

2017 ◽  
Vol 46 (4) ◽  
pp. 1810-1820 ◽  
Author(s):  
Jiao Sima ◽  
Daniel A Bartlett ◽  
Molly R Gordon ◽  
David M Gilbert
Keyword(s):  
De Novo ◽  
Replication Timing ◽  
Bacterial Artificial Chromosomes ◽  
Nuclear Compartment ◽  
Artificial Chromosomes

scholarly journals Telomere-to-telomere assembly of a complete human X chromosome

2019 ◽  
Author(s):  
Karen H. Miga ◽  
Sergey Koren ◽  
Arang Rhie ◽  
Mitchell R. Vollger ◽  
Ariel Gershman ◽  
...  
Keyword(s):  
Human Genome ◽  
X Chromosome ◽  
Satellite Dna ◽  
Tandem Repeats ◽  
Reference Genome ◽  
De Novo ◽  
Hydatidiform Mole ◽  
Segmental Duplications ◽  
High Coverage ◽  
Current Reference

After nearly two decades of improvements, the current human reference genome (GRCh38) is the most accurate and complete vertebrate genome ever produced. However, no one chromosome has been finished end to end, and hundreds of unresolved gaps persist 1,2. The remaining gaps include ribosomal rDNA arrays, large near-identical segmental duplications, and satellite DNA arrays. These regions harbor largely unexplored variation of unknown consequence, and their absence from the current reference genome can lead to experimental artifacts and hide true variants when re-sequencing additional human genomes. Here we present a de novo human genome assembly that surpasses the continuity of GRCh38 2, along with the first gapless, telomere-to-telomere assembly of a human chromosome. This was enabled by high-coverage, ultra-long-read nanopore sequencing of the complete hydatidiform mole CHM13 genome, combined with complementary technologies for quality improvement and validation. Focusing our efforts on the human X chromosome 3, we reconstructed the ∼2.8 megabase centromeric satellite DNA array and closed all 29 remaining gaps in the current reference, including new sequence from the human pseudoautosomal regions and cancer-testis ampliconic gene families (CT-X and GAGE). This complete chromosome X, combined with the ultra-long nanopore data, also allowed us to map methylation patterns across complex tandem repeats and satellite arrays for the first time. These results demonstrate that finishing the human genome is now within reach and will enable ongoing efforts to complete the remaining human chromosomes.

scholarly journals Alpha satellite insertions and the evolutionary landscape of centromeres

2021 ◽  
Author(s):  
Giuliana Giannuzzi ◽  
Glennis A. Logsdon ◽  
Nicolas Chatron ◽  
Danny E. Miller ◽  
Julie Reversat ◽  
...  
Keyword(s):  
Satellite Dna ◽  
Adaptive Sampling ◽  
De Novo ◽  
Regulatory Element ◽  
Higher Order ◽  
Alpha Satellite ◽  
Chromosome 18 ◽  
Alpha Satellite Dna ◽  
Repeat Structure ◽  
Human Genome Assembly

AbstractHuman centromeres are composed of alpha satellite DNA hierarchically organized as higher-order repeats and epigenetically specified by CENP-A binding. Current evolutionary models assert that new centromeres are first epigenetically established and subsequently acquire an alphoid array. We identified during routine prenatal aneuploidy diagnosis by FISH a de novo insertion of alpha satellite DNA array (~50-300 kbp) from the centromere of chromosome 18 (D18Z1) into chromosome 15q26 euchromatin. Although bound by CENP-B, this locus did not acquire centromeric functionality as demonstrated by lack of constriction and absence of CENP-A binding. We characterized the rearrangement by FISH and sequencing using Illumina, PacBio, and Nanopore adaptive sampling which revealed that the insertion was associated with a 2.8 kbp deletion and likely occurred in the paternal germline. Notably, the site was located ~10 Mbp distal from the location where a centromere was ancestrally seeded and then became inactive sometime between 20 and 25 million years ago (Mya), in the common ancestor of humans and apes. Long reads spanning either junction showed that the organization of the alphoid insertion followed the 12-mer higher-order repeat structure of the D18Z1 array. Mapping to the CHM13 human genome assembly revealed that the satellite segment transposed from a specific location of chromosome 18 centromere. The rearrangement did not directly disrupt any gene or predicted regulatory element and did not alter the epigenetic status of the surrounding region, consistent with the absence of phenotypic consequences in the carrier. This case demonstrates a likely rare but new class of structural variation that we name ‘alpha satellite insertion’. It also expands our knowledge about the evolutionary life cycle of centromeres, conveying the possibility that alphoid arrays can relocate near vestigial centromeric sites.

scholarly journals An integrated chromosome-scale genome assembly of the Masai giraffe (Giraffa camelopardalis tippelskirchi)

GigaScience ◽  
2019 ◽  
Vol 8 (8) ◽  
Author(s):  
Marta Farré ◽  
Qiye Li ◽  
Iulia Darolti ◽  
Yang Zhou ◽  
Joana Damas ◽  
...  
Keyword(s):  
Genome Assembly ◽  
De Novo ◽  
Sub Saharan Africa ◽  
Food Sources ◽  
Evolutionary Analysis ◽  
Sequencing Data ◽  
Giraffa Camelopardalis ◽  
Artificial Chromosomes ◽  
Research Fields ◽  
Chromosome Fragments

AbstractBackgroundThe Masai giraffe (Giraffa camelopardalis tippelskirchi) is the largest-bodied giraffe and the world's tallest terrestrial animal. With its extreme size and height, the giraffe's unique anatomical and physiological adaptations have long been of interest to diverse research fields. Giraffes are also critical to ecosystems of sub-Saharan Africa, with their long neck serving as a conduit to food sources not shared by other herbivores. Although the genome of a Masai giraffe has been sequenced, the assembly was highly fragmented and suboptimal for genome analysis. Herein we report an improved giraffe genome assembly to facilitate evolutionary analysis of the giraffe and other ruminant genomes.FindingsUsing SOAPdenovo2 and 170 Gbp of Illumina paired-end and mate-pair reads, we generated a 2.6-Gbp male Masai giraffe genome assembly, with a scaffold N50 of 3 Mbp. The incorporation of 114.6 Gbp of Chicago library sequencing data resulted in a HiRise SOAPdenovo + Chicago assembly with an N50 of 48 Mbp and containing 95% of expected genes according to BUSCO analysis. Using the Reference-Assisted Chromosome Assembly tool, we were able to order and orient scaffolds into 42 predicted chromosome fragments (PCFs). Using fluorescence in situ hybridization, we placed 153 cattle bacterial artificial chromosomes onto giraffe metaphase spreads to assess and assign the PCFs on 14 giraffe autosomes and the X chromosome resulting in the final assembly with an N50 of 177.94 Mbp. In this assembly, 21,621 protein-coding genes were identified using both de novo and homology-based predictions.ConclusionsWe have produced the first chromosome-scale genome assembly for a Giraffidae species. This assembly provides a valuable resource for the study of artiodactyl evolution and for understanding the molecular basis of the unique adaptive traits of giraffes. In addition, the assembly will provide a powerful resource to assist conservation efforts of Masai giraffe, whose population size has declined by 52% in recent years.

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