De Novo Generation of Satellite DNA-Based Artificial Chromosomes by Induced Large-Scale Amplification

2011 ◽  
pp. 111-125 ◽  
Author(s):  
Erika Csonka
Keyword(s):  
Satellite Dna ◽  
Large Scale ◽  
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.

Mammalian artificial chromosome pilot production facility: Large-scale isolation of functional satellite DNA-based artificial chromosomes

Cytometry ◽  
1999 ◽  
Vol 35 (2) ◽  
pp. 129-133 ◽  
Author(s):  
Gary deJong ◽  
Ad�le H. Telenius ◽  
H�kan Telenius ◽  
Carl F. Perez ◽  
Jan I. Drayer ◽  
...  
Keyword(s):  
Satellite Dna ◽  
Large Scale ◽  
Artificial Chromosome ◽  
Production Facility ◽  
Artificial Chromosomes ◽  
Mammalian Artificial Chromosome

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.

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

A Target-Based Method for Designing Heterochiral Cyclic Peptide Binders: De Novo Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
Cyclic Peptide ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

Target-Templated de novo Design of Macrocyclic D-/L-Peptides: Inhibitors of the PD-1/PD-L1 Interaction

2020 ◽  
Author(s):  
Salvador Guardiola ◽  
Monica Varese ◽  
Xavier Roig ◽  
Jesús Garcia ◽  
Ernest Giralt
Keyword(s):  
Protein Interactions ◽  
Cyclic Peptides ◽  
General Framework ◽  
Large Scale ◽  
De Novo ◽  
Inhibitory Effect ◽  
Original Text ◽  
Protein Protein Interactions ◽  
Retraction Notice ◽  
Pharmaceutical Properties

<p>NOTE: This preprint has been retracted by consensus from all authors. See the retraction notice in place above; the original text can be found under "Version 1", accessible from the version selector above.</p><p><br></p><p>------------------------------------------------------------------------</p><p><br></p><p>Peptides, together with antibodies, are among the most potent biochemical tools to modulate challenging protein-protein interactions. However, current structure-based methods are largely limited to natural peptides and are not suitable for designing target-specific binders with improved pharmaceutical properties, such as macrocyclic peptides. Here we report a general framework that leverages the computational power of Rosetta for large-scale backbone sampling and energy scoring, followed by side-chain composition, to design heterochiral cyclic peptides that bind to a protein surface of interest. To showcase the applicability of our approach, we identified two peptides (PD-<i>i</i>3 and PD-<i>i</i>6) that target PD-1, a key immune checkpoint, and work as protein ligand decoys. A comprehensive biophysical evaluation confirmed their binding mechanism to PD-1 and their inhibitory effect on the PD-1/PD-L1 interaction. Finally, elucidation of their solution structures by NMR served as validation of our <i>de novo </i>design approach. We anticipate that our results will provide a general framework for designing target-specific drug-like peptides.<i></i></p>

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

High-Resolution Pachytene Chromosome Mapping of Bacterial Artificial Chromosomes Anchored by Genetic Markers Reveals the Centromere Location and the Distribution of Genetic Recombination Along Chromosome 10 of Rice

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1749-1757 ◽  
Author(s):  
Zhukuan Cheng ◽  
Gernot G Presting ◽  
C Robin Buell ◽  
Rod A Wing ◽  
Jiming Jiang
Keyword(s):  
Large Scale ◽  
Genetic Recombination ◽  
Physical Map ◽  
Rice Chromosome ◽  
Resolving Power ◽  
Pachytene Chromosome ◽  
Chromosome 10 ◽  
Bac Clone ◽  
Fish Mapping ◽  
Artificial Chromosomes

AbstractLarge-scale physical mapping has been a major challenge for plant geneticists due to the lack of techniques that are widely affordable and can be applied to different species. Here we present a physical map of rice chromosome 10 developed by fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BAC) clones on meiotic pachytene chromosomes. This physical map is fully integrated with a genetic linkage map of rice chromosome 10 because each BAC clone is anchored by a genetically mapped restriction fragment length polymorphism marker. The pachytene chromosome-based FISH mapping shows a superior resolving power compared to the somatic metaphase chromosome-based methods. The telomere-centromere orientation of DNA clones separated by 40 kb can be resolved on early pachytene chromosomes. Genetic recombination is generally evenly distributed along rice chromosome 10. However, the highly heterochromatic short arm shows a lower recombination frequency than the largely euchromatic long arm. Suppression of recombination was found in the centromeric region, but the affected region is far smaller than those reported in wheat and barley. Our FISH mapping effort also revealed the precise genetic position of the centromere on chromosome 10.

Abstract 17081: End Stage Mitochondrial Cardiomyopathy And Heart Transplantation Due To Pathogenic Biallelic C1QBP Variants

Circulation ◽  
2021 ◽  
Vol 144 (Suppl_2) ◽  
Author(s):  
Nicholas S Wilcox ◽  
Stuart Prenner ◽  
Marisa Cevasco ◽  
Courtney Condit ◽  
Amy Goldstein ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Heart Transplantation ◽  
Genome Sequencing ◽  
Large Scale ◽  
De Novo ◽  
Late Onset ◽  
Genetic Disorder ◽  
Disease Onset ◽  
Serum Lactate ◽  
Metabolic Decompensation

Case Presentation: A 29-year-old male with LVH diagnosed in childhood was admitted with acute HF. TTE showed LVEF 5-10% and LV thrombi for which he was anticoagulated. He received inappropriate ICD shocks due to T wave oversensing, leading to cardiogenic shock requiring VA-ECMO support. Serum lactate peaked at 17 mmol/L due to cardiac and metabolic decompensation. He underwent heart transplantation (HT) on hospital day (HD) 8 and tolerated standard immunosuppression. First endomyocardial biopsy showed acute cellular rejection requiring pulse steroids. He was discharged on HD 33. Trio whole exome and mitochondrial genome sequencing revealed biallelic variants in complement component 1Q subcomponent-binding protein ( C1QBP ), due to a maternally inherited likely pathogenic variant c.612C>G (p.F204L in exon 5) and an apparently de novo deletion of 17p13.2, spanning exons 4-6 of C1QBP and exon 6 of the RPAIN gene. Mitochondrial genome sequencing of the explanted heart revealed multiple large-scale mitochondrial DNA deletions at 33% heteroplasmy. Discussion: C1QBP variants are associated with mitochondrial and multi-organ dysfunction. Only 12 patients exhibiting biallelic C1QBP variants are reported. Four died in the peripartum period due to fetal hydrops or HF; 5 exhibited early-onset cardiomyopathy (CM); 3 others had late-onset ophthalmoplegia without CM. The p.F204L variant has been reported in 1 patient with compound C1QBP p.F204L/p.C186S heterozygosity who died from hydrops fetalis and a second with p.F204L homozygosity with late-onset ophthalmoplegia and skeletal myopathy without CM. Differences in the size, heteroplasmy, and tissue distribution of mitochondrial genome secondary deletions may explain variability in disease onset and progression. We present the first patient with biallelic pathogenic C1QBP gene variants with mitochondrial CM to undergo HT and highlight the diagnosis and management of an exceptionally uncommon genetic disorder.

Sign in / Sign up

Export Citation Format

Share Document