scholarly journals Targeting Chromosome Trisomy for Chromosome Editing

2021 ◽  
Author(s):  
Takuya Abe ◽  
Yuya Suzuki ◽  
Kouji Hirota
Keyword(s):  
Large Scale ◽  
Cellular Proliferation ◽  
Regulatory Protein ◽  
Chromosome Loss ◽  
Chromosome 2 ◽  
Telomere Repeat ◽  
Artificial Chromosomes ◽  
Counter Selection ◽  
Additional Chromosome ◽  
Dt40 Cells

Abstract A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection makers. Upon construction of cells carrying makers targeted in one of the trisomic chromosome 2s, cells that have lost makers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes. (197 words)

scholarly journals Targeting chromosome trisomy for chromosome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuya Abe ◽  
Yuya Suzuki ◽  
Teppei Ikeya ◽  
Kouji Hirota
Keyword(s):  
Large Scale ◽  
Cellular Proliferation ◽  
Regulatory Protein ◽  
Chromosome Loss ◽  
Chromosome 2 ◽  
Telomere Repeat ◽  
Artificial Chromosomes ◽  
Counter Selection ◽  
Additional Chromosome ◽  
Dt40 Cells

AbstractA trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

scholarly journals Ploidy reduction in Saccharomyces cerevisiae

2007 ◽  
Vol 4 (1) ◽  
pp. 91-94 ◽  
Author(s):  
Aleeza C Gerstein ◽  
Rachel M McBride ◽  
Sarah P Otto
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Large Scale ◽  
Previous Experiment ◽  
Comparative Genomic ◽  
Chromosome Loss ◽  
Culture Experiment ◽  
Additional Chromosome ◽  
Evolution Experiment ◽  
One Step ◽  
Insight Into

Large-scale transitions in genome size from tetraploid to diploid were observed during a previous 1800-generation evolution experiment in Saccharomyces cerevisiae . Whether the transitions occurred via a one-step process (tetraploid to diploid) or through multiple steps (through ploidy intermediates) remained unclear. To provide insight into the mechanism involved, we investigated whether triploid-sized cells sampled from the previous experiment could also undergo ploidy loss. A batch culture experiment was conducted for approximately 200 generations, starting from four triploid-sized colonies and one contemporaneous tetraploid-sized colony. Ploidy reduction towards diploidy was observed in both triploid and tetraploid lines. Comparative genomic hybridization indicated the presence of aneuploidy in both the founder and the evolved colonies. The specific aneuploidies involved suggest that chromosome loss was not haphazard but that nearly full sets of chromosomes were lost at once, with some additional chromosome mis-segregation events. These results suggest the existence of a mitotic mechanism allowing the elimination of an entire set of chromosomes in S. cerevisiae , thereby reducing the ploidy level.

Large-scale cloning of human chromosome 2-specific yeast artificial chromosomes (YACs) using an interspersed repetitive sequences (IRS)-PCR approach

Genomics ◽  
1995 ◽  
Vol 26 (2) ◽  
pp. 178-191 ◽  
Author(s):  
Jing Liu ◽  
Vincent P. Stanton ◽  
T.Mary Fujiwara ◽  
Jian-Xue Wang ◽  
Rebeca Rezonzew ◽  
...  
Keyword(s):  
Human Chromosome ◽  
Large Scale ◽  
Repetitive Sequences ◽  
Chromosome 2 ◽  
Artificial Chromosomes ◽  
Yeast Artificial Chromosomes

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.

Segregation of recessive phenotypes in somatic cell hybrids: role of mitotic recombination, gene inactivation, and chromosome nondisjunction

1981 ◽  
Vol 1 (4) ◽  
pp. 336-346
Author(s):  
C E Campbell ◽  
R G Worton
Keyword(s):  
Somatic Cell ◽  
Chinese Hamster ◽  
Mitotic Recombination ◽  
Chinese Hamster Cell ◽  
Gene Inactivation ◽  
Chromosome Loss ◽  
Resistance Locus ◽  
Chromosome 2 ◽  
Somatic Cell Hybrids ◽  
Cell Hybrids

Somatic cell hybrids heterozygous at the emetine resistance locus (emtr/emt+) or the chromate resistance locus (chrr/chr+) are known to segregate the recessive drug resistance phenotype at high frequency. We have examined mechanisms of segregation in Chinese hamster cell hybrids heterozygous at these two loci, both of which map to the long arm of Chinese hamster chromosome 2. To follow the fate of chromosomal arms through the segregation process, our hybrids were also heterozygous at the mtx (methotrexate resistance) locus on the short arm of chromosome 2 and carried cytogenetically marked chromosomes with either a short-arm deletion (2p-) or a long-arm addition (2q+). Karyotype and phenotype analysis of emetine- or chromate-resistant segregants from such hybrids allowed us to distinguish four potential segregation mechanisms: (i) loss of the emt+- or chr+-bearing chromosome; (ii) mitotic recombination between the centromere and the emt or chr loci, giving rise to homozygous resistant segregants; (iii) inactivation of the emt+ or chr+ alleles; and (iv) loss of the emt+- or chr+-bearing chromosome with duplication of the homologous chromosome carrying the emtr or chrr allele. Of 48 independent segregants examined, only 9 (20%) arose by simple chromosome loss. Two segregants (4%) were consistent with a gene inactivation mechanism, but because of their rarity, other mechanisms such as mutation or submicroscopic deletion could not be excluded. Twenty-one segregants (44%) arose by either mitotic recombination or chromosome loss and duplication; the two mechanisms were not distinguishable in that experiment. Finally, in hybrids allowing these two mechanisms to be distinguished, 15 segregants (31%) arose by chromosome loss and duplication, and none arose by mitotic recombination.

scholarly journals Crystal structure of a tankyrase 1–telomere repeat factor 1 complex

2016 ◽  
Vol 72 (4) ◽  
pp. 320-327 ◽  
Author(s):  
Bo Li ◽  
Ruihong Qiao ◽  
Zhizhi Wang ◽  
Weihong Zhou ◽  
Xin Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Large Scale ◽  
Sparse Matrix ◽  
Critical Role ◽  
Three Dimensional ◽  
Mitotic Cell ◽  
Ankyrin Repeat ◽  
Dimensional Structure ◽  
Telomere Repeat ◽  
Tankyrase 1

Telomere repeat factor 1 (TRF1) is a subunit of shelterin (also known as the telosome) and plays a critical role in inhibiting telomere elongation by telomerase. Tankyrase 1 (TNKS1) is a poly(ADP-ribose) polymerase that regulates the activity of TRF1 through poly(ADP-ribosyl)ation (PARylation). PARylation of TRF1 by TNKS1 leads to the release of TRF1 from telomeres and allows telomerase to access telomeres. The interaction between TRF1 and TNKS1 is thus important for telomere stability and the mitotic cell cycle. Here, the crystal structure of a complex between the N-terminal acidic domain of TRF1 (residues 1–55) and a fragment of TNKS1 covering the second and third ankyrin-repeat clusters (ARC2-3) is presented at 2.2 Å resolution. The TNKS1–TRF1 complex crystals were optimized using an `oriented rescreening' strategy, in which the initial crystallization condition was used as a guide for a second round of large-scale sparse-matrix screening. This crystallographic and biochemical analysis provides a better understanding of the TRF1–TNKS1 interaction and the three-dimensional structure of the ankyrin-repeat domain of TNKS.

scholarly journals A Case Report and Genetic Characterization of a Massive Acinic Cell Carcinoma of the Parotid with Delayed Distant Metastases

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Anthony C. Nichols ◽  
Michelle Chan-Seng-Yue ◽  
John Yoo ◽  
Sumit K. Agrawal ◽  
Maud H. W. Starmans ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Cell Carcinoma ◽  
Large Scale ◽  
Cellular Proliferation ◽  
Genetic Characterization ◽  
Distant Metastases ◽  
Acinic Cell Carcinoma ◽  
Nonsynonymous Mutation ◽  
Acinic Cell

We describe the presentation, management, and clinical outcome of a massive acinic cell carcinoma of the parotid gland. The primary tumor and blood underwent exome sequencing which revealed deletions in CDKN2A as well as PPP1R13B, which induces p53. A damaging nonsynonymous mutation was noted in EP300, a histone acetylase which plays a role in cellular proliferation. This study provides the first insights into the genetic underpinnings of this cancer. Future large-scale efforts will be necessary to define the mutational landscape of salivary gland malignancies to identify therapeutic targets and biomarkers of treatment failure.

scholarly journals Genome Assemblies of the Warthog and Kenyan Domestic Pig Provide Insights into Suidae Evolution and Candidate Genes for African Swine Fever Tolerance

2021 ◽  
Author(s):  
Wen Feng ◽  
Lei Zhou ◽  
Pengju Zhao ◽  
Heng Du ◽  
Chenguang Diao ◽  
...  
Keyword(s):  
Large Scale ◽  
Genetic Resistance ◽  
African Swine Fever ◽  
Gene Families ◽  
Specific Gene ◽  
Chromosome 2 ◽  
Sequencing Data ◽  
Domestic Pig ◽  
Phacochoerus Africanus ◽  
Contraction And Expansion

As warthog (Phacochoerus africanus) has innate immunity against African swine fever (ASF), it is critical to understanding the evolutionary novelty of warthog to explain its specific ASF resistance. Here, we present two completed new genomes of one warthog and one Kenyan domestic pig, as the fundamental genomic references to decode the genetic mechanism on ASF tolerance. Our results indicated, multiple genomic variations, including gene losses, independent contraction and expansion of specific gene families, likely moulded warthog's genome to adapt the environment. Importantly, the analysis of presence and absence of genomic sequences revealed that, the warthog genome had a DNA sequence absence of the lactate dehydrogenase B (LDHB) gene on chromosome 2 compared to the reference genome. The overexpression and siRNA of LDHB indicated that its inhibition on the replication of ASFV. The Combining with large scale sequencing data of 123 pigs from all over world, contraction and expansion of TRIM genes families revealed that TRIM family genes in the warthog genome were potentially responsible for its tolerance to ASF. Our results will help further improve the understanding of genetic resistance ASF in pigs.

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.

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