scholarly journals How a Replication Origin and Matrix Attachment Region Accelerate Gene Amplification under Replication Stress in Mammalian Cells

PLoS ONE ◽  
2014 ◽  
Vol 9 (7) ◽  
pp. e103439 ◽  
Author(s):  
Shun-suke Tanaka ◽  
Sho-hei Mitsuda ◽  
Noriaki Shimizu
Keyword(s):  
Gene Amplification ◽  
Mammalian Cells ◽  
Replication Origin ◽  
Replication Stress ◽  
Matrix Attachment Region ◽  
Attachment Region

scholarly journals Gene Amplification and the Extrachromosomal Circular DNA

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1533
Author(s):  
Noriaki Shimizu
Keyword(s):  
Gene Amplification ◽  
Malignant Tumors ◽  
Human Cancer ◽  
Replication Initiation ◽  
Matrix Attachment Region ◽  
Genome Plasticity ◽  
Sequence Element ◽  
Circular Dna ◽  
Attachment Region ◽  
Human Malignant Tumors

Oncogene amplification is closely linked to the pathogenesis of a broad spectrum of human malignant tumors. The amplified genes localize either to the extrachromosomal circular DNA, which has been referred to as cytogenetically visible double minutes (DMs), or submicroscopic episome, or to the chromosomal homogeneously staining region (HSR). The extrachromosomal circle from a chromosome arm can initiate gene amplification, resulting in the formation of DMs or HSR, if it had a sequence element required for replication initiation (the replication initiation region/matrix attachment region; the IR/MAR), under a genetic background that permits gene amplification. In this article, the nature, intracellular behavior, generation, and contribution to cancer genome plasticity of such extrachromosomal circles are summarized and discussed by reviewing recent articles on these topics. Such studies are critical in the understanding and treating human cancer, and also for the production of recombinant proteins such as biopharmaceuticals by increasing the recombinant genes in the cells.

Nucleoskeleton and initiation of DNA replication in metazoan cells

1998 ◽  
Vol 111 (24) ◽  
pp. 3663-3673 ◽  
Author(s):  
J.M. Ortega ◽  
M.L. DePamphilis
Keyword(s):  
Dna Replication ◽  
Mammalian Cells ◽  
S Phase ◽  
Matrix Attachment Region ◽  
Dhfr Gene ◽  
Specific Sequence ◽  
Egg Extract ◽  
Attachment Region ◽  
Nonionic Detergent ◽  
Nuclear Matrix Attachment Region

To determine whether or not initiation sites for DNA replication in mammalian cells are defined by association with nuclear structure, attachments between the nucleoskeleton and the hamster DHFR gene initiation zone were examined. Nucleoskeletons were prepared by encapsulating cells in agarose and then extracting them with a nonionic detergent in a physiological buffer. The fraction of DNA that remained following endonuclease digestion was resistant to salt, sensitive to Sarkosyl, and essentially unchanged by glutaraldehyde crosslinking. Although newly replicated DNA was preferentially attached to the nucleoskeleton, no specific sequence was preferentially attached within a 65 kb locus containing the DHFR gene, two origins of bi-directional replication and at least one nuclear matrix attachment region. Instead, the entire region went from preferentially unattached to preferentially attached as cells progressed from G1 to late S-phase. Thus, initiation sites in mammalian chromosomes are not defined by attachments to the nucleoskeleton. To further assess the relationship between the nucleoskeleton and DNA replication, plasmid DNA containing the DHFR initiation region was replicated in a Xenopus egg extract. All of the DNA associated with the nucleoskeleton prior to S-phase without preference for a particular sequence and was released upon mitosis. However, about half of this DNA was trapped rather than bound to the nucleoskeleton. Thus, attachments to the nucleoskeleton can form in the absence of either DNA replication or transcription, but if they are required for replication, they are not maintained once replication is completed.

scholarly journals An efficient protein production system via gene amplification on a human artificial chromosome and the chromosome transfer to CHO cells

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Takahito Ohira ◽  
Koichi Miyauchi ◽  
Narumi Uno ◽  
Noriaki Shimizu ◽  
Yasuhiro Kazuki ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Gene Amplification ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Artificial Chromosome ◽  
Human Artificial Chromosome ◽  
Matrix Attachment Region ◽  
Amplification Method ◽  
Anti Vegf ◽  
Green Fluorescent

AbstractGene amplification methods play a crucial role in establishment of cells that produce high levels of recombinant protein. However, the stability of such cell lines and the level of recombinant protein produced continue to be suboptimal. Here, we used a combination of a human artificial chromosome (HAC) vector and initiation region (IR)/matrix attachment region (MAR) gene amplification method to establish stable cells that produce high levels of recombinant protein. Amplification of Enhanced green fluorescent protein (EGFP) was induced on a HAC carrying EGFP gene and IR/MAR sequences (EGFP MAR-HAC) in CHO DG44 cells. The expression level of EGFP increased approximately 6-fold compared to the original HAC without IR/MAR sequences. Additionally, anti-vascular endothelial growth factor (VEGF) antibody on a HAC (VEGF MAR-HAC) was also amplified by utilization of this IR/MAR-HAC system, and anti-VEGF antibody levels were approximately 2-fold higher compared with levels in control cells without IR/MAR. Furthermore, the expression of anti-VEGF antibody with VEGF MAR-HAC in CHO-K1 cells increased 2.3-fold compared with that of CHO DG44 cells. Taken together, the IR/MAR-HAC system facilitated amplification of a gene of interest on the HAC vector, and could be used to establish a novel cell line that stably produced protein from mammalian cells.

scholarly journals The Landscape of Non-Viral Gene Augmentation Strategies for Inherited Retinal Diseases

2021 ◽  
Vol 22 (5) ◽  
pp. 2318
Author(s):  
Lyes Toualbi ◽  
Maria Toms ◽  
Mariya Moosajee
Keyword(s):  
Viral Vectors ◽  
Matrix Attachment Region ◽  
Great Promise ◽  
Viral Gene ◽  
Retinal Diseases ◽  
Gene Copies ◽  
Augmentation Strategies ◽  
Viral Gene Therapy ◽  
Effective Option ◽  
Attachment Region

Inherited retinal diseases (IRDs) are a heterogeneous group of disorders causing progressive loss of vision, affecting approximately one in 1000 people worldwide. Gene augmentation therapy, which typically involves using adeno-associated viral vectors for delivery of healthy gene copies to affected tissues, has shown great promise as a strategy for the treatment of IRDs. However, the use of viruses is associated with several limitations, including harmful immune responses, genome integration, and limited gene carrying capacity. Here, we review the advances in non-viral gene augmentation strategies, such as the use of plasmids with minimal bacterial backbones and scaffold/matrix attachment region (S/MAR) sequences, that have the capability to overcome these weaknesses by accommodating genes of any size and maintaining episomal transgene expression with a lower risk of eliciting an immune response. Low retinal transfection rates remain a limitation, but various strategies, including coupling the DNA with different types of chemical vehicles (nanoparticles) and the use of electrical methods such as iontophoresis and electrotransfection to aid cell entry, have shown promise in preclinical studies. Non-viral gene therapy may offer a safer and effective option for future treatment of IRDs.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

scholarly journals Screening of a Protein That Interacts with the Matrix Attachment Region-Binding Protein fromDunaliella salina

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Rui Yang ◽  
Zhaoxi Li ◽  
Yan Lin ◽  
Baosheng Yang ◽  
Tianyun Wang
Keyword(s):  
Binding Protein ◽  
Matrix Attachment Region ◽  
Regulatory Function ◽  
Glutathione S Transferase ◽  
Reading Frame ◽  
Cis Acting ◽  
Binding Partner ◽  
The Matrix ◽  
Attachment Region ◽  
Regulated Functions

We isolated the matrix attachment region-binding protein (MBP) DMBP-1 fromDunaliella salinain our previous studies. MBPs are part of the cis-acting protein family cluster. The regulatory function possibly works through the interaction of the MBPs with each other. In the present study, DMBP-1 was used as the bait in screening theD. salinacDNA library for DMBP-1 interactors that could potentially mediate the DMBP-1-regulated functions. A novel MBP, namely, DMBP-2, was identified as a DMBP-1 binding partner. The cDNA of DMBP-1 was 823 bp long and contained a 573 bp open reading frame, which encoded a polypeptide of 191 amino acids. The interaction between DMBP-2 and DMBP-1 was further confirmed through glutathione S-transferase pull-down assays.

scholarly journals Critical roles of the immunoglobulin intronic enhancers in maintaining the sequential rearrangement of IgH and Igk loci

2006 ◽  
Vol 203 (7) ◽  
pp. 1721-1732 ◽  
Author(s):  
Matthew A. Inlay ◽  
Tongxiang Lin ◽  
Heather H. Gao ◽  
Yang Xu
Keyword(s):  
B Cells ◽  
Developmental Stage ◽  
Heavy Chain ◽  
Light Chain ◽  
Matrix Attachment Region ◽  
Cis Elements ◽  
Wild Type ◽  
Intronic Enhancer ◽  
Attachment Region

V(D)J recombination of immunoglobulin (Ig) heavy (IgH) and light chain genes occurs sequentially in the pro– and pre–B cells. To identify cis-elements that dictate this order of rearrangement, we replaced the endogenous matrix attachment region/Igk intronic enhancer (MiEκ) with its heavy chain counterpart (Eμ) in mice. This replacement, denoted EμR, substantially increases the accessibility of both Vκ and Jκ loci to V(D)J recombinase in pro–B cells and induces Igk rearrangement in these cells. However, EμR does not support Igk rearrangement in pre–B cells. Similar to that in MiEκ−/− pre–B cells, the accessibility of Vκ segments to V(D)J recombinase is considerably reduced in EμR pre–B cells when compared with wild-type pre–B cells. Therefore, Eμ and MiEκ play developmental stage-specific roles in maintaining the sequential rearrangement of IgH and Igk loci by promoting the accessibility of V, D, and J loci to the V(D)J recombinase.

scholarly journals Radiation down-regulates replication origin activity throughout the S phase in mammalian cells

1999 ◽  
Vol 27 (3) ◽  
pp. 803-809 ◽  
Author(s):  
J. M. Larner ◽  
H. Lee ◽  
P. A. Dijkwel ◽  
R. D. Little ◽  
C. L. Schildkraut ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Replication Origin ◽  
S Phase
Sign in / Sign up

Export Citation Format

Share Document