scholarly journals IN SILICO OLIGONUCLEOTIDE PRIMER DESIGN FOR Campylobacter jejuni cytolethal distending toxin B GENE AMPLIFICATION

2021 ◽  
Vol 4 (1) ◽  
pp. 53
Author(s):  
Rian Ka Praja
Keyword(s):  
Campylobacter Jejuni ◽  
In Silico ◽  
Oligonucleotide Primer ◽  
Cytolethal Distending Toxin ◽  
Dna Double Strand Breaks ◽  
Toxin B ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
M Phase ◽  
Reverse Primer

<p>Cytolethal distending toxin B (cdtB) is a genotoxinexpressed by <em>Campylobacter jejuni</em>. cdtB is a DNasethatinduces DNA double-strand breaks (DSB) in the nucleus causing cell cycle arrest at the G2/M phase and apoptosis. This study aimed to design and analyze in silico primer pairs to amplify cdtB gene of<em>C. jejuni</em>.The cdtB gene sequence with accession number AY445094.1was retrieved from GenBank NCBI and primer pairs were designed by using Primer-BLAST. Further analysis of primer quality such asself dimer, hairpin, repeats, and runwere done by NetPrimer. The results showed that forward primer pair 3 (5’-AGCAAGTGGAGTGTTAGCGT-3’) and reverse primer pair 3 (5’- TTGGAGTGGCTGTTCTTGGT-3’) met requirements as an ideal primer set to amplify cdtB gene in the term of primer length, Tm and GC% with a product length of 103 bp.In addition, based on NetPrimer analysis results, this primer pair had no self dimer, hairpin, repeats, and run. It can be concluded that a primer set to amplify cdtB gene of <em>C. jejuni</em>has been successfully designed<em>.</em>However, a wet experiment is needed to run this primer set in the laboratory setting.</p><p> </p><p>Keywords: <em>Campylobacter jejuni</em>, cdtB gene, in silico, primer.</p>

scholarly journals Campylobacter jejuni Cytolethal Distending Toxin Causes a G2-Phase Cell Cycle Block

1998 ◽  
Vol 66 (5) ◽  
pp. 1934-1940 ◽  
Author(s):  
Chris A. Whitehouse ◽  
Paul B. Balbo ◽  
Everett C. Pesci ◽  
Daniel L. Cottle ◽  
Peter M. Mirabito ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Campylobacter Jejuni ◽  
Hela Cells ◽  
Chromatin Condensation ◽  
Phase Cell ◽  
Specific Cell ◽  
Cytolethal Distending Toxin ◽  
Cycle Arrest ◽  
M Phase

ABSTRACT Cytolethal distending toxin (CDT) from the diarrheagenic bacteriumCampylobacter jejuni was shown to cause a rapid and specific cell cycle arrest in HeLa and Caco-2 cells. Within 24 h of treatment, CDT caused HeLa cells to arrest with a 4N DNA content, indicative of cells in G2 or early M phase. Immunofluorescence studies indicated that the arrested cells had not entered M phase, since no evidence of tubulin reorganization or chromatin condensation was visible. CDT treatment was also shown to cause HeLa cells to accumulate the inactive, tyrosine-phosphorylated form of CDC2. These results indicated that CDT treatment results in a failure to activate CDC2, which leads to cell cycle arrest in G2. This mechanism of action is novel for a bacterial toxin and provides a model for the generation of diarrheal disease byC. jejuni and other diarrheagenic bacteria that produce CDT.

scholarly journals Mitotic cells can repair DNA double-strand breaks via a homology-directed pathway

2020 ◽  
Vol 62 (1) ◽  
pp. 25-33
Author(s):  
Yuki Sakamoto ◽  
Tetsuya Kokuta ◽  
Ai Teshigahara ◽  
Kenta Iijima ◽  
Hiroyuki Kitao ◽  
...  
Keyword(s):  
Mitotic Cell ◽  
S Phase ◽  
Double Strand Breaks ◽  
G1 Phase ◽  
Dna Double Strand Breaks ◽  
Major Pathway ◽  
Strand Breaks ◽  
Homology Directed Repair ◽  
Repair Efficiency ◽  
M Phase

Abstract The choice of repair pathways of DNA double-strand breaks (DSBs) is dependent upon the cell cycle phases. While homologous recombination repair (HRR) is active between the S and G2 phases, its involvement in mitotic DSB repair has not been examined in detail. In the present study, we developed a new reporter assay system to detect homology-directed repair (HDR), a major pathway used for HRR, in combination with an inducible DSB-generation system. As expected, the maximal HDR activity was observed in the late S phase, along with minimal activity in the G1 phase and at the G1/S boundary. Surprisingly, significant HDR activity was observed in M phase, and the repair efficiency was similar to that observed in late S phase. HDR was also confirmed in metaphase cells collected with continuous colcemid exposure. ChIP assays revealed the recruitment of RAD51 to the vicinity of DSBs in M phase. In addition, the ChIP assay for gamma-H2AX and phosphorylated DNA-PKcs indicated that a part of M-phase cells with DSBs could proceed into the next G1 phase. These results provide evidence showing that a portion of mitotic cell DSBs are undoubtedly repaired through action of the HDR repair pathway.

scholarly journals The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection

Parasitology ◽  
2013 ◽  
Vol 140 (4) ◽  
pp. 547-559 ◽  
Author(s):  
RITA DE CÁSSIA VIVEIROS DA SILVEIRA ◽  
MARCELO SANTOS DA SILVA ◽  
VINÍCIUS SANTANA NUNES ◽  
ARINA MARINA PEREZ ◽  
MARIA ISABEL NOGUEIRA CANO
Keyword(s):  
S Phase ◽  
Leishmania Amazonensis ◽  
Nuclear Accumulation ◽  
Dna Double Strand Breaks ◽  
Telomeric Dna ◽  
Strand Breaks ◽  
Cycle Arrest ◽  
Telomere Protection ◽  
Damage Response

SUMMARYWe have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

scholarly journals Loss of p53 suppresses replication-stress-induced DNA breakage in G1/S checkpoint deficient cells

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Bente Benedict ◽  
Tanja van Harn ◽  
Marleen Dekker ◽  
Simone Hermsen ◽  
Asli Kucukosmanoglu ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cancer Cells ◽  
Replication Stress ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Origin Firing ◽  
Strand Breaks ◽  
Dna Breakage ◽  
Cycle Arrest

In cancer cells, loss of G1/S control is often accompanied by p53 pathway inactivation, the latter usually rationalized as a necessity for suppressing cell cycle arrest and apoptosis. However, we found an unanticipated effect of p53 loss in mouse and human G1-checkpoint-deficient cells: reduction of DNA damage. We show that abrogation of the G1/S-checkpoint allowed cells to enter S-phase under growth-restricting conditions at the expense of severe replication stress manifesting as decelerated DNA replication, reduced origin firing and accumulation of DNA double-strand breaks. In this system, loss of p53 allowed mitogen-independent proliferation, not by suppressing apoptosis, but rather by restoring origin firing and reducing DNA breakage. Loss of G1/S control also caused DNA damage and activation of p53 in an in vivo retinoblastoma model. Moreover, in a teratoma model, loss of p53 reduced DNA breakage. Thus, loss of p53 may promote growth of incipient cancer cells by reducing replication-stress-induced DNA damage.

scholarly journals Fusion Tyrosine Kinases Induce Drug Resistance by Stimulation of Homology-Dependent Recombination Repair, Prolongation of G2/M Phase, and Protection from Apoptosis

2002 ◽  
Vol 22 (12) ◽  
pp. 4189-4201 ◽  
Author(s):  
Artur Slupianek ◽  
Grazyna Hoser ◽  
Ireneusz Majsterek ◽  
Agnieszka Bronisz ◽  
Maciej Malecki ◽  
...  
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Tyrosine Kinases ◽  
Transformed Cells ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Recombination Repair ◽  
M Phase ◽  
Induced Apoptosis ◽  
In Cells

ABSTRACT Fusion tyrosine kinases (FTKs) such as BCR/ABL, TEL/ABL, TEL/JAK2, TEL/PDGFβR, TEL/TRKC(L), and NPM/ALK arise from reciprocal chromosomal translocations and cause acute and chronic leukemias and non-Hodgkin's lymphoma. FTK-transformed cells displayed drug resistance against the cytostatic drugs cisplatin and mitomycin C. These cells were not protected from drug-mediated DNA damage, implicating activation of the mechanisms preventing DNA damage-induced apoptosis. Various FTKs, except TEL/TRKC(L), can activate STAT5, which may be required to induce drug resistance. We show that STAT5 is essential for FTK-dependent upregulation of RAD51, which plays a central role in homology-dependent recombinational repair (HRR) of DNA double-strand breaks (DSBs). Elevated levels of Rad51 contributed to the induction of drug resistance and facilitation of the HRR in FTK-transformed cells. In addition, expression of antiapoptotic protein Bcl-xL was enhanced in cells transformed by the FTKs able to activate STAT5. Moreover, cells transformed by all examined FTKs displayed G2/M delay upon drug treatment. Individually, elevated levels of Rad51, Bcl-xL, or G2/M delay were responsible for induction of a modest drug resistance. Interestingly, combination of these three factors in nontransformed cells induced drug resistance of a magnitude similar to that observed in cells expressing FTKs activating STAT5. Thus, we postulate that RAD51-dependent facilitation of DSB repair, antiapoptotic activity of Bcl-xL, and delay in progression through the G2/M phase work in concert to induce drug resistance in FTK-positive leukemias and lymphomas.

scholarly journals Through Its Nonstructural Protein NS1, Parvovirus H-1 Induces Apoptosis via Accumulation of Reactive Oxygen Species

2010 ◽  
Vol 84 (12) ◽  
pp. 5909-5922 ◽  
Author(s):  
Georgi Hristov ◽  
Melanie Krämer ◽  
Junwei Li ◽  
Nazim El-Andaloussi ◽  
Rodrigo Mora ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Nonstructural Protein ◽  
Reactive Oxygen ◽  
Double Strand Breaks ◽  
Oxygen Species ◽  
Ns1 Protein ◽  
Dna Double Strand Breaks ◽  
Caspase 9 ◽  
Strand Breaks ◽  
Cycle Arrest

ABSTRACT The rat parvovirus H-1 (H-1PV) attracts high attention as an anticancer agent, because it is not pathogenic for humans and has oncotropic and oncosuppressive properties. The viral nonstructural NS1 protein is thought to mediate H-1PV cytotoxicity, but its exact contribution to this process remains undefined. In this study, we analyzed the effects of the H-1PV NS1 protein on human cell proliferation and cell viability. We show that NS1 expression is sufficient to induce the accumulation of cells in G2 phase, apoptosis via caspase 9 and 3 activation, and cell lysis. Similarly, cells infected with wild-type H-1PV arrest in G2 phase and undergo apoptosis. Furthermore, we also show that both expression of NS1 and H-1PV infection lead to higher levels of intracellular reactive oxygen species (ROS), associated with DNA double-strand breaks. Antioxidant treatment reduces ROS levels and strongly decreases NS1- and virus-induced DNA damage, cell cycle arrest, and apoptosis, indicating that NS1-induced ROS are important mediators of H-1PV cytotoxicity.

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