scholarly journals Estimation of the Mutagenic Potential of 8-Oxog in Nuclear Extracts of Mouse Cells Using the “Framed Mirror” Method

2020 ◽  
Vol 3 (1) ◽  
pp. 3
Author(s):  
Leonid V. Gening ◽  
Alexandr A. Volodin ◽  
Konstantin Y. Kazachenko ◽  
Irina V. Makarova ◽  
Vyacheslav Z. Tarantul
Keyword(s):  
Mutation Frequency ◽  
Restriction Site ◽  
Restriction Analysis ◽  
Mutagenic Potential ◽  
Adult Mice ◽  
Nuclear Extracts ◽  
Ecori Restriction ◽  
Mouse Cells

We propose an improved earlier described “mirror” method for detecting in cell nuclear extracts mutations that arise in DNA during its replication due to the misincorporation of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). This method is based on the synthesis of a complementary chain (“mirror”) by nuclear extracts of different mice organs on a template containing 8-oxoG and dideoxycytidine residue (ddC) at the 3′‑end. The “mirror” was amplified by PCR using primers part of which was non-complementary to the template. It allowed obtaining the “framed mirror” products. The misincorporation of dAMP in “framed mirror” products forms an EcoRI restriction site. The restriction analysis of double-stranded “framed mirror” products allows a quantification of the mutation frequency in nuclear extracts. The data obtained show that the mutagenic potential of 8-oxoG markedly varied in different organs of adult mice and embryos.

scholarly journals A Pseudo-Full Mutation Identified in Fragile X Assay Reveals a Novel Base Change Abolishing an EcoRI Restriction Site

2008 ◽  
Vol 10 (5) ◽  
pp. 469-474 ◽  
Author(s):  
Shujian Liang ◽  
Harold N. Bass ◽  
Hanlin Gao ◽  
Caroline Astbury ◽  
Mehdi R. Jamehdor ◽  
...  
Keyword(s):  
Restriction Site ◽  
Fragile X ◽  
Base Change ◽  
Full Mutation ◽  
Ecori Restriction

scholarly journals Bop1 Is a Mouse WD40 Repeat Nucleolar Protein Involved in 28S and 5.8S rRNA Processing and 60S Ribosome Biogenesis

2000 ◽  
Vol 20 (15) ◽  
pp. 5516-5528 ◽  
Author(s):  
Žaklina Strezoska ◽  
Dimitri G. Pestov ◽  
Lester F. Lau
Keyword(s):  
Ribosome Biogenesis ◽  
Ribosomal Subunit ◽  
Weight Fraction ◽  
Rnase A ◽  
Mutant Form ◽  
Rrna Processing ◽  
Ribonucleoprotein Complexes ◽  
Nuclear Extracts ◽  
Mouse Tissues ◽  
Mouse Cells

ABSTRACT We have identified and characterized a novel mouse protein, Bop1, which contains WD40 repeats and is highly conserved through evolution. bop1 is ubiquitously expressed in all mouse tissues examined and is upregulated during mid-G1 in serum-stimulated fibroblasts. Immunofluorescence analysis shows that Bop1 is localized predominantly to the nucleolus. In sucrose density gradients, Bop1 from nuclear extracts cosediments with the 50S-80S ribonucleoprotein particles that contain the 32S rRNA precursor. RNase A treatment disrupts these particles and releases Bop1 into a low-molecular-weight fraction. A mutant form of Bop1, Bop1Δ, which lacks 231 amino acids in the N- terminus, is colocalized with wild-type Bop1 in the nucleolus and in ribonucleoprotein complexes. Expression of Bop1Δ leads to cell growth arrest in the G1phase and results in a specific inhibition of the synthesis of the 28S and 5.8S rRNAs without affecting 18S rRNA formation. Pulse-chase analyses show that Bop1Δ expression results in a partial inhibition in the conversion of the 36S to the 32S pre-rRNA and a complete inhibition of the processing of the 32S pre-rRNA to form the mature 28S and 5.8S rRNAs. Concomitant with these defects in rRNA processing, expression of Bop1Δ in mouse cells leads to a deficit in the cytosolic 60S ribosomal subunits. These studies thus identify Bop1 as a novel, nonribosomal mammalian protein that plays a key role in the formation of the mature 28S and 5.8S rRNAs and in the biogenesis of the 60S ribosomal subunit.

scholarly journals Nitric Oxide as an Endogenous Mutagen for Sendai Virus without Antiviral Activity

2004 ◽  
Vol 78 (16) ◽  
pp. 8709-8719 ◽  
Author(s):  
Jun Yoshitake ◽  
Takaaki Akaike ◽  
Teruo Akuta ◽  
Fumio Tamura ◽  
Tsutomu Ogura ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Viral Replication ◽  
Mutation Frequency ◽  
Sendai Virus ◽  
No Synthase ◽  
Mutagenic Potential ◽  
Gfp Gene ◽  
Sw480 Cells

ABSTRACT Nitric oxide (NO) may affect the genomes of various pathogens, and this mutagenesis is of particular interest for viral pathogenesis and evolution. Here, we investigated the effect of NO on viral replication and mutation. Exogenous or endogenous NO had no apparent antiviral effect on influenza A virus and Sendai virus. The mutagenic potential of NO was analyzed with Sendai virus fused to a green fluorescent protein (GFP) gene (GFP-SeV). GFP-SeV was cultured in SW480 cells transfected with a vector expressing inducible NO synthase (iNOS). The mutation frequency of GFP-SeV was examined by measuring loss of GFP fluorescence of the viral plaques. GFP-SeV mutation frequency in iNOS-SW480 cells was much higher than that in parent SW480 cells and was reduced to the level of mutation frequency in the parent cells by treatment with an NO synthase (NOS) inhibitor. Immunocytochemistry showed generation of more 8-nitroguanosine in iNOS-SW480 cells than in SW480 cells without iNOS transfection. Authentic 8-nitroguanosine added exogenously to GFP-SeV-infected CV-1 cells increased the viral mutation frequency. Profiles of the GFP gene mutations induced by 8-nitroguanosine appeared to resemble those of mutations occurring in mouse lungs in vivo. A base substitution that was characteristic of both mutants (those induced by 8-nitroguanosine and those occurring in vivo) was a C-to-U transition. NO-dependent oxidative stress in iNOS-SW480 cells was also evident. Together, the results indicate unambiguously that NO has mutagenic potential for RNA viruses such as Sendai virus without affecting viral replication, possibly via 8-nitroguanosine formation and cellular oxidative stress.

Identification of an EcoRI restriction site for a rapid and precise determination of β-asarone-free Acorus calamus cytotypes

2005 ◽  
Vol 66 (5) ◽  
pp. 507-514 ◽  
Author(s):  
Cinzia M. Bertea ◽  
Chiara M.M. Azzolin ◽  
Simone Bossi ◽  
Giovanni Doglia ◽  
Massimo E. Maffei
Keyword(s):  
Restriction Site ◽  
Precise Determination ◽  
Acorus Calamus ◽  
Ecori Restriction

scholarly journals A Three Amino Acid Deletion in Glycoprotein IIIa Is Responsible for Type I Glanzmann's Thrombasthenia: Importance of Residues Ile325Pro326Gly327 for β3 Integrin Subunit Association

Blood ◽  
1997 ◽  
Vol 90 (2) ◽  
pp. 669-677 ◽  
Author(s):  
Marie-Christine Morel-Kopp ◽  
Cécile Kaplan ◽  
Valérie Proulle ◽  
Vincent Jallu ◽  
Chantal Melchior ◽  
...  
Keyword(s):  
Amino Acid ◽  
Restriction Site ◽  
Restriction Analysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Type I ◽  
Integrin Subunit ◽  
Wild Type ◽  
Amino Acid Deletion ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract Glanzmann's thrombasthenia (GT) is a recessive autosomal bleeding disorder characterized by abnormal platelet aggregation due to a qualitative or quantitative defect of the glycoprotein (GP) IIb-IIIa complex (integrin αIIbβ3). We describe a new mutation in the GPIIIa gene responsible for type I GT in a consanguineous Algerian family. A discordance between phenotyping and genotyping of the GPIIIa-related HPA-1 platelet alloantigen system in three family members heterozygous for the disease suggested a genetic defect in the GPIIIa gene and a normal GPIIb gene. Sequence analysis of amplified genomic DNA fragments showed a 6-bp deletion in exon 7 of the GPIIIa gene resulting in the amino acid deletion/substitution (Ile325Pro326Gly327 → Met) and creating a new BspHI restriction site. Expression of the mutated integrin β3 subunit cDNA in Chinese hamster ovary cells showed that the cDNA gene was transcribed into a full-length β3 protein with an apparent molecular weight identical to wild-type β3 and accumulated as a single-chain molecule in the cell cytoplasm. The absence of heterodimeric complex formation of the mutant β3 protein with endogeneous αv was shown by immunoprecipitation experiments, intracellular immunofluorescent labeling, and a semiquantitative enzyme-linked immunosorbent assay using the αvβ3 complex-specific monoclonal antibodies LM609 and 23C6. Substitution of the methionine residue by a proline, present at position 326 of wild-type β3, did not restore the ability of the recombinant mutant β3 protein to associate with αv, suggesting that the Ile-Pro-Gly motif is located in a β3 domain important for integrin subunit interaction. The association of a BspHI restriction site with this newly identified mutation has allowed allele-specific restriction analysis of Algerian GT individuals and the identification of two new unrelated type I patients exhibiting the same mutation, suggesting that the described mutation might be significant in this population and that BspHI restriction analysis will provide a useful screening assay for antenatal diagnosis and genetic counselling.

scholarly journals Age-Dependent Poliovirus Replication in the Mouse Central Nervous System Is Determined by Internal Ribosome Entry Site-Mediated Translation

2006 ◽  
Vol 80 (6) ◽  
pp. 2589-2595 ◽  
Author(s):  
Steven Kauder ◽  
Sherry Kan ◽  
Vincent R. Racaniello
Keyword(s):  
Spinal Cord ◽  
Internal Ribosome Entry Site ◽  
Species Specificity ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Adult Mice ◽  
Poliovirus Receptor ◽  
Mouse Cells ◽  
Brain And Spinal Cord

ABSTRACT Mouse cells are not permissive for the replication of human rhinovirus type 2 (HRV2). To determine the role of the HRV2 internal ribosome entry site (IRES) in determining species specificity, a recombinant poliovirus (P1/HRV2) was constructed by substituting the poliovirus IRES with the IRES from HRV2. This recombinant virus replicated in all human and murine cell lines examined, demonstrating that the HRV2 IRES does not limit viral replication in transformed murine cells. P1/HRV2 replicated in the brain and spinal cord in neonatal but not adult mice transgenic for the poliovirus receptor, CD155. Passage of P1/HRV2 in mice led to selection of a virus that caused paralysis in neonatal mice. To determine the relationship between HRV2 IRES-mediated translation and replication of P1/HRV2 in mice, recombinant human adenoviruses were used to express bicistronic mRNAs in murine organs. The results demonstrate that the HRV2 IRES mediates translation in organs of neonatal but not adult mice. These findings show that HRV2 IRES-mediated translation is a determinant of virus replication in the murine brain and spinal cord and suggest that the IRES determines the species specificity of HRV2 infection.

scholarly journals Modification of recombinant human epidermal growth factor (rh-EGF) expression vector by site-directed mutagenesis for therapeutic protein production

2019 ◽  
Vol 24 (1) ◽  
pp. 8
Author(s):  
Achmad Rodiansyah ◽  
Riyona Desvy Pratiwi ◽  
Sabighoh Zanjabila ◽  
Asrul Muhamad Fuad
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Expression Vector ◽  
Restriction Site ◽  
Stop Codon ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Epidermal Growth Factor ◽  
Epidermal Growth ◽  
Ecori Restriction

Recombinant human epidermal growth factor (rh-EGF) has high value in therapies for h-EGF deficiency-related diseases. The expression of the h-EGF gene was designed by using the pET21b(+) vector and Escherichia coli BL21(DE3) as the expression host. In a previous study, the sequence of a 6xHis tag without any restriction sites was fused to the h-EGF gene, yet it was not possible to obtain a purified and single rh-EGF by this approach. In this study, we modified the rh-EGF expression vector using site-directed mutagenesis (SDM) to remove the sequence of the 6xHis tag. The vector modification was carried out by inserting a stop codon and the EcoRI restriction site, along with deleting the 6xHis tag sequence. The results of PCR showed non-specific bands, while 2-step cycles PCR produced one non-specific band, and 3-step cycles PCR produced two non-specific bands. After purification of the PCR products, the SDM-recombinant plasmids treated for template plasmid-free product were transformed into E. coli DH5a. Even though the transformation efficiency was low, the planned gene mutations including the deletion of the 6xHis tag and insertion of the stop codon and EcoRI restriction site in plasmid pET21b(+) were successfully carried out. When using this modified vector in expression studies, rh-EGF of a similar size to that of the rh-EGF standard and approximately 1 kDa smaller than the rh-EGF-6xHis of the previous study was obtained.

scholarly journals Rapid diagnosis of beta-thalassemia mutations in Chinese by naturally and amplified created restriction sites

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 2092-2096 ◽  
Author(s):  
JG Chang ◽  
PH Chen ◽  
SS Chiou ◽  
LS Lee ◽  
LI Perng ◽  
...  
Keyword(s):  
Restriction Site ◽  
Globin Gene ◽  
Restriction Enzymes ◽  
Chinese Patients ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Simple Method ◽  
Rare Mutations ◽  
Restriction Sites ◽  
Ecori Restriction

Abstract We developed a rapid and simple method to diagnose the molecular defects of beta-thalassemia in Chinese patients. This method involves the selective amplification of a DNA fragment from human beta globin gene with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. To detect the 4-nucleotide deletion of codon 41–42, we introduced a single mismatch nucleotide into the 3′ end of the upstream primer to create an artificial Taq I restriction site. With a similar approach, an artificial Rsa I site was generated to detect the nucleotide 654 mutation (C-->T) of IVS-2, and Alu I restriction site was created to detect the codon 17 mutation (A-->T), and EcoRI restriction site was created for the -28 mutation (A-->G), a Rsa I restriction site was created for the nucleotide 5 mutation (G-- >C) of IVS-1, and a Spe I restriction site was created to distinguish the codon 71 (+T) and codon 71/72 (+A) mutations from a normal sequence. The other eight rare mutations that occur in the genes of the Chinese people naturally create or abolish restriction sites. Using this kind of approach, we are able to provide a simple, rapid, accurate, and nonradioactive method to detect the genetic defects of beta-thalassemia in the Chinese population. It should be used not only for routine screening but also for prenatal diagnosis.

scholarly journals Transfected DNA is mutated in monkey, mouse, and human cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 1951-1960 ◽  
Author(s):  
J S Lebkowski ◽  
R B DuBridge ◽  
E A Antell ◽  
K S Greisen ◽  
M P Calos
Keyword(s):  
Mammalian Cells ◽  
Time Course ◽  
Mutation Frequency ◽  
Spontaneous Mutation ◽  
Bacterial Cells ◽  
Shuttle Vectors ◽  
High Mutation Frequency ◽  
Mouse Cells ◽  
Nih 3T3 ◽  
Viral Sequences

Papovavirus-based shuttle vectors containing the bacterial lacI gene were used to show that a mutation frequency in the range of 1% occurs in lacI when such vectors are transfected into COS7 and CV-1 simian cells, NIH 3T3, 3T6, L, and C127 mouse cells, and human 293 and HeLa cells. This frequency is approximately four orders of magnitude higher than the spontaneous mutation frequency in either mammalian or bacterial cells. The mutations are predominantly base substitutions and deletions and also include insertions from the mammalian genome. Time course experiments argue that mutagenesis occurs soon after arrival of the DNA into the nucleus. However, replication of the vector is not required since mutations occur even when the vector lacks all viral sequences. The high mutation frequency appears to be the characteristic outcome of transfection of DNA into mammalian cells.

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