scholarly journals An in vitro assay for frameshift mutations: hotspots for deletions of 1 bp by Klenow-fragment polymerase share a consensus DNA sequence.

Genetics ◽  
1988 ◽  
Vol 118 (2) ◽  
pp. 181-191
Author(s):  
J G de Boer ◽  
L S Ripley
Keyword(s):  
Bacteriophage T4 ◽  
Consensus Sequence ◽  
Dna Repeats ◽  
Klenow Fragment ◽  
Purine Base ◽  
Vitro Assay ◽  
Reading Frame ◽  
Dna Primers

Abstract The fidelity of in vitro DNA synthesis catalyzed by the large fragment of DNA polymerase I was examined. The templates, specifically designed to detect shifts to the +1 or to the -1 reading frame, are composites of M13mp8 and bacteriophage T4 rIIB DNA and were designed to assist in the identification of the types of frameshifts that are the specific consequence of DNA polymerization errors. In vitro polymerization by the Klenow fragment produced only deletions, rather than the mixture of duplications and deletions characteristic of in vivo frameshifts. The most frequent frameshifts were deletions of 1 bp opposite a template purine base. Hotspots for these deletions occurred when the template purine immediately preceded the template sequence TT. The highest mutation frequencies were seen when the TTPu consensus sequence was adjacent to G:C rich sequences in the 3' direction. The nature of the consensus sequence itself distinguishes this 1-bp deletion mechanism from those operating in DNA repeats and attributed to the misalignment of DNA primers during synthesis. Deletions that were larger than 1 or 2 bp isolated after in vitro replication were consistent with the misalignment of the primer. Deletions of 2 bp and complex frameshifts (the replacement of AA by C) were also found. Mechanisms that may account for these mutations are discussed.

scholarly journals Telomere DNA G-quadruplex folding within actively extending human telomerase

2019 ◽  
Vol 116 (19) ◽  
pp. 9350-9359 ◽  
Author(s):  
Linnea I. Jansson ◽  
Jendrik Hentschel ◽  
Joseph W. Parks ◽  
Terren R. Chang ◽  
Cheng Lu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Time Series Data ◽  
Resonance Energy ◽  
Series Data ◽  
Dna Repeats ◽  
Telomere Repeat ◽  
G Quadruplex ◽  
Dna Primers

Telomerase reverse transcribes short guanine (G)-rich DNA repeat sequences from its internal RNA template to maintain telomere length. G-rich telomere DNA repeats readily fold into G-quadruplex (GQ) structures in vitro, and the presence of GQ-prone sequences throughout the genome introduces challenges to replication in vivo. Using a combination of ensemble and single-molecule telomerase assays, we discovered that GQ folding of the nascent DNA product during processive addition of multiple telomere repeats modulates the kinetics of telomerase catalysis and dissociation. Telomerase reactions performed with telomere DNA primers of varying sequence or using GQ-stabilizing K+ versus GQ-destabilizing Li+ salts yielded changes in DNA product profiles consistent with formation of GQ structures within the telomerase–DNA complex. Addition of the telomerase processivity factor POT1–TPP1 altered the DNA product profile, but was not sufficient to recover full activity in the presence of Li+ cations. This result suggests GQ folding synergizes with POT1–TPP1 to support telomerase function. Single-molecule Förster resonance energy transfer experiments reveal complex DNA structural dynamics during real-time catalysis in the presence of K+ but not Li+, supporting the notion of nascent product folding within the active telomerase complex. To explain the observed distributions of telomere products, we globally fit telomerase time-series data to a kinetic model that converges to a set of rate constants describing each successive telomere repeat addition cycle. Our results highlight the potential influence of the intrinsic folding properties of telomere DNA during telomerase catalysis, and provide a detailed characterization of GQ modulation of polymerase function.

scholarly journals SEQUENCE AND TRANSCRIPTS OF THE BACTERIOPHAGE T4 DNA REPAIR GENE uvsY

Genetics ◽  
1986 ◽  
Vol 114 (4) ◽  
pp. 1061-1079
Author(s):  
Michael E Gruidl ◽  
Gisela Mosig
Keyword(s):  
Bacteriophage T4 ◽  
Open Reading Frames ◽  
Repair Gene ◽  
Protein Marker ◽  
Reading Frame ◽  
Phage T4 ◽  
Dna Repair Gene ◽  
A Minor

ABSTRACT We have cloned, sequenced and analyzed transcription of the phage T4 uvsY gene. This gene is transcribed from a single gp MotA-dependent middle promoter to give a major transcript of approximately 930 nucleotides and a minor transcript of approximately 620 nucleotides. All in vivo and in vitro uvsY transcripts show anomalous migration in agarose gels. The uvsY transcript contains an open reading frame coding for an 137 amino acid [15.8 kilodaltons (kD)] UvsY protein and two unidentified open reading frames, ORF UvsY.-1 (9.0 kD) and ORF UvsY.-2 (6.0 kD). Our DNA sequence differs in only three places from that published by Takahashi et al.However, one of these changes alters the predicted carboxy terminus of the UvsY protein. Marker rescue experiments map gene 25 to the region upstream of uvsY. Gene 25 is likely, although not certain, to correspond to an ORF that is found upstream from uvsY and is translated in the same direction.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals TNF-α Triggers RIP1/FADD/Caspase-8-Mediated Apoptosis of Astrocytes and RIP3/MLKL-Mediated Necroptosis of Neurons Induced by Angiostrongylus cantonensis Infection

2021 ◽  
Author(s):  
Hongli Zhou ◽  
Minyu Zhou ◽  
Yue Hu ◽  
Yanin Limpanon ◽  
Yubin Ma ◽  
...  
Keyword(s):  
Cell Death ◽  
Enrichment Analysis ◽  
Angiostrongylus Cantonensis ◽  
Gene Set Enrichment Analysis ◽  
Caspase 8 ◽  
Cns Injury ◽  
Vitro Assay ◽  
Tnf Α

AbstractAngiostrongylus cantonensis (AC) can cause severe eosinophilic meningitis or encephalitis in non-permissive hosts accompanied by apoptosis and necroptosis of brain cells. However, the explicit underlying molecular basis of apoptosis and necroptosis upon AC infection has not yet been elucidated. To determine the specific pathways of apoptosis and necroptosis upon AC infection, gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) analysis for gene expression microarray (accession number: GSE159486) of mouse brain infected by AC revealed that TNF-α likely played a central role in the apoptosis and necroptosis in the context of AC infection, which was further confirmed via an in vivo rescue assay after treating with TNF-α inhibitor. The signalling axes involved in apoptosis and necroptosis were investigated via immunoprecipitation and immunoblotting. Immunofluorescence was used to identify the specific cells that underwent apoptosis or necroptosis. The results showed that TNF-α induced apoptosis of astrocytes through the RIP1/FADD/Caspase-8 axis and induced necroptosis of neurons by the RIP3/MLKL signalling pathway. In addition, in vitro assay revealed that TNF-α secretion by microglia increased upon LSA stimulation and caused necroptosis of neurons. The present study provided the first evidence that TNF-α was secreted by microglia stimulated by AC infection, which caused cell death via parallel pathways of astrocyte apoptosis (mediated by the RIP1/FADD/caspase-8 axis) and neuron necroptosis (driven by the RIP3/MLKL complex). Our research comprehensively elucidated the mechanism of cell death after AC infection and provided new insight into targeting TNF-α signalling as a therapeutic strategy for CNS injury.

scholarly journals Isolation in a single step of a highly enriched murine hematopoietic stem cell population with competitive long-term repopulating ability

Blood ◽  
1989 ◽  
Vol 74 (3) ◽  
pp. 930-939 ◽  
Author(s):  
SJ Szilvassy ◽  
PM Lansdorp ◽  
RK Humphries ◽  
AC Eaves ◽  
CJ Eaves
Keyword(s):  
Simple Procedure ◽  
Hematopoietic Cells ◽  
Stem Cell Population ◽  
Proliferative Potential ◽  
Vitro Assay ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract A simple procedure is described for the quantitation and enrichment of murine hematopoietic cells with the capacity for long-term repopulation of lymphoid and myeloid tissues in lethally irradiated mice. To ensure detection of the most primitive marrow cells with this potential, we used a competitive assay in which female recipients were injected with male “test” cells and 1 to 2 x 10(5) “compromised” female marrow cells with normal short-term repopulating ability, but whose long-term repopulating ability had been reduced by serial transplantation. Primitive hematopoietic cells were purified by flow cytometry and sorting based on their forward and orthogonal light-scattering properties, and Thy-1 and H-2K antigen expression. Enrichment profiles for normal marrow, and marrow of mice injected with 5-fluorouracil (5- FU) four days previously, were established for each of these parameters using an in vitro assay for high proliferative potential, pluripotent colony-forming cells. When all four parameters were gated simultaneously, these clonogenic cells were enriched 100-fold. Both day 9 and day 12 CFU-S were copurified; however, the purity (23%) and enrichment (75-fold) of day 12 CFU-S in the sorted population was greater with 5-FU-treated cells. Five hundred of the sorted 5-FU marrow cells consistently repopulated recipient lymphoid and myeloid tissues (greater than 50% male, 1 to 3 months post-transplant) when co-injected with 1 to 2 x 10(5) compromised female marrow cells, and approximately 100 were sufficient to achieve the same result in 50% of recipients under the same conditions. This relatively simple purification and assay strategy should facilitate further analysis of the heterogeneity and regulation of stem cells that maintain hematopoiesis in vivo.

scholarly journals DIGESTIBLE ENERGY IN FORAGE BYIN VIVOANDIN VITROASSAYS

1970 ◽  
Vol 50 (3) ◽  
pp. 557-562 ◽  
Author(s):  
J. E. TROELSEN
Keyword(s):  
Energy Analysis ◽  
Energy Content ◽  
In Vitro Assay ◽  
Pure Species ◽  
Vitro Assay ◽  
Digestible Energy ◽  
The Relationship ◽  
Cattle And Sheep

Forage of six pure species was harvested for hay at several maturity stages during four years. The digestible energy content of 102 different lots of hay was determined by feeding to four groups of sheep during the same period, and by in vitro digestions and energy analysis of the undigested residues. The relationship between digestible energy content assayed by the two methods was highly significant (r = 0.85) and did not differ between years and species. Exclusion from regression of the hays containing less than 2 or more than 3 digestible kcal/g revealed that the in vitro assay could reproduce the in vivo digestible energy value with a standard deviation of 0.31 in over 70% of the hays. This represented the maturity and quality range of forage commonly fed to cattle and sheep. The in vitro assay therefore appeared promising for commercial quality determinations.

scholarly journals Poly(ADP-ribosyl)ation of p53 In Vitro and In Vivo Modulates Binding to its DNA Consensus Sequence

Neoplasia ◽  
2001 ◽  
Vol 3 (3) ◽  
pp. 179-188 ◽  
Author(s):  
Cynthia M. Simbulan-Rosenthal ◽  
Dean S. Rosenthal ◽  
RuiBai Luo ◽  
Raed Samara ◽  
Mira Jung ◽  
...  
Keyword(s):  
Consensus Sequence

scholarly journals Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

1989 ◽  
Vol 9 (11) ◽  
pp. 5073-5080 ◽  
Author(s):  
M Kozak
Keyword(s):  
Context Effects ◽  
Consensus Sequence ◽  
Negative Regulator ◽  
Late Event ◽  
Free Translation ◽  
Initiator Codon ◽  
Translation Systems ◽  
Short Versions

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

scholarly journals SBA1 Encodes a Yeast Hsp90 Cochaperone That Is Homologous to Vertebrate p23 Proteins

1998 ◽  
Vol 18 (7) ◽  
pp. 3727-3734 ◽  
Author(s):  
Yifang Fang ◽  
Albert E. Fliss ◽  
Jie Rao ◽  
Avrom J. Caplan
Keyword(s):  
Hormone Receptors ◽  
Pcr Amplification ◽  
Steroid Hormone Receptors ◽  
Loss Of Function ◽  
Vitro Assay ◽  
Growth Defects ◽  
Double Deletion ◽  
Affinity Isolation

ABSTRACT The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone. TheSBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37°C. A double deletion of SBA1and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects. Affinity isolation of histidine-tagged Sba1p (Sba1His6) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, purified Sba1His6 bound to Hsp90 only in the presence of adenosine 5′-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, interaction between purified Sba1His6 and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1His6, and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized. In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.

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