scholarly journals Bacteriophage-Based Genetic System for Selection of Nonsplicing Inteins

2004 ◽  
Vol 70 (5) ◽  
pp. 3158-3162 ◽  
Author(s):  
Isaac K. O. Cann ◽  
Kensey R. Amaya ◽  
Maurice W. Southworth ◽  
Francine B. Perler

ABSTRACT A genetic selection system that detects splicing and nonsplicing activities of inteins was developed based on the ability to rescue a T4 phage strain with a conditionally inactive DNA polymerase. This phage defect can be complemented by expression of plasmid-encoded phage RB69 DNA polymerase. Insertion of an intein gene into the active site of the RB69 DNA polymerase gene renders polymerase activity and phage viability dependent on protein splicing. The effectiveness of the system was tested by screening for thermosensitive splicing mutants. Development of genetic systems with the potential of identifying protein splicing inhibitors is a first step towards controlling proliferation of pathogenic microbes harboring inteins in essential proteins.

2000 ◽  
Vol 46 (5) ◽  
pp. 620-624 ◽  
Author(s):  
Caroline J Fuery ◽  
Helen L Impey ◽  
Natalie J Roberts ◽  
Tanya L Applegate ◽  
Robyn L Ward ◽  
...  

Abstract Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. Methods: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. Results: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.


2019 ◽  
Vol 4 (8) ◽  
pp. 2398-2403 ◽  
Author(s):  
Xingxing Zhang ◽  
Qiang Liu ◽  
Yan Jin ◽  
Baoxin Li

1974 ◽  
Vol 29 (1-2) ◽  
pp. 72-75 ◽  
Author(s):  
B. Dietzschold ◽  
O.R. Kaaden ◽  
S. Ueberschaer ◽  
F. Weiland ◽  
O. C. Straub

Abstract Typical C-type oncorna virus particles as shown by electron microscopy have been purified from the supernatant of cultured lymphocytes from bovine leukosis. In the purified C-particle fraction a DNA-polymerase activity was detected. Using several synthetic RNA-or DNA-homopolymers and 70S Friend virus RNA the template response of this bovine leukosis cell particle DNA polymerase was compared with those of feline leukaemia virus DNA polymerase and DNA polymerase from normal bovine lymphocytes. The DNA polymerase detected in the viral preparation of bovine leukosis is suggested to be an oncorna-virus-specific enzyme.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Simone Savoia ◽  
Andrea Albera ◽  
Alberto Brugiapaglia ◽  
Liliana Di Stasio ◽  
Alessio Cecchinato ◽  
...  

Abstract Background The possibility of assessing meat quality traits over the meat chain is strongly limited, especially in the context of selective breeding which requires a large number of phenotypes. The main objective of this study was to investigate the suitability of portable infrared spectrometers for phenotyping beef cattle aiming to genetically improving the quality of their meat. Meat quality traits (pH, color, water holding capacity, tenderness) were appraised on rib eye muscle samples of 1,327 Piemontese young bulls using traditional (i.e., reference/gold standard) laboratory analyses; the same traits were also predicted from spectra acquired at the abattoir on the intact muscle surface of the same animals 1 d after slaughtering. Genetic parameters were estimated for both laboratory measures of meat quality traits and their spectra-based predictions. Results The prediction performances of the calibration equations, assessed through external validation, were satisfactory for color traits (R2 from 0.52 to 0.80), low for pH and purge losses (R2 around 0.30), and very poor for cooking losses and tenderness (R2 below 0.20). Except for lightness and purge losses, the heritability estimates of most of the predicted traits were lower than those of the measured traits while the genetic correlations between measured and predicted traits were high (average value 0.81). Conclusions Results showed that NIRS predictions of color traits, pH, and purge losses could be used as indicator traits for the indirect genetic selection of the reference quality phenotypes. Results for cooking losses were less effective, while the NIR predictions of tenderness were affected by a relatively high uncertainty of estimate. Overall, genetic selection of some meat quality traits, whose direct phenotyping is difficult, can benefit of the application of infrared spectrometers technology.


2012 ◽  
Vol 56 (11) ◽  
pp. 6009-6013 ◽  
Author(s):  
Giulia Muratore ◽  
Beatrice Mercorelli ◽  
Laura Goracci ◽  
Gabriele Cruciani ◽  
Paul Digard ◽  
...  

ABSTRACTAL18, an inhibitor of human cytomegalovirus DNA polymerase, was serendipitously found to also block the interaction between the PB1 and PA polymerase subunits of influenza A virus. Furthermore, AL18 effectively inhibited influenza A virus polymerase activity and the overall replication of influenza A and B viruses. A molecular model to explain the binding of AL18 to both cytomegalovirus and influenza targets is proposed. Thus, AL18 represents an interesting lead for the development of new antivirals.


1969 ◽  
Vol 57 (2-3) ◽  
pp. 257-262 ◽  
Author(s):  
Y RABINOWITZ ◽  
I MCCLUSKEY ◽  
P WONG ◽  
B WILHITE

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