scholarly journals Copy-Paste Mutagenesis: A Method for Large-Scale Alteration of Viral Genomes

2019 ◽  
Vol 20 (4) ◽  
pp. 913 ◽  
Author(s):  
Jiajia Tang ◽  
Renke Brixel ◽  
Wolfram Brune
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Point Mutations ◽  
Plasmid Vector ◽  
Donor Strain ◽  
Bacterial Artificial Chromosomes ◽  
New Era ◽  
Phenotypic Differences ◽  
Viral Genomes ◽  
Artificial Chromosomes

The cloning of the large DNA genomes of herpesviruses, poxviruses, and baculoviruses as bacterial artificial chromosomes (BAC) in Escherichia coli has opened a new era in viral genetics. Several methods of lambda Red-mediated genome engineering (recombineering) in E. coli have been described, which are now commonly used to generate recombinant viral genomes. These methods are very efficient at introducing deletions, small insertions, and point mutations. Here we present Copy-Paste mutagenesis, an efficient and versatile strategy for scarless large-scale alteration of viral genomes. It combines gap repair and en passant mutagenesis procedures and relies on positive selection in all crucial steps. We demonstrate that this method can be used to generate chimeric strains of human cytomegalovirus (HCMV), the largest human DNA virus. Large (~15 kbp) genome fragments of HCMV strain TB40/E were tagged with an excisable marker and cloned (copied) in a low-copy plasmid vector by gap repair recombination. The cloned fragment was then excised and inserted (pasted) into the HCMV AD169 genome with subsequent scarless removal of the marker by en passant mutagenesis. We have done four consecutive rounds of this procedure, thereby generating an AD169-TB40/E chimera containing 60 kbp of the donor strain TB40/E. This procedure is highly useful for identifying gene variants responsible for phenotypic differences between viral strains. It can also be used for repair of incomplete viral genomes, and for modification of any BAC-cloned sequence. The method should also be applicable for large-scale alterations of bacterial genomes.

scholarly journals Cross-Species RNAi Rescue Platform in Drosophila melanogaster

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 1165-1173 ◽  
Author(s):  
Shu Kondo ◽  
Matthew Booker ◽  
Norbert Perrimon
Keyword(s):  
Cell Culture ◽  
Drosophila Melanogaster ◽  
Large Scale ◽  
Transgenic Animals ◽  
Selection Marker ◽  
Bacterial Artificial Chromosomes ◽  
Loss Of Function ◽  
Artificial Chromosomes ◽  
Target Effects

RNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.

scholarly journals Dynamic plasticity of large-scale chromatin structure revealed by self-assembly of engineered chromosome regions

2010 ◽  
Vol 190 (5) ◽  
pp. 761-776 ◽  
Author(s):  
Paul Sinclair ◽  
Qian Bian ◽  
Matt Plutz ◽  
Edith Heard ◽  
Andrew S. Belmont
Keyword(s):  
Self Assembly ◽  
Large Scale ◽  
Tandem Repeats ◽  
Es Cells ◽  
Embryonic Stem ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Lac Operator ◽  
Chromatin Folding ◽  
Chromosome Regions

Interphase chromatin compaction well above the 30-nm fiber is well documented, but the structural motifs underlying this level of chromatin folding remain unknown. Taking a reductionist approach, we analyzed in mouse embryonic stem (ES) cells and ES-derived fibroblasts and erythroblasts the folding of 10–160-megabase pair engineered chromosome regions consisting of tandem repeats of bacterial artificial chromosomes (BACs) containing ∼200 kilobases of mammalian genomic DNA tagged with lac operator (LacO) arrays. Unexpectedly, linear mitotic and interphase chromatid regions formed from noncontiguously folded DNA topologies. Particularly, in ES cells, these model chromosome regions self-organized with distant sequences segregating into functionally distinct, compact domains. Transcriptionally active and histone H3K27me3-modified regions positioned toward the engineered chromosome subterritory exterior, with LacO repeats and the BAC vector backbone localizing within an H3K9me3, HP1-enriched core. Differential compaction of Dhfr and α- and β-globin transgenes was superimposed on dramatic, lineage-specific reorganization of large-scale chromatin folding, demonstrating a surprising plasticity of large-scale chromatin organization.

scholarly journals Transposon-Assisted Cloning and Traceless Mutagenesis of Adenoviruses: Development of a Novel Vector Based on SpeciesD

2006 ◽  
Vol 80 (16) ◽  
pp. 8100-8113 ◽  
Author(s):  
Zsolt Ruzsics ◽  
Markus Wagner ◽  
Andrea Osterlehner ◽  
Jonathan Cook ◽  
Ulrich Koszinowski ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Point Mutations ◽  
Cell Types ◽  
Sequence Information ◽  
Bacterial Artificial Chromosomes ◽  
Artificial Chromosomes ◽  
Coxsackievirus And Adenovirus Receptor ◽  
Species Specific ◽  
Adenovirus Receptor ◽  
Limited Sequence

ABSTRACT Until recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependent tropism of Ad5 stimulated attempts to exploit the natural diversity in tropism of the other 50 known human Ad serotypes. Aiming in particular at immunotherapy and vaccination, we have screened representative serotypes from different Ad species for their ability to infect dendritic cells. Ad19a, an Ad from species D, was selected for development as a new vector for vaccination and cancer gene therapy. To clone and manipulate its genome, we have developed a novel methodology, coined “exposon mutagenesis,” that allows the rapid and precise introduction of virtually any genetic alteration (deletions, point mutations, or insertions) into recombinant Ad bacterial artificial chromosomes. The versatility of the system was exemplified by deleting the E3 region of Ad19a, by specifically knocking out expression of a species-specific E3 gene, E3/49K, and by reinserting E3/49K into an E3 null Ad19a mutant. The technology requires only limited sequence information and is applicable to other Ad species. Therefore, it should be extremely valuable for the analysis of gene functions from any Ad species. In addition, a basic, replication-defective E1- and E3-deleted Ad19a vector expressing GFP (Ad19aGFP) was generated. This new vector based on species D Ads exhibits a very promising tropism for lymphoid and muscle cells and shows great potential as an alternative vector for transduction of cell types that are resistant to or only poorly transduced by conventional Ad5-based vectors.

Computer Analyses in Preventive Health Research

1967 ◽  
Vol 06 (01) ◽  
pp. 8-14 ◽  
Author(s):  
M. F. Collen
Keyword(s):  
Medical Research ◽  
Preventive Medicine ◽  
Health Research ◽  
Large Scale ◽  
Preventive Health ◽  
New Era ◽  
Large Numbers ◽  
Normal Test ◽  
Computer Analyses

The utilization of an automated multitest laboratory as a data acquisition center and of a computer for trie data processing and analysis permits large scale preventive medical research previously not feasible. Normal test values are easily generated for the particular population studied. Long-term epidemiological research on large numbers of persons becomes practical. It is our belief that the advent of automation and computers has introduced a new era of preventive medicine.

scholarly journals Mutational patterns and clonal evolution from diagnosis to relapse in pediatric acute lymphoblastic leukemia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shumaila Sayyab ◽  
Anders Lundmark ◽  
Malin Larsson ◽  
Markus Ringnér ◽  
Sara Nystedt ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Large Scale ◽  
Somatic Mutations ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Point Mutations ◽  
Driver Genes ◽  
Protein Coding ◽  
Pediatric Acute Lymphoblastic Leukemia ◽  
Evolutionary Trajectories

AbstractThe mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

Microcomputers in Political Science

1983 ◽  
Vol 38 ◽  
pp. 1-9
Author(s):  
Herbert F. Weisberg
Keyword(s):  
Data Analysis ◽  
Political Science ◽  
Large Scale ◽  
Turnaround Time ◽  
General Purpose ◽  
Batch Mode ◽  
New Era ◽  
Large Scale Data ◽  
The Social ◽  
Frequency Counts

We are now entering a new era of computing in political science. The first era was marked by punched-card technology. Initially, the most sophisticated analyses possible were frequency counts and tables produced on a counter-sorter, a machine that specialized in chewing up data cards. By the early 1960s, batch processing on large mainframe computers became the predominant mode of data analysis, with turnaround time of up to a week. By the late 1960s, turnaround time was cut down to a matter of a few minutes and OSIRIS and then SPSS (and more recently SAS) were developed as general-purpose data analysis packages for the social sciences. Even today, use of these packages in batch mode remains one of the most efficient means of processing large-scale data analysis.

scholarly journals The SAMI Galaxy Survey: the third and final data release

2021 ◽  
Author(s):  
Scott M Croom ◽  
Matt S Owers ◽  
Nicholas Scott ◽  
Henry Poetrodjojo ◽  
Brent Groves ◽  
...  
Keyword(s):  
Large Scale ◽  
Resolving Power ◽  
Stellar Kinematics ◽  
New Era ◽  
Optical Wavelength ◽  
Astronomical Optics ◽  
Data Release ◽  
Gas Ionization ◽  
Release Data ◽  
First Time

Abstract We have entered a new era where integral-field spectroscopic surveys of galaxies are sufficiently large to adequately sample large-scale structure over a cosmologically significant volume. This was the primary design goal of the SAMI Galaxy Survey. Here, in Data Release 3 (DR3), we release data for the full sample of 3068 unique galaxies observed. This includes the SAMI cluster sample of 888 unique galaxies for the first time. For each galaxy, there are two primary spectral cubes covering the blue (370–570 nm) and red (630–740 nm) optical wavelength ranges at spectral resolving power of R = 1808 and 4304 respectively. For each primary cube, we also provide three spatially binned spectral cubes and a set of standardized aperture spectra. For each galaxy, we include complete 2D maps from parameterized fitting to the emission-line and absorption-line spectral data. These maps provide information on the gas ionization and kinematics, stellar kinematics and populations, and more. All data are available online through Australian Astronomical Optics (AAO) Data Central.

scholarly journals The Impact of Gaia and LSST on Binaries and Exoplanets

2011 ◽  
Vol 7 (S282) ◽  
pp. 33-40
Author(s):  
L. Eyer ◽  
P. Dubath ◽  
N. Mowlavi ◽  
P. North ◽  
A. Triaud ◽  
...  
Keyword(s):  
Radial Velocity ◽  
Large Scale ◽  
Binary Systems ◽  
New Era ◽  
The Subject ◽  
The Impact

AbstractTwo upcoming large scale surveys, the ESA Gaia and LSST projects, will bring a new era in astronomy. The number of binary systems that will be observed and detected by these projects is enormous, estimations range from millions for Gaia to several tens of millions for LSST. We review some tools that should be developed and also what can be gained from these missions on the subject of binaries and exoplanets from the astrometry, photometry, radial velocity and their alert systems.

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