scholarly journals CRISPR-interference-based modulation of mobile genetic elements in bacteria

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Ákos Nyerges ◽  
Balázs Bálint ◽  
Judit Cseklye ◽  
István Nagy ◽  
Csaba Pál ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Industrial Biotechnology ◽  
Chromosome Engineering ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Promising Tool ◽  
Single Plasmid

Abstract Spontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of engineered functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5 and IS150 at up to 38 chromosomal loci in Escherichia coli, in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. The fitness cost of multiple IS-knockdown, detectable in flask-and-shaker systems was readily outweighed by the less frequent inactivation of the transgene, as observed in green fluorescent protein (GFP)-overexpression experiments. In addition, global transcriptomics analysis revealed only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multiple E. coli strains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

scholarly journals CRISPR-interference based modulation of mobile genetic elements in bacteria

2018 ◽  
Author(s):  
Ákos Nyerges ◽  
Balázs Bálint ◽  
Judit Cseklye ◽  
István Nagy ◽  
Csaba Pál ◽  
...  
Keyword(s):  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Industrial Biotechnology ◽  
Chromosome Engineering ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Promising Tool ◽  
Single Plasmid ◽  
Bacterial Chromosomes

ABSTRACTSpontaneous mutagenesis of synthetic genetic constructs by mobile genetic elements frequently results in the rapid loss of advantageous functions. Previous efforts to minimize such mutations required the exceedingly time-consuming manipulation of bacterial chromosomes and the complete removal of insertional sequences (ISes). To this aim, we developed a single plasmid-based system (pCRIS) that applies CRISPR-interference to inhibit the transposition of bacterial ISes. pCRIS expresses multiple guide RNAs to direct inactivated Cas9 (dCas9) to simultaneously silence IS1, IS3, IS5, and IS150at up to 38 chromosomal loci inEscherichia coli,in vivo. As a result, the transposition rate of all four targeted ISes dropped to negligible levels at both chromosomal and episomal targets, increasing the half-life of exogenous protein expression. Most notably, pCRIS, while requiring only a single plasmid delivery performed within a single day, provided a reduction of IS-mobility comparable to that seen in genome-scale chromosome engineering projects. Global transcriptomics analysis revealed nevertheless only minute alterations in the expression of untargeted genes. Finally, the transposition-silencing effect of pCRIS was easily transferable across multipleE. colistrains. The plasticity and robustness of our IS-silencing system make it a promising tool to stabilize bacterial genomes for synthetic biology and industrial biotechnology applications.

scholarly journals Mobile genetic elements: in silico,in vitro,in vivo

2016 ◽  
Vol 25 (5) ◽  
pp. 1027-1031 ◽  
Author(s):  
Irina R. Arkhipova ◽  
Phoebe A. Rice
Keyword(s):  
In Silico ◽  
Mobile Genetic Elements ◽  
Genetic Elements

Mobile genetic elements in Drosophila melanogaster (recent experiments)

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 920-928 ◽  
Author(s):  
Georgii P. Georgiev ◽  
Nickolai A. Tchurikov ◽  
Yurii V. Ilyin ◽  
Sofia G. Georgieva ◽  
Lev J. Mizrokhi ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase Ii ◽  
Mobile Element ◽  
Complete Removal ◽  
Mobile Genetic Elements ◽  
Internal Promoter ◽  
Genetic Elements ◽  
First Case ◽  
Large Numbers ◽  
White Locus

Recent data obtained in the authors' laboratories concerning the behaviour of mobile genetic elements of Drosophila melanogaster are reviewed. It was found that the mobile element jockey represents the typical LINE element. It is efficiently transcribed in D. melanogaster cells in flies and in culture. Transcription is initiated from the +1 nucleotide of jockey and depends on an internal promoter. This is the first case of an internal promoter being used by RNA polymerase II. Several events which take place during the transposition bursts in ctMR2 family of strains were described. Among them are the removal of mobile dispersed genetics (mdg) elements (with solo long terminal repeat (LTR) remaining at the site of excision), complete removal of an mdg element, and reinsertion of the same mdg to the same place either in the presence or in absence of solo LTR sequence. Finally, the formation of deletions was observed. A 462-bp deletion destroying the white locus can be further repaired (w+ reversion). Thus, transposition bursts include many different genetic events. A novel system of prolonged genome destabilization was described. It depends on mobilization of a new mobile element called Stalker. After certain crosses Stalker actively moves for dozens of generations giving rise to large numbers of insertion mutations. Several novel genes were detected using mobilized Stalker. They include a modifier of mdg4 and six enhancers of yellow mutations.Key words: Drosophila melanogaster, mobile elements, transcription.

scholarly journals The Superantigen Gene ypm Is Located in an Unstable Chromosomal Locus of Yersinia pseudotuberculosis

2002 ◽  
Vol 184 (16) ◽  
pp. 4489-4499 ◽  
Author(s):  
Christophe Carnoy ◽  
Stephanie Floquet ◽  
Michael Marceau ◽  
Florent Sebbane ◽  
Stephanie Haentjens-Herwegh ◽  
...  
Keyword(s):  
Insertion Sequence ◽  
Yersinia Pseudotuberculosis ◽  
Gc Content ◽  
Mobile Genetic Elements ◽  
Open Reading Frames ◽  
Chromosomal Locus ◽  
Genetic Elements ◽  
Encoding Genes ◽  
Reading Frames

ABSTRACT Yersinia pseudotuberculosis produces YPM (Y. pseudotuberculosis-derived mitogen), a superantigenic toxin that exacerbates the virulence of the bacterium in vivo. To date, three alleles of the superantigen gene (ypmA, ypmB, and ypmC) have been described. These genes are not found in all Y. pseudotuberculosis strains and have a low GC content, suggesting their location on mobile genetic elements. To elucidate this question, the genetic environment of the superantigen-encoding genes was characterized and 11 open reading frames (ORFs) were defined. Sequence analysis revealed that the ypm genes were not associated with plasmids, phages, transposons, or pathogenicity islands and that the superantigen genes were always located in the chromosome between ORF3 and ORF4. Nonsuperantigenic strains exhibited the same genetic organization of the locus but lacked the ypm gene between ORF3 and ORF4. A new insertion sequence, designated IS1398, which displays features of the Tn3 family, was characterized downstream of the ypmA and ypmC genes. A 13.3-kb region containing the ypm genes was not found in the genome of Y. pestis (CO92 and KIM 5 strains). We experimentally induced deletion of the ypm gene from a superantigen-expressing Y. pseudotuberculosis: using the association of aph(3′)-IIIa and sacB genes, we demonstrated that when these reporter genes were present in the ypm locus, deletion of these genes was about 250 times more frequent than when they were located in another region of the Y. pseudotuberculosis chromosome. These results indicate that unlike other superantigenic toxin genes, the Yersinia ypm genes are not associated with mobile genetic elements but are inserted in an unstable locus of the genome.

scholarly journals Long-Term Persistence of Plasmids Targeted by CRISPR Interference in Bacterial Populations

2021 ◽  
Author(s):  
Viktor Mamontov ◽  
Alexander Martynov ◽  
Natalia Morozova ◽  
Anton Bukatin ◽  
Dmitry B. Staroverov ◽  
...  
Keyword(s):  
Complex Dynamics ◽  
Mobile Genetic Elements ◽  
Genetic Alterations ◽  
Common Mechanism ◽  
Type I ◽  
Bacterial Populations ◽  
Crispr Interference ◽  
Genetic Elements ◽  
Rapid Changes

CRISPR-Cas systems provide prokaryotes with an RNA-guided defense against foreign mobile genetic elements (MGEs) such as plasmids and viruses. A common mechanism by which MGEs avoid interference by CRISPR consists of acquisition of escape mutations in regions targeted by CRISPR. Here, using microbiological, live microscopy, and microfluidics analyses we demonstrated that plasmids can persist in Escherichia coli cells at conditions of continuous targeting by the type I-E CRISPR-Cas system without acquiring any genetic alterations. We used mathematical modeling to show how plasmid persistence in a subpopulation of cells mounting CRISPR interference is achieved due to the stochastic nature of CRISPR interference and plasmid replication events. We hypothesize that the observed complex dynamics provides bacterial populations with long-term benefits due to the presence of mobile genetic elements in some cells, leading to diversification of phenotypes in the entire community and allowing rapid changes in the population structure to meet the demands of a changing environment.

scholarly journals Potential for Isolation of Immortalized Hepatocyte Cell Lines by Liver-DirectedIn VivoGene Delivery of Transposons in Mice

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Masahiro Sato ◽  
Issei Saitoh ◽  
Emi Inada ◽  
Shingo Nakamura ◽  
Satoshi Watanabe
Keyword(s):  
Nucleic Acids ◽  
Cell Lines ◽  
Mobile Genetic Elements ◽  
Isolated Hepatocytes ◽  
Proliferative Potential ◽  
Genetic Elements ◽  
Cellular Immortalization

Isolation of hepatocytes and their culturein vitrorepresent important avenues to explore the function of such cells. However, these studies are often difficult to perform because of the inability of hepatocytes to proliferatein vitro. Immortalization of isolated hepatocytes is thus an important step toward continuousin vitroculture. For cellular immortalization, integration of relevant genes into the host chromosomes is a prerequisite. Transposons, which are mobile genetic elements, are known to facilitate integration of genes of interest (GOI) into chromosomesin vitroandin vivo. Here, we proposed that a combination of transposon- and liver-directed introduction of nucleic acids may confer acquisition of unlimited cellular proliferative potential on hepatocytes, enabling the possible isolation of immortalized hepatocyte cell lines, which has often failed using more traditional immortalization methods.

Fluorescent proteins for in vivo imaging, where's the biliverdin?

2020 ◽  
Vol 48 (6) ◽  
pp. 2657-2667
Author(s):  
Felipe Montecinos-Franjola ◽  
John Y. Lin ◽  
Erik A. Rodriguez
Keyword(s):  
Hydrogen Peroxide ◽  
In Vivo Imaging ◽  
Near Infrared ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescent Imaging ◽  
Infrared Light ◽  
Red Fluorescent Protein ◽  
Color Palette

Noninvasive fluorescent imaging requires far-red and near-infrared fluorescent proteins for deeper imaging. Near-infrared light penetrates biological tissue with blood vessels due to low absorbance, scattering, and reflection of light and has a greater signal-to-noise due to less autofluorescence. Far-red and near-infrared fluorescent proteins absorb light >600 nm to expand the color palette for imaging multiple biosensors and noninvasive in vivo imaging. The ideal fluorescent proteins are bright, photobleach minimally, express well in the desired cells, do not oligomerize, and generate or incorporate exogenous fluorophores efficiently. Coral-derived red fluorescent proteins require oxygen for fluorophore formation and release two hydrogen peroxide molecules. New fluorescent proteins based on phytochrome and phycobiliproteins use biliverdin IXα as fluorophores, do not require oxygen for maturation to image anaerobic organisms and tumor core, and do not generate hydrogen peroxide. The small Ultra-Red Fluorescent Protein (smURFP) was evolved from a cyanobacterial phycobiliprotein to covalently attach biliverdin as an exogenous fluorophore. The small Ultra-Red Fluorescent Protein is biophysically as bright as the enhanced green fluorescent protein, is exceptionally photostable, used for biosensor development, and visible in living mice. Novel applications of smURFP include in vitro protein diagnostics with attomolar (10−18 M) sensitivity, encapsulation in viral particles, and fluorescent protein nanoparticles. However, the availability of biliverdin limits the fluorescence of biliverdin-attaching fluorescent proteins; hence, extra biliverdin is needed to enhance brightness. New methods for improved biliverdin bioavailability are necessary to develop improved bright far-red and near-infrared fluorescent proteins for noninvasive imaging in vivo.

ISSR-PCR markers and mobile genetic elements in horse (Equus caballus) genome

2014 ◽  
pp. 42-57 ◽  
Author(s):  
N.V. Bardukov ◽  
◽  
A.V. Feofilov ◽  
T.T. Glazko ◽  
V.I. Glazko ◽  
...  
Keyword(s):  
Equus Caballus ◽  
Mobile Genetic Elements ◽  
Pcr Markers ◽  
Genetic Elements ◽  
Issr Pcr

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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