scholarly journals Comparison of CRISPR and Marker-Based Methods for the Engineering of Phage T7

Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 193 ◽  
Author(s):  
Aurelija M. Grigonyte ◽  
Christian Harrison ◽  
Paul R. MacDonald ◽  
Ariadna Montero-Blay ◽  
Matthew Tridgett ◽  
...  
Keyword(s):  
Therapeutic Agents ◽  
Bacteriophage T7 ◽  
Type I ◽  
Type Ii ◽  
Selection Mechanism ◽  
Wild Type ◽  
Phage T7 ◽  
Fundamental Biology ◽  
Type Phage ◽  
Host Genes

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trxA have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here, we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fibre mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

scholarly journals Comparison of CRISPR and marker based methods for the engineering of phage T7

2020 ◽  
Author(s):  
Aurelija M. Grigonyte ◽  
Christian Harrison ◽  
Paul R. MacDonald ◽  
Ariadna Montero-Blay ◽  
Matthew Tridgett ◽  
...  
Keyword(s):  
Therapeutic Agents ◽  
Bacteriophage T7 ◽  
Tail Fiber ◽  
Type I ◽  
Selection Mechanism ◽  
Wild Type ◽  
Phage T7 ◽  
Fundamental Biology ◽  
Type Phage ◽  
Host Genes

With the recent rise in interest in using lytic bacteriophages as therapeutic agents, there is an urgent requirement to understand their fundamental biology to enable the engineering of their genomes. Current methods of phage engineering rely on homologous recombination, followed by a system of selection to identify recombinant phages. For bacteriophage T7, the host genes cmk or trx have been used as a selection mechanism along with both type I and II CRISPR systems to select against wild-type phage and enrich for the desired mutant. Here we systematically compare all three systems; we show that the use of marker-based selection is the most efficient method and we use this to generate multiple T7 tail fiber mutants. Furthermore, we found the type II CRISPR-Cas system is easier to use and generally more efficient than a type I system in the engineering of phage T7. These results provide a foundation for the future, more efficient engineering of bacteriophage T7.

scholarly journals Phage T7 DNA mimic protein Ocr is a potent inhibitor of BREX defence

2020 ◽  
Vol 48 (10) ◽  
pp. 5397-5406 ◽  
Author(s):  
Artem Isaev ◽  
Alena Drobiazko ◽  
Nicolas Sierro ◽  
Julia Gordeeva ◽  
Ido Yosef ◽  
...  
Keyword(s):  
Type I ◽  
Wild Type ◽  
Phage Dna ◽  
Phage T7 ◽  
In Trans ◽  
Self Differentiation ◽  
Type Phage ◽  
Defensive Action ◽  
T7 Bacteriophage ◽  
Restriction Modification

Abstract BREX (for BacteRiophage EXclusion) is a superfamily of common bacterial and archaeal defence systems active against diverse bacteriophages. While the mechanism of BREX defence is currently unknown, self versus non-self differentiation requires methylation of specific asymmetric sites in host DNA by BrxX (PglX) methyltransferase. Here, we report that T7 bacteriophage Ocr, a DNA mimic protein that protects the phage from the defensive action of type I restriction–modification systems, is also active against BREX. In contrast to the wild–type phage, which is resistant to BREX defence, T7 lacking Ocr is strongly inhibited by BREX, and its ability to overcome the defence could be complemented by Ocr provided in trans. We further show that Ocr physically associates with BrxX methyltransferase. Although BREX+ cells overproducing Ocr have partially methylated BREX sites, their viability is unaffected. The result suggests that, similar to its action against type I R–M systems, Ocr associates with as yet unidentified BREX system complexes containing BrxX and neutralizes their ability to both methylate and exclude incoming phage DNA.

Two Different UGT1A1 Mutations causing Crigler–Najjar Syndrome types I and II in an Iranian Family

2015 ◽  
Vol 24 (4) ◽  
pp. 523-526 ◽  
Author(s):  
Yoshihiro Maruo ◽  
Mahdiyeh Behnam ◽  
Shinichi Ikushiro ◽  
Sayuri Nakahara ◽  
Narges Nouri ◽  
...  
Keyword(s):  
Serum Bilirubin ◽  
Total Serum Bilirubin ◽  
Total Serum ◽  
Compound Heterozygous ◽  
Type I ◽  
Syndrome Type ◽  
Type Ii ◽  
Wild Type ◽  
Iranian Family ◽  
Udp Glucuronosyltransferase

Background: Crigler–Najjar syndrome type I (CN-1) and type II (CN-2) are rare hereditary unconjugated hyperbilirubinemia disorders. However, there have been no reports regarding the co-existence of CN-1 and CN-2 in one family. We experienced a case of an Iranian family that included members with either CN-1 or CN-2. Genetic analysis revealed a mutation in the bilirubin UDP-glucuronosyltransferase (UGT1A1) gene that resulted in residual enzymatic activity.Case report: The female proband developed severe hyperbilirubinemia [total serum bilirubin concentration (TB) = 34.8 mg/dL] with bilirubin encephalopathy (kernicterus) and died after liver transplantation. Her family history included a cousin with kernicterus (TB = 30.0 mg/dL) diagnosed as CN-1. Her great grandfather (TB unknown) and uncle (TB = 23.0 mg/dL) developed jaundice, but without any treatment, they remained healthy as CN-2. Results: The affected cousin was homozygous for a novel frameshift mutation (c.381insGG, p.C127WfsX23). The affected uncle was compound heterozygous for p.C127WfsX23 and p.V225G linked with A(TA)7TAA. p.V225G-UGT1A1 reduced glucuronidation activity to 60% of wild-type. Thus, linkage of A(TA)7TAA and p.V225G might reduce UGT1A1 activity to 18%–36 % of the wild-type. Conclusion: Genetic and in vitro expression analyses are useful for accurate genetic counseling for a family with a history of both CN-1 and CN-2. Abbreviations: CN-1: Crigler–Najjar syndrome type I; CN-2: Crigler–Najjar syndrome type II; GS: Gilbert syndrome; UGT1A1: bilirubin UDP-glucuronosyltransferase; WT: Wild type; TB: total serum bilirubin.

Role of common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of murine mast cells

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2172-2180 ◽  
Author(s):  
Kotaro Suzuki ◽  
Hiroshi Nakajima ◽  
Norihiko Watanabe ◽  
Shin-ichiro Kagami ◽  
Akira Suto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Cytokine Receptor ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Wild Type ◽  
Peritoneal Mast Cells ◽  
The Common

Abstract The regulatory roles of the common cytokine receptor γ chain (γc)– and Jak3-dependent signaling in the proliferation and survival of mast cells were determined using γc-deficient (γc−) and Jak3-deficient (Jak3−) mice. Although the mast cells in γc− and Jak3− mice were morphologically indistinguishable from those in wild-type mice, the number of peritoneal mast cells was decreased in γc− and Jak3− mice as compared with that in wild-type mice. Among γc-related cytokines, interleukin (IL)-4 and IL-9, but not IL-2, IL-7, or IL-15, enhanced the proliferation and survival of bone marrow–derived mast cells (BMMCs) from wild-type mice. However, the effects of IL-4 and IL-9 were absent in BMMCs from γc− and Jak3−mice. In addition, IL-4Rα, γc, and Jak3, but not IL-2Rβ or IL-7Rα, were expressed in BMMCs. In contrast, IL-13 did not significantly induce the proliferation and survival of BMMCs even from wild-type mice, and IL-13Rα1 was not expressed in BMMCs. Furthermore, IL-4 phosphorylated the 65-kd isoform of Stat6 in BMMCs from wild-type mice but not from γc− and Jak3− mice. These results indicate that γc- and Jak3-dependent signaling is essential for IL-4– and IL-9–induced proliferation and survival of murine mast cells, that the effects of IL-4 are mediated by type I IL-4R and that type II IL-4R is absent on mast cells, and that IL-4 phosphorylates the 65-kd isoform of Stat6 in mast cells in a γc- and Jak3-dependent manner.

scholarly journals Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia

2021 ◽  
Vol 22 (16) ◽  
pp. 9027
Author(s):  
Sarah Legrain ◽  
Dan Su ◽  
Mélanie Gaignage ◽  
Cor Breukel ◽  
Jill Claassens ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Type I Interferon ◽  
Type I ◽  
Type Ii ◽  
Interferon Production ◽  
Activated Macrophages ◽  
Wild Type ◽  
Fcγ Receptors ◽  
Interferon Receptor ◽  
Deficient Mice

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes.

scholarly journals Tailless keratins assemble into regular intermediate filaments in vitro

1990 ◽  
Vol 97 (2) ◽  
pp. 317-324
Author(s):  
M. Hatzfeld ◽  
K. Weber
Keyword(s):  
Intermediate Filaments ◽  
Consensus Sequence ◽  
In Vitro Mutagenesis ◽  
Type I ◽  
Type Ii ◽  
Tail Domain ◽  
Filament Formation ◽  
Wild Type ◽  
Keratin 8

To study the influence of the non alpha-helical tail domain of keratins in filament formation, we prepared a truncated keratin 8 mutant, K8/tailless. Using site-directed in vitro mutagenesis we introduced a stop codon in the position coding for amino acid number 417 of the K8/wild-type sequence, thereby deleting 86 amino acids of the non alpha-helical tail domain but leaving the consensus sequence at the end of the rod domain intact. Expression of the truncated keratin 8 in Escherichia coli allowed us to purify the protein by a two-step procedure. The filament-forming capacity of the truncated K8 with wild-type K18 and K19 was analyzed using in vitro reconstitution. The in vitro assembly studies with K8/tailless and K18 wild-type indicate that the C-terminal tail domain of a type II keratin, including the homologous subdomain H2, is not required for filament formation. Moreover, reconstitution experiments with K8/tailless and K19, a naturally occurring tailless keratin I, show that the tail domains of type I as well as type II keratins are not an essential requirement for in vitro filament formation. Our results suggest that in vitro filament elongation does not depend on interactions between head and tail domains, although the tail domain might have a role in stabilization of intermediate filaments arising from certain keratin pairs.

scholarly journals Comparative Transcriptomic Analysis of Thermally Stressed Arabidopsis thaliana Meiotic Recombination Mutants

2020 ◽  
Author(s):  
Jiyue Huang ◽  
Hongkuan Wang ◽  
Yingxiang Wang ◽  
Gregory Copenhaver
Keyword(s):  
Meiotic Recombination ◽  
Environmental Temperature ◽  
Meiotic Chromosome ◽  
Gene Expression Pattern ◽  
Transcriptomic Analysis ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Promoter Regions ◽  
Thermally Stressed

Abstract Background: Meiosis is a specialized cell division that underpins sexual reproduction in most eukaryotes. During meiosis, interhomolog meiotic recombination facilitates accurate chromosome segregation and generates genetic diversity by shuffling parental alleles in the gametes. The frequency of meiotic recombination in Arabidopsis has a U-shaped curve in response to environmental temperature, and is dependent on the Type I, crossover (CO) interference-sensitive pathway. The mechanisms that modulate recombination frequency in response to temperature are not yet known. Results: In this study, we compare the transcriptomes of thermally-stressed meiotic-stage anthers from msh4 and mus81 mutants that mediate the Type I and Type II meiotic recombination pathways, respectively. We show that heat stress reduces the number of expressed genes regardless of genotype. In addition, msh4 mutants have a distinct gene expression pattern compared to mus81 and wild type controls. Interestingly, ASY1, which encodes a HORMA domain protein that is a component of meiotic chromosome axes, is up-regulated in wild type and mus81 but not in msh4. We also characterize 51 novel, previously unannotated transcripts, and show that their promoter regions are associated with A-rich meiotic recombination hotspot motifs. Conclusions: Our transcriptomic analysis of msh4 and mus81 mutants enhances our understanding of how the Type I and Type II meiotic CO pathway respond to environmental temperature stress and might provide a strategy to manipulate recombination levels in plants.

scholarly journals Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.

1985 ◽  
Vol 5 (8) ◽  
pp. 1984-1992 ◽  
Author(s):  
R Van Buskirk ◽  
T Corcoran ◽  
J A Wagner
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Pc12 Cells ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Dependent Protein Kinase ◽  
Nerve Growth ◽  
Regulatory Subunits ◽  
Dependent Protein

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.

scholarly journals The LymphotoxinβReceptor Is Essential for Upregulation of IFN-Induced Guanylate-Binding Proteins and Survival afterToxoplasma gondiiInfection

2017 ◽  
Vol 2017 ◽  
pp. 1-16 ◽  
Author(s):  
Kristina Behnke ◽  
Ursula R. Sorg ◽  
Helmut E. Gabbert ◽  
Klaus Pfeffer
Keyword(s):  
Type I Interferon ◽  
Parasite Load ◽  
Mortality Rates ◽  
Host Responses ◽  
Interferon Response ◽  
Transcriptional Level ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Guanylate Binding Protein

Lymphotoxinβreceptor (LTβR) signaling plays an important role in efficient initiation of host responses to a variety of pathogens, encompassing viruses, bacteria, and protozoans via induction of the type I interferon response. The present study reveals that afterToxoplasma gondiiinfection, LTβR−/−mice show a substantially reduced survival rate when compared to wild-type mice. LTβR−/−mice exhibit an increased parasite load and a more pronounced organ pathology. Also, a delayed increase of serum IL-12p40 and a failure of the protective IFNγresponse in LTβR−/−mice were observed. Serum NO levels in LTβR−/−animals rose later and were markedly decreased compared to wild-type animals. At the transcriptional level, LTβR−/−animals exhibited a deregulated expression profile of several cytokines known to play a role in activation of innate immunity inT. gondiiinfection. Importantly, expression of the IFNγ-regulated murine guanylate-binding protein (mGBP) genes was virtually absent in the lungs of LTβR−/−mice. This demonstrates clearly that the LTβR is essential for the induction of a type II IFN-mediated immune response againstT. gondii. The pronounced inability to effectively upregulate host defense effector molecules such as GBPs explains the high mortality rates of LTβR−/−animals afterT. gondiiinfection.

scholarly journals A6 THE ROLE OF TUFT CELLS IN INTESTINAL HOMEOSTASIS

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 6-7
Author(s):  
A Zhang ◽  
Y Pang ◽  
S Menzies ◽  
L M Sly
Keyword(s):  
Disease Activity ◽  
Cell Activity ◽  
Sh2 Domain ◽  
Type I ◽  
Type Ii ◽  
Wild Type ◽  
Cell Responses ◽  
Tuft Cell ◽  
Tuft Cells

Abstract Background Intestinal epithelial cells may actively regulate homeostasis by recognizing and responding to extracellular signals. One of these cell types, tuft cells, has been proposed to have a role in secretion, absorption, and reception. However, their role in the intestine has not been fully characterized. We have found that tuft cells express the SH2 domain-containing inositol 5’-phosphatase (SHIP), which was formerly thought to be restricted to hematopoietic cells. SHIP negatively regulates PI3K-mediated cell growth, proliferation, and activation. Tuft cells secrete IL-25, which activates group 2 innate lymphoid cells (ILC2s), leading to type 2 immune responses. Tuft cells may contribute to inflammation in the intestine by increasing ILC2 numbers and/or activation, leading to type II inflammation. Aims My hypothesis is that SHIP inhibits tuft cell responses to innate immune stimuli by limiting PI3K activation. Moreover, SHIP deficiency will increase tuft cell responses to commensal microbes, causing ILC2-mediated type II inflammation. To investigate the role of SHIP in tuft cell responses in vivo, I will use a tuft cell-specific SHIP deficient mouse in the dextran sodium sulfate (DSS)-induced colitis model. Methods We created a mouse deficient in SHIP only in intestinal tuft cells (Fabpcre x SHIPfl/fl) to investigate the impact of SHIP deficiency in tuft cells on responses to luminal microbes. Tuft cell-specific SHIP deficient mice (8-week-old) and their wild type littermates were subjected to DSS-induced colitis for 7 days. Clinical disease activity was monitored daily and gross pathology, including total colon length, was examined at the experimental endpoint. The concentrations of pro-inflammatory type I and type II cytokines were assessed in colonic tissue homogenates via ELISA. Results During DSS-induced colitis, mice with SHIP deficient tuft cells had increased disease activity compared to their wild type littermates, particularly evident in their weight loss. Mice with SHIP deficient tuft cells also had significantly shorter colons than their wild type littermates. IL-25 concentrations (produced by tuft cells) were increased in full thickness colon homogenates from mice with SHIP deficient tuft cells. In contrast, pro-inflammatory cytokines IL-1β, IL-6, and TNF did not differ between genotypes. Thus, increased tuft cell activity due to SHIP deficiency correlated with increased disease severity during DSS-induced colitis. Conclusions SHIP deficiency in intestinal tuft cells leads to increased tuft cell activity and exacerbated colitis during DSS treatment. Tuft cells may contribute to inflammation via IL-25 production, leading to increased type II inflammation by ILC2s. In future studies, we will target IL-25 in this model to determine whether increased tuft cell IL-25 production plays a causal role in disease exacerbation. Funding Agencies NSERC

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