scholarly journals Tuba1a is uniquely important for axon guidance through midline commissural structures

2020 ◽  
Author(s):  
Georgia Buscaglia ◽  
Jayne Aiken ◽  
Katelyn J. Hoff ◽  
Kyle R. Northington ◽  
Emily A. Bates
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Intracellular Transport ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Loss Of Function ◽  
Developmental Guidance ◽  
Genes Encoding ◽  
Developing Neurons

AbstractDeveloping neurons undergo dramatic morphological changes to appropriately migrate and extend axons to make synaptic connections. The microtubule cytoskeleton, made of α/β-tubulin dimers, drives neurite outgrowth, promotes neuronal growth cone responses, and facilitates intracellular transport of critical cargoes during neurodevelopment. TUBA1A constitutes the majority of α-tubulin in the developing brain and mutations to TUBA1A in humans cause severe brain malformations accompanied by varying neurological defects, collectively termed tubulinopathies. Studies of TUBA1A function in vivo have been limited by the presence of multiple genes encoding highly similar tubulin proteins, which prevents TUBA1A-specific antibody generation and makes genetic manipulation challenging. Here we present a novel tagging method for studying and manipulating TUBA1A in cells without impairing tubulin function. Using this tool, we show that a TUBA1A loss-of-function mutation TUBA1AN102D (TUBA1AND), reduced the amount of TUBA1A protein and prevented incorporation of TUBA1A into microtubule polymers. Reduced Tuba1a α-tubulin in heterozygous Tuba1aND/+ mice significantly impacted axon extension and impaired formation of forebrain commissures. Neurons with reduced Tuba1a caused by Tuba1aND had altered microtubule dynamics and slower neuron outgrowth compared to controls. Neurons deficient in Tuba1a failed to localize microtubule associated protein-1b (Map1b) to the developing growth cone, likely impacting reception of developmental guidance cues. Overall, we show that reduced Tuba1a is sufficient to support neuronal migration, but not axon guidance, and provide mechanistic insight as to how TUBA1A tunes microtubule function to support neurodevelopment.

The metalloproteinase ADAM10 is required for retinal ganglion cell axon guidance in the developing visual systems

2007 ◽  
Vol 30 (4) ◽  
pp. 77
Author(s):  
Y. Y. Chen ◽  
C. L. Hehr ◽  
K. Atkinson-Leadbeater ◽  
J. C. Hocking ◽  
S. McFarlane
Keyword(s):  
Ganglion Cell ◽  
Axon Guidance ◽  
Retinal Ganglion Cell ◽  
Growth Cone ◽  
Expression Patterns ◽  
Optic Tract ◽  
Axon Growth ◽  
Proteolytic Processing ◽  
Retinal Ganglion

Background: The growth cone interprets cues in its environment in order to reach its target. We want to identify molecules that regulate growth cone behaviour in the developing embryo. We investigated the role of A disintegrin and metalloproteinase 10 (ADAM10) in axon guidance in the developing visual system of African frog, Xenopus laevis. Methods: We first examined the expression patterns of adam10 mRNA by in situ hybridization. We then exposed the developing optic tract to an ADAM10 inhibitor, GI254023X, in vivo. Lastly, we inhibited ADAM10 function in diencephalic neuroepithelial cells (through which retinal ganglion cell (RGC) axons extend) or RGCs by electroporating or transfecting an ADAM10 dominant negative (dn-adam10). Results: We show that adam10 mRNA is expressed in the dorsal neuroepithelium over the time RGC axons extend towards their target, the optic tectum. Second, pharmacological inhibition of ADAM10 in an in vivo exposed brain preparation causes the failure of RGC axons to recognize their target at low concentrations (0.5, 1 μM), and the failure of the axons to make a caudal turn in the mid-diencephalon at higher concentration (5 μM). Thus, ADAM10 function is required for RGC axon guidance at two key guidance decisions. Finally, molecular inhibition of ADAM10 function by electroporating dn-adam10 in the brain neuroepithelium causes defects in RGC axon target recognition (57%) and/or defects in caudal turn (12%), as seen with the pharmacological inhibitor. In contrast, molecular inhibition of ADAM10 within the RGC axons has no effect. Conclusions: These data argue strongly that ADAM10 acts cell non-autonomously within the neuroepithelium to regulate the guidance of RGC axons. This study shows for the first time that a metalloproteinase acts in a cell non-autonomous fashion to direct vertebrate axon growth. It will provide important insights into candidate molecules that could be used to reform nerve connections if destroyed because of injury or disease. References Hattori M, Osterfield M, Flanagan JG. Regulated cleavage of a contact-mediated axon repellent. Science 2000; 289(5483):1360-5. Janes PW, Saha N, Barton WA, Kolev MV, Wimmer-Kleikamp SH, Nievergall E, Blobel CP, Himanen JP, Lackmann M, Nikolov DB. Adam meets Eph: an ADAM substrate recognition module acts as a molecular switch for ephrin cleavage in trans. Cell 2005; 123(2):291-304. Pan D, Rubin GM. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997; 90(2):271-80.

Computational Analysis of The Effects of Nitrogen Source and Sin1 Knockout on Biosilica Morphology in The Model Diatom Thalassiosira Pseudonana

2020 ◽  
Author(s):  
Szabolcs Horvát ◽  
Adeeba Fathima ◽  
Stefan Görlich ◽  
Carl Modes ◽  
Michael Schlierf ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Electron Micrographs ◽  
Self Assembly ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Thalassiosira Pseudonana ◽  
Inorganic Materials ◽  
Automated Image Analysis ◽  
Loss Of Function ◽  
Knockout Mutant

Abstract Morphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as sole nitrogen source. Furthermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work, will be invaluable for unraveling genotype-phenotype correlations in diatom biosilica morphogenesis.

scholarly journals Flavin Monooxygenases Regulate C. elegans Axon Guidance and Growth Cone Protrusion with UNC-6/Netrin signaling and Rac GTPases

2017 ◽  
Author(s):  
Mahekta Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Molecular Mechanisms ◽  
Direct Oxidation ◽  
Rac Gtpase ◽  
C Elegans ◽  
Growth Cone Collapse ◽  
Rac Gtpases ◽  
Inhibition Of Growth

AbstractThe guidance cue UNC-6/Netrin regulates both attractive and repulsive axon guidance. Our previous work showed that in C. elegans, the attractive UNC-6/Netrin receptor UNC-40/DCC stimulates growth cone protrusion, and that the repulsive receptor, an UNC-5/UNC-40 heterodimer, inhibits growth cone protrusion. We have also shown that inhibition of growth cone protrusion downstream of the UNC-5/UNC-40 repulsive receptor involves Rac GTPases, the Rac GTP exchange factor UNC-73/Trio, and the cytoskeletal regulator UNC-33/CRMP, which mediates Semaphorin-induced growth cone collapse in other systems. The multidomain flavoprotein monooxygenase (FMO) MICAL also mediates growth cone collapse in response to Semaphorin by directly oxidizing F-actin, resulting in depolymerization. The C. elegans genome does not encode a multidomain MICAL-like molecule, but does encode five flavin monooxygenases (FMO-1, -2, -3, -4, and 5) and another molecule, EHBP-1, similar to the non-FMO portion of MICAL.Here we show that FMO-1, FMO-4, FMO-5, and EHBP-1 may play a role in UNC-6/Netrin directed repulsive guidance mediated through UNC-40 and UNC-5 receptors. Mutations in fmo-1, fmo-4, fmo-5, and ehbp-1 showed VD/DD axon guidance and branching defects, and variably enhanced unc-40 and unc-5 VD/DD guidance defects. Developing growth cones in vivo of fmo-1, fmo-4, fmo-5, and ehbp-1 mutants displayed excessive filopodial protrusion, and transgenic expression of FMO-5 inhibited growth cone protrusion. Mutations suppressed growth cone inhibition caused by activated UNC-40 and UNC-5 signaling, and activated Rac GTPase CED-10 and MIG-2, suggesting that these molecules are required downstream of UNC-6/Netrin receptors and Rac GTPases. From these studies, we conclude that FMO-1, FMO-4, FMO-5, and EHBP-1 represent new players downstream of UNC-6/Netrin receptors and Rac GTPases that inhibit growth cone filopodial protrusion in repulsive axon guidance.Author SummaryMolecular mechanisms of axon repulsion mediated by UNC-6/Netrin are not well understood. Inhibition of growth cone lamellipodial and filopodial protrusion is critical to repulsive axon guidance. Previous work identified a novel pathway involving Rac GTPases and the cytoskeletal interacting molecule UNC-33/CRMP required for UNC-6/Netrin-mediated inhibition of growth cone protrusion. In other systems, CRMP mediates growth cone collapse in response to semaphorin. Here we demonstrate a novel role of flavoprotein monooxygenases (FMOs) in repulsive axon guidance and inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling and Rac GTPases. In Drosophila and vertebrates, the multidomain MICAL FMO mediates semaphorin-dependent growth cone collapse by direct oxidation and depolymerization of F-actin. The C. elegans genome does not encode a multidomain MICAL-like molecule, and we speculate that the C. elegans FMOs might have an equivalent role downstream of UNC-6/Netrin signaling. Indeed, we show that EHBP-1, similar to the non-FMO portion of MICAL, also controls repulsive axon guidance and growth cone inhibition, suggesting that in C. elegans, the functions of the multidomain MICAL molecule might be distributed across different molecules. In sum, we show conservation of function of molecules involved in semaphorin growth cone collapse with inhibition of growth cone protrusion downstream of UNC-6/Netrin signaling.

scholarly journals L1CAM promotes ovarian cancer stemness and tumor initiation via FGFR1/SRC/STAT3 signaling

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Marco Giordano ◽  
Alessandra Decio ◽  
Chiara Battistini ◽  
Micol Baronio ◽  
Fabrizio Bianchi ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Tumor Formation ◽  
In Vitro Assays ◽  
Tumor Initiation ◽  
Loss Of Function ◽  
Stat3 Signaling

Abstract Background Cancer stem cells (CSC) have been implicated in tumor progression. In ovarian carcinoma (OC), CSC drive tumor formation, dissemination and recurrence, as well as drug resistance, thus contributing to the high death-to-incidence ratio of this disease. However, the molecular basis of such a pathogenic role of ovarian CSC (OCSC) has been elucidated only to a limited extent. In this context, the functional contribution of the L1 cell adhesion molecule (L1CAM) to OC stemness remains elusive. Methods The expression of L1CAM was investigated in patient-derived OCSC. The genetic manipulation of L1CAM in OC cells provided gain and loss-of-function models that were then employed in cell biological assays as well as in vivo tumorigenesis experiments to assess the role of L1CAM in OC cell stemness and in OCSC-driven tumor initiation. We applied antibody-mediated neutralization to investigate L1CAM druggability. Biochemical approaches were then combined with functional in vitro assays to study the molecular mechanisms underlying the functional role of L1CAM in OCSC. Results We report that L1CAM is upregulated in patient-derived OCSC. Functional studies showed that L1CAM promotes several stemness-related properties in OC cells, including sphere formation, tumor initiation and chemoresistance. These activities were repressed by an L1CAM-neutralizing antibody, pointing to L1CAM as a druggable target. Mechanistically, L1CAM interacted with and activated fibroblast growth factor receptor-1 (FGFR1), which in turn induced the SRC-mediated activation of STAT3. The inhibition of STAT3 prevented L1CAM-dependent OC stemness and tumor initiation. Conclusions Our study implicate L1CAM in the tumorigenic function of OCSC and point to the L1CAM/FGFR1/SRC/STAT3 signaling pathway as a novel driver of OC stemness. We also provide evidence that targeting this pathway can contribute to OC eradication.

scholarly journals RHO-1 and the Rho GEF RHGF-1 interact with UNC-6/Netrin signaling to regulate growth cone protrusion and microtubule organization in C. elegans

2019 ◽  
Author(s):  
Mahekta R. Gujar ◽  
Aubrie M. Stricker ◽  
Erik A. Lundquist
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Neural Circuit ◽  
Growth Cones ◽  
Negative Regulator ◽  
Microtubule Organization ◽  
C Elegans ◽  
Guidance Cue ◽  
Rho Gef

AbstractUNC-6/Netrin is a conserved axon guidance cue that directs growth cone migrations in the dorsal-ventral axis of C. elegans and in the vertebrate spinal cord. UNC-6/Netrin is expressed in ventral cells, and growth cones migrate ventrally toward or dorsally away from UNC-6/Netrin. Recent studies of growth cone behavior during outgrowth in vivo in C. elegans have led to a polarity/protrusion model in directed growth cone migration away from UNC-6/Netrin. In this model, UNC-6/Netrin first polarizes the growth cone via the UNC-5 receptor, leading to dorsally biased protrusion and F-actin accumulation. UNC-6/Netrin then regulates protrusion based on this polarity. The receptor UNC-40/DCC drives protrusion dorsally, away from the UNC-6/Netrin source, and the UNC-5 receptor inhibits protrusion ventrally, near the UNC-6/Netrin source, resulting in dorsal migration. UNC-5 inhibits protrusion in part by excluding microtubules from the growth cone, which are pro-protrusive. Here we report that the RHO-1/RhoA GTPase and its activator GEF RHGF-1 inhibit growth cone protrusion and MT accumulation in growth cones, similar to UNC-5. However, growth cone polarity of protrusion and F-actin were unaffected by RHO-1 and RHGF-1. Thus, RHO-1 signaling acts specifically as a negative regulator of protrusion and MT accumulation, and not polarity. Genetic interactions suggest that RHO-1 and RHGF-1 act with UNC-5, as well as with a parallel pathway, to regulate protrusion. The cytoskeletal interacting molecule UNC-33/CRMP was required for RHO-1 activity to inhibit MT accumulation, suggesting that UNC-33/CRMP might act downstream of RHO-1. In sum, these studies describe a new role of RHO-1 and RHGF-1 in regulation of growth cone protrusion by UNC-6/Netrin.Author SummaryNeural circuits are formed by precise connections between axons. During axon formation, the growth cone leads the axon to its proper target in a process called axon guidance. Growth cone outgrowth involves asymmetric protrusion driven by extracellular cues that stimulate and inhibit protrusion. How guidance cues regulate growth cone protrusion in neural circuit formation is incompletely understood. This work shows that the signaling molecule RHO-1 acts downstream of the UNC-6/Netrin guidance cue to inhibit growth cone protrusion in part by excluding microtubules from the growth cone, which are structural elements that drive protrusion.

scholarly journals Semaphorins in axon regeneration: developmental guidance molecules gone wrong?

2006 ◽  
Vol 361 (1473) ◽  
pp. 1499-1511 ◽  
Author(s):  
R. Jeroen Pasterkamp ◽  
Joost Verhaagen
Keyword(s):  
Signal Transduction ◽  
Nervous System ◽  
Axon Guidance ◽  
Growth Cone ◽  
Axon Regeneration ◽  
Critical Role ◽  
Developmental Guidance ◽  
Receptor Complexes ◽  
Fibrotic Scar ◽  
Intracellular Signal

Semaphorins are developmental axon guidance cues that continue to be expressed during adulthood and are regulated by neural injury. During the formation of the nervous system, repulsive semaphorins guide axons to their targets by restricting and channelling their growth. They affect the growth cone cytoskeleton through interactions with receptor complexes that are linked to a complicated intracellular signal transduction network. Following injury, regenerating axons stop growing when they reach the border of the glial-fibrotic scar, in part because they encounter a potent molecular barrier that inhibits growth cone extension. A number of secreted semaphorins are expressed in the glial-fibrotic scar and at least one transmembrane semaphorin is upregulated in oligodendrocytes surrounding the lesion site. Semaphorin receptors, and many of the signal transduction components required for semaphorin signalling, are present in injured central nervous system neurons. Here, we review evidence that supports a critical role for semaphorin signalling in axon regeneration, and highlight a number of challenges that lie ahead with respect to advancing our understanding of semaphorin function in the normal and injured adult nervous system.

scholarly journals LRRK2 mediates axon development by regulating Frizzled3 phosphorylation and growth cone–growth cone communication

2020 ◽  
Vol 117 (30) ◽  
pp. 18037-18048 ◽  
Author(s):  
Keisuke Onishi ◽  
Runyi Tian ◽  
Bo Feng ◽  
Yiqiong Liu ◽  
Junkai Wang ◽  
...  
Keyword(s):  
Axon Guidance ◽  
Growth Cone ◽  
Planar Cell Polarity ◽  
Growth Cones ◽  
Loss Of Function ◽  
Double Knockout ◽  
Intercellular Interaction ◽  
Commissural Axons ◽  
Lrrk2 G2019s ◽  
Cone Growth

Axon–axon interactions are essential for axon guidance during nervous system wiring. However, it is unknown whether and how the growth cones communicate with each other while sensing and responding to guidance cues. We found that the Parkinson’s disease gene, leucine-rich repeat kinase 2 (LRRK2), has an unexpected role in growth cone–growth cone communication. The LRRK2 protein acts as a scaffold and induces Frizzled3 hyperphosphorylation indirectly by recruiting other kinases and also directly phosphorylates Frizzled3 on threonine 598 (T598). InLRRK1orLRRK2single knockout,LRRK1/2double knockout, andLRRK2 G2019Sknockin, the postcrossing spinal cord commissural axons are disorganized and showed anterior–posterior guidance errors after midline crossing. Growth cones from eitherLRRK2knockout orG2019Sknockin mice showed altered interactions, suggesting impaired communication. Intercellular interaction between Frizzled3 and Vangl2 is essential for planar cell polarity signaling. We show here that this interaction is regulated by phosphorylation of Frizzled3 at T598 and can be regulated by LRRK2 in a kinase activity-dependent way. In theLRRK1/2double knockout orLRRK2 G2019Sknockin, the dopaminergic axon bundle in the midbrain was significantly widened and appeared disorganized, showing aberrant posterior-directed growth. Our findings demonstrate that LRRK2 regulates growth cone–growth cone communication in axon guidance and that both loss-of-function mutation and a gain-of-function mutation (G2019S)cause axon guidance defects in development.

scholarly journals mRNAs Encoding Telomerase Components and Regulators Are Controlled by UPF Genes in Saccharomyces cerevisiae

2003 ◽  
Vol 2 (1) ◽  
pp. 134-142 ◽  
Author(s):  
Jeffrey N. Dahlseid ◽  
Jodi Lew-Smith ◽  
Michael J. Lelivelt ◽  
Shinichiro Enomoto ◽  
Amanda Ford ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Telomere Length ◽  
Dna Sequences ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Telomeric Silencing ◽  
Expression Of Genes ◽  
Mutant Strains ◽  
Genes Encoding

ABSTRACT Telomeres, the chromosome ends, are maintained by a balance of activities that erode and replace the terminal DNA sequences. Furthermore, telomere-proximal genes are often silenced in an epigenetic manner. In Saccharomyces cerevisiae, average telomere length and telomeric silencing are reduced by loss of function of UPF genes required in the nonsense-mediated mRNA decay (NMD) pathway. Because NMD controls the mRNA levels of several hundred wild-type genes, we tested the hypothesis that NMD affects the expression of genes important for telomere functions. In upf mutants, high-density oligonucleotide microarrays and Northern blots revealed that the levels of mRNAs were increased for genes encoding the telomerase catalytic subunit (Est2p), in vivo regulators of telomerase (Est1p, Est3p, Stn1p, and Ten1p), and proteins that affect telomeric chromatin structure (Sas2p and Orc5p). We investigated whether overexpressing these genes could mimic the telomere length and telomeric silencing phenotypes seen previously in upf mutant strains. Increased dosage of STN1, especially in combination with increased dosage of TEN1, resulted in reduced telomere length that was indistinguishable from that in upf mutants. Increased levels of STN1 together with EST2 resulted in reduced telomeric silencing like that of upf mutants. The half-life of STN1 mRNA was not altered in upf mutant strains, suggesting that an NMD-controlled transcription factor regulates the levels of STN1 mRNA. Together, these results suggest that NMD maintains the balance of gene products that control telomere length and telomeric silencing primarily by maintaining appropriate levels of STN1, TEN1, and EST2 mRNA.

scholarly journals Computational analysis of the effects of nitrogen source and sin1 knockout on biosilica morphology in the model diatom Thalassiosira pseudonana

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Szabolcs Horvát ◽  
Adeeba Fathima ◽  
Stefan Görlich ◽  
Michael Schlierf ◽  
Carl D. Modes ◽  
...  
Keyword(s):  
Nitrogen Source ◽  
Electron Micrographs ◽  
Self Assembly ◽  
Genetic Manipulation ◽  
Morphological Changes ◽  
Thalassiosira Pseudonana ◽  
Inorganic Materials ◽  
Automated Image Analysis ◽  
Loss Of Function ◽  
Knockout Mutant

AbstractMorphogenesis of the silica based cell walls of diatoms, a large group of microalgae, is a paradigm for the self-assembly of complex 3D nano- and microscale patterned inorganic materials. In recent years, loss-of-function studies using genetic manipulation were successfully applied for the identification of genes that guide silica morphogenesis in diatoms. These studies revealed that the loss of one gene can affect multiple morphological parameters, and the morphological changes can be rather subtle being blurred by natural variations in morphology even within the same clone. Both factors severely hamper the identification of morphological mutants using subjective by-eye inspection of electron micrographs. Here we have developed automated image analysis for objectively quantifying the morphology of ridge networks and pore densities from numerous electron micrographs of diatom biosilica. This study demonstrated differences in ridge network morphology and pore density in diatoms growing on ammonium rather than nitrate as the sole nitrogen source. Furthermore, it revealed shortcomings in previous by-eye evaluation of the biosilica phenotype of the silicanin-1 knockout mutant. We anticipate that the computational methods established in the present work will be invaluable for unraveling genotype–phenotype correlations in diatom biosilica morphogenesis.

scholarly journals Mono-homologous linear DNA recombination by the non-homologous end-joining pathway as a novel and simple gene inactivation method: a proof of concept study inDietziasp. DQ12-45-1b

2018 ◽  
Author(s):  
Shelian Lu ◽  
Yong Nie ◽  
Meng Wang ◽  
Hong-Xiu Xu ◽  
Dong-Ling Ma ◽  
...  
Keyword(s):  
Genetic Manipulation ◽  
Dna Recombination ◽  
Proof Of Concept ◽  
End Joining ◽  
Circular Dna ◽  
Linear Dna ◽  
Genes Encoding ◽  
Non Homologous End Joining ◽  
Nhej Activity

ABSTRACTNon-homologous end-joining (NHEJ) is critical for genome stability because of its roles in double-strand break repair. Ku and ligase D (LigD) are the crucial proteins in this process, and strains expressing Ku and LigD can cyclize linear DNAin vivo.Herein, we established a proof-of-concept mono-homologous linear DNA recombination for gene inactivation or genome editing by which cyclization of linear DNAin vivoby NHEJ could be used to generate non-replicable circular DNA and could allow allelic exchanges between the circular DNA and the chromosome. We achieved this approach inDietziasp. DQ12-45-1b, which expresses Ku and LigD homologs and presents NHEJ activity. By transforming the strain with a linear DNA mono homolog to the sequence in chromosome, we mutated the genome. This method did not require the screening of suitable plasmids and was easy and time-effective. Bioinformatic analysis showed that more than 20% prokaryotic organisms contain Ku and LigD, suggesting the wide distribution of NHEJ activities. Moreover, theEscherichia colistrain also showed NHEJ activity when the Ku and LigD ofDietziasp. DQ12-45-1b were introduced and expressed in it. Therefore, this method may be a widely applicable genome editing tool for diverse prokaryotic organisms, especially for non-model microorganisms.IMPORTANCEThe non-model gram-positive bacteria lack efficient genetic manipulation systems, but they express genes encoding Ku and LigD. The NHEJ pathway inDietziasp. DQ12-45-1b was evaluated and was used to successfully knockout eleven genes in the genome. Since bioinformatic studies revealed that the putative genes encoding Ku and LigD ubiquitously exist in phylogenetically diverse bacteria and archaea, the mono-homologous linear DNA recombination by the NHEJ pathway could be a potentially applicable genetic manipulation method for diverse non-model prokaryotic organisms.

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