scholarly journals Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 664
Author(s):  
Julie Thomy ◽  
Frederic Sanchez ◽  
Marta Gut ◽  
Fernando Cruz ◽  
Tyler Alioto ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Chromosomal Rearrangements ◽  
Single Copy ◽  
Molecular Techniques ◽  
End Joining ◽  
Structural Variations ◽  
Vector Integration ◽  
Ostreococcus Tauri ◽  
Unicellular Green Alga ◽  
Genomic Studies

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.

scholarly journals Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri

2021 ◽  
Author(s):  
Julie Thomy ◽  
Frederic Sanchez ◽  
Marta Gut ◽  
Fernando Cruz ◽  
Tyler Alioto ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Chromosomal Rearrangements ◽  
Molecular Techniques ◽  
End Joining ◽  
Structural Variations ◽  
Vector Integration ◽  
Ostreococcus Tauri ◽  
Unicellular Green Alga ◽  
Genomic Studies ◽  
Non Homologous End Joining

Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration appears to be random, occurring mainly at a single locus, and thus confirming the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci, and often within the vector sequence itself. From these observations, we speculate that a non-homologous end joining-like mechanism is required during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in ecologically important marine algae.

scholarly journals One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Joseph R. Owen ◽  
Sadie L. Hennig ◽  
Bret R. McNabb ◽  
Tamer A. Mansour ◽  
Justin M. Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bos Taurus ◽  
Single Copy ◽  
Blastocyst Stage ◽  
Target Site ◽  
End Joining ◽  
Bull Calf ◽  
Dividing Cells ◽  
Green Fluorescent ◽  
Donor Template

Abstract Background The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells. Results By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1 kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1 kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26 bp insertion allele, and a complex 38 kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18 kb allele was detected that looks to be a derivative of the 38 kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template. Conclusion The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

scholarly journals The Role of RNA in DNA Breaks, Repair and Chromosomal Rearrangements

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 550
Author(s):  
Matvey Mikhailovich Murashko ◽  
Ekaterina Mikhailovna Stasevich ◽  
Anton Markovich Schwartz ◽  
Dmitriy Vladimirovich Kuprash ◽  
Aksinya Nicolaevna Uvarova ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Gene Mutations ◽  
Nonhomologous End Joining ◽  
Published Data ◽  
Dna Double Strand Breaks ◽  
Dna Breaks ◽  
End Joining ◽  
Homology Directed Repair ◽  
Chromosome Aberration Frequency

Incorrect reparation of DNA double-strand breaks (DSB) leading to chromosomal rearrangements is one of oncogenesis’s primary causes. Recently published data elucidate the key role of various types of RNA in DSB formation, recognition and repair. With growing interest in RNA biology, increasing RNAs are classified as crucial at the different stages of the main pathways of DSB repair in eukaryotic cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). Gene mutations or variation in expression levels of such RNAs can lead to local DNA repair defects, increasing the chromosome aberration frequency. Moreover, it was demonstrated that some RNAs could stimulate long-range chromosomal rearrangements. In this review, we discuss recent evidence demonstrating the role of various RNAs in DSB formation and repair. We also consider how RNA may mediate certain chromosomal rearrangements in a sequence-specific manner.

scholarly journals Evidence for Replicative Repair of DNA Double-Strand Breaks Leading to Oncogenic Translocation and Gene Amplification

2002 ◽  
Vol 196 (4) ◽  
pp. 469-480 ◽  
Author(s):  
Michael J. Difilippantonio ◽  
Simone Petersen ◽  
Hua Tang Chen ◽  
Roger Johnson ◽  
Maria Jasin ◽  
...  
Keyword(s):  
Dna Repair ◽  
Dna Cleavage ◽  
Chromosomal Rearrangements ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Telomere Capture ◽  
Culture Models ◽  
Recombination Activating Gene ◽  
Dna Repair Protein ◽  
Break Induced Replication

Nonreciprocal translocations and gene amplifications are commonly found in human tumors. Although little is known about the mechanisms leading to such aberrations, tissue culture models predict that they can arise from DNA breakage, followed by cycles of chromatid fusion, asymmetric mitotic breakage, and replication. Mice deficient in both a nonhomologous end joining (NHEJ) DNA repair protein and the p53 tumor suppressor develop lymphomas at an early age harboring amplification of an IgH/c-myc fusion. Here we report that these chromosomal rearrangements are initiated by a recombination activating gene (RAG)-induced DNA cleavage. Subsequent DNA repair events juxtaposing IgH and c-myc are mediated by a break-induced replication pathway. Cycles of breakage-fusion-bridge result in amplification of IgH/c-myc while chromosome stabilization occurs through telomere capture. Thus, mice deficient in NHEJ provide excellent models to study the etiology of unbalanced translocations and amplification events during tumorigenesis.

scholarly journals Analysis of junction sequences resulting from integration at nonhomologous loci in Neurospora crassa.

Genetics ◽  
1992 ◽  
Vol 130 (4) ◽  
pp. 737-748
Author(s):  
D K Asch ◽  
G Frederick ◽  
J A Kinsey ◽  
D D Perkins
Keyword(s):  
Neurospora Crassa ◽  
Plasmid Dna ◽  
Repetitive Sequence ◽  
Single Copy ◽  
The Other ◽  
Chromosomal Dna ◽  
End Joining ◽  
Dehydrogenase Gene ◽  
The Right ◽  
Complete Deletion

Abstract We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.

Mutagenesis via Insertional or Restriction Enzyme-Mediated Integration (REMI) as a Tool to Tag Pathogenicity Related Genes in Plant Pathogenic Fungi

1999 ◽  
Vol 380 (7-8) ◽  
pp. 855-864 ◽  
Author(s):  
F. J. Maier ◽  
W. Schäfer
Keyword(s):  
Restriction Enzyme ◽  
Molecular Basis ◽  
Insertional Mutagenesis ◽  
Transformation Efficiency ◽  
Pathogenic Fungi ◽  
Transformation Protocol ◽  
Vector Integration ◽  
Future Developments ◽  
Genetically Determined ◽  
Remi Mutagenesis

Abstract Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.

scholarly journals A Magnesium Transport Protein Related to Mammalian SLC41 and Bacterial MgtE Contributes to Circadian Timekeeping in a Unicellular Green Alga

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 158
Author(s):  
Helen K. Feord ◽  
Frederick E.G. Dear ◽  
Darren J. Obbard ◽  
Gerben van Ooijen
Keyword(s):  
Translational Regulation ◽  
Feedback Loops ◽  
Magnesium Ions ◽  
Sequence Analyses ◽  
Clock Period ◽  
Clock Model ◽  
Ostreococcus Tauri ◽  
Unicellular Green Alga ◽  
Taxonomic Groups ◽  
Daily Changes

Circadian clocks in eukaryotes involve both transcriptional-translational feedback loops, post-translational regulation, and metabolic, non-transcriptional oscillations. We recently identified the involvement of circadian oscillations in the intracellular concentrations of magnesium ions (Mg2+i) that were conserved in three eukaryotic kingdoms. Mg2+i in turn contributes to transcriptional clock properties of period and amplitude, and can function as a zeitgeber to define phase. However, the mechanism—or mechanisms—responsible for the generation of Mg2+i oscillations, and whether these are functionally conserved across taxonomic groups, remain elusive. We employed the cellular clock model Ostreococcus tauri to provide a first study of an MgtE domain-containing protein in the green lineage. OtMgtE shares homology with the mammalian SLC41A1 magnesium/sodium antiporter, which has previously been implicated in maintaining clock period. Using genetic overexpression, we found that OtMgtE contributes to both timekeeping and daily changes in Mg2+i. However, pharmacological experiments and protein sequence analyses indicated that critical differences exist between OtMgtE and either the ancestral MgtE channel or the mammalian SLC41 antiporters. We concluded that even though MgtE domain-containing proteins are only distantly related, these proteins retain a shared role in contributing to cellular timekeeping and the regulation of Mg2+i.

scholarly journals Parp3 promotes long-range end joining in murine cells

2018 ◽  
Vol 115 (40) ◽  
pp. 10076-10081 ◽  
Author(s):  
Jacob V. Layer ◽  
J. Patrick Cleary ◽  
Alexander J. Brown ◽  
Kristen E. Stevenson ◽  
Sara N. Morrow ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Embryonic Stem ◽  
Cell Types ◽  
Strand Break ◽  
Nonhomologous End Joining ◽  
Dna Double Strand Breaks ◽  
End Joining ◽  
Strand Breaks ◽  
Class Switch

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class–switch recombination in primary B cells, and inversions in tail fibroblasts that generateEml4–Alkfusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing ofEml4–Alkjunctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

DNA-DNA Hybridization, Phylogenetic Reconstruction and the Fossil Record

1988 ◽  
Vol 1 ◽  
pp. 75-88 ◽  
Author(s):  
Charles R. Marshall
Keyword(s):  
Dna Hybridization ◽  
Nuclear Dna ◽  
Phylogenetic Reconstruction ◽  
Cost Effective ◽  
Single Copy ◽  
Molecular Techniques ◽  
Phylogenetic Information ◽  
Molecular Approaches ◽  
Molecular Characters ◽  
Rates Of Molecular Evolution

In 1962 Zuckerkandl & Pauling suggested that the amino acid sequence of proteins might evolve in a clock-like fashion and thus may be useful for phylogenetic reconstruction. Since then, many different molecular approaches to phylogenetic reconstruction have been proposed (Wilson et al., 1977). Enthusiasm for the clock hypothesis was dampened by the discovery that rates of molecular evolution for many macromolecules have been highly variable through time (Romero-Herrera et al., 1979). However, the contribution of molecular characters to the study of phylogeny is not necessarily dependent on the notion of a molecular clock and molecular approaches continue to be an important source of phylogenetic information. One of the more powerful and cost-effective molecular techniques for phylogenetic purposes is DNA-DNA hybridization, which measures the single-copy nuclear DNA sequence divergences between species.

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