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Keywords = non-homologous chromosome connections

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23 pages, 11957 KiB  
Article
Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress
by Veronika Schusterbauer, Jasmin E. Fischer, Sarah Gangl, Lisa Schenzle, Claudia Rinnofner, Martina Geier, Christian Sailer, Anton Glieder and Gerhard G. Thallinger
J. Fungi 2022, 8(10), 992; https://doi.org/10.3390/jof8100992 - 21 Sep 2022
Cited by 9 | Viewed by 3878
Abstract
The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous [...] Read more.
The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified de novo off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites. Full article
(This article belongs to the Special Issue Yeast Genetics 2022)
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21 pages, 4730 KiB  
Article
Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL
by Manuel Olazábal-Morán, Miriam Sánchez-Ortega, Laura Martínez-Muñoz, Carmen Hernández, Manuel S. Rodríguez, Mario Mellado and Ana C. Carrera
Cells 2021, 10(11), 2803; https://doi.org/10.3390/cells10112803 - 20 Oct 2021
Cited by 5 | Viewed by 3291
Abstract
3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and [...] Read more.
3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP3-effector AKT were examined after the addition of growth factors. Both epidermal growth factor and serum induced the early activation of AKT and the simultaneous inactivation of PTEN (at ~5 min). This PIP3/AKT peak was followed by a general reduction in AKT activity coincident with the recovery of PTEN phosphatase activity (at ~10–15 min). Subsequent AKT peaks and troughs followed. The fluctuation in AKT activity was linked to that of PTEN; PTEN reconstitution in PTEN-null cells restored AKT fluctuations, while PTEN depletion in control cells abrogated them. The analysis of PTEN activity fluctuations after the addition of growth factors showed its inactivation at ~5 min to be simultaneous with its transient ubiquitination, which was regulated by the ubiquitin E3 ligase cCBL (casitas B-lineage lymphoma proto-oncogene). Protein-protein interaction analysis revealed cCBL to be brought into the proximity of PTEN in a PI3-kinase-dependent manner. These results reveal a mechanism for PI3-kinase/PTEN crosstalk and suggest that cCBL could be new target in strategies designed to modulate PTEN activity in cancer. Full article
(This article belongs to the Section Cell Signaling)
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17 pages, 4716 KiB  
Article
Meiotic Chromosome Contacts as a Plausible Prelude for Robertsonian Translocations
by Sergey Matveevsky, Oxana Kolomiets, Aleksey Bogdanov, Elena Alpeeva and Irina Bakloushinskaya
Genes 2020, 11(4), 386; https://doi.org/10.3390/genes11040386 - 2 Apr 2020
Cited by 15 | Viewed by 4599
Abstract
Robertsonian translocations are common chromosomal alterations. Chromosome variability affects human health and natural evolution. Despite the significance of such mutations, no mechanisms explaining the emergence of such translocations have yet been demonstrated. Several models have explored possible changes in interphase nuclei. Evidence for [...] Read more.
Robertsonian translocations are common chromosomal alterations. Chromosome variability affects human health and natural evolution. Despite the significance of such mutations, no mechanisms explaining the emergence of such translocations have yet been demonstrated. Several models have explored possible changes in interphase nuclei. Evidence for non-homologous chromosomes end joining in meiosis is scarce, and is often limited to uncovering mechanisms in damaged cells only. This study presents a primarily qualitative analysis of contacts of non-homologous chromosomes by short arms, during meiotic prophase I in the mole vole, Ellobius alaicus, a species with a variable karyotype, due to Robertsonian translocations. Immunocytochemical staining of spermatocytes demonstrated the presence of four contact types for non-homologous chromosomes in meiotic prophase I: (1) proximity, (2) touching, (3) anchoring/tethering, and (4) fusion. Our results suggest distinct mechanisms for chromosomal interactions in meiosis. Thus, we propose to change the translocation mechanism model from ‘contact first’ to ‘contact first in meiosis’. Full article
(This article belongs to the Special Issue Mechanisms Driving Karyotype Evolution and Genomic Architecture)
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