Kooperativer Bibliotheksverbund

Berlin Brandenburg

and
and

Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
Filter
Type of Medium
Language
Year
  • 1
    Language: English
    In: Yeast, Jan, 2019, Vol.36(1), p.43(9)
    Description: One of the central hypotheses in the theory of codon usage evolution is that in highly expressed genes, particular codon usage patterns arise because they facilitate efficient gene expression and are thus selected for in evolution. Here, we use plasmid copy number assays and growth rate measurements to explore details of the relationship between codon usage, gene expression level, and selective pressure in Saccharomyces cerevisiae. We find that when high expression levels are required, optimal codon usage is beneficial and provides a fitness advantage, consistent with evolutionary theory. However, when high expression levels are not required, optimal codon usage is surprisingly and strongly selected against. We show that this selection acts at the level of protein synthesis, and we exclude a number of molecular mechanisms as the source for this negative selective pressure including nutrient and ribosome limitations and proteotoxicity effects. These findings deepen our understanding of the evolution of codon usage bias, as well as the design of recombinant protein expression systems.
    Keywords: Saccharomyces Cerevisiae ; Growth Rate ; Molecular Modelling ; Gene Expression ; Gene Expression ; Evolution ; Yeast ; Fitness ; Protein Biosynthesis ; Copy Number ; Evolutionary Genetics ; Interferon ; Codon Usage ; Evolution ; Plasmid Copy Number ; Recombinant Gene Expression ; Selective Pressure ; Yeast;
    ISSN: 0749-503X
    E-ISSN: 10970061
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 2
    Language: English
    In: PLoS ONE, 2011, Vol.6(12), p.e28271
    Description: Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. ; As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. ; Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.
    Keywords: Research Article ; Biology ; Engineering ; Molecular Biology ; Biotechnology ; Cell Biology ; Biochemistry
    E-ISSN: 1932-6203
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 3
    Language: English
    In: The Biochemical journal, 05 October 2017, Vol.474(20), pp.3439-3454
    Description: TorsinA (TorA) is an AAA+ (ATPases associated with diverse cellular activities) ATPase linked to dystonia type 1 (DYT1), a neurological disorder that leads to uncontrollable muscular movements. Although DYT1 is linked to a 3 bp deletion in the C-terminus of TorA, the biological function of TorA remains to be established. Here, we use the yeast as a tractable model to explore TorA function. We demonstrate that TorA can protect yeast cells against different forms of environmental stress and show that in the absence of the molecular disaggregase Hsp104, TorA can refold heat-denatured luciferase in an ATP-dependent manner. However, this activity requires TorA to be translocated to the cytoplasm from the endoplasmic reticulum in order to access and process cytoplasmic protein aggregates. Furthermore, mutational or chemical inactivation of the ATPase activity of TorA blocks this activity. We also find that TorA can inhibit the propagation of certain conformational variants of [], the aggregated prion form of the endogenous Sup35 protein. Finally, we show that while cellular localisation remains unchanged in the dystonia-linked TorA mutant ΔE302-303, the ability of this mutant form of TorA to protect against cellular stress and to facilitate protein refolding is impaired, consistent with it being a loss-of-function mutation.
    Keywords: Hsp104 ; Saccharomyces Cerevisiae ; Torsina ; Molecular Chaperone ; Prion ; Cytosol -- Metabolism ; Molecular Chaperones -- Metabolism ; Saccharomyces Cerevisiae -- Metabolism ; Stress, Physiological -- Physiology
    ISSN: 02646021
    E-ISSN: 1470-8728
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 4
    In: Adam, Ilectra and Jossé, Lyne and Tuite, Mick F (2017) Human TorsinA can function in the yeast cytosol as a molecular chaperone. Biochemical Journal, . BCJ20170395.
    Description: TorsinA (TorA) is an AAA+ ATPAse linked to dystonia type 1 (DYT1), a neurological disorder that leads to uncontrollable muscular movements. Although DYT1 is linked to a 3bp deletion in the C terminus of TorA, the biological function of TorA remains to be established. Here we use the yeast Saccharomyces cerevisiae as a tractable in vivo model to explore TorA function. We demonstrate that TorA can protect yeast cells against different forms of environmental stress and show that in the absence of the molecular disaggregase Hsp104, TorA can refold heat-denatured luciferase in vivo in an ATP-dependent manner. However, this activity requires TorA to be translocated to the cytoplasm from the ER in order to access and process cytoplasmic protein aggregates. Furthermore, mutational or chemical inactivation of the ATPase activity of TorA blocks this activity. We also find that TorA can inhibit the propagation of certain conformational variants of [ PSI +], the aggregated prion form of the endogenous Sup35 protein. Finally, we show that while cellular localisation remains unchanged in the dystonia-linked TorA mutant ∆E302-303, the ability of this mutant form of TorA to protect against cellular stress and to facilitate protein refolding, is impaired, consistent with it being a loss of function mutation .
    ISSN: 0264-6021
    Source: University of Kent
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 5
    In: FEMS Yeast Research, 2011, Vol. 11(1), pp.133-150
    Description: At 5 μg mL −1 , T-2 toxin significantly upregulated the transcription of 281 genes and downregulated 86. Strongly upregulated genes included those involved in redox activity, mitochondrial functions, the response to oxidative stress, and cytoplasmic rRNA transcription and processing. Highly repressed genes have roles in mitochondrial biogenesis, and the expression and stability of cytoplasmic rRNAs. T-2 toxin inhibition of growth was greater in a medium requiring respiration, and was antagonized by antioxidants. T-2 toxin treatment induced reactive oxygen species, caused nucleolytic damage to DNA, probably mitochondrial, and externalization of phosphatidylserine. Deletion mutations causing respiratory deficiency substantially increased toxin tolerance, and deletion of some TOR (target of rapamycin) pathway genes altered T-2 toxin sensitivity. Deletion of FMS1 , which plays an indirect role in cytoplasmic protein synthesis, markedly increased toxin tolerance. Overall, the findings suggest that T-2 toxin targets mitochondria, generating oxy-radicals and repressing mitochondrial biogenesis genes, thus inducing oxidative stress and redox enzyme genes, and triggering changes associated with apoptosis. The large transcriptional changes in genes needed for rRNA transcription and expression and the effects of deletion of FMS1 are also consistent with T-2 toxin damage to the cytoplasmic translational mechanism, although it is unclear how this relates to the mitochondrial effects.
    Keywords: T - 2 Toxin ; 〈Kwd〉〈Italic〉Saccharomyces Cerevisiae〈/Italic〉〈/Kwd〉 ; Transcriptomics ; Ros ; Mitochondrion
    ISSN: 15671356
    E-ISSN: 1567-1364
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 6
    In: Biotechnology Journal, October 2018, Vol.13(10), pp.n/a-n/a
    Description: are abundantly expressed in differentiated cells, including CHO models. The microRNA family reportedly inhibit the expression of , implicated in development, and expression of the oncogene . Here, the authors show that fragments of the 3'UTR (1, 2, 3) can successfully repress luciferase reporter gene expression (top panel) and expression (bottom panel) models. The authors harnessed this to design a novel system with attenuated expression of the selection marker to increase stringency and improve recombinant protein yield from CHO cells.
    Keywords: X3'Utr ; Antibody Production ; Chinese Hamster Ovary Cho ; Dhfr Selection ; Microrna
    ISSN: 1860-6768
    E-ISSN: 1860-7314
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 7
    In: Hayes, Nandini V. L. and Jossé, Lyne and Smales, Christopher Mark and Carden, Martin J. (2011) Modulation of Phosducin-Like Protein 3 (PhLP3) Levels Promotes Cytoskeletal Remodelling in a MAPK and RhoA-Dependent Manner. PLoS ONE, 6 (12). e28271.
    Description: Background Phosducin-like protein 3 (PhLP3) forms a ternary complex with the ATP-dependent molecular chaperone CCT and its folding client tubulin. In vitro studies suggest PhLP3 plays an inhibitory role in β-tubulin folding while conversely in vivo genetic studies suggest PhLP3 is required for the correct folding of β-tubulin. We have a particular interest in the cytoskeleton, its chaperones and their role in determining cellular phenotypes associated with high level recombinant protein expression from mammalian cell expression systems. Methodology/Principal Findings As studies into PhLP3 function have been largely carried out in non mammalian systems, we examined the effect of human PhLP3 over-expression and siRNA silencing using a single murine siRNA on both tubulin and actin systems in mammalian Chinese hamster ovary (CHO) cell lines. We show that over-expression of PhLP3 promotes an imbalance of α and β tubulin subunits, microtubule disassembly and cell death. In contrast, β-actin levels are not obviously perturbed. On-the-other-hand, RNA silencing of PhLP3 increases RhoA-dependent actin filament formation and focal adhesion formation and promotes a dramatic elongated fibroblast-like change in morphology. This was accompanied by an increase in phosphorylated MAPK which has been associated with promoting focal adhesion assembly and maturation. Transient overexpression of PhLP3 in knockdown experiments rescues cells from the morphological change observed during PhLP3 silencing but mitosis is perturbed, probably reflecting a tipping back of the balance of PhLP3 levels towards the overexpression state. Conclusions Our results support the hypothesis that PhLP3 is important for the maintenance of β-tubulin levels in mammalian cells but also that its modulation can promote actin-based cytoskeletal remodelling by a mechanism linked with MAPK phosphorylation and RhoA-dependent changes. PhLP3 levels in mammalian cells are thus finely poised and represents a novel target for engineering industrially relevant cell lines to evolve lines more suited to suspension or adherent cell growth.
    Keywords: Q Science
    ISSN: 1932-6203
    Source: University of Kent
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 8
    Language: English
    In: Biotechnology and Bioengineering, 15 February 2010, Vol.105(3), pp.556-566
    Description: Cultured mammalian cells, particularly Chinese hamster ovary (CHO) cells, are widely exploited as hosts for the production of recombinant proteins, but often yields are limiting. Such limitations may be due in part to the misfolding and subsequent degradation of the heterologous proteins. Consequently we have determined whether transiently co‐expressing yeast and/or mammalian chaperones that act to disaggregate proteins, in CHO cell lines, improve the levels of either a cytoplasmic (Fluc) or secreted (Gluc) form of luciferase or an immunoglobulin IgG4 molecule. Over‐expression of the yeast ‘protein disaggregase’ Hsp104 in a CHO cell line increased the levels of Fluc more significantly than for Gluc although levels were not further elevated by over‐expression of the yeast or mammalian Hsp70/40 chaperones. Over‐expression of TorsinA, a mammalian protein related in sequence to yeast Hsp104, but located in the ER, significantly increased the level of secreted Gluc from CHO cells by 2.5‐fold and to a lesser extent the secreted levels of a recombinant IgG4 molecule. These observations indicate that the over‐expression of yeast Hsp104 in mammalian cells can improve recombinant protein yield and that over‐expression of TorsinA in the ER can promote secretion of heterologous proteins from mammalian cells. Biotechnol. Bioeng. 2010; 105: 556–566. © 2009 Wiley Periodicals, Inc.
    Keywords: Molecular Chaperone ; Hsp104 ; Protein Secretion ; Luciferase ; Protein Disaggregation ; Torsina
    ISSN: 0006-3592
    E-ISSN: 1097-0290
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 9
    In: Miller, Christopher N. and Jossé, Lyne and Tsaousis, Anastasios D. (2018) Localization of a Fe-S biosynthesis in Cryptosporidium mitosome. Journal of Eukaryotic Microbiology, .
    Description: Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron-sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron-sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.
    Keywords: QR Microbiology
    ISSN: 1066-5234
    Source: University of Kent
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
  • 10
    Language: English
    In: The Journal of eukaryotic microbiology, November 2018, Vol.65(6), pp.913-922
    Description: Cryptosporidium is a protozoan, apicomplexan, parasite that poses significant risk to humans and animals, as a common cause of potentially fatal diarrhea in immunodeficient hosts. The parasites have evolved a number of unique biological features that allow them to thrive in a highly specialized parasitic lifestyle. For example, the genome of Cryptosporidium parvum is highly reduced, encoding only 3,805 proteins, which is also reflected in its reduced cellular and organellar content and functions. As such, its remnant mitochondrion, dubbed a mitosome, is one of the smallest mitochondria yet found. While numerous studies have attempted to discover the function(s) of the C. parvum mitosome, most of them have been focused on in silico predictions. Here, we have localized components of a biochemical pathway in the C. parvum mitosome, in our investigations into the functions of this peculiar mitochondrial organelle. We have shown that three proteins involved in the mitochondrial iron-sulfur cluster biosynthetic pathway are localized in the organelle, and one of them can functionally replace its yeast homolog. Thus, it seems that the C. parvum mitosome is involved in iron-sulfur cluster biosynthesis, supporting the organellar and cytosolic apoproteins. These results spearhead further research on elucidating the functions of the mitosome and broaden our understanding in the minimalistic adaptations of these organelles.
    Keywords: Apicomplexans ; Immunofluorescence Assay ; Iron-Sulfur Clusters ; Mitochondria ; Mitosomes
    ISSN: 10665234
    E-ISSN: 1550-7408
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
Close ⊗
This website uses cookies and the analysis tool Matomo. Further information can be found on the KOBV privacy pages