scholarly journals Yeast transformation efficiency is enhanced by TORC1- and eisosome-dependent signalling

2018 ◽  
Author(s):  
Sheng-Chun Yu ◽  
Florian Kuemmel ◽  
Maria-Nefeli Skoufou-Papoutsaki ◽  
Pietro D Spanu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Gene Therapy ◽  
Molecular Mechanisms ◽  
Transformation Efficiency ◽  
Fold Increase ◽  
Dna Uptake ◽  
Yeast Transformation ◽  
Transformation Medium ◽  
Membrane Compartment

ABSTRACTTransformation of baker’s yeast (Saccharomyces cerevisiae) plays a key role in several experimental techniques, yet the molecular mechanisms underpinning transformation are still unclear. Addition of amino acids to the growth and transformation medium increases transformation efficiency. Here, we show that target of rapamycin complex 1 (TORC1) activated by amino acids enhances transformation via ubiquitin-mediated endocytosis. We created mutants of the TORC1 pathway, α-arrestins, and eisosome-related genes. Our results demonstrate that the TORC1-Npr1-Art1/Rsp5 pathway regulates yeast transformation. Based on our previous study, activation of this pathway results in a 13-fold increase in transformation efficiency, or greater. Additionally, we suggest DNA is taken up by domains at the membrane compartment of Can1 (MCC) in the plasma membrane formed by eisosomes. Yeast studies on transformation could be used as a platform to understand the mechanism of DNA uptake in mammalian systems, which is clinically relevant to optimise gene therapy.

scholarly journals Nonrecombinant meiosis I nondisjunction in Saccharomyces cerevisiae induced by tRNA ochre suppressors.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 81-95 ◽  
Author(s):  
E J Louis ◽  
J E Haber
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Chromosome ◽  
Stop Codon ◽  
Fold Increase ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Nonsense Mutations ◽  
Chromosome Iii ◽  
Meiosis I ◽  
Chromosome Nondisjunction

Abstract The presence of the tRNA ochre suppressors SUP11 and SUP5 is found to induce meiosis I nondisjunction in the yeast Saccharomyces cerevisiae. The induction increases with increasing dosage of the suppressor and decreases in the presence of an antisuppressor. The effect is independent of the chromosomal location of SUP11. Each of five different chromosomes monitored exhibited nondisjunction at frequencies of 0.1%-1.1% of random spores, which is a 16-160-fold increase over wild-type levels. Increased nondisjunction is reflected by a marked increase in tetrads with two and zero viable spores. In the case of chromosome III, for which a 50-cM map interval was monitored, the resulting disomes are all in the parental nonrecombinant configuration. Recombination along chromosome III appears normal both in meioses that have no nondisjunction and in meioses for which there was nondisjunction of another chromosome. We propose that a proportion of one or more proteins involved in chromosome pairing, recombination or segregation are aberrant due to translational read-through of the normal ochre stop codon. Hygromycin B, an antibiotic that can suppress nonsense mutations via translational read-through, also induces nonrecombinant meiosis I nondisjunction. Increases in mistranslation, therefore, increase the production of aneuploids during meiosis. There was no observable effect of SUP11 on mitotic chromosome nondisjunction; however some disomes caused SUP11 ade2-ochre strains to appear white or red, instead of pink.

scholarly journals Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

Marine Drugs ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 181
Author(s):  
Kun Qiao ◽  
Caiyun Jiang ◽  
Min Xu ◽  
Bei Chen ◽  
Wenhui Qiu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Protein Domain ◽  
Type D ◽  
Protein Toxin ◽  
Factor Type ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

scholarly journals Effect of Time and Legume Type on Germination-Induced Proteolysis of Lentils and Faba Beans

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 4
Author(s):  
Sara Bautista-Expósito ◽  
Elena Peñas ◽  
Albert Vanderberg ◽  
Juana Frias ◽  
Cristina Martínez-Villaluenga
Keyword(s):  
Amino Acids ◽  
Protease Activity ◽  
Protein Quality ◽  
Principal Component ◽  
Fold Increase ◽  
Protein Digestibility ◽  
Essential Amino Acids ◽  
Seedling Development ◽  
Positive Effects ◽  
Faba Beans

Legumes are alternative protein sources that have been successfully used to develop diverse meatless foods. Although these plant-based products have a lower impact on the environment than equivalent animal-based products, they have lower protein digestibility. Germination could be a useful bioprocess to enhance protein digestibility in legumes, although its effect at different times of seedling development has been little studied in lentils and faba beans. This work investigated the effect of germination time (4 and 6 days after full seed imbibition) on the proteins of three types of Canadian lentils (“gray zero tannin”, G; “caviar black”, B; and “red dehulled”, D) and faba beans (“zero vicin/convicin”, F). Germination increased total nitrogen (4–14% increase) and total levels of some amino acids: Asp in all the sprouts studied; Ser, Pro, Ala, Cys, His and Lys in G; and Met and Tyr in B. A concurrent degradation of the 7S and 11S globulin subunits, the accumulation of peptides below 20 kDa and free essential and non-essential amino acids (4- to 6-fold increase) were observed after germination in all the legumes studied. These effects were attributable to the increased protease activity observed after sprouting. Trypsin inhibitory activity was lower in legume sprouts, except for D, where a small increase was detected. Time, legume type and their interaction showed significant effects on the parameters studied. Germination effects were generally more remarkable at longer stages of seedling development. Among the legumes studied, D showed a differential behavior characterized by a faster protein degradation and release of small peptides, probably due to its higher protease activity as indicated by principal component analysis. These results evidence the positive effects of germination on the protein digestibility of different lentil types and faba beans. The protein quality of plant-based foods could be improved through the selection of legume species with higher germination-induced proteolytic rates and optimized germination times.

scholarly journals Neuron-specific activation of necroptosis signaling in multiple sclerosis cortical grey matter

2021 ◽  
Vol 141 (4) ◽  
pp. 585-604 ◽  
Author(s):  
Carmen Picon ◽  
Anusha Jayaraman ◽  
Rachel James ◽  
Catriona Beck ◽  
Patricia Gallego ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Signaling Pathway ◽  
Cortical Neurons ◽  
Grey Matter ◽  
Molecular Mechanisms ◽  
Fold Increase ◽  
Cell Types ◽  
Meningeal Inflammation ◽  
Cortical Grey Matter

AbstractSustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II–III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I–III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.

scholarly journals Mating of 2 Laboratory Saccharomyces cerevisiae Strains Resulted in Enhanced Production of 2-Phenylethanol by Biotransformation of L-Phenylalanine

2017 ◽  
Vol 27 (2) ◽  
pp. 81-90 ◽  
Author(s):  
Jolanta Mierzejewska ◽  
Aleksandra Tymoszewska ◽  
Karolina Chreptowicz ◽  
Kamil Krol
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Batch Culture ◽  
Fold Increase ◽  
Biotechnological Production ◽  
Aromatic Alcohol ◽  
Enhanced Production ◽  
Yeast Strains ◽  
First Time

2-Phenylethanol (2-PE) is an aromatic alcohol with a rosy scent which is widely used in the food, fragrance, and cosmetic industries. Promising sources of natural 2-PE are microorganisms, especially yeasts, which can produce 2-PE by biosynthesis and biotransformation. Thus, the first challenging goal in the development of biotechnological production of 2-PE is searching for highly productive yeast strains. In the present work, 5 laboratory <i>Saccharomyces cerevisiae</i> strains were tested for the production of 2-PE. Thereafter, 2 of them were hybridized by a mating procedure and, as a result, a new diploid, <i>S. cerevisiae</i> AM1-d, was selected. Within the 72-h batch culture in a medium containing 5 g/L of <smlcap>L</smlcap>-phenylalanine, AM1-d produced 3.83 g/L of 2-PE in a shaking flask. In this way, we managed to select the diploid <i>S. cerevisiae</i> AM1-d strain, showing a 3- and 5-fold increase in 2-PE production in comparison to parental strains. Remarkably, the enhanced production of 2-PE by the hybrid of 2 yeast laboratory strains is demonstrated here for the first time.

scholarly journals Feeding a Saccharomyces cerevisiae fermentation product improves udder health and immune response to a Streptococcus uberis mastitis challenge in mid-lactation dairy cows

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
M. Vailati-Riboni ◽  
D. N. Coleman ◽  
V. Lopreiato ◽  
A. Alharthi ◽  
R. E. Bucktrout ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Somatic Cell ◽  
Pathway Analysis ◽  
Molecular Mechanisms ◽  
Somatic Cell Score ◽  
Molecular Networks ◽  
Epithelial Tissue ◽  
Udder Health ◽  
Streptococcus Uberis ◽  
Fermentation Product

Abstract Background We aimed to characterize the protective effects and the molecular mechanisms of action of a Saccharomyces cerevisiae fermentation product (NTK) in response to a mastitis challenge. Eighteen mid-lactation multiparous Holstein cows (n = 9/group) were fed the control diet (CON) or CON supplemented with 19 g/d NTK for 45 d (phase 1, P1) and then infected in the right rear quarter with 2500 CFU of Streptococcus uberis (phase 2, P2). After 36-h, mammary gland and liver biopsies were collected and antibiotic treatment started until the end of P2 (9 d post challenge). Cows were then followed until day 75 (phase 3, P3). Milk yield (MY) and dry matter intake (DMI) were recorded daily. Milk samples for somatic cell score were collected, and rectal and udder temperature, heart and respiration rate were recorded during the challenge period (P2) together with blood samples for metabolite and immune function analyses. Data were analyzed by phase using the PROC MIXED procedure in SAS. Biopsies were used for transcriptomic analysis via RNA-sequencing, followed by pathway analysis. Results DMI and MY were not affected by diet in P1, but an interaction with time was recorded in P2 indicating a better recovery from the challenge in NTK compared with CON. NTK reduced rectal temperature, somatic cell score, and temperature of the infected quarter during the challenge. Transcriptome data supported these findings, as NTK supplementation upregulated mammary genes related to immune cell antibacterial function (e.g., CATHL4, NOS2), epithelial tissue protection (e.g. IL17C), and anti-inflammatory activity (e.g., ATF3, BAG3, IER3, G-CSF, GRO1, ZFAND2A). Pathway analysis indicated upregulation of tumor necrosis factor α, heat shock protein response, and p21 related pathways in the response to mastitis in NTK cows. Other pathways for detoxification and cytoprotection functions along with the tight junction pathway were also upregulated in NTK-fed cows. Conclusions Overall, results highlighted molecular networks involved in the protective effect of NTK prophylactic supplementation on udder health during a subclinical mastitic event.

scholarly journals SEN1, a positive effector of tRNA-splicing endonuclease in Saccharomyces cerevisiae.

1992 ◽  
Vol 12 (5) ◽  
pp. 2154-2164 ◽  
Author(s):  
D J DeMarini ◽  
M Winey ◽  
D Ursic ◽  
F Webb ◽  
M R Culbertson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Gene Product ◽  
Signal Sequence ◽  
Null Alleles ◽  
Nucleoside Triphosphate ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal

The SEN1 gene, which is essential for growth in the yeast Saccharomyces cerevisiae, is required for endonucleolytic cleavage of introns from all 10 families of precursor tRNAs. A mutation in SEN1 conferring temperature-sensitive lethality also causes in vivo accumulation of pre-tRNAs and a deficiency of in vitro endonuclease activity. Biochemical evidence suggests that the gene product may be one of several components of a nuclear-localized splicing complex. We have cloned the SEN1 gene and characterized the SEN1 mRNA, the SEN1 gene product, the temperature-sensitive sen1-1 mutation, and three SEN1 null alleles. The SEN1 gene corresponds to a 6,336-bp open reading frame coding for a 2,112-amino-acid protein (molecular mass, 239 kDa). Using antisera directed against the C-terminal end of SEN1, we detect a protein corresponding to the predicted molecular weight of SEN1. The SEN1 protein contains a leucine zipper motif, consensus elements for nucleoside triphosphate binding, and a potential nuclear localization signal sequence. The carboxy-terminal 1,214 amino acids of the SEN1 protein are essential for growth, whereas the amino-terminal 898 amino acids are dispensable. A sequence of approximately 500 amino acids located in the essential region of SEN1 has significant similarity to the yeast UPF1 gene product, which is involved in mRNA turnover, and the mouse Mov-10 gene product, whose function is unknown. The mutation that creates the temperature-sensitive sen1-1 allele is located within this 500-amino-acid region, and it causes a substitution for an amino acid that is conserved in all three proteins.

scholarly journals Bulked segregant analysis reveals candidate genes responsible for dwarf formation in woody oilseed crop castor bean

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zaiqing Wang ◽  
Anmin Yu ◽  
Fei Li ◽  
Wei Xu ◽  
Bing Han ◽  
...  
Keyword(s):  
Candidate Genes ◽  
Castor Bean ◽  
Woody Plants ◽  
Molecular Mechanisms ◽  
Ricinus Communis ◽  
Bulked Segregant Analysis ◽  
Oilseed Crop ◽  
Yeast Transformation ◽  
Dwarf Phenotype ◽  
Genetic Breeding

AbstractPlant dwarfism is a desirable agronomic trait in non-timber trees, but little is known about the physiological and molecular mechanism underlying dwarfism in woody plants. Castor bean (Ricinus communis) is a typical woody oilseed crop. We performed cytological observations within xylem, phloem and cambia tissues, revealing that divergent cell growth in all tissues might play a role in the dwarf phenotype in cultivated castor bean. Based on bulked segregant analyses for a F2 population generated from the crossing of a tall and a dwarf accession, we identified two QTLs associated with plant height, covering 325 candidate genes. One of these, Rc5NG4-1 encoding a putative IAA transport protein localized in the tonoplast was functionally characterized. A non-synonymous SNP (altering the amino acid sequence from Y to C at position 218) differentiated the tall and dwarf plants and we confirmed, through heterologous yeast transformation, that the IAA uptake capacities of Rc5NG4-1Y and Rc5NG4-1C were significantly different. This study provides insights into the physiological and molecular mechanisms of dwarfing in woody non-timber economically important plants, with potential to aid in the genetic breeding of castor bean and other related crops.

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