scholarly journals Genomic Sequencing of sod1D Yeast That Escape Spore Death

2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. 1015-1015
Author(s):  
Temiloluwa Adanlawo ◽  
Helen Vander Wende
Keyword(s):  
Atp Synthase ◽  
Single Point ◽  
Zinc Ion ◽  
Yeast Cells ◽  
Single Point Mutation ◽  
Sod1 Gene ◽  
Rich Media ◽  
Yeast Meiosis ◽  
The Relationship ◽  
Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that impacts nerve cells and the spinal cord, which in some cases are linked to mutations in the Superoxide Dismutase 1 (SOD1) gene. Sod1 is an antioxidant within cells that converts reactive oxygen from superoxide into water using a copper and zinc ion to deactivate the oxygen. When the SOD1 gene is deleted, yeast cells are still able to undergo meiotic divisions and generate four spores, but the spores that are produced are inviable. However, we see that randomly, sod1∆ spores can grow on rich media. This leads us to hypothesize that somewhere in the genome, there is a suppressor mutation that allows these cells to grow. We tested this hypothesis by preparing samples for whole genome sequencing. By comparing the genomic sequences from our suppressor mutants to wild-type controls, we’re able to identify a single point mutation within a gene called NCA2, which codes for a protein that regulates expression of Fo-F1 ATP synthase subunits 6 and 8. Given this result, we are now working to try and understand the relationship between the sod1∆ spore death phenotype and the modulation of ATP synthase activity. In summary, the results from our work have the potential to further help us understand what role Sod1 plays in yeast meiosis and may be able to give us a deeper understanding for ALS cases that are linked to Sod1.

scholarly journals The mutational landscape of a prion-like domain

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Benedetta Bolognesi ◽  
Andre J. Faure ◽  
Mireia Seuma ◽  
Jörn M. Schmiedel ◽  
Gian Gaetano Tartaglia ◽  
...  
Keyword(s):  
Cellular Systems ◽  
Genetic Interactions ◽  
Yeast Cells ◽  
Insoluble Protein ◽  
Cellular Toxicity ◽  
Powerful Approach ◽  
Unstructured Proteins ◽  
The Relationship ◽  
Lateral Sclerosis

Abstract Insoluble protein aggregates are the hallmarks of many neurodegenerative diseases. For example, aggregates of TDP-43 occur in nearly all cases of amyotrophic lateral sclerosis (ALS). However, whether aggregates cause cellular toxicity is still not clear, even in simpler cellular systems. We reasoned that deep mutagenesis might be a powerful approach to disentangle the relationship between aggregation and toxicity. We generated >50,000 mutations in the prion-like domain (PRD) of TDP-43 and quantified their toxicity in yeast cells. Surprisingly, mutations that increase hydrophobicity and aggregation strongly decrease toxicity. In contrast, toxic variants promote the formation of dynamic liquid-like condensates. Mutations have their strongest effects in a hotspot that genetic interactions reveal to be structured in vivo, illustrating how mutagenesis can probe the in vivo structures of unstructured proteins. Our results show that aggregation of TDP-43 is not harmful but protects cells, most likely by titrating the protein away from a toxic liquid-like phase.

scholarly journals Hec1p, an Evolutionarily Conserved Coiled-Coil Protein, Modulates Chromosome Segregation through Interaction with SMC Proteins

1999 ◽  
Vol 19 (8) ◽  
pp. 5417-5428 ◽  
Author(s):  
Lei Zheng ◽  
Yumay Chen ◽  
Wen-Hwa Lee
Keyword(s):  
Mammalian Cells ◽  
Stop Codon ◽  
Single Point ◽  
Critical Role ◽  
Coiled Coil ◽  
Yeast Cells ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Lethal Phenotype ◽  
Smc Proteins

ABSTRACT hsHec1p, a Homo sapiens coiled-coil-enriched protein, plays an important role in M-phase progression in mammalian cells. ASaccharomyces cerevisiae protein, identical to Tid3p/Ndc80p and here designated scHec1p, has similarities in structure and biological function to hsHec1p. Budding yeast cells deleted in the scHEC1/NDC80 allele are not viable, but this lethal phenotype can be rescued by hsHEC1 under control of the endogenous scHEC1 promoter. At the nonpermissive temperature, significant mitotic delay, chromosomal missegregation, and decreased viability were observed in yeast cells with temperature-sensitive (ts) alleles of hsHEC1. In the hshec1-113 ts mutant, we found a single-point mutation changing Trp395 to a stop codon, which resulted in the expression of a C-terminally truncated 45-kDa protein. The binding of this mutated protein, hshec1-113p, to five identified hsHec1p-associated proteins was unchanged, while its binding to human SMC1 protein and yeast Smc1p was ts. Hec1p also interacts with Smc2p, and the binding of the mutated hshec1-113p to Smc2p was not ts. Overexpression of either hsHEC1 or scHEC1 suppressed the lethal phenotype of smc1-2 and smc2-6 at nonpermissive temperatures, suggesting that the interactions between Hec1p and Smc1p and -2p are biologically significant. These results suggest that Hec1 proteins play a critical role in modulating chromosomal segregation, in part, through their interactions with SMC proteins.

scholarly journals Widespread Mutations in Voltage-Gated Sodium Channel Gene of Cimex lectularius (Hemiptera: Cimicidae) Populations in Paris

2021 ◽  
Vol 18 (2) ◽  
pp. 407
Author(s):  
Mohammad Akhoundi ◽  
Dahlia Chebbah ◽  
Denis Sereno ◽  
Anthony Marteau ◽  
Julie Jan ◽  
...  
Keyword(s):  
Single Point ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Homozygous Mutation ◽  
Knockdown Resistance ◽  
Bed Bugs ◽  
Bed Bug ◽  
Channel Gene ◽  
Blood Sucking ◽  
The Status

Bed bugs, Cimex lectularius and C. hemipterus, are common blood-sucking ectoparasites of humans with a large geographical distribution, worldwide. In France, little is known about the status of bed bugs’ infestation and their resistance to insecticides, particularly, pyrethroids. Here, we aimed to find mutations in the kdr gene, known to be involved in resistance to insecticides. We gathered bed bugs from various infested locations, including 17 private houses, 12 HLM building complex, 29 apartments, 2 EHPAD, and 2 immigrants’ residences. A total of 1211 bed bugs were collected and morphologically identified as C. lectularius. Two fragments of the kdr gene, encompassing codons V419L and L925I, were successfully amplified for 156 specimens. We recorded sense mutation in the first amplified fragment (kdr1) in 89 out of 156 (57%) samples, in which in 61 out of 89 (68.5%) sequences, a change of valine (V) into leucine (L) V419L was observed. Within the second fragment (kdr2), a homozygous mutation was recorded in 73 out of 156 (46.7%) specimens at the codon 925. At this position, 43 out of 73 (58.9%) specimens had a sense mutation leading to the replacement of leucine (L) by isoleucine (I). Among 162 mutant sequences analyzed (89 for the kdr1 fragment and 73 for the kdr2 one), we detected single point mutation in 26.6%, while 73.4% presented the mutation in both kdr1 and kdr2 fragments. All modifications recorded in bed bug populations of Paris are described to be involved in the knockdown resistance (kdr) against pyrethroids.

scholarly journals A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

2021 ◽  
Vol 7 (7) ◽  
pp. 553
Author(s):  
Bin Gao ◽  
Shunyi Zhu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Single Point ◽  
Binding Domain ◽  
Host Cells ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Microsporum Canis ◽  
Receptor Binding Domain ◽  
Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

scholarly journals Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bingran Wang ◽  
Tiancheng Lou ◽  
Lingling Wei ◽  
Wenchan Chen ◽  
Longbing Huang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Single Point ◽  
Sodium Dodecyl ◽  
Cross Resistance ◽  
Molecular Characteristics ◽  
Single Point Mutation ◽  
Wide Range ◽  
Significant Difference ◽  
Leaf Blights ◽  
High Level

AbstractAlternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

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