scholarly journals Mouse hair significantly lightened through replacement of the cysteine residue in the N-terminal domain of Mc1r using the CRISPR/Cas9 system

2020 ◽  
Author(s):  
Hitoshi Suzuki ◽  
Gohta Kinoshita ◽  
Takeru Tsunoi ◽  
Mitsuki Noju ◽  
Kimi Araki
Keyword(s):  
Coat Color ◽  
Receptor Gene ◽  
Genetic System ◽  
Cysteine Residue ◽  
Dorsal Side ◽  
Single Amino Acid ◽  
Mouse Strains ◽  
Loss Of Function ◽  
Body Coloration ◽  
Color Variant

Abstract A loss-of-function mutation in the melanocortin 1 receptor gene (MC1R), which switches off the eumelanin production, causes yellowish coat color variants in mammals. In a wild population of sables (Martes zibellina) in Hokkaido, Japan, the mutation responsible for a bright yellow coat color variant was inferred to be a cysteine replacement at codon 35 of the N-terminal extracellular domain of the Mc1r receptor. In the present study, we validated these findings by applying genome editing on Mc1r in mouse strains C3H/HeJ and C57BL/6N, altering the codon for cysteine (Cys33Phe). The resulting single amino acid substitution (Cys33Phe) and unintentionally generated frameshift mutations yielded a color variant exhibiting substantially brighter body color, indicating that the Cys35 replacement produced sufficient MC1R loss of function to confirm that this mutation is responsible for producing the Hokkaido sable yellow color variant. Notably, the yellowish mutant mouse phenotype exhibited brown coloration in subapical hair on the dorsal side in both the C3H/HeJ and C57BL/6N strains, despite the inability of the latter to produce the agouti signaling protein (Asip). This darker hair and body coloration was not apparent in the Hokkaido sable variant, implying the presence of an additional genetic system shaping yellowish hair variability.

scholarly journals A Novel Pinkish-White Flower Color Variant Is Caused by a New Allele of Flower Color Gene W1 in Wild Soybean (Glycine soja)

Agronomy ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 1001
Author(s):  
Jagadeesh Sundaramoorthy ◽  
Gyu Tae Park ◽  
Hyun Jo ◽  
Jeong-Dong Lee ◽  
Hak Soo Seo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Functional Activity ◽  
Glycine Soja ◽  
Wild Soybean ◽  
Flower Color ◽  
Loss Of Function ◽  
Protein Variation ◽  
Color Variant ◽  
Color Gene

The enzyme flavonoid 3′,5′-hydroxylase (F3′5′H) plays an important role in producing anthocyanin pigments in soybean. Loss of function of the W1 locus encoding F3′5′H always produces white flowers. However, few color variations have been reported in wild soybean. In the present study, we isolated a new color variant of wild soybean accession (IT261811) with pinkish-white flowers. We found that the flower’s pinkish-white color is caused by w1-s3, a single recessive allele of W1. The SNP detected in the mutant caused amino acid substitution (A304S) in a highly conserved SRS4 domain of F3′5′H proteins. On the basis of the results of the protein variation effect analyzer (PROVEAN) tool, we suggest that this mutation may lead to hypofunctional F3′5′H activity rather than non-functional activity, which thereby results in its pinkish-white color.

scholarly journals The Parkinson’s Disease-Associated Protein DJ-1 Protects Dictyostelium Cells from AMPK-Dependent Outcomes of Oxidative Stress

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1874
Author(s):  
Suwei Chen ◽  
Sarah J. Annesley ◽  
Rasha A. F. Jasim ◽  
Paul R. Fisher
Keyword(s):  
Oxidative Stress ◽  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Mitochondrial Dysfunction ◽  
Mitochondrial Respiration ◽  
Cysteine Residue ◽  
Reduced Form ◽  
Dependent Manner ◽  
Loss Of Function ◽  
Cellular Pathology

Mitochondrial dysfunction has been implicated in the pathology of Parkinson’s disease (PD). In Dictyostelium discoideum, strains with mitochondrial dysfunction present consistent, AMPK-dependent phenotypes. This provides an opportunity to investigate if the loss of function of specific PD-associated genes produces cellular pathology by causing mitochondrial dysfunction with AMPK-mediated consequences. DJ-1 is a PD-associated, cytosolic protein with a conserved oxidizable cysteine residue that is important for the protein’s ability to protect cells from the pathological consequences of oxidative stress. Dictyostelium DJ-1 (encoded by the gene deeJ) is located in the cytosol from where it indirectly inhibits mitochondrial respiration and also exerts a positive, nonmitochondrial role in endocytosis (particularly phagocytosis). Its loss in unstressed cells impairs endocytosis and causes correspondingly slower growth, while also stimulating mitochondrial respiration. We report here that oxidative stress in Dictyostelium cells inhibits mitochondrial respiration and impairs phagocytosis in an AMPK-dependent manner. This adds to the separate impairment of phagocytosis caused by DJ-1 knockdown. Oxidative stress also combines with DJ-1 loss in an AMPK-dependent manner to impair or exacerbate defects in phototaxis, morphogenesis and growth. It thereby phenocopies mitochondrial dysfunction. These results support a model in which the oxidized but not the reduced form of DJ-1 inhibits AMPK in the cytosol, thereby protecting cells from the adverse consequences of oxidative stress, mitochondrial dysfunction and the resulting AMPK hyperactivity.

scholarly journals Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2005
Author(s):  
Irene Vorontsova ◽  
James E. Hall ◽  
Thomas F. Schilling ◽  
Noriaki Nagai ◽  
Yosuke Nakazawa
Keyword(s):  
Cell Adhesion ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Adhesion Assay ◽  
Loss Of Function ◽  
Adhesive Properties ◽  
The Difference ◽  
In Vitro Adhesion ◽  
Cell To Cell Adhesion

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

scholarly journals Identification of separate domains in the adenovirus E1A gene for immortalization activity and the activation of virus early genes.

1986 ◽  
Vol 6 (10) ◽  
pp. 3470-3480 ◽  
Author(s):  
E Moran ◽  
B Zerler ◽  
T M Harrison ◽  
M B Mathews
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Point Mutations ◽  
Apparent Molecular Weight ◽  
Amino Acid Position ◽  
Cysteine Residue ◽  
Transformation Function ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
E1a Gene

The transformation and early adenovirus gene transactivation functions of the E1A region were analyzed with deletion and point mutations. Deletion of amino acids from position 86 through 120 had little effect on the lytic or transforming functions of the E1A products, while deletion of amino acids from position 121 through 150 significantly impaired both functions. The sensitivity of the transformation function to alterations in the region from amino acid position 121 to 150 was further indicated by the impairment of transforming activity resulting from single amino acid substitutions at positions 124 and 135. Interestingly, conversion of a cysteine residue at position 124 to glycine severely impaired the transformation function without affecting the early adenovirus gene activating functions. Single amino acid substitutions in a different region of the E1A gene had the converse effect. All the mutants produced polypeptides of sufficient stability to be detected by Western immunoblot analysis. The single amino acid substitutions at positions 124 and 135, although impairing the transformation functions, did not detectably alter the formation of the higher-apparent-molecular-weight forms of the E1A products.

scholarly journals A point mutation in the extracellular domain activates LET-23, the Caenorhabditis elegans epidermal growth factor receptor homolog.

1996 ◽  
Vol 16 (2) ◽  
pp. 529-537 ◽  
Author(s):  
W S Katz ◽  
G M Lesa ◽  
D Yannoukakos ◽  
T R Clandinin ◽  
J Schlessinger ◽  
...  
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Caenorhabditis Elegans ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Point Mutation ◽  
Growth Factor Receptor ◽  
Extracellular Domain ◽  
Cysteine Residue ◽  
Loss Of Function ◽  
Epidermal Growth

The let-23 gene encodes a Caenorhabditis elegans homolog of the epidermal growth factor receptor (EGFR) necessary for vulval development. We have characterized a mutation of let-23 that activates the receptor and downstream signal transduction, leading to excess vulval differentiation. This mutation alters a conserved cysteine residue in the extracellular domain and is the first such point mutation in the EGFR subfamily of tyrosine kinases. Mutation of a different cysteine in the same subdomain causes a strong loss-of-function phenotype, suggesting that cysteines in this region are important for function and nonequivalent. Vulval precursor cells can generate either of two subsets of vulval cells (distinct fates) in response to sa62 activity. The fates produced depended on the copy number of the mutation, suggesting that quantitative differences in receptor activity influence the decision between these two fates.

scholarly journals Melanocortin-4 Receptor Gene Mutations in Obese Slovak Children

2015 ◽  
pp. 883-890 ◽  
Author(s):  
D. STANIKOVA ◽  
M. SUROVA ◽  
L. TICHA ◽  
M. PETRASOVA ◽  
D. VIRGOVA ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Dna Analysis ◽  
Receptor Gene ◽  
Gene Mutations ◽  
Loss Of Function ◽  
Obese Children ◽  
Mc4r Gene ◽  
Melanocortin 4 Receptor ◽  
Melanocortin 4 Receptor Gene ◽  
Common Etiology

The most common etiology of non-syndromic monogenic obesity are mutations in gene for the Melanocortin-4 receptor (MC485) with variable prevalence in different countries (1.2-6.3 % of obese children). The aim of our study was 1) to search for MC4R mutations in obese children in Slovakia and compare their prevalence with other European countries, and 2) to describe the phenotype of the mutation carriers. DNA analysis by direct Sanger sequencing of the coding exons and intron/exon boundaries of the MC4R gene was performed in 268 unrelated Slovak children and adolescents with body mass index above the 97th percentile for age and sex and obesity onset up to 11 years (mean 4.3±2.8 years). Two different previously described heterozygous loss of function MC4R variants (i.e. p.Ser19Alafs*34, p.Ser127Leu) were identified in two obese probands, and one obese (p.Ser19Alafs*34), and one lean (p.Ser127Leu) adult family relatives. No loss of function variants were found in lean controls. The prevalence of loss-of-function MC4R variants in obese Slovak children was 0.7 %, what is one of the lowest frequencies in Europe.

scholarly journals Familial Hypercholesterolemia: Three “under” (Understood, Underdiagnosed, and Undertreated) Disease

2021 ◽  
Author(s):  
Vladimir O. Konstantinov
Keyword(s):  
Familial Hypercholesterolemia ◽  
Time Course ◽  
Ldl Cholesterol ◽  
Genetic Disorders ◽  
Receptor Gene ◽  
Ldl Receptor ◽  
Hdl Cholesterol ◽  
Blood Cholesterol ◽  
Healthy Population ◽  
Loss Of Function

Familial hypercholesterolemia (FH) is one of the most prevalent genetic disorders leading to premature atherosclerosis and coronary heart disease. The main cause of FH is a mutation in the LDL-receptor gene that leads to loss of function of these receptors causing high levels of blood cholesterol. The diagnosis of FH is not very easy. Wide screenings are needed to reveal high levels of LDL cholesterol among “healthy” population. If the patient has MI or stroke at an early age, high levels of LDL cholesterol, and tendon xanthomas, the diagnosis of FH becomes much more clear. Genetic testing is a gold standard in the diagnosis of FH. There are several factors, influencing the time course of FH. Smoking males with low levels of HDL cholesterol have an extremely higher risk of death than nonsmoking females with high HDL cholesterol. Management of FH includes low cholesterol diet, statin and ezetimibe treatment, PCSK inhibitors, and LDL aphaeresis. Early and effective treatment influences much the prognosis in FH patients.

scholarly journals Spemann organizer transcriptome induction by early beta-catenin, Wnt, Nodal, and Siamois signals in Xenopus laevis

2017 ◽  
Vol 114 (15) ◽  
pp. E3081-E3090 ◽  
Author(s):  
Yi Ding ◽  
Diego Ploper ◽  
Eric A. Sosa ◽  
Gabriele Colozza ◽  
Yuki Moriyama ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Gene Signature ◽  
Dorsal Side ◽  
Beta Catenin ◽  
Loss Of Function ◽  
Spemann Organizer ◽  
Signature Genes ◽  
A Genome ◽  
Vertebrate Model ◽  
Chloride Treatment

The earliest event in Xenopus development is the dorsal accumulation of nuclear β-catenin under the influence of cytoplasmic determinants displaced by fertilization. In this study, a genome-wide approach was used to examine transcription of the 43,673 genes annotated in the Xenopus laevis genome under a variety of conditions that inhibit or promote formation of the Spemann organizer signaling center. Loss of function of β-catenin with antisense morpholinos reproducibly reduced the expression of 247 mRNAs at gastrula stage. Interestingly, only 123 β-catenin targets were enriched on the dorsal side and defined an early dorsal β-catenin gene signature. These genes included several previously unrecognized Spemann organizer components. Surprisingly, only 3 of these 123 genes overlapped with the late Wnt signature recently defined by two other groups using inhibition by Dkk1 mRNA or Wnt8 morpholinos, which indicates that the effects of β-catenin/Wnt signaling in early development are exquisitely regulated by stage-dependent mechanisms. We analyzed transcriptome responses to a number of treatments in a total of 46 RNA-seq libraries. These treatments included, in addition to β-catenin depletion, regenerating dorsal and ventral half-embryos, lithium chloride treatment, and the overexpression of Wnt8, Siamois, and Cerberus mRNAs. Only some of the early dorsal β-catenin signature genes were activated at blastula whereas others required the induction of endomesoderm, as indicated by their inhibition by Cerberus overexpression. These comprehensive data provide a rich resource for analyzing how the dorsal and ventral regions of the embryo communicate with each other in a self-organizing vertebrate model embryo.

scholarly journals Screening for Presenilin Inhibitors Using the Free-Living Nematode, Caenorhabditis elegans

2004 ◽  
Vol 9 (2) ◽  
pp. 147-152 ◽  
Author(s):  
Brenda R. Ellerbrock ◽  
Eileen M. Coscarelli ◽  
Mark E. Gurney ◽  
Timothy G. Geary
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput Screening ◽  
Screening Method ◽  
Genetic System ◽  
Body Cavity ◽  
Egg Laying ◽  
The Body ◽  
Loss Of Function ◽  
C Elegans ◽  
Automated Method

Caenorhabditis elegans contains 3 homologs of presenilin genes that are associated with Alzheimer s disease. Loss-of-function mutations in C. elegans genes cause a defect in egg laying. In humans, loss of presenilin-1 (PS1) function reduces amyloid-beta peptide processing from the amyloid protein precursor. Worms were screened for compounds that block egg laying, phenocopying presenilin loss of function. To accommodate even relatively high throughput screening, a semi-automated method to quantify egg laying was devised by measuring the chitinase released into the culture medium. Chitinase is released by hatching eggs, but little is shed into the medium from the body cavity of a hermaphrodite with an egg laying deficient ( egl) phenotype. Assay validation involved measuring chitinase release from wild-type C. elegans (N2 strain), sel-12 presenilin loss-of-function mutants, and 2 strains of C. elegans with mutations in the egl-36K+ channel gene. Failure to find specific presenilin inhibitors in this collection likely reflects the small number of compounds tested, rather than a flaw in screening strategy. Absent defined biochemical pathways for presenilin, this screening method, which takes advantage of the genetic system available in C. elegans and its historical use for anthelminthic screening, permits an entry into mechanism-based discovery of drugs for Alzheimer s disease. ( Journal of Biomolecular Screening 2004:147-152)

Abstract 408: Essential Domain Transition Involving Central Helical Repeats Prevent Lipid-Free ApoA-I from Acquiring Lipid

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Ricquita Pollard ◽  
Brian Fulp ◽  
Michael Thomas ◽  
Mary Sorci-Thomas
Keyword(s):  
Three Dimensional ◽  
Density Lipoprotein ◽  
Particle Formation ◽  
Cysteine Residue ◽  
Lipid Binding ◽  
Structural Topology ◽  
Loss Of Function ◽  
Mobility Shift ◽  
Helix Bundle ◽  
Plasma High Density

Plasma high density lipoprotein (HDL) concentration is negatively correlated with the occurrence of coronary heart disease in the human population. Because apoA-I is the main protein constituent of HDL, a thorough understanding of apoA-I structural topology is essential for elucidating its ability to package and mobilize cholesterol for catabolism. To determine which of the 10 helical repeats within apoA-I participates in the structural transitions that drive unfolding of the 4-helix bundle, we created several loss of function mutations. Published three-dimensional coordinates of full-length lipid-free apoA-I were used to predict amino acids having spatial separations of 3-5Å within the 4-helix bundle. Based on these predictions, we proposed that specific targeted double cysteine residue substitutions could form disulfide linkages and prevent “opening” of a critical domain required for unfolding of the apoA-I 4-helix bundle when exposed to lipid. To test the importance of helical repeats 4, 5, 6 and 7, double cysteine mutants D103C-R177C apoA-I and F104C-H162C apoA-I were created, expressed and purified using established procedures. Mass spectrometry combined with MS/MS sequencing was used to verify the “locked” disulfide form of each double cysteine substitution mutants. Using 20% SDS-PAGE we show that electrophoretic mobility-shift distinguishes between “locked” or -DTT versus “unlocked” or +DTT form for each of the mutant apoA-I proteins. Particle formation was tested for each mutant by measuring the formation of recombinant HDL (rHDL) using cholate dialysis, as well as, the formation of nascent HDL (nHDL) from ABCA1 expressing cells. Examination of the size of rHDL and nHDL particles formed suggests that unfolding of lipid-free apoA-I to acquire lipid involves the “locked” or restricted helical repeats 4-7. In conclusion, when both double cysteine apoA-I mutants exist in their “locked” conformation evidence of impaired particle formation was observed confirming the existence of the apoA-I 4 helix bundle in lipid free state and the role of central helical repeats 4-7 in lipid binding and particle formation.

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