A Single Point Mutation Gln716His on the α2 Integrin: The New Platelet Alloantigen Casa.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3400-3400
Author(s):  
Gerald Bertrand ◽  
Vincent Jallu ◽  
Dominique Kervran ◽  
Corinne Martageix ◽  
Cecile Kaplan-Gouet
Keyword(s):  
Amino Acid ◽  
Cho Cells ◽  
Single Point ◽  
Low Frequency ◽  
Caucasian Population ◽  
Single Amino Acid ◽  
Nucleotide Sequence Analysis ◽  
Single Point Mutation ◽  
Severe Thrombocytopenia ◽  
Transfusion Therapy

Abstract We report here the identification and characterization of a new low-frequency platelet alloantigen Casa involved in a case of neonatal alloimmune thrombocytopenia (NAIT). A 29-year-old mother gave birth to a full-term male infant who exhibited petechiae at birth. Nine hours post-delivery the platelet counts revealed a severe thrombocytopenia (16.109platelets/L) leading to platelet transfusion therapy associated with IVIG. The outcome was uneventful. Blood samples from the parents and infant were referred to our laboratory for investigation because of suspected NAIT. Maternal serum showed a specific positive reaction with the antigen-capture assay (MAIPA) only when it was tested with the paternal platelets and the monoclonal antibodies Gi9 (Immunotech, Marseille, France), P16 (NIBSC, Bristol, UK), and AK7 (Abcys, Paris, France) directed against the GPIa-IIa (a2b1 integrin). Nucleotide sequence analysis of GPIa cDNA of the father and newborn showed a nucleotide substitution at position 2235 (2235G>T according to the International Nomenclature). This substitution induces a Q716H amino acid change in the GPIa mature protein, located outside the I domain involved in cell-adhesion for collagen. In vitro analysis of recombinant CHO cells expressing wild-type or mutant (Q716H) human GPIa allowed us to demonstrate that this single amino-acid substitution is responsible and sufficient for inducing Casa antigen expression. Adhesion of CHO cells to collagen coated on microtiter plates was not modified by the Cas polymorphism, nor by the maternal anti Casa alloantibodies, indicating that the mutation does not affect the function of the integrin a2b1. PCR-SSP was developed for Casa genotyping. In a Caucasian population study none of the 100 unrelated blood donors was found to be Casa carrier. In conclusion, the Casa antigen described here, implicated in a case of severe neonatal thrombocytopenia is a low-frequency platelet antigen in the Caucasian population. This study highlights the high polymorphism of the GPIa gene.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

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.

scholarly journals Intrinsic resistance to terbinafine among human and animal isolates of Trichophyton mentagrophytes related to amino acid substitution in the squalene epoxidase

Infection ◽  
2020 ◽  
Vol 48 (6) ◽  
pp. 889-897 ◽  
Author(s):  
Dominik Łagowski ◽  
Sebastian Gnat ◽  
Aneta Nowakiewicz ◽  
Marcelina Osińska ◽  
Mariusz Dyląg
Keyword(s):  
Amino Acid ◽  
Fungal Infections ◽  
Single Point ◽  
Antifungal Susceptibility ◽  
Point Mutations ◽  
Trichophyton Mentagrophytes ◽  
Single Amino Acid ◽  
Squalene Epoxidase ◽  
Intrinsic Resistance ◽  
Resistant Strains

Abstract Background Dermatomycoses are the most common fungal infections in the world affecting a significant part of the human and animal population. The majority of zoophilic infections in humans are caused by Trichophyton mentagrophytes. Currently, the first-line drug for both oral and topical therapy is terbinafine. However, an increasing number of cases that are difficult to be cured with this drug have been noted in Europe and Asia. Resistance to terbinafine and other allylamines is very rare and usually correlated with point mutations in the squalene epoxidase gene resulting in single amino acid substitutions in the enzyme, which is crucial in the ergosterol synthesis pathway. Purpose Here, we report terbinafine-resistant T. mentagrophytes isolates among which one was an etiological factor of tinea capitis in a man and three were obtained from asymptomatic foxes in Poland. Methods We used the CLSI protocol to determine antifungal susceptibility profiles of naftifine, amphotericin B, griseofulvin, ketoconazole, miconazole, itraconazole, voriconazole, and ciclopirox. Moreover, the squalene epoxidase gene of the terbinafine-resistant strains was sequenced and analysed. Results In the genomes of all four resistant strains exhibiting elevated MICs to terbinafine (16 to 32 µg/ml), single-point mutations leading to Leu393Phe substitution in the squalene epoxidase enzyme were revealed. Among the other tested substances, a MIC50 value of 1 µg/ml was shown only for griseofulvin. Conclusion Finally, our study revealed that the terbinafine resistance phenomenon might not be acquired by exposure to the drug but can be intrinsic. This is evidenced by the description of the terbinafine-resistant strains isolated from the asymptomatic animals.

Chemical structure of light chains: amino acid sequence of type K Bence-Jones proteins

1966 ◽  
Vol 166 (1003) ◽  
pp. 124-137 ◽  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Sequence Data ◽  
Single Point ◽  
Structural Difference ◽  
Light Chains ◽  
Single Point Mutation ◽  
Amino Terminal ◽  
Type K ◽  
Bence Jones Proteins

Bence-Jones proteins are the light chains of the autologous myeloma globulin and are analogous to the light chains of normal human immunoglobulins. Peptide mapping has demonstrated that Bence-Jones proteins share a fixed portion of their sequence (the ‘constant’ portion) and also have a mutable part (the ‘variable’ portion). Sequence analysis and ordering of the tryptic and chymotryptic peptides has provided the tentative complete amino acid sequence of one Bence-Jones protein of antigenic type K. Comparison with partial sequence data for other type K Bence-Jones proteins has revealed many structural differences in the amino terminal half of the molecules, but only one structural difference in the carboxyl terminal half. The latter is strongly correlated with the Inv genetic factor. The points of interchange in the amino terminal half occur in clusters close to the half cystine residues and the ‘switch peptide’ (positions 102 through 105), after which the sequence becomes essentially invariant. This suggests that the major areas subject to sequence variation are part of a single topographical region which may define a portion of the antigen combining site in the light chains of antibodies. Many, but not all, the amino acid interchanges are compatible with a single point mutation. As yet, no single mutational theory suffices to explain the manifold differences in structure of the light chains. Such structural variation, however, could result from the presence of many related genes.

scholarly journals A Single Amino Acid Mutation in the Plum pox virus Helper Component-Proteinase Gene Abolishes Both Synergistic and RNA Silencing Suppression Activities

2005 ◽  
Vol 95 (8) ◽  
pp. 894-901 ◽  
Author(s):  
Pablo González-Jara ◽  
Felix A. Atencio ◽  
Belén Martínez-García ◽  
Daniel Barajas ◽  
Francisco Tenllado ◽  
...  
Keyword(s):  
Amino Acid ◽  
Point Mutation ◽  
Rna Silencing ◽  
Recombinant Virus ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Plum Pox Virus ◽  
Helper Component ◽  
Silencing Suppression ◽  
Helper Component Proteinase

The effects on symptom expression of single amino acid mutations in the central region of the Plum pox virus (PPV) helper component-proteinase (HC-Pro) gene were analyzed in Nicotiana benthamiana using Potato virus X (PVX) recombinant viruses. PVX recombinant virus expressing the wild-type variant of PPV HC-Pro induced the expected enhancement of PVX pathogenicity, manifested as necrosis and plant death. Recombinant virus expressing a variant of PPV HC-Pro containing a single point mutation ( HCL134H) was unable to induce this synergistic phenotype. The RNA silencing suppressor activity of PPV HC-Pro was demonstrated in a transient silencing suppression assay. In contrast, the HCL134H mutant showed no such activity. These results indicate that a unique point mutation in PPV HC-Pro impaired its ability to suppress RNA silencing and abolished its capacity to induce synergism, and clearly shows for the first time the link between these two functions in potyvirus HC-Pro. Additionally, we compared the effects on virus accumulation in N. benthamiana plants infected with either the PVX recombinant constructs or with native viruses in double infection experiments. PVX (+) and (-) strand genomic RNA accumulated at similar levels in plants infected with PVX recombinants, leading to an increase in PVX pathology, compared with plants infected with PVX alone. This finding confirms that the enhancement of pathogenicity associated with synergistic interaction is not a consequence of more efficient PVX replication due to RNA silencing suppression by PPV HC-Pro.

scholarly journals A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

1998 ◽  
Vol 140 (1) ◽  
pp. 91-99 ◽  
Author(s):  
Malini Vashishtha ◽  
Thomas Phalen ◽  
Marianne T. Marquardt ◽  
Jae S. Ryu ◽  
Alice C. Ng ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Semliki Forest Virus ◽  
Single Point ◽  
Cellular Membrane ◽  
Spike Protein ◽  
Single Amino Acid ◽  
Progeny Virus ◽  
Single Point Mutation ◽  
Protein Subunit ◽  
Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

scholarly journals Mutations in oxyR Resulting in Peroxide Resistance in Xanthomonas campestris

2000 ◽  
Vol 182 (13) ◽  
pp. 3846-3849 ◽  
Author(s):  
Skorn Mongkolsuk ◽  
Wirongrong Whangsuk ◽  
Mayuree Fuangthong ◽  
Suvit Loprasert
Keyword(s):  
Amino Acid ◽  
Xanthomonas Campestris ◽  
Resistant Mutant ◽  
The Other ◽  
Single Amino Acid ◽  
Silent Mutation ◽  
Expression Vectors ◽  
Nucleotide Sequence Analysis ◽  
Wild Type ◽  
Single Amino Acid Change

ABSTRACT A spontaneous Xanthomonas campestris pv. phaseoli H2O2-resistant mutant emerged upon selection with 1 mM H2O2. In this report, we show that growth of this mutant under noninducing conditions gave high levels of catalase, alkyl hydroperoxide reductase (AhpC and AhpF), and OxyR. The H2O2 resistance phenotype was abolished inoxyR-minus derivatives of the mutant, suggesting that elevated levels and mutations in oxyR were responsible for the phenotype. Nucleotide sequence analysis of the oxyRmutant showed three nucleotide changes. These changes resulted in one silent mutation and two amino acid changes, one at a highly conserved location (G197 to D197) and the other at a nonconserved location (L301 to R301) in OxyR. Furthermore, these mutations in oxyRaffected expression of genes in the oxyR regulon. Expression of an oxyR-regulated gene, ahpC, was used to monitor the redox state of OxyR. In the parental strain, a high level of wild-type OxyR repressed ahpC expression. By contrast, expression of oxyR5 from the X. campestris pv. phaseoli H2O2-resistant mutant and its derivative oxyR5G197D with a single-amino-acid change on expression vectors activatedahpC expression in the absence of inducer. The other single-amino-acid mutant derivative of oxyR5L301R had effects on ahpC expression similar to those of the wild-type oxyR. However, when the two single mutations were combined, as in oxyR5, these mutations had an additive effect on activation of ahpC expression.

Hereditary Protein C Deficiency Associated with Mutations in Exon IX of the Protein C Gene

1994 ◽  
Vol 72 (02) ◽  
pp. 203-208 ◽  
Author(s):  
R G Doig ◽  
C G Begley ◽  
K M McGrath
Keyword(s):  
Amino Acid ◽  
Protein C ◽  
Novel Mutation ◽  
Single Point ◽  
Enzyme Analysis ◽  
Restriction Enzyme Analysis ◽  
Single Point Mutation ◽  
Nucleotide Position ◽  
Type I ◽  
Protein C Deficiency

SummaryThis report describes five families with symptomatic hereditary protein C deficiency. Using a polymerase chain reaction (PCR)-based method, the entire coding sequence and intron-exon boundaries of the protein C gene was amplified from genomic DNA. In each family a single point mutation in the protein C gene was identified. Two unrelated families were found to share the same mutation, while the other three had different mutations. In the first two families with type I protein C deficiency the normal cytosine residue at nucleotide position 8551 in the protein C gene was replaced by thymidine leading to substitution of the normal proline residue at amino acid position 279 by leucine. In the third family with type I deficiency a previously undescribed mutation was identified. In this family the guanosine residue at position 8559 was replaced by adenosine (glycine 282 substituted by serine). In the fourth family, also with type I deficiency, guanosine 8589 was replaced by adenosine (glycine 292 substituted by serine). The fifth family had type II deficiency and in affected members cytosine 8769 was replaced by thymidine (arginine 352 substituted by tryptophan). All these mutations lead to amino acid substitutions in the serine protease domain of the mature protein. All were able to be confirmed by restriction enzyme analysis of PCR-derived DNA. In addition the novel mutation at nucleotide position 8559 was also demonstrable using single strand conformation polymorphism (SSCP) analysis of PCR-derived DNA. These mutations were likely examples of deamination of methylated cytosine occurring in cytosine-phosphate-guanosine (CpG) dinucleotide sequences. These findings confirm the genetic heterogeneity of hereditary protein C deficiency in these families.

Single amino acid substitution in human platelet glycoprotein Ibβ is responsible for the formation of the platelet-specific alloantigen Iya

Blood ◽  
2000 ◽  
Vol 95 (5) ◽  
pp. 1849-1855 ◽  
Author(s):  
Ulrich J. H. Sachs ◽  
Volker Kiefel ◽  
Micaela Böhringer ◽  
Vahid Afshar-Kharghan ◽  
Hartmut Kroll ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Human Platelet ◽  
Population Analysis ◽  
Severe Case ◽  
Single Amino Acid Substitution ◽  
Single Amino Acid ◽  
Platelet Glycoprotein ◽  
Nucleotide Sequence Analysis ◽  
Platelet Surface

We recently described a new low-frequency platelet alloantigen on the human platelet glycoprotein (GP) Ib-IX complex, termed Iya, which was implicated in a severe case of neonatal alloimmune thrombocytopenia. Immunoprecipitation studies with trypsin-treated platelets indicated that the Iyaalloantigenic determinants are formed by the membrane-associated remnant moiety of GP Ib (GP Ibr) together with GP Ibβ and GP IX. To elucidate the molecular basis underlying the Iya alloantigen, we amplifiedGPIbr, GPIbβ, andGPIX genes by polymerase chain reaction (PCR). Nucleotide-sequence analysis of these 3 genes showed a G to A transition at position 141 on GPIbβ gene in a subject positive for Iya. This transition resulted in a Gly15Glu dimorphism on the N-terminal domain ofGPIbβ. This finding was confirmed by genotyping analysis of 6 Iya-positive subjects by restriction fragment length polymorphism (RFLP) studies using NarI endonuclease. In 300 randomly selected healthy blood donors, one Iya-positive individual was found. Phenotypes determined by monoclonal antibody-specific immobilization of platelet antigens assay and genotypes determined by RFLP were identical in this population. Analysis of Iya-positive platelets showed that the point mutation affected neither the degree of surface expression nor the function of the GP Ib-GP Ibβ-IX complex on the platelet surface. Transient expression of the GP Ib-IX complex in CHO cells using wild-type GP Ibβ (Gly15) or mutant GP Ibβ (Glu15) allowed us to demonstrate that this single amino acid substitution is sufficient to induce Iya epitope(s).

scholarly journals Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

2005 ◽  
Vol 16 (8) ◽  
pp. 3810-3820 ◽  
Author(s):  
Mark S. Miller ◽  
Jessica M. Esparza ◽  
Andrew M. Lippa ◽  
Fordyce G. Lux ◽  
Douglas G. Cole ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Point ◽  
Intraflagellar Transport ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Dominant Effect ◽  
Flagellar Assembly ◽  
Gene Maps

The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.

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