Analysis of the molecular basis of calmodulin defects that affect ion channel-mediated cellular responses: site-specific mutagenesis and microinjection.

The ability of microinjected calmodulin to temporarily restore an ion channel-mediated behavioral phenotype of a calmodulin mutant in Paramecium tetraurelia (cam1) is dependent on the amino acid side chain that is present at residue 101, even when there is extensive variation in the rest of the amino acid sequence. Analysis of conservation of serine-101 in calmodulin suggests that the ability of calmodulin to regulate this ion channel-associated cell function may be a biological role of calmodulin that is widely distributed phylogenetically. A series of mutant calmodulins that differ only at residue-101 were produced by in vitro site-specific mutagenesis and expression in Escherichia coli, purified to chemical homogeneity, and tested for their ability to temporarily restore a wild-type behavioral phenotype to cam1 (pantophobiacA1) Paramecium. Calmodulins with glycine-101 or tyrosine-101 had minimal activity; calmodulins with phenylalanine-101 or alanine-101 had no detectable activity. However, as a standard of comparison, all of the calmodulins were able to activate a calmodulin-regulated enzyme, myosin light chain kinase, that is sensitive to point mutations elsewhere in the calmodulin molecule. Overall, these results support the hypothesis that the structural features of calmodulin required for the transduction of calcium signals varies with the particular pathway that is being regulated and provide insight into why inherited mutations of calmodulin at residue 101 are nonlethal and selective in their phenotypic effects.

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Characterization of an enhanced antigenic change in the pandemic 2009 H1N1 influenza virus haemagglutinin

Journal of General Virology ◽

10.1099/vir.0.061598-0 ◽

2014 ◽

Vol 95 (5) ◽

pp. 1033-1042 ◽

Cited By ~ 5

Author(s):

Blanca García-Barreno ◽

Teresa Delgado ◽

Sonia Benito ◽

Inmaculada Casas ◽

Francisco Pozo ◽

...

Keyword(s):

Influenza Virus ◽

Amino Acid ◽

Influenza A ◽

Point Mutations ◽

Antigenic Site ◽

Single Amino Acid ◽

Antigenic Structure ◽

Human Antibody ◽

2009 H1n1

Murine hybridomas producing neutralizing mAbs specific to the pandemic influenza virus A/California/07/2009 haemagglutinin (HA) were isolated. These antibodies recognized at least two different but overlapping new epitopes that were conserved in the HA of most Spanish pandemic isolates. However, one of these isolates (A/Extremadura/RR6530/2010) lacked reactivity with the mAbs and carried two unique mutations in the HA head (S88Y and K136N) that were required simultaneously to eliminate reactivity with the murine antibodies. This unusual requirement directly illustrates the phenomenon of enhanced antigenic change proposed previously for the accumulation of simultaneous amino acid substitutions at antigenic sites of the influenza A virus HA during virus evolution (Shih et al., Proc Natl Acad Sci USA, 104 , 6283–6288, 2007). The changes found in the A/Extremadura/RR6530/2010 HA were not found in escape mutants selected in vitro with one of the mAbs, which contained instead nearby single amino acid changes in the HA head. Thus, either single or double point mutations may similarly alter epitopes of the new antigenic site identified in this work in the 2009 H1N1 pandemic virus HA. Moreover, this site is relevant for the human antibody response, as shown by competition of mAbs and human post-infection sera for virus binding. The results are discussed in the context of the HA antigenic structure and challenges posed for identification of sequence changes with possible antigenic impact during virus surveillance.

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One-step multiple site-specific base editing by direct embryo injection for precision and pyramid pig breeding

10.1101/2020.08.26.267948 ◽

2020 ◽

Author(s):

Ruigao Song ◽

Yu Wang ◽

Qiantao Zheng ◽

Jing Yao ◽

Chunwei Cao ◽

...

Keyword(s):

Stop Codon ◽

Point Mutations ◽

Embryonic Cells ◽

Site Specific ◽

Editing Efficiency ◽

Base Editing ◽

One Step ◽

Pig Performance ◽

Novel Alleles

AbstractPrecise and simultaneous acquisition of multiple beneficial alleles in the genome to improve pig performance are pivotal for making elite breeders. Cytidine base editors (CBEs) have emerged as powerful tools for site-specific single nucleotide replacement. Here, we compare the editing efficiency of four CBEs in porcine embryonic cells and embryos to show that hA3A-BE3-Y130F and hA3A-eBE3-Y130F consistently results in higher base-editing efficiency and lower toxic effects to in vitro embryo development. We also show that zygote microinjection of hA3A-BE3-Y130F results in one-step generation of pigs (3BE pigs) harboring C-to-T point mutations, including a stop codon in CD163 and in MSTN and induce beneficial allele in IGF2. The 3BE pigs showed improved growth performance, hip circumference, food conversion rate. Our results demonstrate that CBEs can mediate high throughput genome editing by direct embryo microinjection. Our approach allows immediate introduction of novel alleles for beneficial traits in transgene-free animals for pyramid breeding.

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Engineering an acetyllysine reader with photocrosslinking amino acid for interactome profiling

Chemical Communications ◽

10.1039/d1cc04611j ◽

2021 ◽

Author(s):

Babu Sudhamalla ◽

Anirban Roy ◽

Soumen Barman ◽

Jyotirmayee Padhan

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Protein Interactions ◽

Unnatural Amino Acids ◽

Protein Protein Interactions ◽

Site Specific ◽

Powerful Approach

The site-specific installation of light-activable crosslinker unnatural amino acids offers a powerful approach to trap transient protein-protein interactions both in vitro and in vivo. Herein, we engineer a bromodomain to...

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Mutations in Plasmodium falciparumCytochrome b That Are Associated with Atovaquone Resistance Are Located at a Putative Drug-Binding Site

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.8.2100-2108.2000 ◽

2000 ◽

Vol 44 (8) ◽

pp. 2100-2108 ◽

Cited By ~ 232

Author(s):

Michael Korsinczky ◽

Nanhua Chen ◽

Barbara Kotecka ◽

Allan Saul ◽

Karl Rieckmann ◽

...

Keyword(s):

Amino Acid ◽

Binding Site ◽

Point Mutations ◽

Drug Binding ◽

Single Amino Acid ◽

Malaria Parasites ◽

Drug Binding Site ◽

Sole Agent ◽

Amino Acid Mutations

ABSTRACT Atovaquone is the major active component of the new antimalarial drug Malarone. Considerable evidence suggests that malaria parasites become resistant to atovaquone quickly if atovaquone is used as a sole agent. The mechanism by which the parasite develops resistance to atovaquone is not yet fully understood. Atovaquone has been shown to inhibit the cytochrome bc 1 (CYTbc 1) complex of the electron transport chain of malaria parasites. Here we report point mutations in Plasmodium falciparum CYT b that are associated with atovaquone resistance. Single or double amino acid mutations were detected from parasites that originated from a cloned line and survived various concentrations of atovaquone in vitro. A single amino acid mutation was detected in parasites isolated from a recrudescent patient following atovaquone treatment. These mutations are associated with a 25- to 9,354-fold range reduction in parasite susceptibility to atovaquone. Molecular modeling showed that amino acid mutations associated with atovaquone resistance are clustered around a putative atovaquone-binding site. Mutations in these positions are consistent with a reduced binding affinity of atovaquone for malaria parasite CYTb.

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A Novel Automethylation Reaction in the Aspergillus nidulans LaeA Protein Generates S-Methylmethionine

Journal of Biological Chemistry ◽

10.1074/jbc.m113.465765 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14032-14045 ◽

Cited By ~ 37

Author(s):

Alexander N. Patananan ◽

Jonathan M. Palmer ◽

Graeme S. Garvey ◽

Nancy P. Keller ◽

Steven G. Clarke

Keyword(s):

Amino Acid ◽

Aspergillus Nidulans ◽

Point Mutations ◽

Pathogenic Fungi ◽

Gene Clusters ◽

Methionine Residue ◽

Cell Extracts ◽

Animal Pathogens

The filamentous fungi in the genus Aspergillus are opportunistic plant and animal pathogens that can adapt to their environment by producing various secondary metabolites, including lovastatin, penicillin, and aflatoxin. The synthesis of these small molecules is dependent on gene clusters that are globally regulated by the LaeA protein. Null mutants of LaeA in all pathogenic fungi examined to date show decreased virulence coupled with reduced secondary metabolism. Although the amino acid sequence of LaeA contains the motifs characteristic of seven-β-strand methyltransferases, a methyl-accepting substrate of LaeA has not been identified. In this work we did not find a methyl-accepting substrate in Aspergillus nidulans with various assays, including in vivo S-adenosyl-[methyl-3H]methionine labeling, targeted in vitro methylation experiments using putative protein substrates, or in vitro methylation assays using whole cell extracts grown under different conditions. However, in each experiment LaeA was shown to self-methylate. Amino acid hydrolysis of radioactively labeled LaeA followed by cation exchange and reverse phase chromatography identified methionine as the modified residue. Point mutations show that the major site of modification of LaeA is on methionine 207. However, in vivo complementation showed that methionine 207 is not required for the biological function of LaeA. LaeA is the first protein to exhibit automethylation at a methionine residue. These findings not only indicate LaeA may perform novel chemistry with S-adenosylmethionine but also provide new insights into the physiological function of LaeA.

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Correlation of In Vitro Susceptibilities to Newer Quinolones of Naturally Occurring Quinolone-Resistant Neisseria gonorrhoeae Strains with Changes in GyrA and ParC

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.3.734-738.2001 ◽

2001 ◽

Vol 45 (3) ◽

pp. 734-738 ◽

Cited By ~ 48

Author(s):

Tiffany R. Shultz ◽

John W. Tapsall ◽

Peter A. White

Keyword(s):

Amino Acid ◽

Neisseria Gonorrhoeae ◽

Southeast Asia ◽

Point Mutations ◽

The Other ◽

Naturally Occurring ◽

Ciprofloxacin Resistance ◽

Amino Acid Mutations ◽

Sydney Australia

ABSTRACT The in vitro activities of ciprofloxacin, trovafloxacin, moxifloxacin, and grepafloxacin against 174 strains of Neisseria gonorrhoeae isolated in Sydney, Australia, were determined. The strains included 84 quinolone-less-sensitive and -resistant N. gonorrhoeae (QRNG) strains for which ciprofloxacin MICs were in the range of 0.12 to 16 μg/ml. The QRNG included strains isolated from patients whose infections were acquired in a number of countries, mostly in Southeast Asia. The gyrA and parCquinolone resistance-determining regions (QRDR) of 18 selected QRNG strains were sequenced, and the amino acid mutations observed were related to the MICs obtained. The activities of moxifloxacin and grepafloxacin against QRNG were comparable to that of ciprofloxacin. Trovafloxacin was more active than the other quinolones against some but not all of the QRNG strains. Increments in ciprofloxacin resistance occurred in a step-wise manner with point mutations initiated ingyrA resulting in amino acid alterations Ser91-to-Phe, Ser91-to-Tyr, Asp95-to-Gly, and Asp95-to-Asn. Single gyrAchanges correlated with ciprofloxacin MICs in the range 0.12 to 1 μg/ml. The Ser91 changes in GyrA were associated with higher MICs and further QRDR changes. QRNG strains for which ciprofloxacin MICs were greater than 1 μg/ml had both gyrA and parCQRDR point mutations. ParC alterations were seen in these isolates only in the presence of GyrA changes and comprised amino acid changes Asp86-to-Asn, Ser87-to-Asn, Ser87-to-Arg, Ser88-to-Pro, Glu91-to-Lys, and Glu91-to-Gln. QRNG strains for which MICs were in the higher ranges had double GyrA mutations, but again only with accompanying ParC alterations. Not only did the nature and combination of GyrA and ParC changes influence the incremental increases in ciprofloxacin MICs, but they seemingly also altered the differential activity of trovafloxacin. Our findings suggest that the newer quinolones of the type examined are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread.

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Site Specific Combinations of Stabilizing and Destabilizing Amino Acid Replacements in Yeast Cytochrome c: In Vivo and in Vitro Effects

Biochemistry ◽

10.1021/bi00021a023 ◽

1995 ◽

Vol 34 (21) ◽

pp. 7103-7112 ◽

Cited By ~ 7

Author(s):

Lisa I. Linske-O'Connell ◽

Fred Sherman ◽

George McLendon

Keyword(s):

Amino Acid ◽

Cytochrome C ◽

Site Specific ◽

In Vitro Effects

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Effects of Mutations on the Aggregation Propensity of the Human Prion-Like Protein hnRNPA2B1

Molecular and Cellular Biology ◽

10.1128/mcb.00652-16 ◽

2017 ◽

Vol 37 (8) ◽

Cited By ~ 20

Author(s):

Kacy R. Paul ◽

Amandine Molliex ◽

Sean Cascarina ◽

Amy E. Boncella ◽

J. Paul Taylor ◽

...

Keyword(s):

Amino Acid ◽

Point Mutations ◽

Low Complexity ◽

Anomalous Behavior ◽

Yeast Prion ◽

Cytoplasmic Inclusions ◽

Content Type ◽

Aggregation Propensity ◽

High Amino Acid

ABSTRACT Hundreds of human proteins contain prion-like domains, which are a subset of low-complexity domains with high amino acid compositional similarity to yeast prion domains. A recently characterized mutation in the prion-like domain of the human heterogeneous nuclear ribonucleoprotein hnRNPA2B1 increases the aggregation propensity of the protein and causes multisystem proteinopathy. The mutant protein forms cytoplasmic inclusions when expressed in Drosophila, the mutation accelerates aggregation in vitro, and the mutant prion-like domain can substitute for a portion of a yeast prion domain in supporting prion activity. To examine the relationship between amino acid sequence and aggregation propensity, we made a diverse set of point mutations in the hnRNPA2B1 prion-like domain. We found that the effects on prion formation in Saccharomyces cerevisiae and aggregation in vitro could be predicted entirely based on amino acid composition. However, composition was an imperfect predictor of inclusion formation in Drosophila; while most mutations showed similar behaviors in yeast, in vitro, and in Drosophila, a few showed anomalous behavior. Collectively, these results demonstrate the significant progress that has been made in predicting the effects of mutations on intrinsic aggregation propensity while also highlighting the challenges of predicting the effects of mutations in more complex organisms.

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Variations in the major envelope glycoprotein GP5 of Czech strains of porcine reproductive and respiratory syndrome virus

Journal of General Virology ◽

10.1099/0022-1317-81-10-2497 ◽

2000 ◽

Vol 81 (10) ◽

pp. 2497-2502 ◽

Cited By ~ 38

Author(s):

Stanislav Indik ◽

Lubomír Valíček ◽

Dieter Klein ◽

Jana Klánová

Keyword(s):

Amino Acid ◽

Nucleotide Sequence ◽

Phylogenetic Tree ◽

Envelope Glycoprotein ◽

Point Mutations ◽

Nucleotide Sequences ◽

Respiratory Syndrome Virus

The major envelope glycoprotein genes (ORF5) of seven Czech isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified and their nucleotide sequences were determined. ORF5 displayed nucleotide and amino acid identities of 87·5–100% and 87·6–100%, respectively, among the isolates. In a phylogenetic tree, all European isolates were grouped in a genotype distinct from that of reference American strains (VR-2332, IAF-Klop). Among the European isolates, two different clades were identified. Two Czech isolates (V-501 and V-503) and Italian strain PRRSV 2156 fell into one clade. The remaining European strains comprised the second clade. Surprisingly, two separately clustered strains (V-501 and V-516) were isolated from the same herd. Additionally, the possible effect of in vitro cultivation on the nucleotide sequence was analysed. Nine point mutations in the ORF5 region resulted from 152 in vitro passages of the V-502 isolate in MARC-145 cells.

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