scholarly journals Define protein variant functions with high-complexity mutagenesis libraries and enhanced mutation detection software

2021 ◽  
Author(s):  
Xiaoping Yang ◽  
Andrew L Hong ◽  
Ted Sharpe ◽  
Andrew O Giacomelli ◽  
Robert E Lintner ◽  
...  
Keyword(s):  
Amino Acid ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Amino Acid Position ◽  
Single Amino Acid ◽  
Library Design ◽  
Protein Variant ◽  
Reading Frame ◽  
Detection Software ◽  
Small Orfs

Open reading frame (ORF) variant libraries have advanced our ability to query the functions of a large number of variants of a protein simultaneously. A variant library targeting a full-length ORF typically consists of all possible single-amino-acid substitutions and a stop codon at each amino-acid position. Because a single codon variation separates a variant from the template ORF, variant quantification presents the most profound challenge. Efforts such as dividing a library into sub-libraries for direct sequencing or using a tag-directed subassembly approach are practical only with small ORFs. Our approach, on the other hand, features single-pool libraries for all genes up to 3600bp (EGFR), and an enhanced variant-detecting toolkit. Having succeeded in processing screens of ~20 ORF variant libraries, this tool calls variants reliably, and also presents variant annotations in datafiles to enable analyses that have in turn reshaped our strategies governing library design, screen deconvolution, sequencing and its analysis.

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 The medaka fish Tol2 transposable element is in an early stage of decay: identification of a nonautonomous copy

Genome ◽  
2021 ◽  
Author(s):  
Sakura Hayashi ◽  
Takuji Tsukiyama ◽  
Atsuo Iida ◽  
Masato Kinoshita ◽  
Akihiko Koga
Keyword(s):  
Amino Acid ◽  
Stop Codon ◽  
Early Stage ◽  
Single Amino Acid ◽  
Base Substitution ◽  
Unique Element ◽  
Medaka Fish ◽  
Medaka Genome ◽  
Single Amino Acid Change ◽  
Local Sequence

The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The <i>Tol2</i> element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here we report the presence of a nonautonomous <i>Tol2</i> copy that was identified by surveying the medaka genome sequence database. This copy contained 3 local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that the transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous <i>Tol2</i> copy. Thus, <i>Tol2</i> is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost whole decay process that progresses in natural populations.

A G-to-A mutation in IVS-3 of the human gamma fibrinogen gene causing afibrinogenemia due to abnormal RNA splicing

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2501-2505 ◽  
Author(s):  
Maurizio Margaglione ◽  
Rosa Santacroce ◽  
Donatella Colaizzo ◽  
Davide Seripa ◽  
Gennaro Vecchione ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rna Splicing ◽  
Messenger Rna ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Autosomal Recessive Disorder ◽  
Chain Gene ◽  
Hemorrhagic Diathesis ◽  
Reading Frame ◽  
Congenital Afibrinogenemia

Abstract Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by a hemorrhagic diathesis of variable severity. Although more than 100 families with this disorder have been described, genetic defects have been characterized in few cases. An investigation of a young propositus, offspring of a consanguineous marriage, with undetectable levels of functional and quantitative fibrinogen, was conducted. Sequence analysis of the fibrinogen genes showed a homozygous G-to-A mutation at the fifth nucleotide (nt 2395) of the third intervening sequence (IVS) of the γ-chain gene. Her first-degree relatives, who had approximately half the normal fibrinogen values and showed concordance between functional and immunologic levels, were heterozygtes. The G-to-A change predicts the disappearance of a donor splice site. After transfection with a construct, containing either the wild-type or the mutated sequence, cells with the mutant construct showed an aberrant messenger RNA (mRNA), consistent with skipping of exon 3, but not the expected mRNA. Sequencing of the abnormal mRNA showed the complete absence of exon 3. Skipping of exon 3 predicts the deletion of amino acid sequence from residue 16 to residue 75 and shifting of reading frame at amino acid 76 with a premature stop codon within exon 4 at position 77. Thus, the truncated γ-chain gene product would not interact with other chains to form the mature fibrinogen molecule. The current findings show that mutations within highly conserved IVS regions of fibrinogen genes could affect the efficiency of normal splicing, giving rise to congenital afibrinogenemia.

scholarly journals Two novel mutations of the calcium-sensing receptor gene affecting the same amino acid position lead to opposite phenotypes and reveal the importance of p.N802 on receptor activity

2013 ◽  
Vol 168 (2) ◽  
pp. K27-K34 ◽  
Author(s):  
Anne-Sophie Lia-Baldini ◽  
Corinne Magdelaine ◽  
Angélique Nizou ◽  
Coraline Airault ◽  
Jean-Pierre Salles ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Direct Sequencing ◽  
Receptor Gene ◽  
Amino Acid Position ◽  
Receptor Activity ◽  
Site Directed Mutagenesis ◽  
Missense Mutations ◽  
Calcium Sensing Receptor ◽  
Calcium Sensing

ObjectiveGain-of-function mutations of the calcium-sensing receptor (CASR) gene have been identified in patients with sporadic or familial autosomal dominant hypocalcemia (ADH). Inactivating mutations of the CASR gene cause familial hypocalciuric hypercalcemia (FHH). Here, we report two novel CASR mutations affecting the same amino acid (p.N802); one causes ADH and the other atypical FHH.Patients and methodsThe first patient, an 11-year-old girl suffering from hypocalcemia, developed nephrocalcinosis when she was only 5 years old. The second patient is a 30-year-old woman who presented with mild hypercalcemia. PCR amplification of CASR coding exons and direct sequencing of PCR products were used to identify mutations. Site-directed mutagenesis was used to generate mutated CASR cDNAs in an expression plasmid. Using the MAPK assay system and transient transfection of Cos-7 cells with wild-type (WT) and mutated CASR, we studied the responses of these mutated receptors to extracellular Ca2+ and to the negative allosteric CASR modulator, NPS2143.ResultsTwo heterozygous missense mutations (p.N802I and p.N802S) affecting a residue in the sixth transmembrane domain of CASR were identified. In functional tests, the response of the p.N802S mutant to calcium was typical of an inactivating mutation. However, the p.N802I mutant had 70% of the maximally stimulated WT receptor activity even in the absence of extracellular calcium. This constitutive activity was only partially inhibited by the inhibitor, NPS2143.ConclusionsThe asparagine at amino acid position 802 appears to be essential for the activity of the CASR protein and is implicated in the mechanism of CASR signaling.

Mutations in LMAN1 and MCFD2 May Account for All Cases of Combined Deficiency of Factor V and Factor VIII.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 724-724
Author(s):  
Bin Zhang ◽  
Beth McGee ◽  
William C. Nichols ◽  
Hugo Guglielmone ◽  
Katherine Downes ◽  
...  
Keyword(s):  
Amino Acid ◽  
Factor Viii ◽  
Transmembrane Protein ◽  
Direct Sequencing ◽  
Regulatory Region ◽  
Von Willebrand Disease ◽  
Single Amino Acid ◽  
Factor V ◽  
Proteolytic Activation ◽  
Combined Deficiency

Abstract Factor V (FV) and factor VIII (FVIII) are two large plasma glycoproteins that function as essential cofactors for the proteolytic activation of prothrombin and factor X, respectively. Efficient biosynthesis of FV and FVIII requires LMAN1 and MCFD2, two proteins localized to the early secretory pathway of the cell. LMAN1 is a 53-kD homo-hexameric transmembrane protein with homology to leguminous mannose-binding lectins. MCFD2 is an EF-hand domain protein that co-localizes with LMAN1 to the ER-Golgi intermediate compartment (ERGIC). MCFD2 interacts with LMAN1 to form a stable, calcium-dependent protein complex that functions as a cargo receptor, ferrying FV and FVIII from the endoplasmic reticulum to the Golgi. Mutations in LMAN1 or MCFD2 cause combined deficiency of factors V and VIII, an autosomal recessive disorder associated with plasma levels of FV and FVIII in the range of 5% to 30% of normal. However, three families were found to have no LMAN1 or MCFD2 mutations, with 2 of these families showing genetic evidence against linkage to either gene, raising the possibility of additional locus heterogeneity and the involvement of a third F5F8D gene. We now report the analysis of 10 previously reported and 9 new F5F8D families. We identified 3 MCFD2 mutations accounting for 6 F5F8D families, and 8 LMAN1 mutations accounting for 8 additional families, including the first-reported single amino acid substitution, replacement of cysteine at amino acid position 475 with arginine (C475R). Cysteine 475 was previously reported to be important in forming an intermolecular disulfide bond required for LMAN1 oligomerization. However, C475R LMAN1 was undetected by Western blot analysis in lymphoblasts derived from a patient hemizygous for this mutation, with only a trace of protein detectable by immunoprecipitation. Thus, the C475R mutation appears to result in an unstable LMAN1 protein that is rapidly degraded. Failure of proteasome inhibitors to increase the intracellular accumulation of this protein suggests an alternative degradation pathway. Finally, two LMAN1 alleles for which no mutations were identified were nonetheless shown to result in no detectable LMAN1 mRNA, indicating a cis-defect in transcription or mRNA stability. Taken together with our previous reports, we have now identified LMAN1 or MCFD2 mutations as the causes of F5F8D in 70 of 75 families. Two of the remaining 5 families are consistent with linkage to the LMAN1 or MCFD2 loci, suggesting mutations in the regulatory region of the genes that were missed by direct sequencing. Reanalysis of the remaining 3 families suggests an initial misdiagnosis, with one reclassified as isolated, mild FV deficiency, and two others as von Willebrand disease. These results suggest that mutations in LMAN1 and MCFD2 account for all cases of F5F8D, with no evidence for a 3rd F5F8D gene.

Mutations within BCR-ABL1 295–312 Define a Novel Region Associated with Poor Prognosis in Patients with Chronic Myelogenous Leukemia (CML) Resistant to Imatinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1936-1936
Author(s):  
Alfonso Quintás-Cardama ◽  
Don L. Gibbons ◽  
Hagop Kantarjian ◽  
Moshe Talpaz ◽  
Jorge Cortes ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Kinase Inhibitor ◽  
Direct Sequencing ◽  
Amino Acid Position ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Low Frequencies

Abstract ABL kinase domain (AKD) mutations are found in 20%–40% of pts with CML who fail therapy with the tyrosine kinase inhibitor (TKI) imatinib. Except for T315I, resistance conferred by most mutations can be overcome by the 2nd generation TKI dasatinib. In pts failing TKI therapy with no detectable AKD mutations by conventional direct sequencing (DS) of the AKD, other mechanisms of resistance (e.g., BCR-ABL1 amplification, SRC overexpression) have been proposed. However, these mechanisms occur at low frequencies in vivo. To ascertain whether imatinib-resistant pts may harbor mutations not detected by standard DS, we evaluated 61 pts with CML after imatinib failure by DNA expansion of specific clones (DESC) followed by DNA sequencing of at least 10 clones. At this point and prior to start of dasatinib therapy, 26 pts were in CP, 14 in AP, and 21 in BP. A total of 118 distinct AKD mutations at 112 amino acid positions were detected (77 previously unreported) in 58/61 (95%) pts. As previously reported, most mutants mapped to 4 AKD regions: P-loop (16%), catalytic domain (17%), 315–317 region (13%), and activation loop (9%). In addition, mutations were also found to cluster at high frequency to 4 novel AKD regions. One of them spans the residues flanked by amino acid positions 295 and 312 and contained 18% of AKD mutations. In addition to 5 pts who developed the highly dasatinib-resistant mutation V299L, 16/61 (25%) other pts harbored mutations within 295–312 prior to dasatinib start. Eleven (69%) of them never achieved any cytogenetic response on dasatinib and this was associated with significantly worse overall survival than that of patients expressing any other AKD mutation (p=0.02), except for T315I (Figure 1). Structurally, 295–312 mutations can potentially interfere with the N-lobe:helix αC interface. Although the exact energetic consequences of mutations at these residues are difficult to predict, structural analysis appears to indicate that these may hinder the outward torsion of the adjacent helix αC, potentially hampering the transition to the inactive conformation (“αC-Glu In” conformation) to which imatinib binds. Alternatively, the 295–312 region may serve as the structural scaffold for amino acid position 299, a direct dasatinib contact residue. Mutations mapping to 295–312 might distort the topography surrounding 299, thus altering the 299/dasatinib interface. Notably, 5/16 (32%) of these pts carried clones expressing more than 1 mutation within 295–312. All but 1 (80%) of these pts are dead. In summary, the use of techniques with higher sensitivity than conventional DS reveals that AKD mutations are highly prevalent (95%) in pts failing imatinib therapy, which could explain TKI resistance in pts not found to carry resistant mutations by conventional DS. We present evidence supporting the deleterious effect of mutations mapping to the novel 295–312 region. Experiments designed to prove these hypotheses are ongoing. Survival of 295–312 mutants Survival of 295–312 mutants

scholarly journals Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing

2018 ◽  
Author(s):  
Kenneth A. Matreyek ◽  
Lea M. Starita ◽  
Jason J. Stephany ◽  
Beth Martin ◽  
Melissa A. Chiasson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Massively Parallel Sequencing ◽  
Dominant Negative ◽  
Massively Parallel ◽  
Single Amino Acid ◽  
Protein Variant ◽  
Parallel Sequencing ◽  
Missense Variants ◽  
Functional Variants ◽  
Amino Acid Variants

ABSTRACTDetermining the pathogenicity of human genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes will likely require generalizable, scalable assays. Here we describe Variant Abundance by Massively Parallel Sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance in a single experiment. We apply VAMP-seq to quantify the abundance of 7,595 single amino acid variants of two proteins, PTEN and TPMT, in which functional variants are clinically actionable. We identify 1,079 PTEN and 805 TPMT single amino acid variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and our abundance data suggest that a PTEN variant accounting for ~10% of PTEN missense variants in melanomas functions via a dominant negative mechanism. Finally, we demonstrate that VAMP-seq can be applied to other genes, highlighting its potential as a generalizable assay for characterizing missense variants.

scholarly journals Variants of β-Lactamase KPC-2 That Are Resistant to Inhibition by Avibactam

2015 ◽  
Vol 59 (7) ◽  
pp. 3710-3717 ◽  
Author(s):  
Krisztina M. Papp-Wallace ◽  
Marisa L. Winkler ◽  
Magdalena A. Taracila ◽  
Robert A. Bonomo
Keyword(s):  
Amino Acid ◽  
Molecular Modeling ◽  
Clavulanic Acid ◽  
Amino Acid Position ◽  
Proton Donor ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type ◽  
Mechanistic Basis ◽  
Dynamics Simulations

ABSTRACTKPC-2 is the most prevalent class A carbapenemase in the world. Previously, KPC-2 was shown to hydrolyze the β-lactamase inhibitors clavulanic acid, sulbactam, and tazobactam. In addition, substitutions at amino acid position R220 in the KPC-2 β-lactamase increased resistance to clavulanic acid. A novel bridged diazabicyclooctane (DBO) non-β-lactam β-lactamase inhibitor, avibactam, was shown to inactivate the KPC-2 β-lactamase. To better understand the mechanistic basis for inhibition of KPC-2 by avibactam, we tested the potency of ampicillin-avibactam and ceftazidime-avibactam against engineered variants of the KPC-2 β-lactamase that possessed single amino acid substitutions at important sites (i.e., Ambler positions 69, 130, 234, 220, and 276) that were previously shown to confer inhibitor resistance in TEM and SHV β-lactamases. To this end, we performed susceptibility testing, biochemical assays, and molecular modeling.Escherichia coliDH10B carrying KPC-2 β-lactamase variants with the substitutions S130G, K234R, and R220M demonstrated elevated MICs for only the ampicillin-avibactam combinations (e.g., 512, 64, and 32 mg/liter, respectively, versus the MICs for wild-type KPC-2 at 2 to 8 mg/liter). Steady-state kinetics revealed that the S130G variant of KPC-2 resisted inactivation by avibactam; thek2/Kratio was significantly lowered 4 logs for the S130G variant from the ratio for the wild-type enzyme (21,580 M−1s−1to 1.2 M−1s−1). Molecular modeling and molecular dynamics simulations suggested that the mobility of K73 and its ability to activate S70 (i.e., function as a general base) may be impaired in the S130G variant of KPC-2, thereby explaining the slowed acylation. Moreover, we also advance the idea that the protonation of the sulfate nitrogen of avibactam may be slowed in the S130G variant, as S130 is the likely proton donor and another residue, possibly K234, must compensate. Our findings show that residues S130 as well as K234 and R220 contribute significantly to the mechanism of avibactam inactivation of KPC-2. Fortunately, the emergence of S130G, K234R, and R220M variants of KPC in the clinic should not result in failure of ceftazidime-avibactam, as the ceftazidime partner is potent againstE. coliDH10B strains possessing all of these variants.

scholarly journals Structural and functional analyses of Saccharomyces cerevisiae wild-type and mutant RNA1 genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 2989-2999 ◽  
Author(s):  
H M Traglia ◽  
N S Atkinson ◽  
A K Hopper
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Open Reading Frame ◽  
Single Amino Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Reading Frame ◽  
Genomic Deletions ◽  
Single Amino Acid Change ◽  
Carboxy Terminal

The yeast gene RNA1 has been defined by the thermosensitive rna1-1 lesion. This lesion interferes with the processing and production of all major classes of RNA. Each class of RNA is affected at a distinct and presumably unrelated step. Furthermore, RNA does not appear to exit the nucleus. To investigate how the RNA1 gene product can pleiotropically affect disparate processes, we undertook a structural analysis of wild-type and mutant RNA1 genes. The wild-type gene was found to contain a 407-amino-acid open reading frame that encodes a hydrophilic protein. No clue regarding the function of the RNA1 protein was obtained by searching banks for similarity to other known gene products. Surprisingly, the rna1-1 lesion was found to code for two amino acid differences from wild type. We found that neither single-amino-acid change alone resulted in temperature sensitivity. The carboxy-terminal region of the RNA1 open reading frame contains a highly acidic domain extending from amino acids 334 to 400. We generated genomic deletions that removed C-terminal regions of this protein. Deletion of amino acids 397 to 407 did not appear to affect cell growth. Removal of amino acids 359 to 397, a region containing 24 acidic residues, caused temperature-sensitive growth. This allele, rna1-delta 359-397, defines a second conditional lesion of the RNA1 locus. We found that strains possessing the rna1-delta 359-397 allele did not show thermosensitive defects in pre-rRNA or pre-tRNA processing. Removal of amino acids 330 to 407 resulted in loss of viability.

scholarly journals Mapping Attenuation Determinants in Enterovirus-D68

Viruses ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 867
Author(s):  
Ming Te Yeh ◽  
Sara Capponi ◽  
Adam Catching ◽  
Simone Bianco ◽  
Raul Andino
Keyword(s):  
Amino Acid ◽  
Amino Acid Position ◽  
Single Amino Acid ◽  
Type I ◽  
Virulence Determinants ◽  
Genetic Changes ◽  
Infectious Clones ◽  
Viral Virulence ◽  
Single Amino Acid Change ◽  
Enterovirus D68

Enterovirus (EV)-D68 has been associated with epidemics in the United Sates in 2014, 2016 and 2018. This study aims to identify potential viral virulence determinants. We found that neonatal type I interferon receptor knockout mice are susceptible to EV-D68 infection via intraperitoneal inoculation and were able to recapitulate the paralysis process observed in human disease. Among the EV-D68 strains tested, strain US/MO-14-18949 caused no observable disease in this mouse model, whereas the other strains caused paralysis and death. Sequence analysis revealed several conserved genetic changes among these virus strains: nucleotide positions 107 and 648 in the 5′-untranslated region (UTR); amino acid position 88 in VP3; 1, 148, 282 and 283 in VP1; 22 in 2A; 47 in 3A. A series of chimeric and point-mutated infectious clones were constructed to identify viral elements responsible for the distinct virulence. A single amino acid change from isoleucine to valine at position 88 in VP3 attenuated neurovirulence by reducing virus replication in the brain and spinal cord of infected mice.

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