scholarly journals Adenine Base Editor Creates Novel Substitution Mutations in eIF4G Gene of Rice

2021 ◽  
Vol 108 (special) ◽  
Author(s):  
Yaiphabi Kumam ◽  
◽  
Rajadurai G ◽  
Kumar K K ◽  
Varanavasiappan S ◽  
...  
Keyword(s):  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Missense Mutations ◽  
Amino Acid Residues ◽  
Single Nucleotide ◽  
Base Editing ◽  
Rice Tungro Disease ◽  
Substitution Mutations ◽  
Resistant Genotypes ◽  
Adenine Base

Two single nucleotide polymorphic mutations and deletion affecting Y1059V1060V1061 amino acid residues in a host translation initiation factor four gamma (eIF4G) gene in rice are reported to confer resistance to rice tungrospherical virus in resistant genotypes. A CRISPR-based adenine base editing vector was used to target these residues in a susceptible indica cultivar, ASD16.Agrobacterium-mediated transformation of ASD16 generated 16 missense mutants and two deletion mutants. Substitution mutations occurred at A5> G5 and A4> G4, where 5.5 % and 3.37 % of adenosines got converted to guanosines, respectively. The mutantsgenerated had missense mutations affecting the YVV residues and the residues immediately adjacent to YVV.Thus,these novel mutationsare promising candidates in imparting resistance against rice tungro disease.

scholarly journals Characterization of Substitution Mutations of eIF4G Gene Generated through Adenine Base Editors in Rice

2021 ◽  
Vol 108 (september) ◽  
Author(s):  
Yaiphabi Kumam ◽  
◽  
Rajadurai C ◽  
Kumar K K ◽  
Varanavasiappan S ◽  
...  
Keyword(s):  
Rice Genome ◽  
Rice Cultivar ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Base Substitution ◽  
Amino Acid Residues ◽  
Substitution Mutations ◽  
Mutation Efficiency ◽  
Adenine Base

Adenine base editor (ABE) creates A to G transitions within its editing window. In the present study, an ABE was used to target a stretch of six amino acid residues, VLFPNL in translation initiation factor four gamma (eIF4G) gene of rice. Agrobacterium-mediated transformation of rice cultivar ASD16 resulted in T0 events with high mutation efficiency of 89.29 %. Substitution mutations of A > G occurred within the editing window of four to eight bases at A7 > G7 (74.67 %) and A4 > G4 (2.46 %). Non-canonical substitutions of G > C/A was also observed at G15 > C15 (9.29 %) and G8 > A8 (1.15 %). A total of 15 missense base substitution events affecting the target residue was identified. Taken together, the present study showed that ABEs create unexpected base substitutions besides efficient canonical editing of A > G in the rice genome

scholarly journals Single Nucleotide Polymorphisms in a Gene for Translation Initiation Factor (eIF4G) of Rice (Oryza sativa) Associated with Resistance to Rice tungro spherical virus

2010 ◽  
Vol 23 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Jong-Hee Lee ◽  
Muhammad Muhsin ◽  
Genelou A. Atienza ◽  
Do-Yeon Kwak ◽  
Suk-Man Kim ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Recessive Gene ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Rice Tungro Bacilliform Virus ◽  
Nucleotide Polymorphisms ◽  
Rice Tungro Spherical Virus ◽  
Single Nucleotide ◽  
Rice Tungro Disease ◽  
Backcross Populations

Rice tungro disease (RTD) is a serious constraint to rice production in South and Southeast Asia. RTD is caused by Rice tungro spherical virus (RTSV) and Rice tungro bacilliform virus. Rice cv. Utri Merah is resistant to RTSV. To identify the gene or genes involved in RTSV resistance, the association of genotypic and phenotypic variations for RTSV resistance was examined in backcross populations derived from Utri Merah and rice germplasm with known RTSV resistance. Genetic analysis revealed that resistance to RTSV in Utri Merah was controlled by a single recessive gene (tsv1) mapped within an approximately 200-kb region between 22.05 and 22.25 Mb of chromosome 7. A gene for putative translation initiation factor 4G (eIF4Gtsv1) was found in the tsv1 region. Comparison of eIF4Gtsv1 gene sequences among susceptible and resistant plants suggested the association of RTSV resistance with one of the single nucleotide polymorphism (SNP) sites found in exon 9 of the gene. Examination of the SNP site in the eIF4Gtsv1 gene among various rice plants resistant and susceptible to RTSV corroborated the association of SNP or deletions in codons for Val1060-1061 of the predicted eIF4Gtsv1 with RTSV resistance in rice.

scholarly journals Cytosine base editor 4 but not adenine base editor generates off-target mutations in mouse embryos

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Genome Wide ◽  
Wide Range ◽  
Family Based ◽  
Correct Point ◽  
Adenine Base

AbstractDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms. However, the genome-wide off-target effects introduced by base editors in the mammalian genome have been examined in only one study. Here, we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editors (ABE) in mouse embryos using unbiased whole-genome sequencing of a family-based trio cohort. The same sgRNA was used for BE4 and ABE. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls. Therefore, an optimization of cytosine base editors is required to improve its fidelity. While the remarkable fidelity of ABE has implications for a wide range of applications, the occurrence of rare aberrant C-to-T conversions at specific target sites needs to be addressed.

scholarly journals Mutational Analysis of Plant Cap-Binding Protein eIF4E Reveals Key Amino Acids Involved in Biochemical Functions and Potyvirus Infection

2008 ◽  
Vol 82 (15) ◽  
pp. 7601-7612 ◽  
Author(s):  
Sylvie German-Retana ◽  
Jocelyne Walter ◽  
Bénédicte Doublet ◽  
Geneviève Roudet-Tavert ◽  
Valérie Nicaise ◽  
...  
Keyword(s):  
Amino Acids ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Point Mutations ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Natural Resistance ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation

ABSTRACT The eukaryotic translation initiation factor 4E (eIF4E) (the cap-binding protein) is involved in natural resistance against several potyviruses in plants. In lettuce, the recessive resistance genes mo1 1 and mo1 2 against Lettuce mosaic virus (LMV) are alleles coding for forms of eIF4E unable, or less effective, to support virus accumulation. A recombinant LMV expressing the eIF4E of a susceptible lettuce variety from its genome was able to produce symptoms in mo1 1 or mo1 2 varieties. In order to identify the eIF4E amino acid residues necessary for viral infection, we constructed recombinant LMV expressing eIF4E with point mutations affecting various amino acids and compared the abilities of these eIF4E mutants to complement LMV infection in resistant plants. Three types of mutations were produced in order to affect different biochemical functions of eIF4E: cap binding, eIF4G binding, and putative interaction with other virus or host proteins. Several mutations severely reduced the ability of eIF4E to complement LMV accumulation in a resistant host and impeded essential eIF4E functions in yeast. However, the ability of eIF4E to bind a cap analogue or to fully interact with eIF4G appeared unlinked to LMV infection. In addition to providing a functional mutational map of a plant eIF4E, this suggests that the role of eIF4E in the LMV cycle might be distinct from its physiological function in cellular mRNA translation.

scholarly journals A Novel Function of the MA-3 Domains in Transformation and Translation Suppressor Pdcd4 Is Essential for Its Binding to Eukaryotic Translation Initiation Factor 4A

2004 ◽  
Vol 24 (9) ◽  
pp. 3894-3906 ◽  
Author(s):  
Hsin-Sheng Yang ◽  
Myung-Haing Cho ◽  
Halina Zakowicz ◽  
Glenn Hegamyer ◽  
Nahum Sonenberg ◽  
...  
Keyword(s):  
Amino Acid ◽  
Translation Initiation ◽  
Binding Activity ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Amino Acid Residues ◽  
Eukaryotic Translation Initiation Factor ◽  
Stem Loop ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Αn α-helical MA-3 domain appears in several translation initiation factors, including human eukaryotic translation initiation factor 4G (eIF4G) and DAP-5/NAT1/p97, as well as in the tumor suppressor Pdcd4. The function of the MA-3 domain is, however, unknown. C-terminal eIF4G (eIG4Gc) contains an MA-3 domain that is located within the eIF4A-binding region, suggesting a role for eIF4A binding. Interestingly, C-terminal DAP-5/NAT1/p97 contains an MA-3 domain, but it does not bind to eIF4A. Mutation of amino acid residues conserved between Pdcd4 and eIF4Gc but not in DAP-5/NAT1/p97 to the amino acid residues found in the DAP-5/NAT1/p97 indicates that some of these amino acid residues within the MA-3 domain are critical for eIF4A-binding activity. Six Pdcd4 mutants (Pdcd4E249K, Pdcd4D253A, Pdcd4D414K, Pdcd4D418A, Pdcd4E249K,D414K, and Pdcd4D253A,D418A) lost >90% eIF4A-binding activity. Mutation of the corresponding amino acid residues in the eIF4Gc also produced similar results, as seen for Pdcd4. These results demonstrate that the MA-3 domain is important for eIF4A binding and explain the ability of Pdcd4 or eIF4Gc but not DAP-5/NAT1/p97 to bind to eIF4A. Competition experiments indicate that Pdcd4 prevents ca. 60 to 70% of eIF4A binding to eIF4Gc at a Pdcd4/eIF4A ratio of 1:1, but mutants Pdcd4D253A and Pdcd4D253A,D418A do not. Translation of stem-loop structured mRNA is susceptible to inhibition by wild-type Pdcd4 but not by Pdcd4D253A, Pdcd4D418A, or Pdcd4D235A,D418A. Together, these results indicate that not only binding to eIF4A but also prevention of eIF4A binding to the MA-3 domain of eIF4Gc contributes to the mechanism by which Pdcd4 inhibits translation.

scholarly journals Identification of Domains and Residues within the ɛ Subunit of Eukaryotic Translation Initiation Factor 2B (eIF2Bɛ) Required for Guanine Nucleotide Exchange Reveals a Novel Activation Function Promoted by eIF2B Complex Formation

2000 ◽  
Vol 20 (11) ◽  
pp. 3965-3976 ◽  
Author(s):  
Edith Gomez ◽  
Graham D. Pavitt
Keyword(s):  
Catalytic Activity ◽  
Complex Formation ◽  
Translation Initiation ◽  
Translation Initiation Factor ◽  
The Other ◽  
Initiation Factor ◽  
Missense Mutations ◽  
Nucleotide Exchange ◽  
Eukaryotic Translation ◽  
Eukaryotic Translation Initiation

ABSTRACT Eukaryotic translation initiation factor 2B (eIF2B) is the guanine nucleotide exchange factor for protein synthesis initiation factor 2 (eIF2). Composed of five subunits, it converts eIF2 from a GDP-bound form to the active eIF2-GTP complex. This is a regulatory step of translation initiation. In vitro, eIF2B catalytic function can be provided by the largest (epsilon) subunit alone (eIF2Bɛ). This activity is stimulated by complex formation with the other eIF2B subunits. We have analyzed the roles of different regions of eIF2Bɛ in catalysis, in eIF2B complex formation, and in binding to eIF2 by characterizing mutations in the Saccharomyces cerevisiaegene encoding eIF2Bɛ (GCD6) that impair the essential function of eIF2B. Our analysis of nonsense mutations indicates that the C terminus of eIF2Bɛ (residues 518 to 712) is required for both catalytic activity and interaction with eIF2. In addition, missense mutations within this region impair the catalytic activity of eIF2Bɛ without affecting its ability to bind eIF2. Internal, in-frame deletions within the N-terminal half of eIF2Bɛ disrupt eIF2B complex formation without affecting the nucleotide exchange activity of eIF2Bɛ alone. Finally, missense mutations identified within this region do not affect the catalytic activity of eIF2Bɛ alone or its interactions with the other eIF2B subunits or with eIF2. Instead, these missense mutations act indirectly by impairing the enhancement of the rate of nucleotide exchange that results from complex formation between eIF2Bɛ and the other eIF2B subunits. This suggests that the N-terminal region of eIF2Bɛ is an activation domain that responds to eIF2B complex formation.

scholarly journals Translational control of nicotine-evoked synaptic potentiation in mice and neuronal responses in human smokers by eIF2α

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Andon N Placzek ◽  
David L Molfese ◽  
Sanjeev Khatiwada ◽  
Gonzalo Viana Di Prisco ◽  
Wei Huang ◽  
...  
Keyword(s):  
Translational Control ◽  
Drugs Of Abuse ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Neuronal Responses ◽  
Gene Encoding ◽  
Single Nucleotide ◽  
Adult Mice ◽  
Reduced Activity ◽  
Synaptic Effects

Adolescents are particularly vulnerable to nicotine, the principal addictive component driving tobacco smoking. In a companion study, we found that reduced activity of the translation initiation factor eIF2α underlies the hypersensitivity of adolescent mice to the effects of cocaine. Here we report that nicotine potentiates excitatory synaptic transmission in ventral tegmental area dopaminergic neurons more readily in adolescent mice compared to adults. Adult mice with genetic or pharmacological reduction in p-eIF2α-mediated translation are more susceptible to nicotine’s synaptic effects, like adolescents. When we investigated the influence of allelic variability of the Eif2s1 gene (encoding eIF2α) on reward-related neuronal responses in human smokers, we found that a single nucleotide polymorphism in the Eif2s1 gene modulates mesolimbic neuronal reward responses in human smokers. These findings suggest that p-eIF2α regulates synaptic actions of nicotine in both mice and humans, and that reduced p-eIF2α may enhance susceptibility to nicotine (and other drugs of abuse) during adolescence.

Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice

Science ◽  
2019 ◽  
pp. eaaw7166 ◽  
Author(s):  
Shuai Jin ◽  
Yuan Zong ◽  
Qiang Gao ◽  
Zixu Zhu ◽  
Yanpeng Wang ◽  
...  
Keyword(s):  
High Fidelity ◽  
Single Nucleotide Variants ◽  
Disease Treatment ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Base Editing ◽  
Guide Rna ◽  
Genome Wide ◽  
Adenine Base

Cytosine and adenine base editors (CBEs and ABEs) are promising new tools for achieving the precise genetic changes required for disease treatment and trait improvement. However, genome-wide and unbiased analyses of their off-target effects in vivo are still lacking. Our whole genome sequencing (WGS) analysis of rice plants treated with BE3, high-fidelity BE3 (HF1-BE3), or ABE revealed that BE3 and HF1-BE3, but not ABE, induce substantial genome-wide off-target mutations, which are mostly the C→T type of single nucleotide variants (SNVs) and appear to be enriched in genic regions. Notably, treatment of rice with BE3 or HF1-BE3 in the absence of single-guide RNA also results in the rise of genome-wide SNVs. Thus, the base editing unit of BE3 or HF1-BE3 needs to be optimized in order to attain high fidelity.

scholarly journals Cytosine but not adenine base editor generates mutations in mice

2019 ◽  
Author(s):  
Hye Kyung Lee ◽  
Harold E. Smith ◽  
Chengyu Liu ◽  
Michaela Willi ◽  
Lothar Hennighausen
Keyword(s):  
Point Mutations ◽  
Living Cells ◽  
Mouse Embryos ◽  
Single Nucleotide Variants ◽  
Single Nucleotide ◽  
Base Editing ◽  
Wide Range ◽  
Family Based ◽  
Correct Point ◽  
Adenine Base

ABSTRACTDeaminase base editing has emerged as a tool to install or correct point mutations in the genomes of living cells in a wide range of organisms and its ultimate success therapeutically depends on its accuracy. Here we have investigated the fidelity of cytosine base editor 4 (BE4) and adenine base editor (ABE) in mouse embryos using unbiased whole genome sequencing of a family-based trio cohort. We demonstrate that BE4-edited mice carry an excess of single-nucleotide variants and deletions compared to ABE-edited mice and controls.

scholarly journals Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica Isolates Inciting Karnal Bunt of Wheat

2021 ◽  
Vol 7 (2) ◽  
pp. 103
Author(s):  
Malkhan Singh Gurjar ◽  
Rashmi Aggarwal ◽  
Shekhar Jain ◽  
Sapna Sharma ◽  
Jagmohan Singh ◽  
...  
Keyword(s):  
Multilocus Sequence Typing ◽  
Sequence Similarity ◽  
Uttar Pradesh ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Karnal Bunt ◽  
Polymorphism Analysis ◽  
Nucleotide Polymorphisms ◽  
Tilletia Indica ◽  
Single Nucleotide

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), β-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse.

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