scholarly journals Targeted Mutagenesis of the Female-Suppressor SyGI Gene in Tetraploid Kiwifruit by CRISPR/CAS9

Plants ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 62
Author(s):  
Gloria De Mori ◽  
Giusi Zaina ◽  
Barbara Franco-Orozco ◽  
Raffaele Testolin ◽  
Emanuele De Paoli ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Genomic Region ◽  
Targeted Mutagenesis ◽  
Stable Gene ◽  
Putative Gene ◽  
Knock Out ◽  
Long Time ◽  
Time Required ◽  
A Minor

Kiwifruit belong to the genus Actinidia with 54 species apparently all functionally dioecious. The sex-determinants of the type XX/XY, with male heterogametic, operate independently of the ploidy level. Recently, the SyGI protein has been described as the suppressor of female development. In the present study, we exploited the CRISPR/Cas9 technology by targeting two different sites in the SyGI gene in order to induce a stable gene knock-out in two tetraploid male accessions of Actinidia chinensis var. chinensis. The two genotypes showed a regenerative efficiency of 58% and 73%, respectively. Despite not yet being able to verify the phenotypic effects on the flower structure, due to the long time required by tissue-cultured kiwifruit plants to flower, we obtained two regenerated lines showing near fixation of a unique modification in their genome, resulting in both cases in the onset of a premature stop codon, which induces the putative gene knock-out. Evaluation of gRNA1 locus for both regenerated plantlets resulted in co-amplification of a minor variant differing from the target region for a single nucleotide. A genomic duplication of the region in proximity of the Y genomic region could be postulated.

scholarly journals Molecular parallelisms between pigmentation in the avian iris and the integument of ectothermic vertebrates

PLoS Genetics ◽  
2021 ◽  
Vol 17 (2) ◽  
pp. e1009404
Author(s):  
Pedro Andrade ◽  
Małgorzata A. Gazda ◽  
Pedro M. Araújo ◽  
Sandra Afonso ◽  
Jacob. A. Rasmussen ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Stop Codon ◽  
Expression Profiles ◽  
Single Gene ◽  
Skin Pigmentation ◽  
Premature Stop Codon ◽  
Gene Expression Profiles ◽  
Genomic Region ◽  
Pigment Cells ◽  
Eye Color

Birds exhibit striking variation in eye color that arises from interactions between specialized pigment cells named chromatophores. The types of chromatophores present in the avian iris are lacking from the integument of birds or mammals, but are remarkably similar to those found in the skin of ectothermic vertebrates. To investigate molecular mechanisms associated with eye coloration in birds, we took advantage of a Mendelian mutation found in domestic pigeons that alters the deposition of yellow pterin pigments in the iris. Using a combination of genome-wide association analysis and linkage information in pedigrees, we mapped variation in eye coloration in pigeons to a small genomic region of ~8.5kb. This interval contained a single gene, SLC2A11B, which has been previously implicated in skin pigmentation and chromatophore differentiation in fish. Loss of yellow pigmentation is likely caused by a point mutation that introduces a premature STOP codon and leads to lower expression of SLC2A11B through nonsense-mediated mRNA decay. There were no substantial changes in overall gene expression profiles between both iris types as well as in genes directly associated with pterin metabolism and/or chromatophore differentiation. Our findings demonstrate that SLC2A11B is required for the expression of pterin-based pigmentation in the avian iris. They further highlight common molecular mechanisms underlying the production of coloration in the iris of birds and skin of ectothermic vertebrates.

scholarly journals Within-population variability in a moth sex pheromone blend, part 2: selection towards fixation

2019 ◽  
Vol 6 (3) ◽  
pp. 182050 ◽  
Author(s):  
Astrid T. Groot ◽  
Michiel van Wijk ◽  
Ernesto Villacis-Perez ◽  
Peter Kuperus ◽  
Gerhard Schöfl ◽  
...  
Keyword(s):  
Communication Systems ◽  
Sexual Communication ◽  
Stop Codon ◽  
Single Point ◽  
Premature Stop Codon ◽  
Behavioural Response ◽  
Population Variation ◽  
Single Point Mutation ◽  
Knock Out ◽  
Female Pheromone

To understand how variation in sexual communication systems evolves, the genetic architecture underlying sexual signals and responses needs to be identified. Especially in animals where mating signals are important for mate recognition, and signals and responses are governed by independently assorting genes, it is difficult to envision how signals and preferences can (co)evolve. Moths are a prime example of such animals. In the noctuid moth Heliothis virescens , we found within-population variation in the female pheromone. In previous selection experiments followed by quantitative trait locus (QTL) analysis and expression analysis of candidate desaturase genes, we developed a model involving a trans -acting repressor of the delta-11-desaturase. In our current study with new selection lines, we fixed the most extreme phenotype and found a single underlying mutation: a premature stop codon in the first coding exon of delta-11-desaturase, which we could trace back to its origin in the laboratory. Interestingly, we found no pleiotropic effects of this knock-out mutation on the male physiological or behavioural response, or on growth or fertility. This finding is in contrast to Drosophila melanogaster , where a single desaturase gene affects both female pheromone production and male behavioural response, but similar to other Lepidoptera where these traits are under independent genetic control. To our knowledge, this is the first time that a single point mutation has been identified that underlies the phenotypic variation in the pheromone signal of a moth.

scholarly journals Identification and characteristics of the structural gene for the Drosophila eye colour mutant sepia, encoding PDA synthase, a member of the Omega class glutathione S-transferases

2006 ◽  
Vol 398 (3) ◽  
pp. 451-460 ◽  
Author(s):  
Jaekwang Kim ◽  
Hyunsuk Suh ◽  
Songhee Kim ◽  
Kiyoung Kim ◽  
Chiyoung Ahn ◽  
...  
Keyword(s):  
Structural Gene ◽  
Stop Codon ◽  
Gel Filtration ◽  
Premature Stop Codon ◽  
Genomic Region ◽  
Gene Encoding ◽  
Colour Mutant ◽  
Eye Colour ◽  
Glutathione S Transferases

The eye colour mutant sepia (se1) is defective in PDA {6-acetyl-2-amino-3,7,8,9-tetrahydro-4H-pyrimido[4,5-b]-[1,4]diazepin-4-one or pyrimidodiazepine} synthase involved in the conversion of 6-PTP (2-amino-4-oxo-6-pyruvoyl-5,6,7,8-tetrahydropteridine; also known as 6-pyruvoyltetrahydropterin) into PDA, a key intermediate in drosopterin biosynthesis. However, the identity of the gene encoding this enzyme, as well as its molecular properties, have not yet been established. Here, we identify and characterize the gene encoding PDA synthase and show that it is the structural gene for sepia. Based on previously reported information [Wiederrecht, Paton and Brown (1984) J. Biol. Chem. 259, 2195–2200; Wiederrecht and Brown (1984) J. Biol. Chem. 259, 14121–14127; Andres (1945) Drosoph. Inf. Serv. 19, 45; Ingham, Pinchin, Howard and Ish-Horowicz (1985) Genetics 111, 463–486; Howard, Ingham and Rushlow (1988) Genes Dev. 2, 1037–1046], we isolated five candidate genes predicted to encode GSTs (glutathione S-transferases) from the presumed sepia locus (region 66D5 on chromosome 3L). All cloned and expressed candidates exhibited relatively high thiol transferase and dehydroascorbate reductase activities and low activity towards 1-chloro-2,4-dinitrobenzene, characteristic of Omega class GSTs, whereas only CG6781 catalysed the synthesis of PDA in vitro. The molecular mass of recombinant CG6781 was estimated to be 28 kDa by SDS/PAGE and 56 kDa by gel filtration, indicating that it is a homodimer under native conditions. Sequencing of the genomic region spanning CG6781 revealed that the se1 allele has a frameshift mutation from ‘AAGAA’ to ‘GTG’ at nt 190–194, and that this generates a premature stop codon. Expression of the CG6781 open reading frame in an se1 background rescued the eye colour defect as well as PDA synthase activity and drosopterins content. The extent of rescue was dependent on the dosage of transgenic CG6781. In conclusion, we have discovered a new catalytic activity for an Omega class GST and that CG6781 is the structural gene for sepia which encodes PDA synthase.

scholarly journals Forward genetics by sequencing EMS variation induced inbred lines

2016 ◽  
Author(s):  
Charles Addo-Quaye ◽  
Elizabeth Buescher ◽  
Norman Best ◽  
Vijay Chaikam ◽  
Ivan Baxter ◽  
...  
Keyword(s):  
Stop Codon ◽  
False Positive Rate ◽  
Premature Stop Codon ◽  
Genomic Region ◽  
Forward Genetics ◽  
Reference Sequence ◽  
Variant Discovery ◽  
Validation Population ◽  
Giberellic Acid ◽  
Eukaryotic Organisms

ABSTRACTIn order to leverage novel sequencing techniques for cloning genes in eukaryotic organisms with complex genomes, the false positive rate of variant discovery must be controlled for by experimental design and informatics. We sequenced five lines from three pedigrees of EMS mutagenized Sorghum bicolor, including a pedigree segregating a recessive dwarf mutant. Comparing the sequences of the lines, we were able to identify and eliminate error prone positions. One genomic region contained EMS mutant alleles in dwarfs that were homozygous reference sequence in wild-type siblings and heterozygous in segregating families. This region contained a single non-synonymous change that cosegregated with dwarfism in a validation population and caused a premature stop codon in the sorghum ortholog encoding the giberellic acid biosynthetic enzyme ent-kaurene oxidase. Application of exogenous giberillic acid rescued the mutant phenotype. Our method for mapping did not require outcrossing and introduced no segregation variance. This enables work when line crossing is complicated by life history, permitting gene discovery outside of genetic models.This inverts the historical approach of first using recombination to define a locus and then sequencing genes. Our formally identical approach first sequences all the genes and then seeks co-segregation with the trait. Mutagenized lines lacking obvious phenotypic alterations are available for an extention of this approach: mapping with a known marker set in a line that is phenotypically identical to starting material for EMS mutant generation.

Ectopic Transcript Analysis Indicates that Allelic Exclusion is an Important Cause of Type I Protein C Deficiency in Patients with Nonsense and Frameshift Mutations in the PROC Gene

1996 ◽  
Vol 75 (06) ◽  
pp. 870-876 ◽  
Author(s):  
José Manuel Soria ◽  
Lutz-Peter Berg ◽  
Jordi Fontcuberta ◽  
Vijay V Kakkar ◽  
Xavier Estivill ◽  
...  
Keyword(s):  
Splice Site ◽  
Protein C ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Allelic Exclusion ◽  
Polymorphism Analysis ◽  
Type I ◽  
Protein C Deficiency ◽  
Nonsense Mutations ◽  
Stop Codons

SummaryNonsense mutations, deletions and splice site mutations are a common cause of type I protein C deficiency. Either directly or indirectly by altering the reading frame, these' lesions generate or may generate premature stop codons and could therefore be expected to result in premature termination of translation. In this study, the possibility that such mutations could instead exert their pathological effects at an earlier stage in the expression pathway, through “allelic exclusion” at the RNA level, was investigated. Protein C (PROC) mRNA was analysed in seven Spanish type I protein C deficient patients heterozygous for two nonsense mutations, a 7bp deletion, a 2bp insertion and three splice site mutations. Ectopic RNA transcripts from patient and control lymphocytes were analysed by RT-PCR and direct sequencing of amplified PROC cDNA fragments. The nonsense mutations and the deletion were absent from the cDNAs indicating that only mRNA derived from the normal allele had been expressed. Similarly for the splice site mutations, only normal PROC cDNAs were obtained. In one case, exclusion of the mutated allele could be confirmed by polymorphism analysis. In contrast to these six mutations, the 2 bp insertion was not associated with loss of mRNA from the mutated allele. In this case, cDNA analysis revealed the absence of 19 bases from the PROC mRNA consistent with the generation and utilization of a cryptic splice site 3’ to the site of mutation, which would result in a frameshift and a premature stop codon. It is concluded that allelic exclusion is a common causative mechanism in those cases of type I protein C deficiency which result from mutations that introduce premature stop codons

Effect of Vial shaking on Loading Time of Epirubicin into Drug-Eluting Bead

2015 ◽  
Vol 5 (3) ◽  
Author(s):  
Kenji Ikeda ◽  
Yusuke Kawamura ◽  
Masahiro Kobayashi ◽  
Taito Fukushima ◽  
Yushi Sorin ◽  
...  
Keyword(s):  
Antitumor Agent ◽  
Loading Rates ◽  
Vortex Mixer ◽  
Long Time ◽  
Group A ◽  
Drug Eluting ◽  
Large Hepatocellular Carcinoma ◽  
Group B ◽  
Time Required ◽  
Short Time

Background: Although DC Bead has been useful in treatment of multiple and large hepatocellular carcinoma, loading time of doxorubicin into the DC Bead takes a long time of 30-120 minutes. Epirubicin is also used as an antitumor agent together with DC Bead, but its loading efficiency was not sufficiently elucidated. Methods: To shorten loading time of epirubicin into DC Bead (100-300µm, 300-500µm, 500-700µm), we examined the following three methods after mixing the drug: (a) let stand in room temperature, (b) agitated for 30 seconds with Vortex mixer, and (c) sonicated for 30 seconds with ultrasonic cleaner. After loading of epirubicin by each method, supernatant concentration for epirubicin was assayed at 5, 10, 30, 60, and 120 minutes. Results: Epirubicin loading rates for small bead (100-300µm) at 5 minutes were 82.9 % in group a, 93.8% in group b, and 79.9 % in group c. Similarly, medium bead (300-500µm), 40.1% in group a, 65.7% in group b and 45.5% in group c, respectively. In large-sized bead (500-700µm), loaded rates of epirubicin were 38.8% in group a, 59.0% in group b and 48.0% in group c. Agitation of mixture of epirubicin and DC Bead with Vortex mixer significantly shortened the loading time, but sonication did not affect the time required. Microscopic examination did not lead to any morphological change of microspheres in all the methods. Conclusions: Short time of agitation with Vortex mixer reduced the necessary time for loading of epirubicin in every standard of DC Bead.

Induction of Translational Readthrough across the Thalassemia-Causing Premature Stop Codon in β-Globin-Encoding mRNA

Biochemistry ◽  
2019 ◽  
Vol 59 (1) ◽  
pp. 80-84 ◽  
Author(s):  
Debaleena Kar ◽  
Karthi Sellamuthu ◽  
Sangeetha Devi Kumar ◽  
Sandeep M. Eswarappa
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Translational Readthrough

scholarly journals A Novel Truncating Mutation in HOMER2 Causes Nonsyndromic Progressive DFNA68 Hearing Loss in a Spanish Family

Genes ◽  
2021 ◽  
Vol 12 (3) ◽  
pp. 411
Author(s):  
María Lachgar ◽  
Matías Morín ◽  
Manuela Villamar ◽  
Ignacio del Castillo ◽  
Miguel Ángel Moreno-Pelayo
Keyword(s):  
Hearing Loss ◽  
Stop Codon ◽  
Coiled Coil ◽  
Premature Stop Codon ◽  
Scaffolding Protein ◽  
Common Mechanism ◽  
Chinese Family ◽  
Truncating Mutation ◽  
Spanish Family ◽  
Sensory Defect

Nonsyndromic hereditary hearing loss is a common sensory defect in humans that is clinically and genetically highly heterogeneous. So far, 122 genes have been associated with this disorder and 50 of them have been linked to autosomal dominant (DFNA) forms like DFNA68, a rare subtype of hearing impairment caused by disruption of a stereociliary scaffolding protein (HOMER2) that is essential for normal hearing in humans and mice. In this study, we report a novel HOMER2 variant (c.832_836delCCTCA) identified in a Spanish family by using a custom NGS targeted gene panel (OTO-NGS-v2). This frameshift mutation produces a premature stop codon that may lead in the absence of NMD to a shorter variant (p.Pro278Alafs*10) that truncates HOMER2 at the CDC42 binding domain (CBD) of the coiled-coil structure, a region that is essential for protein multimerization and HOMER2-CDC42 interaction. c.832_836delCCTCA mutation is placed close to the previously identified c.840_840dup mutation found in a Chinese family that truncates the protein (p.Met281Hisfs*9) at the CBD. Functional assessment of the Chinese mutant revealed decreased protein stability, reduced ability to multimerize, and altered distribution pattern in transfected cells when compared with wild-type HOMER2. Interestingly, the Spanish and Chinese frameshift mutations might exert a similar effect at the protein level, leading to truncated mutants with the same Ct aberrant protein tail, thus suggesting that they can share a common mechanism of pathogenesis. Indeed, age-matched patients in both families display quite similar hearing loss phenotypes consisting of early-onset, moderate-to-profound progressive hearing loss. In summary, we have identified the third variant in HOMER2, which is the first one identified in the Spanish population, thus contributing to expanding the mutational spectrum of this gene in other populations, and also to clarifying the genotype–phenotype correlations of DFNA68 hearing loss.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

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