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 Functional evaluation of human ClC-2 chloride channel mutations associated with idiopathic generalized epilepsies

2004 ◽  
Vol 19 (1) ◽  
pp. 74-83 ◽  
Author(s):  
María Isabel Niemeyer ◽  
Yamil R. Yusef ◽  
Isabel Cornejo ◽  
Carlos A. Flores ◽  
Francisco V. Sepúlveda ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Atp Depletion ◽  
Functional Evaluation ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Hek 293 ◽  
Channel Expression ◽  
Generalized Epilepsy

The ClC-2 Cl− channel has been postulated to play a role in the inhibitory GABA response in neurons or to participate in astrocyte-dependent extracellular electrolyte homeostasis. Three different mutations in the CLCN2 gene, encoding the voltage-dependent homodimeric ClC-2 channel, have been associated with idiopathic generalized epilepsy (IGE). We study their function in vitro by patch clamp and confocal microscopy in transiently transfected HEK-293 cells. A first mutation predicts a premature stop codon (M200fsX231). An altered splicing, due to an 11-bp deletion in intron 2 (IVS2-14del11), predicts exon 3 skipping (Δ74–117). A third is a missense mutation (G715E). M200fsX231 and Δ74–117 are nonfunctional and do not affect the function of the normal (wild type, WT) channel. Neither M200fsX231 nor Δ74–117 reach the plasma membrane. Concerning the IVS2-14del11 mutation, we find no difference in the proportion of exon-skipped to normally spliced mRNA using a minigene approach and, on this basis, predict no alteration in channel expression in affected individuals. G715E has voltage dependence and intracellular Cl− dependence indistinguishable from WT channels. ClC-2 channels are shown to be sensitive to intracellular replacement of ATP by AMP, which accelerates the opening and closing kinetics. This effect is diminished in the G715E mutant and not significant in WT+G715E coexpression. We do not know whether, in a situation of cellular ATP depletion, this might become pathological in individuals carrying the mutation. We postulate that loss of function mutation M200fsX231 of ClC-2 might contribute to the IGE phenotype through a haploinsufficiency mechanism.

scholarly journals Membrane blebbing as an assessment of functional rescue of dysferlin-deficient human myotubes via nonsense suppression

2010 ◽  
Vol 109 (3) ◽  
pp. 901-905 ◽  
Author(s):  
Bingjing Wang ◽  
Zhaohui Yang ◽  
Becky K. Brisson ◽  
Huisheng Feng ◽  
Zhiqian Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Nonsense Suppression ◽  
Muscle Membrane ◽  
Membrane Repair ◽  
Vitro Assay ◽  
Membrane Blebbing ◽  
Human Myotubes

Mutations that result in the loss of the protein dysferlin result in defective muscle membrane repair and cause either a form of limb girdle muscular dystrophy (type 2B) or Miyoshi myopathy. Most patients are compound heterozygotes, often carrying one allele with a nonsense mutation. Using dysferlin-deficient mouse and human myocytes, we demonstrated that membrane blebbing in skeletal muscle myotubes in response to hypotonic shock requires dysferlin. Based on this, we developed an in vitro assay to assess rescue of dysferlin function in skeletal muscle myotubes. This blebbing assay may be useful for drug discovery/validation for dysferlin deficiency. With this assay, we demonstrate that the nonsense suppression drug, ataluren (PTC124), is able to induce read-through of the premature stop codon in a patient with a R1905X mutation in dysferlin and produce sufficient functional dysferlin (∼15% of normal levels) to rescue myotube membrane blebbing. Thus ataluren is a potential therapeutic for dysferlin-deficient patients harboring nonsense mutations.

scholarly journals SAT-286 TSH Synthesis and Secretion Are Unperturbed in Male IRS4 Knockout Mice

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Emilie Brule ◽  
Anita Boelen ◽  
Daniel J Bernard
Keyword(s):  
Knockout Mice ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Whole Body ◽  
Gene Encoding ◽  
Central Hypothyroidism ◽  
Guide Rna ◽  
Downstream Effectors ◽  
Tsh Secretion ◽  
Hpt Axis

Abstract It was recently reported that mutations in the insulin receptor substrate 4 (IRS4) gene cause a novel form of X-linked congenital central hypothyroidism (OMIM 300904). To date, four different mutations, three frameshift and one nonsense, have been reported, with two affected male patients showing decreased basal, pulsatile, and total thyroid-stimulation hormone (TSH) secretion (PMID 30061370). Members of the IRS family canonically act as scaffold proteins between tyrosine kinase receptors and their downstream effectors. IRS4/Irs4 expression is enriched in the pituitary; however, its role in the hypothalamic-pituitary-thyroid (HPT) axis has not been studied in detail. We generated novel whole-body Irs4-knockout mouse lines using CRISPR-Cas9. A specific guide RNA was used to target the Cas9 enzyme to the 5’ end of the single exon Irs4 gene. A two-nucleotide deletion was introduced into Irs4, resulting in a frameshift and premature stop codon. We hypothesized that like IRS4 deficient patients, these mice would exhibit central hypothyroidism. Given that Irs4 is X-linked, we focused our initial characterization on males. Under normal laboratory conditions, Irs4 knockout mice do not exhibit differences in pituitary expression of Tshb, which encodes one of the subunits of the TSH heterodimer. Expression of the gene encoding the thyrotropin-releasing hormone (TRH) receptor, Trhr1, is also unperturbed in these knockout mice. Additionally, there are no differences in their serum thyroid hormones, T3 (triiodothyronine) and T4 (thyroxine). When Irs4 knockout males were placed on a low-iodine diet supplemented with propylthiouracil (PTU) for 3 weeks and rendered hypothyroid, their serum TSH increased similarly to wild-type males. Overall, Irs4 knockout males do not exhibit central hypothyroidism or phenocopy IRS4 deficient patients. Compensation by another IRS protein may explain euthyroidism in these mice.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

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 Correction of NR2E3 Associated Enhanced S-cone Syndrome Patient-specific iPSCs using CRISPR-Cas9

Genes ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 278 ◽  
Author(s):  
Laura Bohrer ◽  
Luke Wiley ◽  
Erin Burnight ◽  
Jessica Cooke ◽  
Joseph Giacalone ◽  
...  
Keyword(s):  
Photoreceptor Cell ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Model Systems ◽  
Patient Specific ◽  
Rod Photoreceptor ◽  
Cone Photoreceptor ◽  
Retinal Cells ◽  
Enhanced S Cone Syndrome

Enhanced S-cone syndrome (ESCS) is caused by recessive mutations in the photoreceptor cell transcription factor NR2E3. Loss of NR2E3 is characterized by repression of rod photoreceptor cell gene expression, over-expansion of the S-cone photoreceptor cell population, and varying degrees of M- and L-cone photoreceptor cell development. In this study, we developed a CRISPR-based homology-directed repair strategy and corrected two different disease-causing NR2E3 mutations in patient-derived induced pluripotent stem cells (iPSCs) generated from two affected individuals. In addition, one patient’s iPSCs were differentiated into retinal cells and NR2E3 transcription was evaluated in CRISPR corrected and uncorrected clones. The patient’s c.119-2A>C mutation caused the inclusion of a portion of intron 1, the creation of a frame shift, and generation of a premature stop codon. In summary, we used a single set of CRISPR reagents to correct different mutations in iPSCs generated from two individuals with ESCS. In doing so we demonstrate the advantage of using retinal cells derived from affected patients over artificial in vitro model systems when attempting to demonstrate pathophysiologic mechanisms of specific mutations.

scholarly journals Release of Nonstop Ribosomes Is Essential

mBio ◽  
2014 ◽  
Vol 5 (6) ◽  
Author(s):  
Heather A. Feaga ◽  
Patrick H. Viollier ◽  
Kenneth C. Keiler
Keyword(s):  
Messenger Rna ◽  
Caulobacter Crescentus ◽  
Stop Codon ◽  
Selective Advantage ◽  
Gene Encoding ◽  
Content Type ◽  
Bacterial Phyla ◽  
Almost All

ABSTRACTBacterial ribosomes frequently translate to the 3′ end of an mRNA without terminating at a stop codon. Almost all bacteria use the transfer-messenger RNA (tmRNA)-basedtrans-translation pathway to release these “nonstop” ribosomes and maintain protein synthesis capacity.trans-translation is essential in some species, but in others, such asCaulobacter crescentus,trans-translation can be inactivated. To determine whytrans-translation is dispensable inC. crescentus, a Tn-seq screen was used to identify genes that specifically alter growth in cells lackingssrA, the gene encoding tmRNA. One of these genes,CC1214, was essential in ΔssrAcells. Purified CC1214 protein could release nonstop ribosomesin vitro. CC1214 is a homolog of theEscherichia coliArfB protein, and using the CC1214 sequence, ArfB homologs were identified in the majority of bacterial phyla. Most species in whichssrAhas been deleted contain an ArfB homolog, suggesting that release of nonstop ribosomes may be essential in most or all bacteria.IMPORTANCEGenes that are conserved across large phylogenetic distances are expected to confer a selective advantage. The genes required fortrans-translation,ssrAandsmpB, have been found in >99% of sequenced bacterial genomes, suggesting that they are broadly important. However, these genes can be deleted in some species without loss of viability. The identification and characterization ofC. crescentusArfB reveals whytrans-translation is not essential inC. crescentusand suggests that many other bacteria are likely to use ArfB to survive whentrans-translation is compromised.

Loss of cysteine 584 impairs the storage and release, but not the synthesis of von Willebrand factor

2014 ◽  
Vol 112 (12) ◽  
pp. 1159-1166 ◽  
Author(s):  
Viviana Daidone ◽  
Giovanni Barbon ◽  
Elena Pontara ◽  
Grazia Cattini ◽  
Lisa Gallinaro ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Von Willebrand Disease ◽  
Hek293 Cells ◽  
Loss Of Function ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryCysteines play a key part in von Willebrand factor (VWF) dimerisation and polymerisation, and their loss may severely affect VWF structure and function. We report on three patients with type 3 von Willebrand disease carrying the new c.1751G>T missense mutation that induces the substitution of cysteine 584 by phenylalanine (C584F), and the deletion of seven nucleotides in exon 7 (c.729_735del), producing a premature stop codon at position 454 (E244Lfs*211). VWF was almost undetectable in the patients’ plasma and platelets, while a single, poorly represented, oligomer emerged on plasma VWF multimer analysis. No post-DDAVP increase in VWF and factor VIII was observed. Expressing human recombinant C584F-VWF in HEK293T cells showed that C584F-VWF was synthesised and multimerised but not secreted – apart from the first oligomer, which was slightly represented in the conditioned medium, with a pattern similar to the patients’ plasma VWF. The in vitro expression of the E244Lfs*211–VWF revealed a defective synthesis of the mutated VWF, with a behavior typical of loss of function mutations. Cellular trafficking, investigated in HEK293 cells, indicated a normal C584F-VWF content in the endoplasmic reticulum and Golgi apparatus, confirming the synthesis and multimerisation of C584F-VWF. No pseudo-Weibel Palade bodies were demonstrable, however, suggesting that C584F mutation impairs the storage of C584F-VWF. These findings point to cysteine 584 having a role in the release of VWF and its targeting to pseudo-Weibel Palade bodies in vitro, as well as in its storage and release by endothelial cells in vivo.

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.

Sign in / Sign up

Export Citation Format

Share Document