Mouse Dspp frameshift model of human dentinogenesis imperfecta

AbstractNon-syndromic inherited defects of tooth dentin are caused by two classes of dominant negative/gain-of-function mutations in dentin sialophosphoprotein (DSPP): 5′ mutations affecting an N-terminal targeting sequence and 3′ mutations that shift translation into the − 1 reading frame. DSPP defects cause an overlapping spectrum of phenotypes classified as dentin dysplasia type II and dentinogenesis imperfecta types II and III. Using CRISPR/Cas9, we generated a Dspp−1fs mouse model by introducing a FLAG-tag followed by a single nucleotide deletion that translated 493 extraneous amino acids before termination. Developing incisors and/or molars from this mouse and a DsppP19L mouse were characterized by morphological assessment, bSEM, nanohardness testing, histological analysis, in situ hybridization and immunohistochemistry. DsppP19L dentin contained dentinal tubules but grew slowly and was softer and less mineralized than the wild-type. DsppP19L incisor enamel was softer than normal, while molar enamel showed reduced rod/interrod definition. Dspp−1fs dentin formation was analogous to reparative dentin: it lacked dentinal tubules, contained cellular debris, and was significantly softer and thinner than Dspp+/+ and DsppP19L dentin. The Dspp−1fs incisor enamel appeared normal and was comparable to the wild-type in hardness. We conclude that 5′ and 3′ Dspp mutations cause dental malformations through different pathological mechanisms and can be regarded as distinct disorders.

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Causal Role of Single Nucleotide Polymorphisms within themprFGene of Staphylococcus aureus in Daptomycin Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01184-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5658-5664 ◽

Cited By ~ 48

Author(s):

Soo-Jin Yang ◽

Nagendra N. Mishra ◽

Aileen Rubio ◽

Arnold S. Bayer

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Point Mutations ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Single Nucleotide ◽

Content Type ◽

Reading Frame ◽

A Charge

ABSTRACTSingle nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been commonly observed in daptomycin-resistant (DAPr)Staphylococcus aureusstrains. Such SNPs are usually associated with a gain-in-function phenotype, in terms of either increased synthesis or enhanced translocation (flipping) of lysyl-phosphatidylglycerol (L-PG). However, it is unclear if suchmprFSNPs are causal in DAPrstrains or are merely a biomarker for this phenotype. In this study, we used an isogenic set ofS. aureusstrains: (i) Newman, (ii) its isogenic ΔmprFmutant, and (iii) several intransplasmid complementation constructs, expressing either a wild-type or point-mutated form of themprFORF cloned from two isogenic DAP-susceptible (DAPs)-DAPrstrain pairs (616-701 and MRSA11/11-REF2145). Complementation of the ΔmprFstrain with singly point-mutatedmprFgenes (mprFS295LormprFT345A) revealed that (i) individual and distinct point mutations within themprFORF can recapitulate phenotypes observed in donor strains (i.e., changes in DAP MICs, positive surface charge, and cell membrane phospholipid profiles) and (ii) these gain-in-function SNPs (i.e., enhanced L-PG synthesis) likely promote reduced DAP binding toS. aureusby a charge repulsion mechanism. Thus, for these two DAPrstrains, the definedmprFSNPs appear to be causally related to this phenotype.

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High-fidelity amplified FISH for the detection and allelic discrimination of single mRNA molecules

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1814463116 ◽

2019 ◽

Vol 116 (28) ◽

pp. 13921-13926 ◽

Cited By ~ 10

Author(s):

Salvatore A. E. Marras ◽

Yuri Bushkin ◽

Sanjay Tyagi

Keyword(s):

Single Molecule ◽

Hybridization Chain Reaction ◽

Wild Type ◽

Allelic Discrimination ◽

Single Nucleotide ◽

Tissue Sections ◽

Powerful Approach ◽

Probe System ◽

Single Nucleotide Variations

Amplification of signals by the hybridization chain reaction (HCR) is a powerful approach for increasing signal strength in single-molecule fluorescence in situ hybridization, but probes tagged with an HCR initiator sequence are prone to producing false signals. Here we describe a system of interacting hairpin binary probes in which the HCR initiator sequence is conditionally sequestered. The binding of these probes to a perfectly complementary target unmasks the initiator, enabling the generation of an amplified signal. This probe system can distinguish single-nucleotide variations within single mRNA molecules and produces amplified signals in situ for both mutant and wild-type variants, each in a distinguishable color. This technology will augment studies of imbalanced allelic expression and will be useful for the detection of somatic mutations in cancer biopsies. By tiling these probes along the length of an mRNA target, enhanced signals can be obtained, thereby enabling the scanning of tissue sections for gene expression utilizing lower magnification microscopy, overcoming tissue autofluorescence, and allowing the detection of low-abundance biomarkers in flow cytometry.

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Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.3.c596 ◽

2000 ◽

Vol 279 (3) ◽

pp. C596-C602 ◽

Cited By ~ 47

Author(s):

Jay D. Pal ◽

Xiaoqin Liu ◽

Donna Mackay ◽

Alan Shiels ◽

Viviana M. Berthoud ◽

...

Keyword(s):

Xenopus Oocytes ◽

Dominant Negative ◽

Metabolic Labeling ◽

Loss Of Function ◽

Wild Type ◽

Reading Frame ◽

Gap Junction Channel ◽

Intercellular Channels ◽

Normal Lens ◽

Gap Junctional

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.

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Mutations in SIX1 associated with Branchio-oto-renal Syndrome (BOR) differentially affect otic expression of putative target genes

10.1101/2021.06.11.447956 ◽

2021 ◽

Author(s):

Tanya Mehdizadeh ◽

Himani Datta Majumdar ◽

Sahra Ahsan ◽

Andre Luiz Pasqua Tavares ◽

Sally A Moody

Keyword(s):

Gene Expression ◽

Target Genes ◽

Otic Vesicle ◽

Wild Type ◽

Single Nucleotide ◽

Putative Target ◽

Individual Effects ◽

Factor Interactions ◽

Ability To Drive

Single nucleotide mutations in SIX1 are causative in some individuals diagnosed with branchiootic/branchio-oto-renal (BOR) syndrome. To test whether these mutations have differential effects on otic gene expression, we engineered four BOR mutations in Xenopus six1 and targeted mutant protein expression to the neural crest and cranial placode precursor cells in wild-type embryos. Changes in the otic expression of putative Six1 targets and/or co-factors were monitored by qRT-PCR and in situ hybridization. We found that each mutant had a different combination of effects. The V17E mutant reduced eya2, tspan13, zbtb16 and pa2g4 otic vesicle expression at a frequency indistinguishable from wildtype Six1, but reduced prdm1 more and spry1 less compared to wild-type Six1. For most of these genes, the R110W, W122R and Y129C mutants were significantly less repressive compared to wild-type Six1. Their individual effects varied according to the level at which they were expressed. The R110W, W122R and Y129C mutants also often expanded prdm1 otic expression. Since previous studies showed that all four mutants are transcriptionally deficient and differ in their ability to interact with co-factors such as Eya1, we propose that altered co-factor interactions at the mutated sites differentially interfere with their ability to drive otic gene expression.

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Frequency of Spontaneous Mutations in an Avian Hepadnavirus Infection

Journal of Virology ◽

10.1128/jvi.75.20.9623-9632.2001 ◽

2001 ◽

Vol 75 (20) ◽

pp. 9623-9632 ◽

Cited By ~ 23

Author(s):

Irmgard Pult ◽

Nathan Abbott ◽

Yong-Yuan Zhang ◽

Jesse Summers

Keyword(s):

Nucleotide Substitution ◽

Acute Liver Injury ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Single Nucleotide ◽

Reading Frame ◽

Duck Hepatitis ◽

B Virus ◽

Infected Cells

ABSTRACT In this study, we measured the frequency of revertants of a cytopathic strain of the duck hepatitis B virus that bears a single nucleotide substitution in the pre-S envelope protein open reading frame, resulting in the amino acid substitution G133E. Cytopathic virus mixed with known amounts of a genetically marked wild-type virus was injected into ducklings. Virus outgrowth was accompanied by a coselection of wild-type and spontaneous revertants during recovery of the ducklings from the acute liver injury caused by death of the G133E-infected cells. The frequency of individual revertants in the selected noncytopathic virus population was estimated by determining the ratio of each revertant to the wild-type virus. Spontaneous revertants were found to be present at frequencies of 1 × 10−5 to 6 × 10−5 per G133E genome inoculated. A mathematical model was used to estimate that the mutation rate was 0.8 × 10−5 to 4.5 × 10−5per nucleotide per generation.

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Mismatch repair hierarchy of Pseudomonas putida revealed by mutagenic ssDNA recombineering of the pyrF gene

10.1101/710293 ◽

2019 ◽

Author(s):

Tomas Aparicio ◽

Akos Nyerges ◽

István Nagy ◽

Csaba Pal ◽

Esteban Martínez-García ◽

...

Keyword(s):

Pseudomonas Putida ◽

Mismatch Repair ◽

Dominant Negative ◽

Wild Type ◽

Single Nucleotide ◽

Important Species ◽

E Coli ◽

Different Types ◽

Nucleotide Mismatch ◽

The One

SUMMARYThe mismatch repair (MMR) system is one of the key molecular devices that prokaryotic cells have for ensuring fidelity of DNA replication. While the canonical MMR of E. coli involves 3 proteins (encoded by mutS, mutL and mutH), the soil bacterium Pseudomonads putida has only 2 bona fide homologues (mutS and mutL) and the sensitivity of this abridged system to different types of mismatches is unknown. On this background, sensitivity to MMR of this bacterium was inspected through single stranded (ss) DNA recombineering of the pyrF gene (the prokaryotic equivalent to yeast’s URA3) with mutagenic oligos representative of every possible mispairing under either wild-type conditions, permanent deletion of mutS or transient loss of mutL activity (brought about by the thermoinducible dominant negative allele mutLE36K). Analysis of single nucleotide mutations borne by clones resistant to fluoroorotic acid (5FOA, the target of wild type PyrF) pinpointed prohibited and tolerated single-nucleotide replacements and exposed a clear grading of mismatch recognition. The resulting data unequivocally established the hierarchy A:G< C:C< G:A< C:A, A:A, G:G, T:T, T:G, A:C, C:T< G:T, T:C as the one prevalent in Pseudomonas putida. This information was vital for enabling recombineering strategies aimed at single-nucleotide changes in this biotechnologically important species.Originality-Significance StatementSingle-stranded DNA (ssDNA) recombineering has emerged in recent years as one of the most powerful technologies of genome editing in E. coli and other Enterobacteria. However, the efforts to expand the concept and the methods towards environmental microorganisms such as Pseudomonas putida have been limited thus far by several gaps in our fundamental knowledge of how nucleotide mismatch repair (MMR) operates in such non-model species. One critical bottleneck is the hierarchy of recognition of different types of base mispairings as well as the need of setting up strategies for counteracting MMR and thus enabling tolerance to all types of changes. The work presented here tackles both issues and makes P. putida amenable to sophisticated genetic manipulations that were impossible before.

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Poxviral Regulation of the Host NF-κB Response: the Vaccinia Virus M2L Protein Inhibits Induction of NF-κB Activation via an ERK2 Pathway in Virus-Infected Human Embryonic Kidney Cells

Journal of Virology ◽

10.1128/jvi.00935-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8676-8685 ◽

Cited By ~ 73

Author(s):

Roderick Gedey ◽

Xiao-Lu Jin ◽

Olivia Hinthong ◽

Joanna L. Shisler

Keyword(s):

Vaccinia Virus ◽

Nuclear Translocation ◽

Dominant Negative ◽

Dominant Negative Mutant ◽

Wild Type ◽

Cell Nuclei ◽

Human Embryonic Kidney Cells ◽

Reading Frame ◽

Regulated Gene Expression ◽

In Cells

ABSTRACT Exposure of eukaryotic cells to viruses will activate the host NF-κB transcription factor, resulting in proinflammatory and immune protein production. Vaccinia virus (VV), the prototypic orthopoxvirus, expresses products that inhibit this antiviral event. To identify novel mechanisms responsible for this effect, we made use of a VV deletion mutant (MVA) that stimulates NF-κΒ activation in infected 293T cells. In this virus-host system, the extents of NF-κΒ-regulated gene expression and nuclear translocation were reduced in the presence of either PD 98059 or U0126, two compounds capable of blocking ERK1 and ERK2 phosphorylation. A similar repression was also observed in cells that contained a dominant, nonactive form of ERK2 but not in cells where ERK1 phosphorylation was inhibited via overexpression of a dominant-negative mutant MEK1 protein. Presumably, proteins expressed from a wild-type VV that block ERK2 activity would also inhibit MVA-induced NF-κB activation. Indeed, the expression of one such open reading frame, M2L, supported this prediction. First, ectopic M2L expression hampered ERK2 phosphorylation induced by exposure to phorbol myristate acetate. Second, viral M2L expression via infection of cells with a recombinant MVA construct that stably expressed M2L decreased the phosphorylation of ERK2 compared to that in cells infected with the parental MVA strain. Finally, the recombinant M2L-expressing virus restored the “wild-type” NF-κB-inhibitory phenotype, as indicated by decreased NF-κB migration to infected cell nuclei and interference in transcription. Thus, in 293T cells, VV apparently utilizes its M2L protein to interfere with a step(s) that would otherwise enable ERK2 phosphorylation and the consequential activation of an NF-κΒ response.

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Amplification-free in situ KRAS point mutation detection at 60 copies per mL in urine in a background of 1000-fold wild type

The Analyst ◽

10.1039/c5an02048d ◽

2016 ◽

Vol 141 (4) ◽

pp. 1421-1433 ◽

Cited By ~ 6

Author(s):

Ceyhun E. Kirimli ◽

Wei-Heng Shih ◽

Wan Y. Shih

Keyword(s):

Nucleic Acid ◽

Point Mutation ◽

Mutation Detection ◽

Piezoelectric Plate ◽

Locked Nucleic Acid ◽

Wild Type ◽

Single Nucleotide ◽

Lna Probe

We have examined thein situdetection of a single-nucleotideKRASmutation in urine using a (Pb(Mg1/3Nb2/3)O3)0.65(PbTiO3)0.35(PMN-PT) piezoelectric plate sensor (PEPS) coated with a 17-nucleotide (nt) locked nucleic acid (LNA) probe DNA complementary to theKRASmutation.

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The Role of Alcohol, LPS Toxicity, and ALDH2 in Dental Bony Defects

Biomolecules ◽

10.3390/biom11050651 ◽

2021 ◽

Vol 11 (5) ◽

pp. 651

Author(s):

Hsiao-Cheng Tsai ◽

Che-Hong Chen ◽

Daria Mochly-Rosen ◽

Yi-Chen Ethan Li ◽

Min-Huey Chen

Keyword(s):

Bone Loss ◽

Bacterial Infection ◽

Bone Regeneration ◽

Alcohol Drinking ◽

Bacterial Species ◽

Dominant Negative ◽

Enzyme Inactivation ◽

Wild Type ◽

Micro Computed Tomography ◽

Dominant Negative Mutation

It is estimated that 560 million people carry an East Asian-specific ALDH2*2 dominant-negative mutation which leads to enzyme inactivation. This common ALDH2 polymorphism has a significant association with osteoporosis. We hypothesized that the ALDH2*2 mutation in conjunction with periodontal Porphyromonas gingivalis bacterial infection and alcohol drinking had an inhibitory effect on osteoblasts and bone regeneration. We examined the prospective association of ALDH2 activity with the proliferation and mineralization potential of human osteoblasts in vitro. The ALDH2 knockdown experiments showed that the ALDH2 knockdown osteoblasts lost their proliferation and mineralization capability. To mimic dental bacterial infection, we compared the dental bony defects in wild-type mice and ALDH2*2 knockin mice after injection with purified lipopolysaccharides (LPS), derived from P. gingivalis which is a bacterial species known to cause periodontitis. Micro-computed tomography (micro-CT) scan results indicated that bone regeneration was significantly affected in the ALDH2*2 knockin mice with about 20% more dental bony defects after LPS injection than the wild-type mice. Moreover, the ALDH2*2 knockin mutant mice had decreased osteoblast growth and more dental bone loss in the upper left jaw region after LPS injection. In conclusion, these results indicated that the ALDH2*2 mutation with alcohol drinking and chronic exposure to dental bacterial-derived toxin increased the risk of dental bone loss.

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Identification of a novel homozygous loss-of-function mutation in FUCA1 gene causing severe fucosidosis: A case report

Journal of International Medical Research ◽

10.1177/03000605211005975 ◽

2021 ◽

Vol 49 (4) ◽

pp. 030006052110059

Author(s):

Xinwen Zhang ◽

Shaozhi Zhao ◽

Hongwei Liu ◽

Xiaoyan Wang ◽

Xiaolei Wang ◽

...

Keyword(s):

Amino Acids ◽

Lysosomal Storage Disorder ◽

Autosomal Recessive ◽

Signs And Symptoms ◽

Lysosomal Storage ◽

Loss Of Function ◽

Wild Type ◽

Single Nucleotide ◽

Whole Exome ◽

Exon 1

Fucosidosis is a rare lysosomal storage disorder characterized by deficiency of α-L-fucosidase with an autosomal recessive mode of inheritance. Here, we describe a 4-year-old Chinese boy with signs and symptoms of fucosidosis but his parents were phenotypically normal. Whole exome sequencing (WES) identified a novel homozygous single nucleotide deletion (c.82delG) in the exon 1 of the FUCA1 gene. This mutation will lead to a frameshift which will result in the formation of a truncated FUCA1 protein (p.Val28Cysfs*105) of 132 amino acids approximately one-third the size of the wild type FUCA1 protein (466 amino acids). Both parents were carrying the mutation in a heterozygous state. This study expands the mutational spectrum of the FUCA1 gene associated with fucosidosis and emphasises the benefits of WES for accurate and timely clinical diagnosis of this rare disease.

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