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Lethal Phenotype (lethal + phenotype)
Selected AbstractsNew Approaches for Validation of Lethal Phenotypes and Genetic Reversion in Helicobacter pyloriHELICOBACTER, Issue 1 2001Timothy K. McDaniel Background. Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Materials and Methods. We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results. After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. Conclusions. The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori. [source] Nuclease sensitive element binding protein 1 gene disruption results in early embryonic lethalityJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2006Lin Fan Abstract Nuclease sensitive element binding protein 1 (NSEP1) is a member of the EFIA/NSEP1/YB-1 family of DNA-binding proteins whose members share a cold shock domain; it has also been termed DNA-binding protein B and Y box binding protein-1 because of its recognition of transcriptional regulatory elements. In addition, NSEP1 functions in the translational regulation of renin, ferritin, and interleukin 2 transcripts, and our laboratory has reported that it plays a role in the biosynthesis of selenium-containing proteins. To test the functional importance of NSEP1 in murine embryonic development, we have utilized a clone of ES cells in which the NSEP1 gene had been disrupted by integration of a plasmid gene-trapping vector into the seventh exon. Injection of these cells into C57BL/6 blastocysts resulted in 11 high percentage chimeric mice; crosses to wild type C57BL/6 mice generated 82 F1 agouti mice, indicating germ line transmission of the ES cell clone, but genotyping showed no evidence of the disrupted allele in any of these agouti offspring even though spermatozoa from four of five tested mice contained the targeted allele. Embryos harvested after timed matings of chimeric male mice demonstrated only the wildtype allele in 27 embryos tested at E7.5, E12.5, and E18.5. These results suggest that gene targeting of NSEP1 induces a lethal phenotype in early embryos, due to either haploinsufficiency of NSEP1 or formation of a dominant negative form of the protein. In either case, these data indicate the functional importance of the NSEP1 gene in murine early embryonic development. J. Cell. Biochem. © 2006 Wiley-Liss, Inc. [source] Unique epidermolytic bullous dermatosis with associated lethal cardiomyopathy related to novel desmoplakin mutationsJOURNAL OF CUTANEOUS PATHOLOGY, Issue 5 2009Angeliki Asimaki Background:, Desmoplakin plays a vital role in cell adhesion, linking the transmembrane desmosomal complex to the cytoskeletal network. Clues to the biological significance of desmoplakin have emerged from the embryonic lethal phenotype of null mice and from naturally occurring human desmoplakin mutations, which cause cardiocutaneous phenotypes. Index case:, In this study, we describe a child who presented with the unique constellation of bullous dermatosis, profound plantar keratoderma, alopecia totalis and cardiomyopathy leading to sudden cardiac death at the age of 9 years. Results:, This complex cardiocutaneous phenotype is associated with compound heterozygosity for two novel nonsense desmoplakin mutations. Histological examination of a plantar skin biopsy showed full thickness epidermal acantholysis with superimposed spongiosis, hyperorthokeratosis and focal parakeratosis. Immunohistochemistry and quantitative confocal microscopy showed abnormal tissue distribution and reduced levels of expression for plakoglobin, desmoplakin and connexin 43 at epidermal junctional sites. Conclusions:, Interpretation of the changes in the context of the two mutations provides insight into the mechanism of clinical cell adhesion disease. [source] Trypanosoma brucei pteridine reductase 1 is essential for survival in vitro and for virulence in miceMOLECULAR MICROBIOLOGY, Issue 3 2010Natasha Sienkiewicz Summary Gene knockout and knockdown methods were used to examine essentiality of pteridine reductase (PTR1) in pterin metabolism in the African trypanosome. Attempts to generate PTR1 null mutants in bloodstream form Trypanosoma brucei proved unsuccessful; despite integration of drug selectable markers at the target locus, the gene for PTR1 was either retained at the same locus or elsewhere in the genome. However, RNA interference (RNAi) resulted in complete knockdown of endogenous protein after 48 h, followed by cell death after 4 days. This lethal phenotype was reversed by expression of enzymatically active Leishmania major PTR1 in RNAi lines (oeRNAi) or by addition of tetrahydrobiopterin to cultures. Loss of PTR1 was associated with gross morphological changes due to a defect in cytokinesis, resulting in cells with multiple nuclei and kinetoplasts, as well as multiple detached flagella. Electron microscopy also revealed increased numbers of glycosomes, while immunofluorescence microscopy showed increased and more diffuse staining for glycosomal matrix enzymes, indicative of mis-localisation to the cytosol. Mis-localisation was confirmed by digitonin fractionation experiments. RNAi cell lines were markedly less virulent than wild-type parasites in mice and virulence was restored in the oeRNAi line. Thus, PTR1 may be a drug target for human African trypanosomiasis. [source] Methods in Nutrition Science: Cre/loxP System for Generating Tissue-specific Knockout Mouse ModelsNUTRITION REVIEWS, Issue 6 2004Claudine H. Kos Ph.D. Editor's note: From time to time, we take the opportunity in Nutrition Reviews to highlight a particularly exciting application of sophisticated methodological advances that are relevant to the nutrition research community. In the current issue of Nutrition Reviews, Dr. Claudine Kos has provide a brief review of some of the salient features of the Cre/loxP system for generating tissue-specific knockout mouse models. Hopefully, this review will provide additional background to Dr. George Wolf's Brief Critical Review (page 253) of the use of the Cre/loxP technique by investigators to gain further insight into the function of the peroxysome proliferators-activated receptor-gamma (PPAR-,), as well as promote its further use within experimental nutrition. Alteration of the mouse genome by conventional transgenic and gene-targeted approaches has greatly facilitated studies of gene function. However, a gene alteration expressed in the germ line may cause an embryonic lethal phenotype resulting in no viable mouse to study gene function. Similarly, a gene alteration may exert its effect in multiple different cell and tissue types, creating a complex phenotype in which it is difficult to distinguish direct function in a particular tissue from secondary effects resulting from altered gene function in other tissues. Therefore, methods have been developed to control conditions such as the timing, cell-type, and tissue specificity of gene activation or repression. This brief review provides an overview of the Cre/LoxP system for generating tissue-specific knockout mouse models. [source] Severe perinatal hypophosphatasia due to homozygous deletion of T at nucleotide 1559 in the tissue nonspecific alkaline phosphatase genePRENATAL DIAGNOSIS, Issue 9 2003Hideaki Sawai Abstract Objectives Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of tissue nonspecific alkaline phosphatase (TNSALP) activity. This disorder is caused by various mutations in the TNSALP gene. We report here hypophosphatasia in two siblings, both of them severely affected by the perinatal (lethal) type. Methods We diagnosed the first infant by clinical and radiologic manifestations, and laboratory findings. Laboratory findings were characterized by deficiency of serum alkaline phosphatase. Both parents and the second infant were then analyzed by molecular techniques. Results The radiograph of the first infant showed severe hypomineralization of the skeleton. Molecular analysis of the second infant showed that this condition was caused by a homozygous single T nucleotide deletion at cDNA number 1559 (1559delT). Both parents were heterozygous carriers for this mutation, although they were not consanguineous. Conclusion This mutation has been frequently found in Japanese hypophosphatasia patients, but this is the first observation of a homozygous deletion. This report shows that homozygosity for the 1559delT mutation of the TNSALP gene results in a severe lethal phenotype. Copyright © 2003 John Wiley & Sons, Ltd. [source] The maize viviparous15 locus encodes the molybdopterin synthase small subunitTHE PLANT JOURNAL, Issue 2 2006Masaharu Suzuki Summary A new Zea mays viviparous seed mutant, viviparous15 (vp15), was isolated from the UniformMu transposon-tagging population. In addition to precocious germination, vp15 has an early seedling lethal phenotype. Biochemical analysis showed reduced activities of several enzymes that require molybdenum cofactor (MoCo) in vp15 mutant seedlings. Because MoCo is required for abscisic acid (ABA) biosynthesis, the viviparous phenotype is probably caused by ABA deficiency. We cloned the vp15 mutant using a novel high-throughput strategy for analysis of high-copy Mu lines: We used MuTAIL PCR to extract genomic sequences flanking the Mu transposons in the vp15 line. The Mu insertions specific to the vp15 line were identified by in silico subtraction using a database of MuTAIL sequences from 90 UniformMu lines. Annotation of the vp15 -specific sequences revealed a Mu insertion in a gene homologous to human MOCS2A, the small subunit of molybdopterin (MPT) synthase. Molecular analysis of two allelic mutations confirmed that Vp15 encodes a plant MPT synthase small subunit (ZmCNX7). Our results, and a related paper reporting the cloning of maize viviparous10, demonstrate robust cloning strategies based on MuTAIL-PCR. The Vp15/CNX7, together with other CNX genes, is expressed in both embryo and endosperm during seed maturation. Expression of Vp15 appears to be regulated independently of MoCo biosynthesis. Comparisons of Vp15 loci in genomes of three cereals and Arabidopsis thaliana identified a conserved sequence element in the 5, untranslated region as well as a micro-synteny among the cereals. [source] Regulation of signal transduction by glycosylationINTERNATIONAL JOURNAL OF EXPERIMENTAL PATHOLOGY, Issue 4 2004Robert S. Haltiwanger The incredible diversity found in cell-surface glycoconjugate structures led researchers over 30 years ago to propose that complexity in carbohydrates must play a role in cellular communication. Recent studies from a number of laboratories have confirmed this hypothesis, demonstrating that cell-surface glycoconjugates play significant roles in signal transduction events. One striking example is the effect of O -fucose modifications on the Notch-signalling pathway. Notch is a cell-surface receptor that is essential for proper development. The extracellular domain of Notch contains up to 36-tandem epidermal growth factor-like (EGF) repeats, many of which are predicted to be modified at putative consensus sequences with O -fucose and O -glucose saccharides. Genetic alterations (by knockout or RNAi methodologies) in the enzyme responsible for the addition of O -fucose to Notch, protein O -fucosyltransferase-1, result in severe, embryonic lethal phenotypes resembling Notch mutants. Thus, O -fucosylation appears to be essential for proper Notch function. Elongation of the O -fucose monosaccharide by the ,1,3- N -acetylglucosaminyltransferase, Fringe, modulates Notch function, either increasing or decreasing response from ligands depending on context. Although it is now clear that O -fucose modifications affect Notch signalling, the molecular mechanism by which this occurs is not known. As an initial step in understanding how O -fucose glycans affect Notch function, we are mapping O -fucose modifications to specific sites on Notch. Already, we have demonstrated that O -fucose modifies one of the EGF repeats involved in ligand binding, suggesting that the sugars may play a role in Notch,ligand interactions. Experiments to test the role of O -fucose modifications at specific sites are in progress. We have also found that Fringe modifies O -fucose on some EGF repeats but not others. Our initial analyses suggest that the basis of this specificity is encoded within the sequences of the individual EGF repeats. Site mapping has also confirmed the presence of O -glucose saccharides on Notch. The evolutionarily conserved, predicted O -glucose sites on Notch are as numerous as those for O -fucose, suggesting that the O -glucose modifications will play an equally important role in Notch biology. We have recently identified an enzymatic activity capable of catalyzing the addition of O -glucose to EGF repeats. Purification of the protein O -glucosyltransferase is underway. These and other results will be discussed. [source] The maize Viviparous10/Viviparous13 locus encodes the Cnx1 gene required for molybdenum cofactor biosynthesisTHE PLANT JOURNAL, Issue 2 2006Timothy G. Porch Summary Abscisic acid (ABA), auxin and nitrate are important signaling molecules that affect plant growth responses to the environment. The synthesis or metabolism of these compounds depends on the molybdenum cofactor (MoCo). We show that maize (Zea mays) viviparous10 (vp10) mutants have strong precocious germination and seedling lethal phenotypes that cannot be rescued with tissue culture. We devised a novel PCR-based method to clone a transposon-tagged allele of vp10, and show that Vp10 encodes the ortholog of Cnx1, which catalyzes the final common step of MoCo synthesis. The seedling phenotype of vp10 mutants is consistent with disruptions in ABA and auxin biosynthesis, as well as a disruption in nitrate metabolism. Levels of ABA and auxin are reduced in vp10 mutants, and vp10 seedlings lack MoCo-dependent enzyme activities that are repairable with exogenous molybdenum. vp10 and an Arabidopsis cnx1 mutant, chlorate6 (chl6), have similar defects in aldehyde oxidase (AO) enzyme activity, which is required for ABA synthesis. Surprisingly, chl6 mutants do not show defects in abiotic stress responses. These observations confirm an orthologous function for Cnx1 and Vp10, as well as defining a characteristic viviparous phenotype to identify other maize cnx mutants. Finally, the vp10 mutant phenotype suggests that cnx mutants can have auxin- as well as ABA-biosynthesis defects, while the chl6 mutant phenotype suggests that low levels of AO activity are sufficient for normal abiotic stress responses. [source] Transactivation of BARNASE under the AtLTP1 promoter affects the basal pole of the embryo and shoot development of the adult plant in ArabidopsisTHE PLANT JOURNAL, Issue 5 2001Célia Baroux Summary Genetically controlled expression of a toxin provides a tool to remove a specific structure and consequently study its role during a developmental process. The availability of many tissue-specific promoters is a good argument for the development of such a strategy in plants. We have developed a conditional system for targeted toxin expression and demonstrated its use for generating embryo phenotypes that can bring valuable information about signalling during embryogenesis. The BARNASE gene was expressed in the Arabidopsis embryo under the control of two promoters, one from the cyclin AtCYCB1 gene and one from the AtLTP1 gene (LipidTransferProtein 1). One-hundred percent seed abortion was obtained with the cyclin promoter. Surprisingly however, the embryos displayed a range of lethal phenotypes instead of a single arrested stage as expected from this promoter. We also show that BARNASE expression under the control of the AtLTP1 promoter affects the basal pole of the globular embryo. Together with reporter expression studies, this result suggests a role of the epidermis in controlling the development of the lower tier of the embryo. This defect was not embryo-lethal and we show that the seedlings displayed a severe shoot phenotype correlated to epidermal defects. Therefore, the epidermis does not play an active role during organogenesis in seedlings but is important for the postgermination development of a viable plant. [source] | |||||||||||||