A new Escherichia coli gene,fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

A self-compatible mutant S allele conferring a dominant negative effect on the functional S allele in Ipomoea trifida

A spontaneously occurring self-compatible mutant has been identified in Ipomoea trifida, a species possessing sporophytic self-incompatibility controlled by a single multiallelic S locus. Analysis of the segregation of compatibility/incompatibility phenotypes in selfed and crossed progenies of the self-compatible mutant plant indicated that the self-compatibility trait was caused by a mutation at the S locus; the mutated S allele was therefore designated Sc. RFLP analysis of progeny plants segregating for the Sc allele using the SSP gene (a gene linked closely to the S locus of I. trifida) as a probe confirmed that the mutation was present at the S locus. Self-incompatibility responses were examined in F₁ progenies obtained from crosses between the self-compatible mutant and self-incompatible plants homozygous for one of three S alleles, S ₁ , S ₃ and S ₂₂ , where the dominance relationship is S ₂₂ >S ₁ >S ₃ . All F₁ progeny plants from crosses with S ₂₂ and S ₁ homozygotes were self-incompatible and exhibited the respective phenotypes of each self-incompatible parent (either S ₂₂ or S ₁ ) in both stigma and pollen. However, of the F₁ progeny plants from the cross with the S ₃ homozygote, those carrying the genotype ScS ₃ were all self-compatible and cross-compatible as both female and male parents with the S ₃ homozygote. These results indicate that the dominance relationship between the four S alleles is: S ₂₂ >S ₁ >Sc>S ₃ and so reveal the unexpected finding that the mutated Sc allele is dominant over a functional S ₃ allele. A possible explanation for this observation is that the gene product encoded by the Sc allele confers a dominant negative effect on the S ₃ gene product. Received: 21 June 2000 / Accepted: 18 July 2000     

PanR1, a dominant negative missense allele of the gene encoding TNF-alpha (Tnf), does not impair lymphoid development

A dominant hypomorphic allele of Tnf, PanR1, was identified in a population of G(1) mice born to N-ethyl-N-nitrosourea-mutagenized sires. Macrophages from homozygotes produced no detectable TNF bioactivity, although normal quantities of immunoreactive TNF were secreted. The phenotype was confined to a critical region on mouse chromosome 17, and then ascribed to a C-->A transversion at position 3480 of the Tnf gene, corresponding to the amino acid substitution P138T. As a result of subunit exchange, the protein exerts a dominant-negative effect on normal TNF trimers, interfering with the trimer/receptor interaction. Homozygotes are highly susceptible to infection by Listeria monocytogenes, confirming the essential role of TNF in innate immune defense. However, PanR1 mutant mice show normal architecture of the spleen and Peyer's patches, suggesting that TNF is not essential for the formation of these lymphoid structures.     

A new Escherichia coli gene, fdrA, identified by suppression analysis of dominant negative FtsH mutations

An Escherichia coli membrane protein, FtsH, has been implicated in several cellular processes, including integration of membrane proteins, translocation of secreted proteins, and degradation of some unstable proteins. However, how it takes part in such diverse cellular events is largely unknown. We previously isolated dominant negative ftsH mutations and proposed that FtsH functions in association with some other cellular factor(s). To test this proposal we isolated multicopy suppressors of dominant negative ftsH mutations. One of the multicopy suppressor clones contained an N-terminally truncated version of a new gene that was designated fdrA. The FdrA fragment suppressed both of the phenotypes — increased abnormal translocation of a normally cytoplasmic domain of a model membrane protein and retardation of protein export — caused by dominant negative FtsH proteins. The intact fdrA gene (11.9 min on the chromosome) directed the synthesis of a 60 kDa protein in vitro.     

Use of a dominant rpsL allele conferring streptomycin dependence for positive and negative selection in Thermus thermophilus

A spontaneous rpsL mutant of Thermus thermophilus was isolated in a search for new selection markers for this organism. This new allele, named rpsL1, encodes a K47R/K57E double mutant S12 ribosomal protein that confers a streptomycin-dependent (SD) phenotype to T. thermophilus. Models built on the available three-dimensional structures of the 30S ribosomal subunit revealed that the K47R mutation directly affects the streptomycin binding site on S12, whereas the K57E does not apparently affect this binding site. Either of the two mutations conferred the SD phenotype individually. The presence of the rpsL1 allele, either as a single copy inserted into the chromosome as part of suicide plasmids or in multicopy as replicative plasmids, produced a dominant SD phenotype despite the presence of a wild-type rpsL gene in a host strain. This dominant character allowed us to use the rpsL1 allele not only for positive selection of plasmids to complement a kanamycin-resistant mutant strain, but also more specifically for the isolation of deletion mutants through a single step of negative selection on streptomycin-free growth medium.     

Inhibition of anchorage-independent cell growth, adhesion, and cyclin D1 gene expression by a dominant negative mutant of a tyrosine phosphatase

PTP-S4/TC48 protein tyrosine phosphatase is localized in the nuclear and cytoplasmic membranes. To investigate the role of PTP-S4 in cell growth, adhesion, and transformation, normal and a catalytically inactive mutant form of this phosphatase were expressed in polyoma virus-transformed F111 fibroblast cell line, PyF. Expression of mutant PTP-S4 in PyF cells resulted in strong inhibition of anchorage-independent growth in soft agar but had no significant effect on growth in liquid culture. Tumor formation in nude mice was also reduced by mutant PTP-S4. Expression of normal PTP-S4 in PyF cells significantly increased anchorage-independent cell growth and tumor formation in nude mice. Overexpression of catalytically inactive mutant of PTP-S2/TC45 (a splice variant of PTP-S4 that is nuclear) did not inhibit anchorage-independent growth of PyF cells. Mutant PTP-S4-expressing cells were inhibited in adhesion and spreading on tissue culture plates compared to control cells. Expression of mutant PTP-S4 in PyF cells reduced the levels of cyclin D1 and cyclin A mRNA, whereas cyclin D2 mRNA level was not affected significantly. Expression of antisense cyclin D1 strongly inhibited anchorage-independent growth. Inhibition of anchorage-independent growth by mutant PTP-S4 was overcome to a large extent by coexpression of cyclin D1. These results suggest that mutant PTP-S4 inhibits anchorage-independent growth and adhesion of polyoma virus-transformed cells by interfering with the normal function of PTP-S4 upstream of cyclin D1 gene expression.     

A dominant, recombination-defective allele of Dmc1 causing male-specific sterility

DMC1 is a meiosis-specific homolog of bacterial RecA and eukaryotic RAD51 that can catalyze homologous DNA strand invasion and D-loop formation in vitro. DMC1-deficient mice and yeast are sterile due to defective meiotic recombination and chromosome synapsis. The authors identified a male dominant sterile allele of Dmc1, Dmc1^Mei11, encoding a missense mutation in the L2 DNA binding domain that abolishes strand invasion activity. Meiosis in male heterozygotes arrests in pachynema, characterized by incomplete chromosome synapsis and no crossing-over. Young heterozygous females have normal litter sizes despite having a decreased oocyte pool, a high incidence of meiosis I abnormalities, and susceptibility to premature ovarian failure. Dmc1^Mei11 exposes a sex difference in recombination in that a significant portion of female oocytes can compensate for DMC1 deficiency to undergo crossing-over and complete gametogenesis. Importantly, these data demonstrate that dominant alleles of meiosis genes can arise and propagate in populations, causing infertility and other reproductive consequences due to meiotic prophase I defects.     

Identification and comparative mapping of a dominant potyvirus resistance gene cluster in Capsicum

The dominant gene Pvr7 from Capsicum chinense Jacq. ’PI159236’ confers resistance to the pepper mottle potyvirus (PepMoV) Florida (V1182) strain. This gene is tightly linked to the dominant potyvirus resistance gene Pvr4 with observed recombination frequencies of 0.012 to 0.016. A cleaved amplified polymorphic sequence (CAPS) marker linked to Pvr4 was used to localize Pvr4 and, by extension, Pvr7, to linkage group 10 on an interspecific map of pepper. Our results indicated that Pvr4, Pvr7, and Tsw, a gene conferring resistance to tomato spotted wilt virus, comprise the first identified cluster of dominant disease resistance genes in Capsicum L. This position does not correspond to the locations of dominant potyvirus resistance genes in potato or to the positions of any other mapped solanaceous resistance genes or resistance gene homologues. Received: 20 September 1999 / Accepted: 21 March 2000     

Ligand-independent oligomerization of natriuretic peptide receptors. Identification of heteromeric receptors and a dominant negative mutant.

Activation of many single-transmembrane receptors requires ligand-induced receptor oligomerization. We have examined the oligomerization of the atrial natriuretic peptide receptor, NPR-A, using epitope-tagged receptor in a co-immunoprecipitation assay. Unlike other single-transmembrane receptors, NPR-A oligomerized in a ligand-independent fashion. Extracellular receptor sequences were both necessary and sufficient for oligomer formation. NPR-A was also able to oligomerize with the related natriuretic peptide receptor, NPR-B. A truncated NPR-A lacking most of the cytoplasmic domain blocked activation of the full-length receptor, presumably through formation of an inactive heteromer. These results indicate that oligomerization of this single-transmembrane receptor is important for the transduction of a conformational change across the plasma membrane but are not consistent with models in which natriuretic peptide receptor oligomerization serves merely to bring intracellular domains together.     

Identification and analysis of dominant negative mutants of RAIDD and PIDD

Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.     

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.     

Improving gene set analysis of microarray data by SAM-GS

Background  

Gene-set analysis evaluates the expression of biological pathways, or a priori defined gene sets, rather than that of individual genes, in association with a binary phenotype, and is of great biologic interest in many DNA microarray studies. Gene Set Enrichment Analysis (GSEA) has been applied widely as a tool for gene-set analyses. We describe here some critical problems with GSEA and propose an alternative method by extending the individual-gene analysis method, Significance Analysis of Microarray (SAM), to gene-set analyses (SAM-GS).     

A multivariate extension of the gene set enrichment analysis

A test-statistic typically employed in the gene set enrichment analysis (GSEA) prevents this method from being genuinely multivariate. In particular, this statistic is insensitive to changes in the correlation structure of the gene sets of interest. The present paper considers the utility of an alternative test-statistic in designing the confirmatory component of the GSEA. This statistic is based on a pertinent distance between joint distributions of expression levels of genes included in the set of interest. The null distribution of the proposed test-statistic, known as the multivariate N-statistic, is obtained by permuting group labels. Our simulation studies and analysis of biological data confirm the conjecture that the N-statistic is a much better choice for multivariate significance testing within the framework of the GSEA. We also discuss some other aspects of the GSEA paradigm and suggest new avenues for future research.     

Viscumin modulates neutrophil respiratory burst, caused by a chemotactic peptide

The ability of viscum at different concentrations to modulate the respiratory burst in neutrophils, induced by the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine was studied. This does not exclude the possibility that viscum can interact with the receptor of this peptide. The analysis of the primary structure of viscum revealed elements structurally analogous to the chemotactic peptide. It is assumed that viscum can exhibit the properties an antagonist of the receptor of N-formyl-methionyl-leucyl-phenylalanine, and the mechanism of action of viscum depends on its concentration.     

A new allele of the lurcher gene, lurcher

A new neurological mouse mutation that arose spontaneously in a BALB/cByJ stock displays a semidominant pattern of inheritance. In the heterozygote, this mutation results in an early loss of Purkinje cells in the cerebellum, which is followed by the overt symptom of an ataxic gait first observed at postnatal day 13 (P13). A portion of animals homozygous for the mutation die within P0; the remaining homozygotes die by P25. The mutation maps to mouse Chromosome (Chr) 6 between markers D6Rck314 and D6Rck361, a chromosomal segment that contains the lurcher (Lc) locus. The Lc mutation is also semidominant and has a strikingly similar phenotype. A cross between a new mutant (Nm) heterozygote and an Lc heterozygote yields double heterozygotes, animals that carry both mutations, with a phenotype similar to that of both Nm and Lc homozygotes. The similarity in phenotype, the colocalization of the two loci on mouse Chr 6, and the positive result of the allelism test demonstrate that the new mutation is an allele of the Lc gene. Received: 4 April 1997 / Accepted: 21 April 1997