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Results for “Loss of function”

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PubMed1996

In vivo and in vitro characterization of Escherichia coli protein CheZ gain- and loss-of-function mutants.

Bacterial chemotaxis results from the ability of flagellated bacteria to control the frequency of switching between smooth-swimming and tumbling episodes in response to changes in concentration of extracellular substances. High levels of phosphorylated CheY protein are the intracellular signal for inducing the tumbling mode of swimming. The CheZ protein has been shown to control the level of phosphorylated CheY by regulating its rate of dephosphorylation. To identify functional domains in the CheZ protein, we made mutants by random mutagenesis of the cheZ gene and constructed a series of deletions. The map position and the in vivo and in vitro activity of the resulting gain- or loss-of-function mutant proteins define separate functional domains of the CheZ protein.

Bacterial Proteins↗

PubMed2006

A loss-of-function RNA interference screen for molecular targets in cancer.

The pursuit of novel therapeutic agents in cancer relies on the identification and validation of molecular targets. Hallmarks of cancer include self-sufficiency in growth signals and evasion from apoptosis; genes that regulate these processes may be optimal for therapeutic attack. Here we describe a loss-of-function screen for genes required for the proliferation and survival of cancer cells using an RNA interference library. We used a doxycycline-inducible retroviral vector for the expression of small hairpin RNAs (shRNAs) to construct a library targeting 2,500 human genes. We used retroviral pools from this library to infect cell lines representing two distinct molecular subgroups of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like DLBCL and germinal centre B-cell-like DLBCL. Each vector was engineered to contain a unique 60-base-pair 'bar code', allowing the abundance of an individual shRNA vector within a population of transduced cells to be measured using microarrays of the bar-code sequences. We observed that a subset of shRNA vectors was depleted from the transduced cells after three weeks in culture only if shRNA expression was induced. In activated B-cell-like DLBCL cells, but not germinal centre B-cell-like DLBCL cells, shRNAs targeting the NF-kappaB pathway were depleted, in keeping with the essential role of this pathway in the survival of activated B-cell-like DLBCL. This screen uncovered CARD11 as a key upstream signalling component responsible for the constitutive IkappaB kinase activity in activated B-cell-like DLBCL. The methodology that we describe can be used to establish a functional taxonomy of cancer and help reveal new classes of therapeutic targets distinct from known oncogenes.

Adaptor Proteins, Signal Transducing↗

PubMed2003

A mouse with a loss-of-function mutation in the c-Cbl TKB domain shows perturbed thymocyte signaling without enhancing the activity of the ZAP-70 tyrosine kinase.

The unique tyrosine kinase binding (TKB) domain of Cbl targets phosphorylated tyrosines on activated protein tyrosine kinases (PTKs); this targeting is considered essential for Cbl proteins to negatively regulate PTKs. Here, a loss-of-function mutation (G304E) in the c-Cbl TKB domain, first identified in Caenorhabditis elegans, was introduced into a mouse and its effects in thymocytes and T cells were studied. In marked contrast to the c-Cbl knockout mouse, we found no evidence of enhanced activity of the ZAP-70 PTK in thymocytes from the TKB domain mutant mouse. This finding contradicts the accepted mechanism of c-Cbl-mediated negative regulation, which requires TKB domain targeting of phosphotyrosine 292 in ZAP-70. However, the TKB domain mutant mouse does show aspects of enhanced signaling that parallel those of the c-Cbl knockout mouse, but these involve the constitutive activation of Rac and not enhanced PTK activity. Furthermore, the enhanced signaling in CD4(+)CD8(+) double positive thymocytes appears to be compensated by the selective down-regulation of CD3 on mature thymocytes and peripheral T cells from both strains of mutant c-Cbl mice.

Animals↗

PubMed2003

Epilepsy with auditory features: a LGI1 gene mutation suggests a loss-of-function mechanism.

Autosomal dominant partial epilepsy with auditory features (ADPEAF) is a genetically heterogeneous disorder. Some patients exhibit mutations in the leucine-rich glioma inactivated (LGI1) gene. In an ADPEAF family, a novel mutation in the Lgi1 signal peptide is predicted to interfere with the protein cell sorting, resulting in altered processing. This finding suggests a loss-of-function mechanism for LGI1 gene mutations causing ADPEAF even if other mechanisms cannot be ruled out.

Acoustic Stimulation↗

PubMed2003

The loss of the hemoglobin H2S-binding function in annelids from sulfide-free habitats reveals molecular adaptation driven by Darwinian positive selection.

The hemoglobin of the deep-sea hydrothermal vent vestimentiferan Riftia pachyptila (annelid) is able to bind toxic hydrogen sulfide (H(2)S) to free cysteine residues and to transport it to fuel endosymbiotic sulfide-oxidising bacteria. The cysteine residues are conserved key amino acids in annelid globins living in sulfide-rich environments, but are absent in annelid globins from sulfide-free environments. Synonymous and nonsynonymous substitution analysis from two different sets of orthologous annelid globin genes from sulfide rich and sulfide free environments have been performed to understand how the sulfide-binding function of hemoglobin appeared and has been maintained during the course of evolution. This study reveals that the sites occupied by free-cysteine residues in annelids living in sulfide-rich environments and occupied by other amino acids in annelids from sulfide-free environments, have undergone positive selection in annelids from sulfide-free environments. We assumed that the high reactivity of cysteine residues became a disadvantage when H(2)S disappeared because free cysteines without their natural ligand had the capacity to interact with other blood components, disturb homeostasis, reduce fitness and thus could have been counterselected. To our knowledge, we pointed out for the first time a case of function loss driven by molecular adaptation rather than genetic drift. If constraint relaxation (H(2)S disappearance) led to the loss of the sulfide-binding function in modern annelids from sulfide-free environments, our work suggests that adaptation to sulfide-rich environments is a plesiomorphic feature, and thus that the annelid ancestor could have emerged in a sulfide-rich environment.

Amino Acid Sequence↗

PubMed1998

Long-term follow-up after finger and upper-limb replantation: clinical, angiologic, and lymphographic studies.

This 10-year follow-up study evaluates 25 patients with a total of 57 successfully replanted fingers and six successfully replanted upper limbs. The global functional loss, including loss of range of motion, sensibility, and strength of the hand, was determined using the "Millesi score." The hemodynamic parameters of replanted and control fingers under resting and stress conditions were measured using a laser Doppler flowmeter. The lymphatic system at the site of replantation was examined by fluorescence microlymphography. All patients showed a considerable functional loss, according to the Millesi score, that averaged 56 percent of the normal function of the hand. In order to overcome functional deficit, many patients had developed successful compensatory mechanisms. In general, the patients themselves subjectively rated their functional deficits lower than indicated by the Millesi score. Laser Doppler flowmetry at rest and after arterial occlusion, and capillaroscopy before, during, and after a cold provocation test, revealed subnormal resting flow conditions and significantly decreased vascular capacity in the replanted fingers. Lymphatic drainage capacity was also significantly reduced despite documented reanastomosis between the skin microlymphatic network distal and proximal to the scar (fluorescence microlymphography). The coexistence of functional loss with compensatory mechanisms, decreased reactive hyperemia, and deficit in lymphatic drainage, present in all patients, must be considered as definitive sequelae of the initial injury.

Adolescent↗

PubMed1992

A study of the photoelectrical signal from human nasal cilia under several conditions.

The movement of normal human nasal cilia was analyzed. Ciliary beat was recorded by means of a phase-contrast microscope equipped with a photodetector. The electrical signal was analyzed as follows: i) a power spectrum was calculated in order to measure ciliary beat frequency (CBF), ii) the beat cycles were averaged and the standard deviation of the waveform was computed to determine signal consistency (SC), and iii) the ratio of the duration of the smooth to that of the steep part of the cycles was measured. This was done under three different conditions: 1) normal or "initial", 2) after induction of "function loss", and 3) after "salbutamol stimulation". At "function loss," the cilia beat slower and with less harmony. CBF decreased from an average of 9.0 Hz in the "initial" condition to 5.8 Hz. SC decreased from an average of 5.7 to 1.9. After "salbutamol stimulation", average CBF was partially restored to 7.7 Hz, while average SC increased to 4.4. These findings indicate that in ciliary function studies, SC, as a measure for ciliary beat harmony, may be introduced alongside CBF as a second valuable parameter. In this study we were not able to identify different phases in the signal that might be used as a third parameter to indicate the effective and the recovery stroke.

Albuterol↗

PubMed1996

Both loss-of-function and gain-of-function mutations in snf define a role for snRNP proteins in regulating Sex-lethal pre-mRNA splicing in Drosophila development.

The Drosophila snf gene encodes a protein with functional homology to the mammalian U1A and U2B" snRNP proteins. Studies, based on the analysis of three viable alleles, have suggested a role for snf in establishing the female-specific splicing pattern of the sex determination switch gene, sex-lethal. Here, we show that the non-sex-specific lethal null allele is required for female sex determination, arguing against the formal possibility that the viable alleles disrupt a function unrelated to snf's wild-type function. Moreover, we find snf is required for normal cell growth and/or survival, as expected for a protein involved in a cell-vital process such as RNA splicing. We also show that of the three viable alleles only one, snfJA2, is a partial loss-of-function mutation. The other two viable alleles, snf1621 and snfe8H, encode antimorphic proteins. We find the antimorphic proteins are mislocalized and correlate their mislocalization with their molecular lesions and mutant phenotypes. Finally, we provide genetic evidence that the antimorphic alleles interfere with the autoregulatory splicing function of the Sex-lethal protein. Based on these studies we suggest a model in which the snRNP protein, Snf, functions with Sex-lethal to block recognition of the regulated male-specific exon.

Alleles↗

PubMed1995

Behavioral and pheromonal phenotypes associated with expression of loss-of-function mutations in the sex-lethal gene of Drosophila melanogaster.

We have shown that female-specific functions of the sex determination gene Sex-lethal (Sxl) regulate sexual behavior and synthesis of the three major sex pheromones that have been identified in normal, sexually mature Drosophilia melanogaster males and virgin females. Diplo-X flies, heterozygous in trans for two partial loss-of-function Sxl mutations, elicit less courtship than normal females and produce large quantities of the inhibitory pheromones that normal males synthesize. In addition, the mutant flies fail to synthesize the female-predominant aphrodisiac pheromone or make very small quantities of this compound.

Animals↗

PubMed2000

R127W-HNF-4alpha is a loss of function mutation but not a rare polymorphism and causes Type II diabetes in a Japanese family with MODY1.

AIMS/HYPOTHESIS: Mutations in the hepatocyte nuclear factor (HNF)-4alpha gene cause the type 1 form of maturity-onset diabetes of the young (MODY1). The R127W mutation is a missense mutation located in the T-box region of HNF-4alpha that was first identified in a Japanese family with MODY. We have examined the functional properties of this mutation in order to clarify the molecular basis of MODY1. METHODS: The intracellular localisation, DNA binding ability, transactivation activity and functional synergism with the coactivator CREB-binding protein (CBP) of R127W-HNF-4alpha were investigated. RESULTS: The nuclear import and functional synergy with CBP of R127W-HNF-4alpha were normal. The DNA binding ability of the mutant was decreased as was its transcriptional activation of the HNF-1alpha and L-type pyruvate kinase (PKL) genes. CONCLUSION/INTERPRETATION: The R127W mutation seems to be a loss-of-function mutation.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2005

Brain and bone damage in KARAP/DAP12 loss-of-function mice correlate with alterations in microglia and osteoclast lineages.

Human polycystic lipomembraneous osteodysplasia with sclerosing leukoencephalopathy, also known as Nasu-Hakola disease, has been described to be associated with mutations affecting the immunoreceptor tyrosine-based activation motif-bearing KARAP/DAP12 immunoreceptor gene. Patients present bone fragilities and severe neurological alterations leading to presenile dementia. Here we investigated whether the absence of KARAP/DAP12-mediated signals in loss-of-function (KDelta75) mice also leads to bone and central nervous system pathological features. Histological analysis of adult KDelta75 mice brains revealed a diffuse hypomyelination predominating in anterior brain regions. As this was not accompanied by oligodendrocyte degeneration or microglial cell activation it suggests a developmental defect of myelin formation. Interestingly, in postnatal KDelta75 mice, we observed a dramatic reduction in microglial cell numbers similar to in vitro microglial cell differentiation impairment. Our results raise the intriguing possibility that defective microglial cell differentiation might be responsible for abnormal myelin development. Histomorphometry revealed that bone remodeling is also altered, because of a resorption defect, associated with a severe block of in vitro osteoclast differentiation. In addition, we show that, among monocytic lineages, KARAP/DAP12 specifically controls microglial and osteoclast differentiation. Our results confirm that KARAP/DAP12-mediated signals play an important role in the regulation of both brain and bone homeostasis. Yet, important differences exist between the symptoms observed in Nasu-Hakola patients and KDelta75 mice.

Adaptor Proteins, Signal Transducing↗

PubMed2002

A Robertsonian translocation suppresses a somatic recombination pathway to loss of heterozygosity.

In mammals, loss of APC/Apc gatekeeper function initiates intestinal tumorigenesis. Several different mechanisms have been shown or proposed to mediate functional loss of APC/Apc: mutation in APC/Apc, non-disjunction, homologous somatic recombination and epigenetic silencing. The demonstration that, in the C57BL/6 (B6) Apc(Min/+) mouse model of inherited intestinal cancer, loss of Apc function can occur by loss of heterozygosity (LOH) through somatic recombination between homologs presents an opportunity to search for polymorphisms in the homologous somatic recombination pathway. We report that the Robertsonian translocation Rb(7.18)9Lub (Rb9) suppresses the multiplicity of intestinal adenomas in this mouse model. As the copy number of Rb9 increases, the association with the interphase nucleolus of the rDNA repeats centromeric to the Apc locus on Chromosome 18 is increasingly disrupted. Our analysis shows that homologous somatic recombination is the principal pathway for LOH in adenomas in B6 Apc(Min/+) mice. These studies provide additional evidence that neoplastic growth can initiate in the complete absence of canonical genomic instability.

Adenoma↗

PubMed1999

Melanoma loss-of-function mutants in Xiphophorus caused by Xmrk-oncogene deletion and gene disruption by a transposable element.

The overexpression of the Xmrk oncogene (ONC-Xmrk) in pigment cells of certain Xiphophorus hybrids has been found to be the primary change that results in the formation of malignant melanoma. Spontaneous mutant stocks have been isolated that have lost the ability to induce tumor formation when crossed with Xiphophorus helleri. Two of these loss-of-function mutants were analyzed for genetic defects in ONC-Xmrk's. In the lof-1 mutant a novel transposable element, TX-1, has jumped into ONC-Xmrk, leading to a disruption of the gene and a truncated protein product lacking the carboxyterminal domain of the receptor tyrosine kinase. TX-1 is obviously an active LTR-containing retrotransposon in Xiphophorus that was not found in other fish species outside the family Poeciliidae. Surprisingly, it does not encode any protein, suggesting the existence of a helper function for this retroelement. In the lof-2 mutant the entire ONC-Xmrk gene was found to be deleted. These data show that ONC-Xmrk is indeed the tumor-inducing gene of Xiphophorus and thus the critical constituent of the tumor (Tu) locus.

Amino Acid Sequence↗

PubMed2004

Characterization of a novel loss of function mutation of PAX8 in a familial case of congenital hypothyroidism with in-place, normal-sized thyroid.

Thyroid dysgenesis is the most common cause of congenital hypothyroidism, a relatively frequent disease affecting 1 in 3000-4000 newborns. Whereas most cases are sporadic, mutations in transcription factors implicated in thyroid development have been shown to cause a minority of cases transmitted as monogenic Mendelian diseases. PAX8 is one of these transcription factors, and so far, five mutations have been identified in its paired domain in patients with thyroid dysgenesis. We have identified a novel mutation of PAX8, in the heterozygous state, in a father and his two children both presenting with congenital hypothyroidism associated with an in-place thyroid of normal size at birth. In addition, one of the affected siblings displayed unilateral kidney agenesis. The mutation substitutes a highly conserved serine in position 54 of the DNA-binding domain of the protein (S54G mutation) by a glycine. Functional analyses of the mutant protein (PAX8-S54G) demonstrated that it is unable to bind a specific cis-element of the thyroperoxidase gene promoter in EMSAs and that it has almost completely lost the ability to act in synergy with Titf1 to transactivate transcription from the thyroglobulin promoter/enhancer. These results indicate that loss of function mutations of the PAX8 gene may cause congenital hypothyroidism in the absence of thyroid hypoplasia.

Amino Acid Sequence↗

PubMed1998

Cooperation of p53 loss of function and v-Ha-ras in transformation of mouse keratinocyte cell lines.

We previously demonstrated that after transduction with the v-Ha-ras oncogene and grafting onto nude mouse hosts, primary epidermal keratinocytes with a null mutation in the p53 gene form tumors with increased growth rates and predisposition to malignant conversion relative to p53 wild-type keratinocytes (Weinberg WC, et al., Cancer Res 54:5584-5592, 1994). To further explore the cooperation between p53 loss of function and activation of the ras oncogene, cell lines were established from the normal epidermises of newborn and adult p53-null mice, and parallel subclones were reconstituted with the p53val135 temperature-sensitive mutant. Reconstituted lines C, G, N, and V demonstrated functional p53 transcriptional activator activity at the wild-type-permissive temperature of 32 degrees C, compared with the hygromycin-selected control line X and parental p53-null lines NHK4 and AK1b. Hygromycin-selected subclones, but not the parental lines, made normal skin in vivo; all cell lines made carcinomas after introduction of v-Ha-ras, independent of p53 status. These cell lines were compared in vitro at 32 degrees C to maximize the amount of p53val135 in the wild-type conformation. Expression of v-Ha-ras did not consistently alter p53-mediated transcriptional activity, suggesting tat ras acts downstream or independently of p53. No correlation was observed between p53-mediated transcriptional activity and in vitro growth rates, colony formation after exposure to ultraviolet light, or suppression by normal neighboring keratinocytes. However, keratinocyte cell lines devoid of p53 and expressing v-Ha-ras formed colonies in soft agar; this was blocked at 32 degrees C in all cell lines reconstituted with p53val135. These keratinocyte lines provide a model for exploring the role of p53 and the interaction of p53 and ras in keratinocyte transformation.

Animals↗

PubMed2004

Loss-of-function mutations of the K(+) channel gene KCNJ2 constitute a rare cause of long QT syndrome.

Mutations of the KCNJ2 gene encoding the potassium channel Kir2.1 were previously shown to cause Andersen's syndrome (AS), a multisystem disease manifesting with developmental abnormalities, cardiac arrhythmias and periodic paralyses. We conducted a search for KCNJ2 mutations among 188 unrelated patients suspected to have long QT syndrome (LQTS). The screening was performed by denaturing high-performance liquid chromatography (dHPLC) and DNA sequencing. Two novel mutations of the KCNJ2 gene were detected: a missense threonine to alanine mutation (T75A) in the N-terminal region (family 1) and an in-frame deletion of two amino acids (DeltaFQ163-164) in the M2 transmembrane region (family 2). In addition, a previously described silent polymorphism C1146T was detected. In family 1, some of the affected family members had a history of periodic muscle weakness characteristic of AS, but no dysmorphic features. The mean QTc interval of the affected members were 444 +/- 24 ms (family 1, n=7) and 456 +/- 8 ms (family 2, n=2). The mutations affect functionally important regions of the KCNJ2 channel protein: upon injection of the Xenopus oocytes with the wild type and mutant KCNJ2 constructs, the channel proteins were correctly synthesized and localized to the cell surface, but no measurable inward K(+) current could be detected for the mutant KCNJ2 constructs. In conclusion, we report two novel loss-of-function mutations of the KCNJ2 channel, affecting different domains of the channel protein. Mutations of the KCNJ2 gene should be considered in genetic subclassification of LQTS patients, even in the absence of overt manifestations of AS.

Amino Acid Sequence↗

PubMed2003

An overview of the variables selection methods for the minimum sum of absolute errors regression.

The minimum sum of absolute errors regression is a robust alternative to the least squares regression whenever the errors follow a distribution for which the sample median is a more efficient estimator of location parameter than the sample mean, the errors follow a long tailed distribution, there are outliers in the values of the response variable in the data or the absolute error loss function is more appropriate than the quadratic loss function. Often an initial model may contain a large number of variables. However, in many situations, it is neither necessary nor important to include all the variables in the model. The methods for variable selection for the minimum sum of absolute errors regression are not as well documented and known as for the least squares regression. Our objective is to present an overview of the procedures to fit models with fewer variables and some criteria for selecting a model.

Algorithms↗

PubMed2026

Loss of function of ALDH3B2 transdifferentiates human pancreatic duct cells into β-like cells.

Replenishment of pancreatic β cells is key to a cure for diabetes. β cell regeneration is achieved predominantly by self-replication, especially in rodents, but it was also shown that pancreatic duct cells can transdifferentiate into β cells. How pancreatic duct cells are transdifferentiated and whether we can manipulate transdifferentiation to replenish β cell mass are not well understood. Using a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screen, we found that the loss of function of aldehyde dehydrogenase family 3 member B2 (ALDH3B2) was sufficient to transdifferentiate cell line-based and human pancreatic duct cells into functional β-like cells. The transdifferentiated cells had substantially increased the expression of β cell marker genes, secreted insulin in response to glucose, and lowered blood glucose to near normal for 6 weeks after transplantation into streptozotocin-induced diabetic mice under the kidney capsule. Our study identifies a gene that could potentially be targeted in human pancreatic duct cells to replenish β cell mass for diabetes therapy.

Humans↗