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PubMed1995

Suppressors of thermosensitive mutations in the DNA polymerase delta gene of Saccharomyces cerevisiae.

DNA polymerases (Pol) alpha, delta and epsilon are necessary for replication of nuclear DNA. Pol delta interacts permanently or transiently with numerous accessory proteins whose identification may shed light on the function(s) of Pol delta. In vitro mutagenesis was used to induce thermosensitive (ts) mutations in the DNA polymerase delta gene (POL3). We have attempted to clone two recessive extragenic suppressors of such ts mutants (sdp1 for mutation pol3-14 and sdp5-1 for mutation pol3-11) by transforming thermoresistant haploid strains pol3-14 sdp1 and pol3-11 sdp5-1 with wild-type genomic libraries in singlecopy or multicopy vectors. None of the thermosensitive transformants so obtained was identified as being sdp1 or sdp5-1. Instead, three genes were cloned whose products interfere with the activity of suppressors. One of them is the type 1 protein phosphatase gene, DIS2. Another is a novel gene, ASM4, whose gene product is rich in asparagine and glutamine residues.

Amino Acid Sequence↗
PubMed2000

Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.

Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1997

Upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.

Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. We have shown that FAS transcription rate is induced dramatically when fasted animals are refed with a high carbohydrate diet or when streptozotocin-diabetic mice are given insulin. We also reported that FAS gene transcription was up-regulated by insulin through the proximal promoter region from -71 to -50 and that upstream stimulatory factors (USFs), including USF1 and USF2, interact with this region in vitro. In the present study, by using site-directed mutagenesis of the -71/-50 region and correlating functional assays of the mutated promoter with USF binding activities, we demonstrate that the -65/-60 E-box motif (5'-CATGTG-3') is functionally required for insulin regulation and that USFs are in vivo components of the insulin response complex. Mutation of the -65/-60 E-box sequence abolished insulin response in both transiently and stably transfected 3T3-L1 adipocytes in the -2. 1 kb promoter context, which contains all the necessary regulatory elements of the promoter based on our previous transgenic mice studies, and in the minimal -67 promoter context. Gel mobility shift assays demonstrated that USFs can no longer bind to the -71/-50 promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with the FAS promoter-luciferase reporter constructs increased insulin-stimulated FAS promoter activity. Moreover, cotransfection of dominant negative USF1 and USF2 mutants lacking the DNA binding domain inhibited the insulin stimulation of the FAS promoter activity. On the other hand, site-directed mutagenesis of the -65/-60 E-box surrounding sequences within the overlapped tandem copies of sterol regulatory element-binding protein (SREBP) binding sites prevented SREBP from binding to -71/-50 promoter region in vitro but had no effect on insulin regulation of the FAS promoter in vivo. When rat liver nuclear extracts were used in gel mobility shift assays, only USF-containing protein-DNA complexes that can be supershifted by specific USF antibodies were observed. These results demonstrate that upstream stimulatory factor binding to the E-box at -65 is required for insulin regulation of the fatty acid synthase promoter.

3T3 Cells↗
PubMed2002

Characterization of six mutations in five Spanish patients with mitochondrial acetoacetyl-CoA thiolase deficiency: effects of amino acid substitutions on tertiary structure.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine metabolism. We identified and characterized 6 mutations, DelE85, K124R, A127V, Q145E, G152A, and E345V in 5 Spanish T2-deficient patients. Transient expression of mutant cDNAs was done at 37 and at 30 degrees C. Expression of the Q145E mutant cDNA resulted in about 12.5% normal amount at 37 degrees C and it retained 15% residual T2, indicating that specific activity of Q145E mutant protein was almost normal. This mutation reduced the heat stability of T2 activity. Although no significant residual activity was detected in either the G152A and A127V substitution, mutant proteins were detected, at 12.5% the normal amount at 37 degrees C and one-half normal at 30 degrees C for A127V, and 25 % only at 30 degrees C for G152A. Mutant proteins with Q145E, G152A, or A127V accumulated at 30 degrees C expression were stable for 48 h at 37 degrees C after cycloheximide treatment. Expression of DelE85, K124R, and E345V cDNAs gave neither residual T2 protein nor T2 activity. We constructed an improved tertiary structural model of T2 based on the X-ray crystal structure of acetoacetyl-CoA thiolase of Zoogloea ramigera. On the basis of this model, K124, A127, and G152 are located near the active site, mutations of which might affect catalytic function whereas Q145E, De185E, and E345V are distant from the active site with mutants being expected to destabilize the tertiary structure, especially during protein folding and dimerization.

Acetyl-CoA C-Acyltransferase↗
PubMed2001

Development of new T-vectors containing the luciferase gene. Easy application for direct cloning of a promoter DNA.

For promoter analyses of genes, it is usually necessary to amplify promoter DNA fragments by polymerase chain reaction (PCR) and clone them into a plasmid containing a reporter gene. In the present study we developed a novel plasmid, pGL2-X, which was constructed through a simple procedure of cloning an XcmI cassette from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene into the multicloning site of pGL2-Basic (Promega) pGL2-X was then converted by XcmI digestion into a T-vector which was named pGL2-T. Unfortunately, however, the firefly luciferase gene in pGL2-Basic contains one XcmI restriction site and therefore one base within the recognition site was silent-mutated. The cloning efficiency of the pGL2-T vector was approximately 63% when tested with a PCR product amplified from a promoter region (-501(-)+24) of the murine acetylcholine receptor delta subunit (AchR delta) gene. In C2C12 muscle cells transiently transfected with pGL2-T containing the AchR delta promoter, transcription of the silent-mutated luciferase gene increased 2.2-fold by neuregulin (EGF domain of heregulin beta 1; 100 ng/mL), a known stimulator of AchR delta expression. This result suggested that the pGL2-T vector was biologically functional. Thus, the present study provides an easy method to construct a variety of T-vectors containing different reporter genes.

Base Sequence↗
PubMed1999

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.

Human mastocytosis is characterized by increased mast cells. It usually occurs as a sporadic disease that is often transient and limited in children and persistent or progressive in adults. The c-KIT protooncogene encodes KIT, a tyrosine kinase that is the receptor for mast cell growth factor. Because mutated KIT can transform cells, we examined c-KIT in skin lesions of 22 patients with sporadic mastocytosis and 3 patients with familial mastocytosis. All patients with adult sporadic mastocytosis had somatic c-KIT mutations in codon 816 causing substitution of valine for aspartate and spontaneous activation of mast cell growth factor receptor (P = 0.0001). A subset of four pediatric onset cases with clinically unusual disease also had codon 816 activating mutations substituting valine, tyrosine, or phenylalanine for aspartate. Typical pediatric patients lacked 816 mutations, but limited sequencing showed three of six had a novel dominant inactivating mutation substituting lysine for glutamic acid in position 839, the site of a potential salt bridge that is highly conserved in receptor tyrosine kinases. No c-KIT mutations were found in the entire coding region of three patients with familial mastocytosis. We conclude that c-KIT somatic mutations substituting valine in position 816 of KIT are characteristic of sporadic adult mastocytosis and may cause this disease. Similar mutations causing activation of the mast cell growth factor receptor are found in children apparently at risk for extensive or persistent disease. In contrast, typical pediatric mastocytosis patients lack these mutations and may express inactivating c-KIT mutations. Familial mastocytosis, however, may occur in the absence of c-KIT coding mutations.

Adult↗
PubMed1997

Transient DNA relaxation in Escherichia coli induced by nalidixic acid.

We report here the transient relaxation of plasmid DNA by inhibitors of DNA gyrase in Escherichia coli. Relaxation of plasmid DNA in the presence of nalidixic acid, an inhibitor of the A subunit of DNA gyrase, lasted less than 60 min. The effect of nalidixic acid was not observed in a nalA26 mutant, a strain resistant to nalidixic acid due to a mutation in the gyrA gene. Novobiocin, whose target is the B subunit of DNA gyrase, also caused transient DNA relaxation. Resupercoiling of relaxed DNA in the presence of nalidixic acid was inhibited by pretreatment of the cells with chloramphenicol. As mutations of both the rpoH and recA genes did not affect the resupercoiling reaction, this step seems to require protein synthesis that is independent of both heat shock and SOS responses.

Bacterial Proteins↗
PubMed1987

Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation.

pp60c-src is phosphorylated in vivo at tyrosine 527, a residue not present in pp60v-src (its transforming homolog), and not at tyrosine 416, its site of in vitro autophosphorylation. To test the hypothesis that tyrosine phosphorylation regulates pp60c-src biological activity, we constructed and studied pp60c-src mutants in which Tyr 527 and Tyr 416 were separately or coordinately altered to phenylalanine. Tyr----Phe 527 mutation strongly activated pp60c-src transforming and kinase activities, whereas the additional introduction of a Tyr----Phe 416 mutation suppressed these activities. Tyr----Phe 416 mutation of normal pp60c-src eliminated its partial transforming activity, which suggests that transient or otherwise restricted phosphorylation of Tyr 416 is important for pp60c-src function even though stable phosphorylation is not observed in vivo.

Animals↗
PubMed2004

A mutation in serca underlies motility dysfunction in accordion zebrafish.

Zebrafish acquire the ability for fast swimming early in development. The motility mutant accordion (acc) undergoes exaggerated and prolonged contractions on both sides of the body, interfering with the acquisition of patterned swimming responses. Our whole cell recordings from muscle indicate that the defect is not manifested in neuromuscular transmission. However, imaging of skeletal muscle of larval acc reveals greatly prolonged calcium transients and associated contractions in response to depolarization. Positional cloning of acc identified a serca mutation as the cause of the acc phenotype. SERCA is a sarcoplasmic reticulum transmembrane protein in skeletal muscle that mediates calcium re-uptake from the myoplasm. The mutation in SERCA, a serine to phenylalanine substitution, is likely to result in compromised protein function that accounts for the observed phenotype. Indeed, direct evidence that mutant SERCA causes the motility dysfunction was provided by the finding that wild type fish injected with an antisense morpholino directed against serca, exhibited accordion-like contractions and impaired swimming. We conclude that the motility dysfunction in embryonic and larval accordion zebrafish stems directly from defective calcium transport in skeletal muscle rather than defective CNS drive.

Amino Acid Sequence↗
PubMed2006

MPL mutations in 23 patients suffering from congenital amegakaryocytic thrombocytopenia: the type of mutation predicts the course of the disease.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare inherited bone marrow failure syndrome. Mutations in the gene for the thrombopoietin receptor MPL were defined as the molecular cause in CAMT patients. Extending our sequence analyses from eight to a total of now 23 CAMT patients we could verify our hypothesis of genotype-phenotype correlation in CAMT. Seven different mutations predicted to lead to a complete loss of function of the thrombopoietin receptor were found in 13 patients belonging to group CAMT I with persistently low platelet counts and a fast progression into pancytopenia. Nine different missense mutations were detected in 10 patients of group CAMT II, characterized by a transient increase in platelet counts over 50 nl(-1) during the first years of life. Using in vitro assays with hematopoietic progenitors from patients of both patient groups we could provide experimental evidence for a residual activity of the thrombopoietin receptor in CAMT II patients.

Adolescent↗
PubMedDate not supplied

Behavioral analysis of Drosophila mutants displaying abnormal male courtship.

We describe six recessive autosomal male sterile mutations in Drosophila, generated by mobilization of single P-elements, exhibiting abnormal male courtship behavior. Detailed analysis of courtship behavior elicited by virgin wild type females indicated that five of the six mutants are affected in the early steps of courtship. The sixth mutant is blocked at the step of attempted copulation which occurs later in the courtship sequence. All of the mutants have normal olfactory responses and normal locomotor activity. No defect in the visual modality has been observed for the five mutants affected in the initiation of courtship. The mutant blocked at attempted copulation lacks the 'on' and 'off' transients, but this appears to be due to genetic background rather than the mutation itself. Abnormal spermatogenesis was observed in five of the mutants. Spermatogenic defects vary and include lesions in the proliferation of the germline, in meiosis, and in the differentiation and maturation of the spermatids into motile sperm.

Animals↗
PubMed1976

Skin exfoliation and purulent conjunctivitis in a new mutant-the exfoliative mouse.

A spontaneous autosomal recessive mutation has been named the exfoliative mouse (genotype ex/ex). Exfoliative mice suffer a transient purulent conjunctivitis in their 3rd week and an exfoliative skin disease in their 4th week. Gram-negative bacilli are present in blood and cerebrospinal fluid during the conjunctivitis stage, and in skin during the exfoliative period. The mutant can be differentiated from the ichthyotic mouse mutant.

Animals↗
PubMed1995

Evidence of increased glyceraldehyde-3-phosphate dehydrogenase and fatty acid synthetase promoter activities in transiently transfected adipocytes from genetically obese rats.

Previous studies have shown that the adipose tissue of young genetically obese Zucker rats was characterized by a coordinate overtranscription of lipogenic genes, suggesting that the fa mutation triggers transcription factor(s) acting in common on the promoters of these genes. To test this hypothesis, we developed a system of transient transfection of rat adipocytes with constructs containing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and fatty acid synthetase (FAS) promoters fused to gene reporter CAT. Those transfected cells expressed high levels of promoter-driven chloramphenicol acetyltransferase (CAT) activity through correctly initiated transcription as shown by primer extension analysis. Using this system we found a direct effect of insulin on GAPDH and FAS gene expression in rat adipocytes. In transfected adipocytes from obese compared to lean rats, activity of GAPDH and FAS promoters fused to CAT, was 2.6- and 8-fold increased, respectively. In contrast when reporter gene activity was driven by either phosphoenolpyruvate carboxykinase or beta-actin promoter, no difference could be observed between lean and obese, pointing out the promoter specificity of genotype effect. 5' deletion analysis of GAPDH promoter allowed us to narrow down the fa responsive region to nucleotide -488-329. As assessed by gel retardation and DNase I footprinting analysis, adipocyte nuclear protein interactions to this 159-bp fragment were found to be identical and to footprint the same 20-bp sequence. This study pointed out that overexpression of GAPDH and FAS genes in adipose tissue of genetically obese rats relies on promoter activation, through a 159-bp cis-acting region within the GAPDH promoter. The effects of the fa mutation on trans-acting factors binding to this region remain to be identified.

Adipocytes↗
PubMed2004

To slip or skip, visualizing frameshift mutation dynamics for error-prone DNA polymerases.

Three models describing frameshift mutations are "classical" Streisinger slippage, proposed for repetitive DNA, and "misincorporatation misalignment" and "dNTP-stabilized misalignment," proposed for non-repetitive DNA. We distinguish between models using pre-steady state fluorescence kinetics to visualize transiently misaligned DNA intermediates and nucleotide incorporation products formed by DNA polymerases adept at making small frameshift mutations in vivo. Human polymerase (pol) mu catalyzes Streisinger slippage exclusively in repetitive DNA, requiring as little as a dinucleotide repeat. Escherichia coli pol IV uses dNTP-stabilized misalignment in identical repetitive DNA sequences, revealing that pol mu and pol IV use different mechanisms in repetitive DNA to achieve the same mutational end point. In non-repeat sequences, pol mu switches to dNTP-stabilized misalignment. pol beta generates -1 frameshifts in "long" repeats and base substitutions in "short" repeats. Thus, two polymerases can use two different frameshift mechanisms on identical sequences, whereas one polymerase can alternate between frameshift mechanisms to process different sequences.

Catalysis↗
PubMed2005

In vitro studies of cross-resistance mutations against two hepatitis C virus serine protease inhibitors, VX-950 and BILN 2061.

VX-950 is a potent, small molecule, peptidomimetic inhibitor of the hepatitis C virus (HCV) NS3.4A serine protease and has recently been shown to possess antiviral activity in a phase I trial in patients chronically infected with genotype 1 HCV. In a previous study, we described in vitro resistance mutations against either VX-950 or another HCV NS3.4A protease inhibitor, BILN 2061. Single amino acid substitutions that conferred drug resistance (distinct for either inhibitor) were identified in the HCV NS3 serine protease domain. The dominant VX-950-resistant mutant (A156S) remains sensitive to BILN 2061. The major BILN 2061-resistant mutants (D168V and D168A) are fully susceptible to VX-950. Modeling analysis suggested that there are different mechanisms of resistance for these mutations induced by VX-950 or BILN 2061. In this study, we identified mutants that are cross-resistant to both HCV protease inhibitors. The cross-resistance conferred by substitution of Ala(156) with either Val or Thr was confirmed by characterization of the purified enzymes and reconstituted replicon cells containing the single amino acid substitution A156V or A156T. Both cross-resistance mutations (A156V and A156T) displayed significantly diminished fitness (or replication capacity) in a transient replicon cell system.

Amino Acid Substitution↗
PubMed2006

Proton sensing of CLC-0 mutant E166D.

CLC Cl- channels are homodimers in which each subunit has a proper pore and a (fast) gate. An additional slow gate acts on both pores. A conserved glutamate (E166 in CLC-0) is a major determinant of gating in CLC-0 and is crucially involved in Cl-/H+ antiport of CLC-ec1, a CLC of known structure. We constructed tandem dimers with one wild-type (WT) and one mutant subunit (E166A or E166D) to show that these mutations of E166 specifically alter the fast gate of the pore to which they belong without effect on the fast gate of the neighboring pore. In addition both mutations activate the common slow gate. E166A pores have a large, voltage-independent open probability of the fast gate (popen), whereas popen of E166D pores is dramatically reduced. Similar to WT, popen of E166D was increased by lowering pHint. At negative voltages, E166D presents a persistent inward current that is blocked by p-chlorophenoxy-acetic acid (CPA) and increased at low pHext. The pHext dependence of the persistent current is analogous to a similar steady inward current in WT CLC-0. Surprisingly, however, the underlying unitary conductance of the persistent current in E166D is about an order of magnitude smaller than that of the transient deactivating inward Cl- current. Collectively, our data support the possibility that the mutated CLC-0 channel E166D can assume two distinct open states. Voltage-independent protonation of D166 from the outside favors a low conductance state, whereas protonation from the inside favors the high conductance state.

Animals↗
PubMed2005

A new mutation in exon 7 of NEMO gene: late skewed X-chromosome inactivation in an incontinentia pigmenti female patient with immunodeficiency.

Incontinentia pigmenti is an X-linked genodermatosis, lethal in males. Affected females survive because of X-chromosome dizygosity and negative selection of cells carrying the mutant X-chromosome, and for this reason the skewed X inactivation pattern is often used to confirm the diagnosis. The most frequent mutation is a deletion of part of the NEMO gene (NEMODelta4-10), although other mutations have been reported. Mutations of NEMO which do not abolish NF-kappaB activity totally permit male survival, causing an allelic variant of IP called hypohidrotic ectodermal dysplasia and immunodeficiency (HED-ID). We present a non-classical IP female patient who also suffered transient immunodeficiency because of a late and progressive selection against peripheral blood cells carrying an active mutated X-chromosome. This finding suggests that in the absence of known mutation the X-inactivation studies used in genetic counselling can induce mistakes with some female patients. At the age of 3 years and 6 months, all immunodeficiency signs disappeared, and the X-chromosome inactivation pattern was completely skewed. The low T cell proliferation and CD40L expression corroborate the important role of NEMO/ NF-kappaB pathway in T cell homeostasis. The decreased NEMO protein amount and the impaired IkBalpha degradation suggest that this new mutation, NM_003639: c.1049dupA, causes RNA or protein instability. To our knowledge, this is the first time that selection against the mutated X-chromosome in X-linked disease has been documented in vivo.

Age of Onset↗
PubMed1996

Why do stem cells exist?

Self-renewing tissues have a differentiation hierarchy such that the stem cells are the only permanent residents of the tissue, and it is in these cells that most cancerous mutations arise. The progeny of the stem cells either remain stem cells or enter a transient proliferating cell population that differentiates to produce the functional cells of the tissue. The reason that this differentiation hierarchy exists has not been established. We show here that alternative hierarchies, in which there would be no stem cells, are feasible and biologically plausible. We show that current evidence from somatic mutation frequencies at both transgenic and endogenous loci implicates cell division in the origin of most somatic mutations. We suggest, therefore, that the existence of stem cells is an evolutionary consequence of a selective pressure to avoid cancer by reducing the number of somatic mutations. The stem cell hierarchy reduces the number of cell divisions of those cells that reside permanently in the tissue, which reduces the number of somatic mutations and thus minimizes the cancer rate. In the small intestine, the existence of stem cells reduces the mutant frequency in the stem cells by about one order of magnitude. Since two or more mutations are required to transform a cell, the protective effect may be 100-fold or more. Similar factors may be expected in other tissues.

Age Factors↗