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PubMed2006

The TATA-box sequence in the basal promoter contributes to determining light-dependent gene expression in plants.

A prototype 13-bp TATA-box sequence, TCACTATATATAG, was mutated at each nucleotide position and examined for its function in the core promoter. Specific nucleotides in the first TATA, the second TATA, as well as the flanking sequences influenced promoter function in transient transformation of tobacco (Nicotiana tabacum var Petit Havana) leaves. The effect of a given mutation on reporter gene expression in light versus dark was variable and sometimes contrasting. Some mutations, like T(7) or A(8)-->C or G, completely inactivated the expression of the minimal promoter in light but not in dark. In general, the sequence requirement for dark expression was less stringent than that for light expression. The selective effect of TATA-box mutations on light versus dark expression was exerted on core promoter function in the chromatin-integrated state also. Even in the presence of an upstream light response activator element, TATA-box mutations influenced modulation of the promoter by light. An A at the eighth position was specifically involved in the red light response of the promoter. Selectivity in gene expression was associated with a high level of transcript initiation from a site that was not active in the dark. Nuclear proteins from dark- and light-grown seedlings showed that the sequence variation within the TATA-box governs the formation of alternative transcriptional complexes. The experiments give direct evidence for the role of a core TATA-box sequence in determining the level as well as selectivity of gene expression in plants.

Base Sequence↗
PubMed1997

Constitutively active germline mutation of the thyrotropin receptor gene as a cause of congenital hyperthyroidism.

Congenital hyperthyroidism is a rare, transient disease usually caused by transmission of thyrotropin receptor autoantibodies from the mother with Graves' disease to her child. We report a German women and her two sons who had congenital, but persistent hyperthyroidism without any signs of autoimmunity. Direct sequencing of the polymerase chain reaction-amplified exon 10 of the thyrotropin receptor genomic DNA revealed in the mother and both sons a transition of GCC to GTC, resulting in an exchange of alanine 623 to valine. This germline mutation in a highly conserved region of the thyrotropin receptor resulted in a constitutive activation of the cyclic adenosine monophosphate-generating cascade with resulting hyperthyroidism. Analysis of the family for a corresponding BstXI restriction-site polymorphism revealed heterozygosity for this mutation in the affected family members, but not in the father or other relatives. We conclude that whenever congenital hyperthyroidism is persistent and parameters of autoimmunity are absent, a constitutively active thyrotropin receptor mutation should be considered. Treatment appears to require aggressive means such as total thyroidectomy or ablation by 131iodine because two subtotal thyroidectomies in the mother were insufficient to control the disease.

Adult↗
PubMed1999

Role of a common mutation in the homocysteine regulatory enzyme methylenetetrahydrofolate reductase in ischemic stroke.

A common mutation in methylenetetrahydrofolate reductase (MTHFR), a homocysteine metabolic pathway enzyme, has been associated with increased homocysteine levels and increased risk for premature cardiovascular disease. The purpose of this study was to assess the association between the prevalence of the MTHFR mutation, hyperhomocysteinemia, and subtypes of ischemic stroke in an elderly population comprised of three age-balanced groups of patients. The presence of the C677T MTHFR mutation was determined by a direct polymerase chain reaction-based assay performed on blood samples from 136 patients with acute ischemic stroke, 95 patients with atherosclerotic risk factors for stroke (including some with a history of previous stroke or transient ischemic attack), and 52 healthy control subjects. The prevalence of the homozygous C677T mutation was not significantly higher in the elderly stroke patients (7%) than in the atherosclerotic risk (8%) or healthy elderly control (2%) groups. Plasma homocysteine levels were higher in the acute stroke patient group (14.5+/-4.5 micromol/L) and atherosclerotic risk patient group (14.6+/-6.2 micromol/L) compared with the control subjects (10.3+/-3.1 micromol/ L, P < 0.03). Homozygotes for the C677T MTHFR mutation did not have significantly higher homocysteine levels than non-homozygotes. Moderate hyperhomocysteinemia, though common in older patients with ischemic cerebrovascular disease, is not attributable, at least in this patient group, to a higher prevalence of the C677T MTHFR mutation.

Adult↗
PubMed1996

Molecular basis of a hereditary type I protein S deficiency caused by a substitution of Cys for Arg474.

The molecular basis for a hereditary type I protein S (PS) deficiency was investigated. DNA sequence analysis in the proband showed a novel missense mutation substituting Cys (TGT) for Arg474 (CGT) that is a highly conserved amino acid residue among the related proteins. This missense mutation cosegregated with the type I PS deficiency in this family. Transient expression studies showed that the secretion of the recombinant Cys-mutant PS was markedly decreased compared with that of the recombinant wild-type PS, reproducing the observed phenotype of type I deficiency. Stable expression and pulse-chase experiments demonstrated an intracellular degradation and an impaired secretion of the recombinant Cys-mutant PS. Furthermore, the substitution of Arg474 by Ala or Glu, but not by Lys, markedly reduced the secretion of the recombinant PS mutants in transient expression studies, suggesting that a positively charged basic amino acid might be needed at residue 474 and might play a key role in the protein structure and conformation of the sex hormone binding globulin-homology domain of the PS molecule. We postulate that the loss of the highly conserved Arg474 might be responsible for the type I PS deficiency inherited in this family.

Androgen-Binding Protein↗
PubMed2003

Single mutation in the linker domain confers protein flexibility and camptothecin resistance to human topoisomerase I.

DNA topoisomerase I relaxes supercoiled DNA by the formation of a covalent intermediate in which the active-site tyrosine is transiently bound to the cleaved DNA strand. The antineoplastic agent camptothecin specifically targets DNA topoisomerase I, and several mutations have been isolated that render the enzyme camptothecin-resistant. The catalytic and structural dynamical properties of a human DNA topoisomerase I mutant in which Ala-653 in the linker domain was mutated into Pro have been investigated. The mutant is resistant to camptothecin and in the absence of the drug displays a cleavage-religation equilibrium strongly shifted toward religation. The shift is mainly because of an increase in the religation rate relative to the wild type enzyme, indicating that the unperturbed linker is involved in slowing religation. Molecular dynamics simulation indicates that the Ala to Pro mutation increases the linker flexibility allowing it to sample a wider conformational space. The increase in religation rate of the mutant, explained by means of the enhanced linker flexibility, provides an explanation for the mutant camptothecin resistance.

Alanine↗
PubMed2004

p42/44 Mitogen-activated protein kinase regulated by p53 and nitric oxide in human pulmonary arterial smooth muscle cells.

Although nitric oxide (NO) is known to inhibit vascular smooth muscle cell proliferation, the subcellular molecular mechanisms involved with the inhibitory signal transduction pathways are uncertain. We investigated the effect of exogenous NO on cell proliferation and the expression of p53, p21, and phosphorylated p42/44 mitogen-activated protein kinase (MAPK) in human pulmonary arterial smooth muscle cells (HPASMC). Both S-nitroso-N-acetyl penicillamine and diethylenetriaminelNONOate dose-dependently suppressed [3H]-thymidine incorporation in cultured HPASMC, and induced the expression of p53 and p21 protein. Further, the NO donors transiently increased the phosphorylation of p42/44 MAPK and then suppressed it. Although MAPK kinase inhibitors suppressed [3H]-thymidine incorporation by the cells, no significant change was observed in the expression of p53 and p21. The NO donors also suppressed the activation of p42/44 MAPK evoked by transient transfection of the wild-type p53 gene; however, they failed to suppress the activation of p42/44 MAPK in constitutive-active mutations of the Ras or Raf genes trasnsfected from HPASMC. These results indicate that exogenous NO is able to transiently activate p42/44 MAPK via the induction of p53, and then suppress it via inactivation of the Ras and Raf cascades.

Base Sequence↗
PubMed2001

Characterization of two novel mutations in the glucocorticoid receptor gene in patients with primary cortisol resistance.

OBJECTIVE: Primary glucocorticoid resistance is characterized by decreased sensitivity to cortisol signalling. We have performed genetic analysis of the glucocorticoid receptor (GR) gene in 12 unrelated patients with primary cortisol resistance as defined by a pathological dexamethasone suppression test. METHODS: Exon specific polymerase chain reaction amplification of the GR gene and sequencing of each exon was carried out. The two mutations were characterized in vitro in terms of glucocorticoid driven reporter gene activity in a transient transfection assay and in a ligand binding assay. Molecular modelling of the R477H mutant was performed based on the X-ray structure of the GR-DNA binding domain. RESULTS: Two novel mutations in the GR gene were found: R477H in the DNA-binding domain which is the first reported mutation in that region of the human GR gene and G679S in the ligand binding domain. The R477H mutation showed no transactivating capacity, whereas the G679S mutation had reduced transactivation capacity compared to the wild-type (wt) GR. When tested for ligand binding capacity, the G679S mutation had 50% binding affinity compared to the wt GR. The effect of the point mutation R477H was deduced by a comparison between the wt structure and the model of the mutant. The wt GR has direct and water mediated contact with the phosphate groups of the glucocorticoid responsive element (GRE) whereas, in the model, the mutation R477H has no contact with the GRE. The G679S mutation is located on the surface of the ligand binding domain, at a distance from the steroid-binding site. A previously reported polymorphism, AAT to AAC at amino acid position 766, was found in four of the patients. CONCLUSIONS: In two of 12 patients with clinical glucocorticoid resistance, mutant forms of GR could be found. The glucocorticoid resistance in vivo in these two patients corresponds to impaired function of the two mutated GR forms in two in vitro assays. The relevance of the conservative polymorphism for the glucocorticoid insensitivity noted in these patients remains to be clarified.

Adult↗
PubMed2005

PDGFRA mutations in gastrointestinal stromal tumors: frequency, spectrum and in vitro sensitivity to imatinib.

PURPOSE: Gastrointestinal stromal tumors (GISTs) commonly harbor oncogenic mutations of the KIT tyrosine kinase, which is a target for the kinase inhibitor imatinib. A subset of GISTs, however, contains mutations in the homologous kinase platelet derived growth factor receptor alpha (PDGFRA), and the most common of these mutations is resistant to imatinib in vitro. Little is known of the other types of PDGFRA mutations that occur in GISTs. MATERIALS AND METHODS: We determined the KIT and PDGFRA mutation status of 1,105 unique GISTs using a combination of denaturing high-performance liquid chromatography and direct sequencing. RESULTS: 66 in exon 18, 11 in exon 12, and three in exon 14. Transient expression of representative PDGFRA isoforms in CHO cells revealed imatinib sensitivity of exon 12 mutations (SPDHE566-571R and insertion ER561-562) and an exon 14 substitution (N659K). However, most isoforms with a substitution involving codon D842 in exon 18 (D842V, RD841-842KI, DI842-843IM) were resistant to the drug, with the exception of D842Y. Interestingly, other mutations in exon 18 (D846Y, N848K, Y849K and HDSN845-848P) were all imatinib sensitive. Proliferation studies with BA/F3 cell lines stably expressing selected PDGFRA mutant isoforms supported these findings. CONCLUSION: Including our cases, there are 289 reported PDGFRA-mutant GISTs, of which 181 (62.6%) had the imatinib-resistant substitution D842V. However, our findings suggest that more than one third of GISTs with PDGFRA mutations may respond to imatinib and that mutation screening may be helpful in the management of these tumors.

Animals↗
PubMed1997

Metachromatic leucodystrophy in three families from Nova Scotia, Canada: a recurring mutation in the arylsulphatase A gene.

Metachromatic leucodystrophy (MLD) is a lysosomal storage disease resulting from a deficiency of arylsulphatase A. We have identified a child with infantile onset MLD who is homozygous for an A212V mutation, a mutation previously reported but not further characterised. We have introduced this mutation into an arylsulphatase A expression vector by site directed mutagenesis. Transient expression of this mutant plasmid in COS cells yields very low levels of arylsulphatase A activity consistent with the patient's phenotype. The arylsulphatase A pseudodeficiency also segregates in this family causing difficulty in interpreting enzyme levels in the absence of DNA data. Two other patients from the same province, also carrying the A212V allele, have juvenile and adult onset MLD and are heterozygous for P426L ("A" allele) and I179S alleles respectively, known late onset alleles.

Adult↗
PubMed2005

Biological, biochemical, and kinetic effects of mutations of the cardiomyopathy loop of Dictyostelium myosin II: importance of ALA400.

The cardiomyopathy (CM)-loop of the heavy chain of class-II myosins begins with a highly conserved Arg residue (whose mutation in human beta-cardiac myosin II results in familial hypertrophic cardiomyopathy). The CM-loop of Dictyostelium myosin II (Arg397-Gln407) is essential for its biological functions and biochemical activities. We found that the CM-loop of smooth muscle myosin II substituted partially, and the CM-loop of beta-cardiac myosin II less well, for growth, capping of surface receptors and development, and the actin-activated MgATPase and in vitro motility activities of purified myosins. There was little correlation between the biochemical and biological activities of the two chimeras and 19 point mutants, but only the five mutants with k cat/K actin values equivalent to wild-type myosin supported essentially full biological function. The three point mutations of Arg397 equivalent to those that result in hypertrophic cardiomyopathy in humans had minimal biological effects and different biochemical effects. The A400V mutation rendered full-length wild-type myosin almost completely inactive, both in vitro and in vivo, and the reverse V400A mutation in the cardiac CM-loop chimera restored almost full activity, even though the sequence still differed from wild-type in 7 of 11 positions. Transient kinetic studies of acto-subfragment-1 (S1) showed that the chimeras and the Ala/Val, Val/Ala mutations do not affect the equilibrium or the association and dissociation rate constants for either ATP or ADP binding to acto-S1 or the rate of ATP-induced dissociation of acto-S1. We conclude that the Ala/Val, Val/Ala mutations affect the release of Pi from acto-S1.ADP.Pi. In addition, Val at position 400 substantially reduces the affinity of actin for S1 in the absence of nucleotide.

Actins↗
PubMed1995

Suppression of single and double nonsense mutations introduced into the diphtheria toxin A-chain gene: a potential binary system for toxin gene therapy.

We have previously shown that ablation of specific cells can be achieved through the transcriptionally regulated expression of the diphtheria toxin A-chain (DT-A) gene in both cell culture and transgenic mice. Such targeted toxin gene expression provides a novel approach to cancer and acquired immunodeficiency syndrome (AIDS) therapy. The use of mutants of DT-A with attenuated toxicity may allow targeting of cells for which only moderately selective gene regulatory elements are available. Alternatively, conditional mutants might be used to target cells in which conditions can be established for suppression of the mutation. We have investigated the effects of mutating selected serine codons to amber (TAG) nonsense codons in the DT-A coding sequence. In transient transfection of HeLa cells, DT-A activity was markedly reduced by the introduction of a single amber codon and was virtually eliminated by two amber mutations. Cotransfection of a serine inserting suppressor tRNA expression plasmid substantially restored DT-A expression from both single and double amber mutants. Expression of the same suppressor tRNA also suppressed a previously described amber mutation at the tyrosine codon 28 in DT-A. Thus, nonsense suppression can be used to control the expression of DT-A in mammalian cells, potentially allowing binary control over the targeting of tissues for selective ablation.

Amino Acid Sequence↗
PubMed1994

Synergism between CACGTG (G-box) and CACCTG cis-elements is required for activation of the bean seed storage protein beta-phaseolin gene.

Expression of bean seed storage protein phaseolin is under strict developmental control. Four distinct nuclear proteins recognize in vitro the proximal beta-phaseolin promoter (-295/+45) which confers spatial and temporal regulation of the native gene. Functional significance of these protein-binding sites was evaluated by substitution mutation of the motifs in the promoter, which was fused to GUS reporter gene, and subsequent transient gene expression assay using protoplasts from developing bean cotyledons. DNA-binding protein CAN binds three CANNTG motifs, CACGTG (-248/-243), CACCTG (-163/-158), and CATATG (-100/-95). Substitution mutation of the CACGTG motif, which is commonly known as G-box, reduced the -295 promoter activity by 75%, indicating that the G-box is a major positive cis-element. Mutation analyses also demonstrated that the CACCTG and CATATG motifs act as positive and negative cis-elements, respectively. Substitution mutation of all three CANNTG motifs essentially eliminated the -295 promoter activity. A construct containing the G-box and CACCTG motif resulted in a transcriptional level that is much greater than the sum of the transcriptional levels from the individual cis-elements, demonstrating that the G-box and CACCTG act synergistically. Substitution mutations of two AT-rich sequences, to which a nuclear protein AG-1 binds, showed that these sites function as major negative (-376/-367, -356/-347) or positive (-191/-182) cis-elements, and that the effect of the two AG-1 binding sites was counteractive in the -391 promoter. These results indicate that the three CANNTG motifs and two AG-1-binding sites play critical roles in transcription of the beta-phaseolin gene in cotyledons.

Base Sequence↗
PubMed2001

[Kappa-B nuclear factor and apoptosis of cancerous cells].

The transcriptional factor NF-kappa B is a key regulator of many biological processes such as the immune response, inflammation, development, cell proliferation and apoptosis. We investigated the role of NF-kappa B in the regulation of the pro-apoptotic protein Bax. We observed increased Bax proteins expression in several cancer cell lines stably expressing a mutated I kappa B-alpha that blocks NF-kappa B activity. Transient transfection experiments showed that NF-kappa B inhibited p53-dependent transactivation of the bax promoter, but this effect was not released by concomitant expression of several cofactors including CBP/p300. We also identified, for the first time, an NF-kappa B binding site in the bax gene promoter but the mutation of this site did not affect NF-kappa B induced inhibition of transcription. The mechanisms responsible for NF-kappa B inhibition of the bax promoter thus remain to be elucidated.

Apoptosis↗
PubMed1988

Mutational analysis of the contribution of sequence motifs within the IgH enhancer to tissue specific transcriptional activation.

We have investigated the role of sequence motifs in the immunoglobulin heavy chain (IgH) enhancer on its activity in myeloma and fibroblast cell-lines. In transient transfection assays the transcription stimulatory activity of the enhancer is decreased in myeloma cells by mutating the E motifs 1, 2 and 3, the core motifs C1, C2, C3 and the octamer motif (OC) and in fibroblasts by mutating E2, E3, and C2. Our results suggest that transcription factors binding to E1, C1, C3 and OC contribute in a positive manner to the tissue specificity of the IgH enhancer.

Base Sequence↗
PubMed2005

A new, recurrent mutation of GJB3 (Cx31) in erythrokeratodermia variabilis.

BACKGROUND: Erythrokeratodermia variabilis (EKV) is an autosomal dominant or recessive genodermatosis characterized by the coexistence of randomly occurring, transient, erythematous patches and hyperkeratosis of the skin. The disorder has been mapped to chromosome 1p35.1 but is genetically heterogeneous. EKV may be caused by pathogenic mutations in one of two neighbouring connexin genes, GJB3 and GJB4, encoding the gap junction proteins Cx31 and Cx30.3, respectively. Twelve distinct mutations identified to date cluster either at the cytoplasmic amino-terminus or in the four transmembrane domains. OBJECTIVES: To report a large family with EKV and an unrelated sporadic case. METHODS: DNA amplification and mutation analysis, followed by denaturing high-performance liquid chromatography to confirm the segregation of the mutations in the two families with EKV. RESULTS: A novel, recurrent GJB3 mutation (625C-->T; L209F) was identified in the family with EKV and in the unrelated sporadic case. CONCLUSIONS: This mutation is the first to affect a conserved residue in the cytoplasmic carboxy-terminus of any connexin gene with a cutaneous phenotype, emphasizing its structural and/or functional importance.

Adult↗
PubMed2001

A novel mutation lacking the bromodomain of the transcriptional coactivator p300 in the SiHa cervical carcinoma cell line.

The transcriptional coactivator p300, a histone acetyltransferase (HAT), plays key roles in the regulation of cell proliferation and differentiation. p300 is targeted by viral oncoproteins, and mutations of p300, accompanied by inactivation of the second allele, have been reported in certain types of cancers originating in the epithelium. Here, we identified a homozygous p300 deletion of exons 15--18 in the SiHa cervical carcinoma cell line, which results in an in-frame deletion that causes specific loss of the bromodomain, a conserved domain implicated in the regulation of HAT activity. Furthermore, we show that the mutation severely impaired its ability to activate the p21(WAF1/CIP1) promoter in transient reporter assay. These results suggest a critical role for the bromodomain in p300 functions as a tumor-suppressor gene.

Base Sequence↗
PubMed2001

Recent advances in the understanding of tau protein and movement disorders.

Tau plays an important role in movement disorders. The accumulation of pathological tau is a major substrate of frontotemporal dementia and parkinsonism linked to chromosome 17, progressive supranuclear palsy, and corticobasal degeneration. Over the past year, several new mutations on the tau gene have been found. These mutations have been classified into three groups: (i) mutations in constitutively spliced exons; (ii) mutations in the alternatively spliced exon 10; and (iii) mutations of the exon 10 5' splice site. Some patients presenting with frontotemporal dementia and parkinsonism linked to chromosome 17 transiently respond to levodopa therapy. The significance of Pick bodies was recognized by a recent study on kindred with the Glu342Val tau mutation. In sporadic cases of progressive supranuclear palsy, the presence of the H1 haplotype was found to be a risk factor. Corticobasal degeneration shares a common genetic background with progressive supranuclear palsy. This opens the question of whether corticobasal degeneration represents a separate disorder or a spectrum of disease with progressive supranuclear palsy. However, distinguishing features are observed, and include oculomotor abnormalities, which may help to differentiate these two disorders on clinical grounds. Despite recent advances in the understanding of the tauopathies, there are still no curative therapies available. It is hoped that studies in transgenic tau animal models will lead to the development of successful treatments.

Atrophy↗
PubMed2000

Mutation of conserved aspartates affects maturation of both aspartate mutant and endogenous presenilin 1 and presenilin 2 complexes.

Presenilin (PS1 and PS2) holoproteins are transiently incorporated into low molecular weight (MW) complexes. During subsequent incorporation into a higher MW complex, they undergo endoproteolysis to generate stable N- and C-terminal fragments. Mutation of either of two conserved aspartate residues in transmembrane domains inhibits both presenilin-endoproteolysis and the proteolytic processing of beta-amyloid precursor protein and Notch. We show that although PS1/PS2 endoproteolysis is not required for inclusion into the higher MW N- and C-terminal fragment-containing complex, aspartate mutant holoprotein presenilins are not incorporated into the high MW complexes. Aspartate mutant presenilin holoproteins also preclude entry of endogenous wild type PS1/PS2 into the high MW complexes but do not affect the incorporation of wild type holoproteins into lower MW holoprotein complexes. These data suggest that the loss of function effects of the aspartate mutants result in altered PS complex maturation and argue that the functional presenilin moieties are contained in the high molecular weight complexes.

Aspartic Acid↗