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At least 145 records · Page 8Linked to original sources
PubMed1994

A baculovirus gp64 early promoter is activated by host transcription factor binding to CACGTG and GATA elements.

The early promoter of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus gp64 gene is active when transfected into several insect cell lines and does not require viral gene products for transcription in uninfected cells. Because previous studies have shown that the gp64 early promoter is activated above basal levels in uninfected cells, host transcription factors are likely to play a role in gp64 activation at early times postinfection. By using nuclear extracts from uninfected Sf9 cells for electrophoretic mobility shift analysis of gp64 regulatory regions, host nuclear proteins were shown to bind specifically to the upstream regulatory region of the gp64 early promoter. Host factor binding was mapped to a 24-bp sequence centered approximately 35 bp upstream of the TATA box. Two consensus eukaryotic transcription factor-binding site motifs, GATA and CACGTG, were identified within the 24-bp sequence. Competition assays using oligonucleotides containing either a GATA or a CACGTG motif and similar oligonucleotides with point mutations in these sites showed that each site is required for binding host transcription factors. To investigate the functional significance of host factor binding to GATA and CACGTG motifs, constructs containing point mutations in these motifs were examined in transient expression assays. Mutations in either or both GATA and CACGTG sites decreased reporter activity in transient expression assays, suggesting that binding of host transcription factors to these motifs is important in transcriptional regulation of the gp64 early promoter.

Animals↗
PubMed1992

The dissonance mutation at the no-on-transient-A locus of D. melanogaster: genetic control of courtship song and visual behaviors by a protein with putative RNA-binding motifs.

Genetic and molecular results are here presented revealing that the dissonance (diss) courtship song mutation is an allele of the no-on-transient-A (nonA) locus of Drosophila melanogaster. diss (now called nonAdiss) was originally isolated as a mutant with aberrant pulse song, although it was then noted to exhibit defects in responses to visual stimuli as well. The lack of transient spikes in the electroretinogram (ERG) and optomotor blindness associated with nonAdiss are shown to be similar to the visual abnormalities caused by the original nonA mutations. nonAdiss failed to complement either the ERG or optomotor defects associated with four other nonA mutations. However, all four of these nonA mutants--which were isolated on visual criteria alone--sang a normal courtship song. nonAdiss complemented at least three of the nonA mutations with regard to the singing phenotype, as assessed by a new method for temporal analysis of the male's pulse song. Both visual and song abnormalities caused by nonAdiss were rescued by P-element-mediated transformation with overlapping 11 and 16 kilobase (kb) fragments of genomic DNA (originally cloned from the nonA locus by Jones and Rubin, 1990). Analysis of behavioral phenotypes in transformed flies carrying mutagenized versions of the 11 kb genomic fragment (in a nonAdiss genomic background) localized the rescuing DNA to a region containing an open reading frame that encodes a polypeptide (NONA) with similarity to a family of RNA-binding proteins. Immunohistochemical determination of NONA's spatial and temporal expression revealed that it is localized to the nuclei of cells in many neural and non-neural tissues, at all stages of the life cycle after very early in development. Genetic connections between the control of two quite different behaviors--reproductive and visual--are discussed, along with precedences for generally expressed gene products playing roles in specific behaviors.

Alleles↗
PubMed2001

Muscle-directed gene transfer and transient immune suppression result in sustained partial correction of canine hemophilia B caused by a null mutation.

The X-linked bleeding disorder hemophilia B is caused by absence of functional blood coagulation factor IX (F9) and can be treated by adeno-associated viral (AAV) mediated gene transfer to skeletal muscle. The safety of this approach is currently being evaluated in a phase I clinical trial. Efficacy of this and several other gene therapy strategies has been addressed in hemophilia B dogs, an important preclinical model of the disease. While previously published data demonstrated sustained expression of canine F9 in dogs with a missense mutation in the gene F9, we show here that AAV-mediated canine F9 gene transfer to skeletal muscle of hemophilia B dogs carrying a null mutation of F9 (causing an early stop codon and an unstable mRNA) results in induction of inhibitory anti-canine F9 at comparable vector doses (1 x 10(12) vector genomes/kg). Thus, the risk of inhibitor formation following AAV-mediated F9 gene therapy may be influenced by the nature of the underlying mutation in F9. Transient immune suppression with cyclophosphamide at the time of vector administration blocked formation of anti-canine F9 antibodies in the one animal treated with this approach. Treatment with this combination of gene transfer and transient immune modulation has resulted in sustained expression (>8 months) of canine F9 at levels sufficient for partial correction of coagulation parameters.

Adenoviridae↗
PubMed1997

Genetic basis of dominantly inherited transient bullous dermolysis of the newborn: a splice site mutation in the type VII collagen gene.

Transient bullous dermolysis of the newborn (TBDN) is a blistering disease evident at birth or shortly thereafter, but the blistering tendency decreases with advancing age. The tissue separation in TBDN is below the lamina densa, and electron microscopy has revealed abnormalities in anchoring fibrils. Immunofluorescence staining demonstrates intracellular accumulation of type VII collagen. In this study, we report a G-to-C transversion mutation in the last nucleotide of intron 35 of the type VII collagen gene (COL7A1) in a family with autosomal dominant TBDN in three generations. This nucleotide substitution abolishes the obligatory consensus 3'-acceptor splice site, predicting in-frame skipping of exon 36. Thus, TBDN in this family is caused by a mutation in COL7A1, and is therefore allelic with other variants of dominant dystrophic epidermolysis bullosa.

Child, Preschool↗
PubMed1995

Mutagenesis and the molecular modeling of the rat angiotensin II receptor (AT1).

The molecular interaction involved in the ligand binding of the rat angiotensin II receptor (AT1A) was studied by site-directed mutagenesis and receptor model building. The three-dimensional structure of AT1A was constructed on the basis of a multiple amino acid sequence alignment of seven transmembrane domain receptors and angiotensin II receptors and after the beta 2 adrenergic receptor model built on the template of the bacteriorhodopsin structure. These data indicated that there are conserved residues that are actively involved in the receptor-ligand interaction. Eleven conserved residues in AT1, His166, Arg167, Glu173, His183, Glu185, Lys199, Trp253, His256, Phe259, Thr260, and Asp263, were targeted individually for site-directed mutation to Ala. Using COS-7 cells transiently expressing these mutated receptors, we found that the binding of angiotensin II was not affected in three of the mutations in the second extracellular loop, whereas the ligand binding affinity was greatly reduced in mutants Lys199-->Ala, Trp253-->Ala, Phe259-->Ala, Asp263-->Ala, and Arg167-->Ala. These amino acid residues appeared to provide binding sites for Ang II. The molecular modeling provided useful structural information for the peptide hormone receptor AT1A. Binding of EXP985, a nonpeptide angiotensin II antagonist, was found to be involved with Arg167 but not Lys199.

Amino Acid Sequence↗
PubMed2006

Prolonged Ca2+ and force transients in myosin RLC transgenic mouse fibers expressing malignant and benign FHC mutations.

Clinical studies have revealed that mutations in the ventricular myosin regulatory light chain (RLC) lead to the development of familial hypertrophic cardiomyopathy (FHC), an autosomal dominant disease characterized by left ventricular hypertrophy, myofibrillar disarray and sudden cardiac death. While mutations in other contractile proteins have been studied widely by others, there is no report elucidating the mechanism(s) associated with FHC-linked RLC mutations. In this study, we have assessed the functional consequences of two RLC mutations, R58Q and N47K, in transgenic mice. Clinical phenotypes associated with these mutations included inter-ventricular hypertrophy, abnormal ECG findings and the R58Q mutation caused multiple cases of premature sudden cardiac death. Simultaneous measurements of the ATPase and force in transgenic skinned papillary muscle fibers from mutated versus control mice showed an increase in the Ca(2+) sensitivity of ATPase and steady-state force only in R58Q fibers. The calculated energy cost or rate of dissociation of force generating myosin cross-bridges (ATPase/force ratio) plotted as a function of activation state was the same in all groups of fibers. Both mutations caused prolonged [Ca(2+)] transients in electrically stimulated intact papillary muscles; however, the R58Q mutation also resulted in a significantly prolonged force transient. Our results suggest that the phenotypes of FHC observed in patients harboring these RLC mutations correlate with the extent of physiological changes monitored in transgenic fibers. Cardiac hypertrophy observed in patients is most likely caused by the activation of compensatory mechanisms ensuing from higher workloads due to incomplete relaxation as evidenced by prolonged [Ca(2+)] transients for both N47K and R58Q fibers. Furthermore, the poor prognosis of the R58Q patients may be associated with more severe diastolic dysfunction due to the slower off-rate of Ca(2+) from troponin C leading to longer force and [Ca(2+)] transients and increased Ca(2+) sensitivity of ATPase and force.

Adenosine Triphosphatases↗
PubMed2002

Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants.

BACKGROUND: Germline mutations in the mismatch repair (MMR) genes hMLH1 and hMSH2 can cause hereditary non-polyposis colorectal cancer (HNPCC). However, the functional in vitro analysis of hMLH1 and hMSH2 mutations remains difficult. AIMS: To establish an in vitro method for the functional characterisation of hMLH1 and hMSH2 mutations. METHODS: hMLH1 and hMSH2 wild type (wt) genes and several mutated subclones were transiently transfected in mismatch repair deficient cell lines (HCT-116 and LOVO). Apoptosis, proliferation, and regulation of mRNA expression and protein expression of interacting proteins were analysed by Hoechst staining, AlamarBlue staining, real time polymerase chain reaction, and western blotting, respectively. RESULTS: The protein expression of hMLH1 and hMSH2 mutants was significantly decreased after transfection compared with wild type transfections. The hMLH1 and hMSH2 interacting proteins hPMS2 and hMSH6 became detectable only after transfection of the respective wild type genes. In parallel, hMSH6 mRNA levels were increased in hMSH2 wt transfected cells. However, hPMS2 mRNA levels were independent of the mutation status of its interacting partner hMLH1, indicating a post-transcriptional regulating pathway. In the hMLH1 deficient HCT-116 cell line apoptosis was not affected by transfection of any mismatch repair gene, whereas complementation of hMSH2 deficiency in LOVO cells increased apoptosis. Conversely, proliferative activity of HCT-116 was decreased by complementation with hMLH1wt and unaffected in hMSH2 deficient LOVO cells. CONCLUSION: These data show that the cellular role of the MMR genes and its mutations are assessable in a simple transient transfection system and show the influence of MMR gene regulation on major cell growth regulating mechanisms. This method is applicable for the functional definition of mutations in hMLH1 and hMSH2 genes observed in patients with suspected HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Naturally-occurring missense mutations in the human growth hormone-releasing hormone receptor alter ligand binding.

Growth hormone (GH) releasing hormone (GHRH) is a hypothalamic factor that stimulates GH secretion. It acts by activating a seven transmembrane domain G protein-coupled receptor of 423 amino acids expressed by the somatotroph cells of the pituitary gland (GHRH receptor, GHRH-R). Familial isolated growth hormone deficiency (IGHD) can be caused by mutations in the GHRH-R gene both in humans and mice. We have described six disease-causing missense mutations in this gene in IGHD patients (H137L, L144H, A176V, A222E, F242C, K329E). These mutations are inherited as autosomal recessive traits, and cause impairment of the receptor to transmit GHRH signalling. The aim of this study is to investigate the mechanisms through which these mutations cause receptor malfunction. To this end, we transiently expressed each mutated receptor into Chinese hamster ovary cells. Cells expressing each of the mutated receptors did not show an increase in intracellular cyclic AMP in response to GHRH. Immunoprecipitation and immunofluorescence studies indicated that the amino acid changes do not cause protein degradation, and do not alter the proper insertion of the receptor into the cell membrane. Binding studies with human 125I-GHRH showed that the lack of response to GHRH is due to inability of all the mutated receptors to bind the ligand. These studies demonstrate that abnormal ligand binding is a common mechanism by which naturally occurring missense mutation alter GHRH-R function.

Analysis of Variance↗
PubMed2000

Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.

Mucolipidosis type IV (MLIV) is a developmental neurodegenerative disorder characterized by severe neurologic and ophthalmologic abnormalities. The MLIV gene, ML4 (MCOLN1), has recently been localized to chromosome 19p13.2-13.3 by genetic linkage. Here we report the cloning of a novel transient receptor potential cation channel gene and show that this gene is mutated in patients with the disorder. ML4 encodes a protein, which we propose to call mucolipin, which has six predicted transmembrane domains and is a member of the polycystin II subfamily of the Drosophila transient receptor potential gene family. The role of a potential receptor-stimulated cation channel defect in the pathogenesis of mucolipidosis IV is discussed.

Amino Acid Sequence↗
PubMedDate not supplied

[Arg506 --> Gln mutation of coagulation factor V (factor V Leiden) and transient cerebral ischemia at a young age in 3 members of the same family].

The Arg506 --> Gln coagulation factor V mutation (factor V Leiden) is the most frequent inherited abnormality of blood coagulation which predisposes to venous thromboembolism. Its association with an increased risk of arterial thrombosis is uncertain. We describe 3 members of the same family (a woman and her 2 children) who were heterozygous for the Arg506 --> Gln mutation and who presented cerebral transient ischemic attacks (TIA) at a young age. The patients (with the exception of one smoker) had no risk factors for TIA and no abnormality of the coagulation system other than the Arg506 --> Gln mutation. The observation of the mutation and TIA in 3 members of the same family may suggest the hypothesis of an association between the mutation and arterial thrombosis. This hypothesis must be interpreted with caution, due to the absence of objective instrumental findings in patients with TIA and to the high prevalence of the Arg506 --> Gln mutation in the general population.

Adult↗
PubMed1996

Functional characterization of five V2 vasopressin receptor gene mutations.

COS7 cells were transiently transfected with plasmids encoding mutant forms of the V2 vasopressin receptors corresponding to mutations [Y280C, L292P, R337stop, V277A, and G12E (the latter found in the same kindred with L292P)] recently identified in subjects with X-linked nephrogenic diabetes insipidus (NDI). cAMP response to dDAVP and AVP, saturation binding experiments with [3H]-AVP, immunofluorescence, and indirect ELISA studies were performed to characterize the functional consequences of these mutations. The Y280C, L292P, and R337stop mutant V2 receptors show substantially decreased cell surface expression and are functionally inactive. The V277A mutant receptor, though well expressed at the cell surface as seen by immunofluorescence and ELISA and having a dissociation constant with AVP similar to the wild type receptor, was functionally less active as seen by a substantially decreased receptor number (Bmax) and reduced cAMP stimulation by dDAVP. The G12E mutant was functionally the same as the wild type V2 receptor in both cAMP stimulation and binding. These results provide insight into residues critical for V2 receptor expression and function and also provide direct evidence that Y280C, L292P, R337stop and V277A mutations are the cause of X-linked NDI in affected subjects.

Animals↗
PubMed2002

Isolated peripheral neuropathy in atypical metachromatic leukodystrophy: a recurrent mutation.

BACKGROUND: Metachromatic leukodystrophy (MLD) is a genetic neurodegenerative disorder resulting from a deficiency of arylsulfatase A. Late onset forms are relatively rare. Central nervous system (CNS) involvement is characteristic at all ages. METHODS: A patient in her late 40s with peripheral neuropathy was assessed by EEG, evoked potentials, CT and nerve conduction studies. Nerve and muscle biopsy samples were investigated by electron microscopy. Arylsulfatase A activity in leukocytes and excreted cerebroside sulfate were determined. The arylsulfatase A gene was investigated for mutations using polymerase chain reaction (PCR) and DNA sequencing. The identified mutation was expressed transiently in African green monkey kidney (COS) cells to determine the effect of the mutation on arylsulfatase A activity. RESULTS: Central nervous system functions were normal. Nerve conduction velocities were decreased. Sural nerve biopsy showed inclusions typical of MLD. Arylsulfatase A was less than 5% of normal. A homozygous mutation thr286pro was identified in the arylsulfatase A gene and demonstrated to be deleterious through transient expression studies. CONCLUSIONS: Our patient has a progressive peripheral neuropathy but has apparently intact CNS function at her present age of 57 years. Biochemical, physiological and pathological findings are consistent with a diagnosis of MLD. A homozygous mutation, thr286pro, found in her arylsulfatase A gene, decreased enzyme activity to a level consistent with a late onset form of MLD.

Animals↗
PubMed2003

Nuclear factor 1 is required for both hormone-dependent chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter.

The mouse mammary tumor virus (MMTV) promoter has been used as a model to study how the glucocorticoid receptor (GR) remodels chromatin to allow other transcription factors to bind and activate transcription. To dissect the precise role of nuclear factor 1 (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant MMTV promoter in the context of transiently transfected templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We show that on a transiently transfected template, mutation in the NF1 binding site reduces both basal and hormone-dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a transiently transfected template is independent of mutation in hormone response elements or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a transiently transfected template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR-mediated chromatin remodeling of nucleosome B is reduced and hormone-dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for both the association of BRG1 chromatin remodeling complex and the GR on the promoter in vivo. These results suggest the novel possibility that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site the GR is unable to effectively bind the promoter and recruit the remodeling complex.

Animals↗
PubMed1997

Benign HEXA mutations, C739T(R247W) and C745T(R249W), cause beta-hexosaminidase A pseudodeficiency by reducing the alpha-subunit protein levels.

Two benign mutations, C739T(R247W) and C745T(R249W), in the alpha-subunit of beta-hexosaminidase A (Hex A) have been found in all but one of the currently identified Hex A-pseudodeficient subjects. To confirm the relationship of the benign mutations and Hex A pseudodeficiency and to determine how the benign mutations reduce Hex A activity, we transiently expressed each of the benign mutations, and other mutations associated with infantile, juvenile, and adult onset forms of GM2 gangliosidosis, as Hex S (alphaalpha) and Hex A (alphabeta) in COS-7 cells. The benign mutations decreased the expressed Hex A and Hex S activity toward the synthetic substrate 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide (4-MUGS) by 60-80%, indicating that they are the primary cause of Hex A pseudodeficiency. Western blot analysis showed that the benign mutations decreased the enzymatic activity by reducing the alpha-subunit protein level. No change in heat sensitivity, catalytic activity, or the substrate specificity to the synthetic substrates, 4-methylumbelliferyl-beta-N-acetylglucosaminide or 4-methylumbelliferyl-6-sulfo-beta-N-acetylglucosaminide, was detected. The effects of the benign mutations on Hex A were further analyzed in fibroblasts, and during transient expression, using pulse-chase metabolic labeling. These studies showed that the benign mutations reduced the alpha-subunit protein by affecting its stability in vivo, not by affecting the processing of the alpha-subunit, i.e. phosphorylation, targeting, or secretion. Our studies also demonstrated that these benign mutations could be readily differentiated from disease-causing mutations using a transient expression system.

Adult↗
PubMed2003

Hypermutation as a factor contributing to the acquisition of antimicrobial resistance.

Contrary to what was thought previously, bacteria seem to be, not merely spectators to their own evolution, but, through a variety of mechanisms, able to increase the rate at which mutations occur and, consequently, to increase their chances of becoming resistant to antibiotics. Laboratory studies and mathematical models suggest that, under stressful conditions, such as antibiotic challenge, selective pressure favors mutator strains of bacteria over nonmutator strains. These hypermutable strains have been found in natural bacterial populations at higher frequencies than expected. The presence of mutator strains in the clinical setting may indicate an enhanced risk of acquiring antibiotic resistance through mutational and recombinational events. In addition, some antibiotics are inducers of mechanisms that transiently increase the mutation rate, and thus probably act, not only as mere selectors of antibiotic resistant clones, but also as resistance-promoters.

Anti-Bacterial Agents↗
PubMed1998

Transient bullous dermolysis of the newborn associated with compound heterozygosity for recessive and dominant COL7A1 mutations.

The neonatal skin blistering disorder transient bullous dermolysis of the newborn (TBDN) heals spontaneously or improves dramatically within the first months and years of life. TBDN is characterized by subepidermal blisters, reduced or abnormal anchoring fibrils at the dermo-epidermal junction, and electron-dense inclusions in keratinocytes. These features are partly similar to those in dystrophic epidermolysis bullosa, which is caused by defects in COL7A1 gene encoding collagen VII, the main anchoring fibril protein. TBDN has been grouped separately from dystrophic epidermolysis bullosa based on the pronounced morphologic features and the self-limiting course of the disorder; however, it remains unclear whether it represents a distinct clinical entity with a single etiology. We now report a TBDN patient who is compound heterozygous for a recessive and a dominant glycine substitution mutation in COL7A1. Two point mutations caused amino acid substitutions G1519D and G2251E in the triple helical domain of collagen VII. In the heterozygous state G1519D was silent, and G2251E led to nail dystrophy, but not to skin blistering. In the proband, compound heterozygosity for the mutations caused massive, transitory retention of collagen VII in the epidermis, its reduced deposition at the basement membrane zone, and extensive dermo-epidermal separation at birth. Accordingly, TBDN keratinocytes in vitro accumulated collagen VII intracellularly in the rough endoplasmic reticulum.

Biopsy↗