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PubMed2005

Light-regulated interaction of Dmoesin with TRP and TRPL channels is required for maintenance of photoreceptors.

Recent studies in Drosophila melanogaster retina indicate that absorption of light causes the translocation of signaling molecules and actin from the photoreceptor's signaling membrane to the cytosol, but the underlying mechanisms are not fully understood. As ezrin-radixin-moesin (ERM) proteins are known to regulate actin-membrane interactions in a signal-dependent manner, we analyzed the role of Dmoesin, the unique D. melanogaster ERM, in response to light. We report that the illumination of dark-raised flies triggers the dissociation of Dmoesin from the light-sensitive transient receptor potential (TRP) and TRP-like channels, followed by the migration of Dmoesin from the membrane to the cytoplasm. Furthermore, we show that light-activated migration of Dmoesin results from the dephosphorylation of a conserved threonine in Dmoesin. The expression of a Dmoesin mutant form that impairs this phosphorylation inhibits Dmoesin movement and leads to light-induced retinal degeneration. Thus, our data strongly suggest that the light- and phosphorylation-dependent dynamic association of Dmoesin to membrane channels is involved in maintenance of the photoreceptor cells.

Animals↗
PubMed1985

In vitro mutagenesis of the promoter region for a vaccinia virus gene: evidence for tandem early and late regulatory signals.

A vaccinia virus gene that is expressed throughout the reproductive cycle was found to have two sets of RNA start sites approximately 55 nucleotides apart. The site nearest to the coding segment is used early in infection and the one further upstream is used after DNA replication. A series of 5' to 3' deletions were made in the promoter region, and the truncated DNA segments were then ligated to the coding portion of the procaryotic chloramphenicol acetyltransferase gene to measure expression. The effects of these mutations on chloramphenicol acetyltransferase synthesis were determined in a vaccinia virus helper-dependent transient expression system and by forming infectious vaccinia virus recombinants that contain the chimeric genes. Deletions extending up to 31 nucleotides before the late RNA start site had no effect on either early or late expression. Removal of an additional 15 nucleotides produced a dramatic decrease in late expression but had no effect on early expression. The latter was not diminished until the deletion was extended from 31 to 24 nucleotides before the early RNA start site. These results were confirmed by transcriptional analyses. We concluded that this vaccinia virus gene has two promoters and that the regulatory signals for each are located within 31 nucleotides of their sites of transcription.

Base Sequence↗
PubMed1992

How a mutation in the gene encoding sigma 70 suppresses the defective heat shock response caused by a mutation in the gene encoding sigma 32.

In Escherichia coli, transcription of the heat shock genes is regulated by sigma 32, the alternative sigma factor directing RNA polymerase to heat shock promoters. sigma 32, encoded by rpoH (htpR), is normally present in limiting amounts in cells. Upon temperature upshift, the amount of sigma 32 transiently increases, resulting in the transient increase in transcription of the heat shock genes known as the heat shock response. Strains carrying the rpoH165 nonsense mutation and supC(Ts), a temperature-sensitive suppressor tRNA, do not exhibit a heat shock response. This defect is suppressed by rpoD800, a mutation in the gene encoding sigma 70. We have determined the mechanism of suppression. In contrast to wild-type strains, the level of sigma 32 and the level of transcription of heat shock genes remain relatively constant in an rpoH165 rpoD800 strain after a temperature upshift. Instead, the heat shock response in this strain results from an approximately fivefold decrease in the cellular transcription carried out by the RNA polymerase holoenzyme containing mutant RpoD800 sigma 70 coupled with an overall increase in the translational efficiency of all mRNA species.

Bacterial Proteins↗
PubMed1991

Androgen receptor abnormalities.

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2-8, we identified a G----T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the activation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G----C) results in an aspartic acid----histidine substitution (with negligible androgen binding), whereas the other mutation (G----A) leads to an aspartic acid----asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A----G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional errors in the human androgen receptor have an enormous impact on phenotype and fertility.

Amino Acid Sequence↗
PubMed2005

Identification of a bacterial factor required for actin-based motility of Burkholderia pseudomallei.

Burkholderia pseudomallei is a Gram-negative facultative intracellular pathogen that enters and escapes from eukaryotic cells using the power of actin polymerization. We have identified a bacterial protein (BimA) that is required for the ability of B. pseudomallei to induce the formation of actin tails. BimA contains proline-rich motifs and WH2-like domains and shares limited homology at the C-terminus with the Yersinia autosecreted adhesin YadA. BimA is located at the pole of the bacterial cell at which actin polymerization occurs and mutation of bimA abolished actin-based motility of the pathogen in J774.2 cells. Transient expression of BimA in HeLa cells resulted in F-actin clustering reminiscent of that seen on WASP overexpression. Antibody-mediated clustering of a CD32 chimera in which the cytoplasmic domain was replaced with BimA resulted in localization of the chimera to the tips of F-actin enriched membrane protrusions. We report that purified truncated BimA protein binds monomeric actin in a concentration-dependent manner in cosedimentation assays and that BimA stimulates actin polymerization in vitro in a manner independent of the cellular Arp2/3 complex.

Actins↗
PubMed1998

Requirement of corepressor binding of thyroid hormone receptor mutants for dominant negative inhibition.

Resistance to thyroid hormone (RTH) is mainly caused by dominant negative inhibition of wild type thyroid hormone receptor (TR) function due to mutations in the ligand binding domain of the TR beta. Because no RTH mutant was identified in the hinge region of the TR, the contribution of this region for dominant negative inhibition was explored. In transient transfection assays, the dominant negative activity of a RTH-type mutant (P453X) was abolished by an introduction of a mutation into the hinge region (P214R). Although this hinge mutation did not alter homo- or heterodimerization with retinoid X receptor (RXR), its association with nuclear receptor corepressor (N-CoR) was impaired. These results indicate that association of corepressor with the RTH mutants through the hinge region is crucial for their dominant negative activity.

Binding Sites↗
PubMed1998

A point mutation in the human cytomegalovirus DNA polymerase gene selected in vitro by cidofovir confers a slow replication phenotype in cell culture.

In cell culture, cidofovir (CDV) was used to select a human cytomegalovirus (HCMV) strain with decreased drug susceptibility. The genotypic characterization of this virus revealed a single base substitution resulting in a K513N amino acid alteration in the viral DNA polymerase (UL54). Performed in parallel, the selection of HCMV for replication in the presence of ganciclovir (GCV) selected an M460V substitution in the phosphotransferase (UL97), as well as a K513N/V812L double substitution in DNA polymerase. Neither of the two DNA polymerase mutations has been previously identified in HCMV drug-resistant strains. To precisely elucidate their role in drug resistance, corresponding recombinant mutant viruses were generated by recombination of nine overlapping viral DNA fragments. The K513N recombinant virus showed 13- and 6.5-fold decreased susceptibility to CDV and GCV in vitro, respectively, compared with the wild-type recombinant virus. Mutation V812L was associated with a moderate (2-3-fold) decrease in susceptibility to CDV, GCV, foscarnet, and adefovir. A multiplicative interaction of the K513N and V812L mutations with regard to the profile and level of drug resistance was demonstrated in recombinant virus expressing both mutations. In vitro replication kinetic experiments revealed that the K513N mutation significantly decreased HCMV replication capacity. Consistent with this finding, the K513N mutant DNA polymerase exhibited reduced specific activity in comparison with the wild-type enzyme and was severely impaired in its 3'-5' exonuclease function. Unexpectedly, the K513N mutant enzyme showed no decrease in susceptibility to CDV-diphosphate or GCV-triphosphate. However, the K513N mutation decreased the susceptibility to CDV and GCV of the oriLyt plasmid replication in the transient transfection/infection assay, suggesting that the DNA replication of the K513N mutant virus is less sensitive to the corresponding inhibitors.

Amino Acid Sequence↗
PubMed2004

Rapid and transient activation of transcription of the ERF3 gene by wounding in tobacco leaves: possible involvement of NtWRKYs and autorepression.

This study investigated the regulatory mechanism of rapid and transient induction of a transcriptional repressor ERF3 gene by wounding in tobacco (Nicotiana tabacum) leaves. Deletion and mutation analysis of the promoter region have suggested that the proximal W boxes (TGAC(C/T)) and a GCC box, respectively, may be involved in the positive and negative regulation of wound-induced expression of the ERF3 gene. Electrophoretic mobility shift assays indicated that wounding enhanced the specific binding activity of nuclear factors to the W boxes. NtWRKY1, -2, and -4, which are tobacco group I WRKYs, interacted specifically with the W boxes and activated transcription via the W boxes. On the other hand, deletion of the GCC box from NsERF3 promoter-GUS reporter gene caused a delay in down-regulation of transcription after wound induction. In addition, ERF3 repressed transcription via the NsERF3 promoter activated by NtWRKYs. These results suggest the possible involvement of NtWRKYs and autorepression in the rapid and transient expression of the ERF3 gene by wounding.

Base Sequence↗
PubMed1988

Biological activities of oligonucleotides spanning the F9 point mutation within the enhancer region of polyomavirus DNA.

A mutant of polyomavirus, F441, selected to grow in undifferentiated mouse F9 embryonal carcinoma cells, carries a single-base change in the enhancer region at nucleotide (nt) 5233 of the viral genome. Enhancers of most of the F9 mutants have a duplicated segment of viral DNA encompassing nt 5233. The minimum duplicated segment of all the known F9 mutants is from nt 5218 to nt 5239. We prepared oligonucleotides spanning the sequence from nt 5218 through nt 5239 of the genome of the wild type and F441 and examined the biological activities of the oligonucleotides by a transient assay of chloramphenicol acetyltransferase (CAT) gene expression in F9 cells. The oligonucleotide harboring the F441 mutation was shown to increase cat gene expression in F9 cells when linked at an upstream position in both orientations. When dimerized at an upstream position, the F441 oligonucleotide showed even higher cat gene expression enhancing activity. In contrast, no such effects were observed with the oligonucleotide of the wild-type sequence. In addition, the F441 oligonucleotide, but not the wild-type sequence, could inhibit the activity of whole enhancer fragment of F441 when cotransfected into F9 cells in excess amounts. On the basis of the results obtained, we suggest that the segment of F441 enhancer encompassing the point mutation contains a target for a cellular factor(s) which acts in a positive manner to increase the transcription of a gene in undifferentiated mouse F9 cells.

Base Sequence↗
PubMed1999

Translational effects of mutations and polymorphisms in a repressive upstream open reading frame of the human cytomegalovirus UL4 gene.

The human cytomegalovirus (HCMV) gpUL4 mRNA contains a 22-codon upstream open reading frame (uORF2), the peptide product of which represses downstream translation by blocking translation termination at its own stop codon and by causing ribosomes to stall on the mRNA. A distinctive feature of this unusual mechanism is its strict dependence on the uORF2 peptide sequence. To delineate sequence elements that function in the inhibitory mechanism, deletions and missense mutations affecting the previously uncharacterized amino-terminal region of uORF2 were analyzed in transient-transfection and infection assays. These experiments identified multiple codons in this region that are necessary for inhibition of downstream translation by uORF2 and, in conjunction with previous results, demonstrated that amino acids dispersed throughout the uORF2 peptide participate in the repressive mechanism. In contrast to the highly conserved carboxy terminus, the amino-terminal portion of the uORF2 peptide is polymorphic. A survey of uORF2 sequences in HCMV clinical isolates revealed that although most have uORF2 sequences that are predicted to retain the uORF2 inhibitory activity, approximately 15% contain polymorphisms at codons that are essential for full inhibition by uORF2. Consistent with predictions based on analyses of engineered mutations, two viral isolates with uORF2 sequences that do not inhibit downstream translation in transfection assays expressed much more gpUL4 protein but similar levels of UL4 mRNA compared to the levels produced by the prototypic laboratory strain HCMV (Towne) and another clinical isolate with an inhibitory variant uORF2. These results demonstrate that uORF2 is polymorphic in sequence and repressive activity and suggest that the uORF2 regulatory mechanism, although prevalent among natural HCMV isolates, is not absolutely essential for viral replication.

Amino Acid Sequence↗
PubMed1991

Two new arylsulfatase A (ARSA) mutations in a juvenile metachromatic leukodystrophy (MLD) patient.

Fragments of the arylsulfatase A (ARSA) gene from a patient with juvenile-onset metachromatic leukodystrophy (MLD) were amplified by PCR and ligated into MP13 cloning vectors. Clones hybridizing with cDNA for human ARSA were selected, examined for appropriate size inserts, and used to prepare single-stranded phage DNA. Examination of the entire coding and most of the intronic sequence revealed two putative disease-related mutations. One, a point mutation in exon 3, resulted in the substitution of isoleucine by serine. Introduction of this alteration into the normal ARSA cDNA sequence resulted in a substantial decrease in ARSA activity on transient expression in cultured baby hamster kidney cells. About 5% of the control expression was observed, suggesting a small residual activity in the mutated ARSA. The second mutation, a G-to-A transition, occurred in the other allele and resulted in an altered splice-recognition sequence between exon 7 and the following intron. The mutation also resulted in the loss of a restriction site. Apparently normal levels of mRNA were generated from this allele, but no ARSA activity or immuno-cross-reactive material could be detected. A collection of DNA samples from known or suspected MLD patients, members of their families, and normal controls was screened for these mutations. Four additional individuals carrying each of the mutations were found among the nearly 100 MLD patients in the sample. Gene segregation in the original patient's family was consistent with available clinical and biochemical data. No individuals homozygous for either of these two mutations were identified. However, combinations with other MLD mutations suggest that the point mutation in exon 3 does result in some residual enzyme activity and is associated with late-onset forms of the disease. The splice-site mutation following exon 7 produces late-infantile MLD when combined with other enzyme-null mutations, implying that it is completely silent enzymatically.

Adolescent↗
PubMed1989

Mutation of glycine 49 to valine in the alpha subunit of GS results in the constitutive elevation of cyclic AMP synthesis.

The G-protein GS couples hormone-activated receptors with adenylyl cyclase and stimulates increased cyclic AMP synthesis. Transient expression in COS-1 cells of cDNAs coding for the GS alpha-subunit (alpha S) or alpha S cDNAs having single amino acid mutations Gly49----Val or Gly225----Thr elevated cyclic AMP levels, resulting in the activation of cyclic AMP dependent protein kinase. Stable expression in Chinese hamster ovary cells of alpha S Val49 cDNA resulted in a small constitutive elevation of cyclic AMP that was sufficient to persistently activate cyclic AMP dependent protein kinase activity 1.5-2-fold over basal activity. Stable expression of wild-type alpha S or alpha S Thr225 in Chinese hamster ovary cells was less effective in sustaining elevated cyclic AMP synthesis and kinase activation compared to alpha SVal49.

Animals↗
PubMed2003

Disruption of the Cys5-Cys7 disulfide bridge in the platelet glycoprotein Ibbeta prevents the normal maturation and surface exposure of GPIb-IX complexes.

This work aimed at elucidating the molecular genetic defect in two related patients with Bernard-Soulier syndrome (BSS) phenotype. Flow cytometric analysis revealed undetectable levels of platelet glycoproteins (GP), Ibalpha and IX, although plasma glycocalicin was detectable in both cases. The complete sequencing of GPIbalpha, GPIbbeta, and GPIX revealed the presence of a single point mutation, a G to A substitution, in codon 30 of GPIbbeta, that changes Cys5 to Tyr. The parents and sibling of the patients, heterozygotes for this mutation, were asymptomatic and they all showed a reduced platelet content of GPIbalpha and GPIX. Transient transfection of the mutant GPIbalpha subunit failed to render surface expression of GPIbalpha and exerted a dominant-negative effect on the surface exposure of the GPIb-IX complex. Metabolic labelling and immunoprecipitation analysis of transfected cells indicated that [5Tyr]GPIbbeta may associate with GPIX and GPIbalpha, but the maturation of the GPIb-IX complex is impaired. Substitution of either Cys5 or Cys7 by Ala failed to show surface expression of GPIb-IX, suggesting that the Cys5- Cys7 disulfide loop in GPIbbeta is essential for the efficient processing and trafficking of GPIb-IX complexes toward the plasma membrane. Our findings indicate that the identified novel GPIbbeta mutation is responsible for the BSS phenotype of the patients and provide an explanation for the molecular mechanism underlying the reduced platelet content of GPIb-IX complex in the heterozygous individuals.

Adult↗
PubMed1999

Genetic factors affecting the impact of DNA polymerase delta proofreading activity on mutation avoidance in yeast.

Base selectivity, proofreading, and postreplication mismatch repair are important for replication fidelity. Because proofreading plays an important role in error correction, we have investigated factors that influence its impact in the yeast Saccharomyces cerevisiae. We have utilized a sensitive mutation detection system based on homonucleotide runs of 4 to 14 bases to examine the impact of DNA polymerase delta proofreading on mutation avoidance. The contribution of DNA polymerase delta proofreading on error avoidance was found to be similar to that of DNA polymerase epsilon proofreading in short homonucleotide runs (A4 and A5) but much greater than the contribution of DNA polymerase epsilon proofreading in longer runs. We have identified an intraprotein interaction affecting mutation prevention that results from mutations in the replication and the proofreading regions, resulting in an antimutator phenotype relative to a proofreading defect. Finally, a diploid strain with a defect in DNA polymerase delta proofreading exhibits a higher mutation rate than a haploid strain. We suggest that in the diploid population of proofreading defective cells there exists a transiently hypermutable fraction that would be inviable if cells were haploids.

Base Sequence↗
PubMed2006

Transient cardiomyopathy in a patient with congenital contractural arachnodactyly (Beals syndrome).

We report on an infant with Beals syndrome (congenital contractural arachnodactyly [CCA], MIM 121050) with transient cardiomyopathy showing ballon-like dilatation of the left ventricle that was similar to noncompaction. The patients father and two of his brothers were also found to have CCA without cardiovascular complications. CCA, which is caused by a mutation of the gene for fibrillin 2 protein is similar to Marfan syndrome (MIM 154700), which is caused by a mutation of fibrillin 1 but produces a life-threatening cardiovascular complications. This is the first report of CCA with transient cardiomyopathy. We discuss the mechanism of the spontaneous improvement of cardiomyopathy in this case on the basis of expression of the responsible gene.

Cardiomyopathies↗
PubMed2004

Lipid metabolism in zebrafish.

Forward genetics is an unbiased methodology to discover new genes or functions of genes. At the present, the zebrafish is one of the few vertebrate systems where large-scale forward genetic studies are practical. Fluorescent lipid labeling of zebrafish larvae derived from families created from ENU-mutagenized fish enabled us to perform a large scale in vivo screen to identify mutants with perturbed lipid processing. With the aid of the zebrafish genome project, positional cloning of mutated genes with abnormal lipid metabolism can be accelerated. MO- and gripNA-based transient gene silencing is feasible in zebrafish embryos and provides a reverse genetic screening strategy to search for important lipid regulators. The advantages of using zebrafish as a vertebrate model to study lipid metabolism include its rapid external development and its optical clarity that enables the monitoring of biological processes. Large scale, high-throughput drug screening in vivo, especially for drugs that inhibit lipid absorption, can be easily achieved in this model. These zebrafish-based assays are important tools to understand aspects of lipid biology with significant clinical implications.

4-Chloro-7-nitrobenzofurazan↗