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PubMed1990

Chromosomal structure is altered by mutations that suppress or enhance position effect variegation.

We examined the genetic, morphological, and molecular effects of position effect variegation in Drosophila, and the effects of mutations that either suppress [Su(var)] or enhance [E(var)] this phenomenon. All eight Su(var) mutations examined strongly suppress the inactivation of variegating alleles of the genes white [In(l) wm4], brown [In(2R) bwVDe2] and Stubble [T(2; 3) SbV]. The E(var) mutation enhances variegation of these loci. The chromosomal region 3C-E (26 bands) which includes the white locus is usually packaged as heterochromatin in salivary glands of the variegating strain wm4. Addition of any of the Su(var) mutations restores a more euchromatic morphology to this region. In situ hybridization to polytene chromosomes and DNA blot analyses of gene copy number demonstrate that the DNA of the w+ gene is less accessible to its probe in the variegating wm4 strain than it is in the wild-type or variegation-suppressed strains. Blot analysis of larval salivary gland DNA indicates that the white gene copy number does not vary among the strains. Hence, the differences in binding of the w+ gene probe in the variegating and variegation-suppressed strains reflect differences in chromosomal packaging rather than alterations in gene number. The effects of variegation and the Su(var) mutations on chromatin structure were analyzed further by DNAse I digestion and DNA blot hybridization. In contrast to their dramatic effects on chromosomal morphology and gene expression, the Su(var) mutations had negligible effects on nuclease sensitivity of the white gene chromatin. We suggest that the changes in gene expression resulting from position effect variegation and the action of the Su(var) mutations involve alterations in chromosomal packaging.

Animals↗
PubMed2001

Characterization of a glycine receptor domain that controls the binding and gating mechanisms of the beta-amino acid agonist, taurine.

The beta-amino acid, taurine, is a full agonist of the human glycine receptor alpha1 subunit when recombinantly expressed in a mammalian (HEK293) cell line, but a partial agonist of the same receptor when expressed in Xenopus oocytes. Several residues in the Ala101-Thr112 domain have previously been identified as determinants of beta-amino acid binding and gating mechanisms in Xenopus oocyte-expressed receptors. The present study used the substituted cysteine accessibility method to investigate the role of this domain in controlling taurine-specific binding and gating mechanisms of glycine receptors recombinantly expressed in mammalian cells. Asn102 and Glu103 are identified as taurine and glycine binding sites, whereas Ala101 is eliminated as a possible binding site. The N102C mutation also abolished the antagonistic actions of taurine, indicating that this site does not discriminate between the putative agonist- and antagonist-bound conformations of beta-amino acids. The effects of mutations from Lys104-Thr112 indicate that the mechanism by which this domain controls beta-amino acid-specific binding and gating processes differs substantially depending on whether the receptor is expressed in mammalian cells or Xenopus oocytes. Thr112 is the only domain element in mammalian cell-expressed GlyRs which was demonstrated to discriminate between glycine and taurine.

Amino Acid Substitution↗
PubMed2022

High tumor amplification burden is associated with TP53 mutations in the pan-cancer setting.

Next-generation sequencing data is fundamentally changing the clinical management of patients with cancer. The most frequent genomic alterations in malignancy are mutations and amplifications, with a subset of tumors having multiple amplifications - "amplificators". We sought to understand the molecular correlates of high tumor amplification burden in a pan-cancer context. Using both national registries and a single-institution dataset, our results demonstrate that cancers with TP53 mutations (as compared to those with wild-type TP53) exhibited significantly higher tumor amplification burden across all datasets. Amplifications, generally associated with overexpression, may be potentially actionable secondary consequences of TP53 mutations.

Biomarkers, Tumor↗
PubMed1996

Mutations in the peptidyl transferase center of 23 S rRNA reveal the site of action of sparsomycin, a universal inhibitor of translation.

Sparsomycin is a universal and powerful inhibitor of peptide bond formation which, in contrast to many other ribosome-targeted antibiotics, does not produce footprints on rRNA. A mutant of an archaeon Halobacterium halobium has been isolated that exhibits resistance to sparsomycin. Resistant cells possessed a mutation in the 23 S rRNA, where C2518 (C2499 in Escherichia coli) was substituted by U. Introduction of the C2518U mutation into the chromosomal 23 S rRNA gene of wild-type H. halobium rendered cells resistant to sparsomycin, demonstrating that a single nucleotide alteration in the rRNA is sufficient to confer resistance. Accordingly, ribosomes containing mutant 23 S rRNA exhibited increased tolerance to sparsomycin in vitro. Mutations of two other nucleotide positions in the peptidyl transferase center, C2471 and U2519 (C2452 and U2500 in E. coli), conferred resistance to low concentrations of sparsomycin. The location of the sparsomycin resistance mutations reveals the possible site of drug binding and/or action. Our findings provide further support for the idea that rRNA may be directly involved in interaction with antibiotics and the catalysis of the peptide bond formation.

Base Sequence↗
PubMed2001

Efficient virus extinction by combinations of a mutagen and antiviral inhibitors.

The effect of combinations of the mutagenic base analog 5-fluorouracil (FU) and the antiviral inhibitors guanidine hydrochloride (G) and heparin (H) on the infectivity of foot-and-mouth disease virus (FMDV) in cell culture has been investigated. Related FMDV clones differing up to 10(6)-fold in relative fitness in BHK-21 cells have been compared. Systematic extinction of intermediate fitness virus was attained with a combination of FU and G but not with the mutagen or the inhibitor alone. Systematic extinction of high-fitness FMDV required the combination of FU, G, and H. FMDV showing high relative fitness in BHK-21 cells but decreased replicative ability in CHO cells behaved as a low-fitness virus with regard to extinction mutagenesis in CHO cells. This confirms that relative fitness, rather than a specific genomic sequence, determines the FMDV response to enhanced mutagenesis. Mutant spectrum analysis of several genomic regions from a preextinction population showed a statistically significant increase in the number of mutations compared with virus passaged in parallel in the absence of FU and inhibitors. Also, in a preextinction population the types of mutations that can be attributed to the mutagenic action of FU were significantly more frequent than other mutation types. The results suggest that combinations of mutagenic agents and antiviral inhibitors can effectively drive high-fitness virus into extinction.

Animals↗
PubMed1997

Molecular studies on the voltage dependence of dihydropyridine action on L-type Ca2+ channels. Critical involvement of tyrosine residues in motif IIIS6 and IVS6.

The interaction site(s) of dihydropyridine (DHP) antagonists and agonists have been identified by site-directed mutagenesis and localized on motifs IIIS5, IIIS6, and IVS6 of L-type voltage-gated calcium channels. In this study, we investigated the voltage-dependent action of DHPs with mutants of the IIIS6 and IVS6 segments of a cardiac calcium channel. Tyrosine residues in both motifs (Tyr1178 and Tyr1489) strongly contributed to the action of DHP agonists and antagonists. When these two sites were mutated, the communication between the voltage sensor and the DHP interaction site(s) was substantially impaired. In contrast, mutants of a nearby Ile (Ile1182) had much less influence on DHP agonist and antagonist interaction, and the voltage dependence of DHP antagonists was very similar to that of the wild type. The effect of a mutating of Ile1182, on agonist or antagonist action, however, depended strongly on the type of amino acid change. When Ile1182 was substituted with alanine, small changes were noted for DHP agonist and antagonist action. Changing this site into phenylalanine, however, significantly decreased the action of the DHP antagonist. These data show that Ile1182 can preferentially interact with DHP antagonists, but has a lesser contribution in agonist interaction. Thus, even though the agonist and antagonist interaction sites for DHPs with L-type calcium channels may overlap, some amino acids in this site may exhibit a preference for either DHP enantiomers.

Animals↗
PubMed1994

The sevenmaker gain-of-function mutation in p42 MAP kinase leads to enhanced signalling and reduced sensitivity to dual specificity phosphatase action.

A mammalian mutant MAP kinase, D319N ERK2, analogous to Drosophila melanogaster sevenmaker (rlsem) gain-of-function mutation was shown to have an increased sensitivity to low levels of signalling in vivo. However, the mutation does not lead to an elevated basal kinase activity and still requires activation by MAP kinase kinase (MAPKK) as does wild type ERK2. This increased responsiveness seen in vivo is not due to an increased ability to phosphorylate substrates but appears to reflect a reduced sensitivity to a MAP kinase phosphatase CL100.

3T3 Cells↗
PubMed2004

[Pituitary-adrenal function in female mice with mutation Agouti yellow (A(y))].

Agouti protein is a paracrine signaling factor modulating action of ACTH and alpha-MSH. Dominant mutation Ay causes ectopic, ubiquitous expression of Agouti protein in mice. It was shown that Ay mutation increased stress-induced hypothalamo-adrenal activity in male mice. There is a sex difference in the hypothalamo-pituitary-adrenal axis in rodents. The aim of this study was to test effects of ectopic overexpression of Agouti protein on pituitary-adrenal function in female mice. Female mice of C57Bl/6J strain with Ay mutation (Ay/alpha) and with mutation nonagouti (alpha/alpha; lack of Agouti protein) were used. Ay/alpha-females had an increased blood level of corticosterone and ACTH after 10-minute restriction as compared with alpha/alpha-females. The adrenal threshold sensitivity and reaction to exogenous ACTH in vivo suggests that increased corticosterone reaction to emotional stress is caused by increased pituitary stimulation.

Adrenal Glands↗
PubMed2006

Upregulation of HERG channels by the serum and glucocorticoid inducible kinase isoform SGK3.

Human ether-a-go-go (HERG) channels participate in the repolarization of the cardiac action potential. Loss of function mutations of HERG lead to delayed cardiac repolarization reflected by prolonged QT interval. HERG channels are regulated through a signaling cascade involving phosphatidylinositol 3 (PI3) kinase. Downstream targets of PI3 kinase include the serum and glucocorticoid inducible kinase (SGK) and protein kinase B (PKB) isoforms. The present study has been performed to explore whether SGK1 and SGK3 participate in the regulation of HERG channel activity. HERG was expressed in Xenopus oocytes with or without additional expression of SGK1 or SGK3. Chemiluminescence was employed to determine HERG plasma membrane protein abundance. Coexpression of SGK3 but not of SGK1 in Xenopus oocytes resulted in an increase of steady state current (I(HERG)) and enhanced cell membrane protein abundance without affecting gating kinetics of the channel. Replacement of serine by alanine at the two SGK consensus sites decreased I(HERG) but neither mutation abolished the stimulating effect of SGK3. In conclusion, SGK3 participates in the regulation of HERG by increasing HERG protein abundance in the plasma membrane and may thus modify the duration of the cardiac action potential.

Action Potentials↗
PubMed1998

[Structure of the retinoic acid receptor and its functional analysis using a mutated receptor].

RA shows a variety of actions including the teratogenicity at pharmacological doses, but its physiological roles remain unclear. Recently, receptors for RA, RARs, have been identified and shown to belong to the nuclear receptor superfamily. We introduced a mutation, which was originally identified in the thyroid hormone receptor gene, causing dominantly inherited thyroid hormone resistance, to the equivalent position in RAR, and showed that the mutated RAR demonstrated a dominant-negative phenotypes. We next expressed the mutated RAR specifically in chondrogenic cells in mice. From the analysis of mice phenotypes, we unveiled the chondrogenic cells as a novel vital RA target in skeletal development. The results simultaneously indicated that RA specifies cervical identities through the regulation of homeobox genes.

Amino Acid Sequence↗
PubMed2000

Molecular genetics of arrhythmias--a new paradigm.

The molecular genetic background of inherited cardiac arrhythmias has only recently been uncovered. This late development in comparison to other inherited cardiac disorders has partly been due to the high mortality and early disease onset of these arrhythmias resulting in mostly small nucleus families. Thus, traditional genetic linkage studies, which are based on the genetic information obtained from large multi-generation families, were made difficult. Inherited arrhythmogenic disorders can be divided into 'primary electrical disorders' (e.g., long-QT [LQT] syndrome) in which a detectable, organic heart disease is not evident, and into inherited diseases of the myocardial structure (e.g., hypertrophic cardiomyopathies) in which the arrhythmias occur combined with the structural alterations. To date, all inherited arrhythmogenic disorders in which the causative genes have been identified turned out to be channelopathies, since the genes encode channel subunits that regulate important ion currents that tune the cardiac action potential. The discovery of the genetic bases of the LQT syndrome became a new methodologic paradigm; because with the use of 'classical' genetic linkage strategies (named [positional] candidate strategies) not only the causative genes have been found, but moreover, functional components with a previously unknown but fundamental role for a normal repolarization process were discovered. Disease mutations turned out to be not only a family-specific event with a distinct phenotype and the potential of an additional diagnostic tool, but also, when expressed in heterologous expression systems, characterize the defective ion channel in a topological way and lead to a more specific understanding of ion channel function. Most, if not all, primary electrical cardiac disorders show a high genetic diversity. For the LQT syndromes, sixth disease loci and the responsible gene have been recently discovered (so-called locus or genetic heterogeneity). Within all disease genes, the mutations are spread over the entire gene (allelic heterogeneity); in addition, more than one disease mutation may be present. This complexity requires, at least, complete mutation analysis of all LQT genes before medical advice should be given. Meanwhile, genotype-phenotype correlations in large families are being used to evaluate intergene, interfamilial and intrafamilial differences in the clinical phenotype, reflecting gene specific, gene-site specific and individual consequences of a given mutation. A widespread phenotypic heterogeneity even within mutation carriers in the same family raises the importance of modifying factors and genes that are mostly unknown to date. The reduced penetrance and variable expressivity associated with the LQT mutations remain still to be explained. First insights into the complex actions of mutations are being extracted, from expression data; these preliminary results may lead to potential implications for a specific (gene-site directed) therapy. This paper discusses the current data on molecular genetics and genotype-phenotype correlations in LQT syndrome and related disorders and the potential implications for diagnosis and treatment.

Arrhythmias, Cardiac↗
PubMed2001

Response to mivacurium in a patient compound heterozygous for a novel and a known silent mutation in the butyrylcholinesterase gene: genotyping by sequencing.

BACKGROUND: Patients who are homozygous for the atypical mutation, compound heterozygous for atypical and silent mutations, or homozygous for silent mutations (SS) respond to mivacurium with extensively prolonged neuromuscular block. Although important, exact phenotyping of these patients is difficult. This article presents the pharmacodynamics and pharmacokinetics of a normal dose of mivacurium in a patient with phenotype SS, including a pedigree analysis and delineation of the molecular genetic method used to identify the genotype. METHODS: The neuromuscular block following administration of mivacurium, at a dose of 0.14 mg/kg, was monitored in a 30-yr-old healthy man with use of a mechanosensor and mechanomyography, and times to different levels of recovery were measured. Venous samples for determination of the mivacurium isomers were collected during the interval 134-494 min after administration of mivacurium, and the terminal half-lives were calculated. Butyrylcholinesterase activity, phenotype, and genotype were determined for both the patient and the family. Complete nucleotide sequencing was used to identify the genotype. RESULTS: A train-of-four ratio of 0.75 was reached 469 min after the injection of mivacurium. The terminal elimination half-lives of the mivacurium isomers, cis-trans and trans-trans, were 90 min. Complete nucleotide sequencing revealed two point mutations, the known silent variant S7 and a previously undescribed mutation of amino acid residue 170 introducing a stop codon. CONCLUSIONS: The patient was compound heterozygous for silent mutations in the butyrylcholinesterase gene. The response to mivacurium was an extensively prolonged duration of action. Identification of the rare silent mutations presupposes access to modern molecular genetic methods such as complete nucleotide sequencing.

Adult↗
PubMed2000

Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function.

The recent unraveling of structures of genes for the gonadotropin subunits and gonadotropin receptors has provided reproductive endocrinologists with new tools to study normal and pathological functions of the hypothalamic-pituitary-gonadal axis. Rare inactivating mutations that produce distinctive phenotypes of isolated LH or FSH deficiency have been discovered in gonadotropin subunit genes. In addition, there is a common polymorphism in the LHbeta subunit gene with possible clinical significance as a contributing factor to pathologies of LH-dependent gonadal functions. Both activating and inactivating mutations have been detected in the gonadotropin receptor genes, a larger number in the LH receptor gene, but so far only a few in the gene for the FSH receptor. These mutations corroborate and extend our knowledge of clinical consequences of gonadotropin resistance and inappropriate gonadotropin action. The information obtained from human mutations has been complemented by animal models with disrupted or inappropriately activated gonadotropin ligand or receptor genes. These clinical and experimental genetic disease models form a powerful tool for exploring the physiology and pathophysiology of gonadotropin function and provide an excellent example of the power of molecular biological approaches in the study of pathogenesis of diseases.

Amino Acid Sequence↗
PubMed2003

A novel T608R missense mutation in insulin receptor substrate-1 identified in a subject with type 2 diabetes impairs metabolic insulin signaling.

Naturally occurring mutations in insulin receptor substrate-1 (IRS-1) have previously been implicated in impaired insulin action. We now report a novel mutation in IRS-1 with substitution of Arg for Thr(608) that was identified in a patient with type 2 diabetes mellitus. We detected the T608R mutation in 1 of 136 chromosomes from diabetic patients and in 0 of 120 chromosomes from nondiabetic controls, suggesting that this is a rare IRS-1 variant. Conservation of Thr(608) in human, monkey, rat, mouse, and chicken IRS-1 sequences is consistent with a crucial function for this residue. Moreover, Thr(608) is located near the YMXM motif containing Tyr(612) that is important for binding and activation of phosphoinositol 3-kinase (PI 3-kinase). To investigate whether the T608R mutation impairs insulin signaling, we transiently transfected NIH-3T3(IR) cells with hemagglutinin-tagged wild-type or T608R mutant IRS-1 constructs. Recombinant IRS-1 immunoprecipitated from transfected cells treated with or without insulin was subjected to immunoblotting for the p85 regulatory subunit of PI 3-kinase as well as a PI 3-kinase assay. As expected, in control cells transfected with wild-type IRS-1, insulin stimulation caused an increase in p85 coimmunoprecipitated with IRS-1 as well as a 10-fold increase in IRS-1-associated PI 3-kinase activity. Interestingly, when cells transfected with IRS1-T608R were stimulated with insulin, both the amount of p85 coimmunoprecipitated with IRS1-T608R as well as the associated PI 3-kinase activity were approximately 50% less than those observed with wild-type IRS-1. Moreover, in rat adipose cells, overexpression of IRS1-T608R resulted in significantly less translocation of GLUT4 to the cell surface than comparable overexpression of wild-type IRS-1. We conclude that a naturally occurring substitution of Arg for Thr(608) in IRS-1 is a rare human mutation that may contribute to insulin resistance by impairing metabolic signaling through PI 3-kinase-dependent pathways.

3T3 Cells↗
PubMed1987

[Mutants of Saccharomyces cerevisiae characterized by increased level of induced mutagenesis. I. Isolation and preliminary characterization of mutants].

6 mutants with enhanced nitrous acid-induced reversibility of the ade2-42 allele were isolated and designated hm (high mutagenesis). Apart from sensitivity to the mutagenic exposure to nitrous acid, hm mutants were also spontaneous mutators and hypermutable under the action of UV-light and 6-N-hydroxyaminopurine. All these effects were detected not only when analysing reversibility of the ade2-42 allele, but also when scoring forward mutations in the ADE1, ADF2 genes. Gamma-mutagenesis, however, was not affected by hm mutations.

Alleles↗
PubMed2004

Biallelic mutation of SOCS-1 impairs JAK2 degradation and sustains phospho-JAK2 action in the MedB-1 mediastinal lymphoma line.

Primary mediastinal B-cell lymphoma (PMBL) is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene because expression profiling showed high Janus kinase-2 (JAK2) transcript levels and JAK2 was found to be constitutively phosphorylated in mediastinal B-cell lymphomas. We confirm that in the MedB-1 mediastinal B-cell line, harboring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not overexpressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the suppressor of cytokine signaling-1 (SOCS-1) gene in this cell, which abrogates SOCS box function of the protein. Ectopic expression of wild-type (wt) SOCS-1 in MedB-1 leads to growth arrest and dramatic reduction of phospho-JAK2 and its downstream partner phospho-signal transducer and activator of transcription-5 (phospho-STAT5). Ultimately, the target gene cyclin D1 is repressed in transfectants while RB1, which is silenced in MedB-1, is induced. We conclude that, in MedB-1, action of phospho-JAK2 is sustained due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, SOCS-1 mutations are also present in the parental tumor of MedB-1 and were detected in 9 of 20 PMBLs.

Alleles↗
PubMed2006

Understanding anthelmintic resistance: the need for genomics and genetics.

Anthelmintic resistance is a major problem for the control of many parasitic nematode species and has become a major constraint to livestock production in many parts of the world. In spite of its increasing importance, there is still a poor understanding of the molecular and genetic basis of resistance. It is unclear which mutations contribute most to the resistance phenotype and how resistance alleles arise, are selected and spread in parasite populations. The main strategy used to identify mutations responsible for anthelmintic resistance has been to undertake experimental studies on candidate genes. These genes have been chosen predominantly on the basis of our knowledge of drug mode-of-action and the identification of mutations that can confer resistance in model organisms. The application of these approaches to the analysis of benzimidazole and ivermectin resistance is reviewed and the reasons for their relative success or failure are discussed. The inherent limitation of candidate gene studies is that they rely on very specific and narrow assumptions about the likely identity of resistance-associated genes. In contrast, forward genetic and functional genomic approaches do not make such assumptions, as illustrated by the successful application of these techniques in the study of insecticide resistance. Although there is an urgent need to apply these powerful approaches to anthelmintic resistance research, the basic methodologies and resources are still lacking. However, these are now being developed for the trichostrongylid nematode Haemonchus contortus and the current progress and research priorities in this area are discussed.

Animals↗
PubMed1989

Genotypic variation for a quantitative character maintained under stabilizing selection without mutations: epistasis.

A model of the gene action on a quantitative character is suggested. The model takes into account epistasis by combining multiplicative with the traditional additive approximation of the action of loci. It is demonstrated on the basis of this model that a high level of genotypic variation can be maintained in a population for a quantitative character under stabilizing selection in the absence of mutations, if there is epistasis. It is also shown that a large amount of additive variation as well as high heritability can be "hidden" in such a population and "released" if stabilizing selection is relaxed.

Computer Simulation↗