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PubMed1970

Catabolite-insensitive revertants of lac promoter mutants.

The maximum rate of expression of the lac operon is severely reduced in lac promoter mutants. Revertants of these mutations which produce higher levels of enzyme were isolated. Some of these revertants had lost sensitivity to catabolite repression and transient repression. The mutations responsible for these losses took place at sites very close to the original promoter mutations. From these results we conclude that the promoter itself is the target site for both catabolite and transient repression of the lac operon.

Chromosome Mapping↗
PubMed1996

Familial Alzheimer's disease-linked mutations at Val717 of amyloid precursor protein are specific for the increased secretion of A beta 42(43).

In some pedigrees of familial Alzheimer's disease (FAD), three mutations of beta amyloid precursor protein (APP) have been found at the Val717 residue (to Ile, Phe, or Cly) and these mutations increase the secretion of A beta 42(43). To study the specificity of the effects of these mutations on APP processing, we transiently expressed APP genes with mutations of Val717 to Lys, Ser, Glu, or Cys in COS cells. The three familial AD-linked mutations increased the levels or ratios of A beta 42(43), whereas the secretion of A beta 40 was decreased. Other mutations irrelevant to FAD except Val717 to Lys had little effect on the ratio of A beta 42(43). Substitution to Lys decreased the secretion of A beta 42(43); substitution to Glu or Gly decreased the amount of intracellular C-terminal fragment produced by alpha-secretase, whereas it was increased by mutations to Phe, Cys, or Lys. However, the levels of secretion of soluble APP were constant, but a substitution to Glu reduced it. These results suggest a specific role of the Val717 residue in APP processing and, especially, in gamma-cleavage.

Alzheimer Disease↗
PubMed1996

Identification of a carbohydrate response element in rat S14 gene.

To characterize the mechanism by which carbohydrate feeding regulates S14 gene transcription, we created mutations within footprinted sequences of the carbohydrate response region of the S14 gene and either transiently transfected these mutations into rat primary hepatocytes or end-labeled them for gel mobility shift assays. The wild type DNA gave a 2.9 +/- 1.0-fold response to raising the media glucose concentration. Mutations within the 3' footprint eliminated the glucose response and also eliminated hepatic nuclear factor binding. However, mutations within the 5' footprint did not inhibit the glucose responsiveness and had no effect on hepatic nuclear binding. The gel mobility shift assays were confirmed by appropriate competition gel assays. The 3' footprint contains a sequence similar to the USF (upstream stimulating factor) binding site (CACGTG) that has been suggested to play a role in carbohydrate regulation. However, the gel shift pattern with purified USF1 is distinctly different from that of rat hepatic nuclear extract. These studies demonstrate that the sequence GGCACCCGTGT is required for the carbohydrate response in the S14 gene.

Animals↗
PubMed2000

Experimental analysis of molecular events during mutational periodic selections in bacterial evolution.

A fundamental feature of bacterial evolution is a succession of adaptive mutational sweeps when fitter mutants take over a population. To understand the processes involved in mutational successions, Escherichia coli continuous cultures were analyzed for changes at two loci where mutations provide strong transport advantages to fitness under steady-state glucose limitation. Three separate sweeps, observed as classic periodic selection events causing a change in the frequency of neutral mutations (in fhuA causing phage T5 resistance), were identified with changes at particular loci. Two of the sweeps were associated with a reduction in the frequency of neutral mutations and the concurrent appearance of at least 13 alleles at the mgl or mlc loci, respectively. These mgl and mlc polymorphisms were of many mutational types, so were not the result of a mutator or directed mutation event. The third sweep observed was altogether distinct and involved hitchhiking between T5 resistance and advantageous mgl mutations. Moreover, the hitchhiking event coincided with an increase in mutation rates, due to the transient appearance of a strong mutator in the population. The spectrum of mgl mutations among mutator isolates was distinct and due to mutS. The mutator-associated periodic selection also resulted in mgl and fhuA polymorphism in the sweeping population. These examples of periodic selections maintained significant genotypic diversity even in a rapidly evolving culture, with no individual "winner clone" or genotype purging the population.

Adenosine Triphosphatases↗
PubMed1999

Three steroidogenic factor-1 binding elements are required for constitutive and cAMP-regulated expression of the human adrenocorticotropin receptor gene.

In the present study, we characterized two new SF-1 binding sites, SF-209 and SF-98, in the promoter of the human ACTH receptor (hACTH-R) gene. Both sites, together with the previously described SF-35 site, are required for full constitutive activity of this gene. This was demonstrated by the use of constructs containing part of the promoter upstream of the luciferase gene and carrying mutation in one of these sites, to transiently transfect H295R cells. Mutations of either SF-35, SF-98, or SF-209 induced a decrease of luciferase activity. This effect was amplified when two or three elements were mutated together in the same construct. Only SF-35 and SF-98 seem to play a major role in the cAMP-induced regulation of the hACTH-R gene, since mutation of either one of these sites reduced the forskolin induction of luciferase activity by 50%. When both elements were mutated, no stimulation was obtained over the control. This indicates that SF-1 protein must bind to both sites for the cAMP response.

Binding Sites↗
PubMed1983

Paradoxical fixation of deleterious alleles in two-locus systems with epistasis.

The dynamics of four deterministic models of interaction between two Mendelian loci are explored numerically. At one locus there are two hypostatic alleles: h, the wild type and H, a deleterious mutant that in either heterozygous or homozygous state (depending on the specifics of the model) produces an abnormal phenotype. At another arbitrarily linked epistatic locus there are two alleles: e, the wild type with no effect on the expression of the H locus, and E, an epistatic mutant that in heterozygous or homozygous state (again, depending on the specifics of the model) blocks the expression of H. The parameters are the initial gamete frequencies, the recombination fraction, the genotypic viabilities, and the forward and back mutation rates at each locus. The He haplotype is eventually eliminated (unless back mutation occurs) from the population. If mutation is ignored, the evolutionary outcome is determined by the initial gamete frequencies and is either (1) an edge equilibrium comprising one pair of haplotypes only (he and He, or he and hE, or hE and HE, or He and HE, or, if there is no recombination, he and HE or hE and He); or (2) a corner equilibrium consisting of a single gametic type. Given that the forward mutation rates at both loci are greater than the back mutation rates, then the outcome is always the corner equilibrium in which HE is universal (apart from transient perturbations by mutation). In the process of fixation, the phenotypic impact of the deleterious allele H becomes neutralized by the epistatic allele E. The rate at which the initially harmful gene replaces the wild type gene depends on the recombination fraction, the genotypic viabilities, and the mutation rates.

Alleles↗
PubMed1996

Galpha12 differentially regulates Na+-H+ exchanger isoforms.

Activation of several GTPases stimulates Na+-H+ exchange, resulting in an increased efflux of intracellular H+. These GTPases include alpha subunits of the heterotrimeric G proteins Gq and G13, as well as the low molecular weight GTP-binding proteins Ras, Cdc42, and Rho (Hooley, R., Yu, C.-Y., Simon, M., and Barber, D. L. (1996) J. Biol. Chem. 271, 6152-6158). GTPases coupled to the inhibition of Na+-H+ exchange, however, have not been identified. Several neurotransmitters, including somatostatin and dopamine, inhibit Na+-H+ exchange through a guanine-nucleotide-dependent mechanism, suggesting the involvement of a GTPase. In this study we determined that mutational activation of the alpha subunit of G12 inhibits the ubiquitously expressed Na+-H+ exchanger isoform, NHE1. Transient expression of mutationally activated Galpha12 inhibited serum- and Galpha13-stimulated NHE1 activity in HEK293 cells and CCL39 fibroblasts. In addition, in NHE-deficient AP1 cells stably expressing specific NHE isoforms, mutationally activated Galpha12 inhibited NHE1 activity but stimulated activities of the Na+-H+ exchanger (NHE) isoforms NHE2 and NHE3. In contrast, mutationally activated Galpha13, another member of the Galpha12/13 family, stimulated all three NHE isoforms. Although previous studies have identified a parallel action of Galpha12 and Galpha13 in regulating MAP (mitogen-activated protein) kinases and cell growth, these GTPases have opposing effects on NHE1 activity.

Animals↗
PubMed1999

The arginine 276 anchor for NADP(H) dictates fluorescence kinetic transients in 3 alpha-hydroxysteroid dehydrogenase, a representative aldo-keto reductase.

Fluorescence stopped-flow studies were conducted with recombinant rat liver 3 alpha-HSD, an aldo-keto reductase (AKR) that plays critical roles in steroid hormone inactivation, to characterize the binding of nicotinamide cofactor, the first step in the kinetic mechanism. Binding of NADP(H) involved two events: the fast formation of a loose complex (E.NADP(H)), followed by a conformational change in enzyme structure leading to a tightly bound complex (E.NADP(H)), which was observed as a fluorescence kinetic transient. Binding of NAD(H) was not characterized by a similar kinetic transient, implying a difference in the mode of binding of the two cofactors. Unlike previously characterized AKRs, the rates associated with the formation and decay of E.NADP(H) and E.NADP(H) were much faster than kcat for the oxidoreduction of various substrates, indicating that binding and release of cofactor is not rate-limiting overall in 3 alpha-HSD. Mutation of Arg 276, a highly conserved residue in AKRs that forms a salt bridge with the adenosine 2'-phosphate of NADP(H), resulted in large changes in Km and Kd for NADP(H) that were not observed with NAD(H). The loss in free energy associated with the increase in Kd for NADP(H) is consistent with the elimination of an electrostatic link. Importantly, this mutation abolished the kinetic transient associated with NADPH binding. Thus, anchoring of the adenosine 2'-phosphate of NADPH by Arg 276 appears to be obligatory for the fluorescence kinetic transients to be observed. The removal of Trp 86, a residue involved in fluorescence energy transfer with NAD(P)H, also abolished the kinetic transient, but mutation of Trp 227, a residue on a mobile loop associated with cofactor binding, did not. It is concluded that in 3 alpha-HSD, the time dependence of the change in Trp 86 fluorescence is due to cofactor anchoring, and thus, Trp 86 is a distal reporter of this event. Further, the loop movement that accompanies cofactor binding is spectrally silent.

3-Hydroxysteroid Dehydrogenases↗
PubMed2002

Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy.

Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)-mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 x 10(12) vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.

Animals↗
PubMed2001

The chum salmon insulin-like growth factor II promoter requires Sp1 for its activation by C/EBPbeta.

The chum salmon insulin-like growth factor II (IGF-II) gene is highly expressed in liver tissue. In this study we demonstrate that two transcription factors, Sp1 and C/EBPbeta, are involved in the enhanced expression of the salmon IGF-II gene. The presence of the fish homolog for C/EBPbeta in salmon liver RNA was confirmed by Northern blotting. The sIGF-II promoter was activated up to 20-fold by co-transfection with C/EBPbeta. The functional importance of four out of the five putative C/EBPbeta binding sites was demonstrated with mutational analysis in transient transfection assays. The transcription factor Sp1 binds to two sites within the salmon IGF-II promoter. Interestingly, mutation of the Sp1 binding sites decreases not only the basal IGF-II promoter activity but also the C/EBPbeta-induced transactivation. These results demonstrate that liver-enriched C/EBPbeta and ubiquitously expressed Sp1 each activate the sIGF-II promoter and that Sp1 is required for full transactivation of the sIGF-II gene by C/EBPbeta. This suggests that C/EBPbeta and Sp1 act in synergy.

Animals↗
PubMedDate not supplied

Neonatal reductions in osteoclast number and function account for the transient nature of osteopetrosis in the rat mutation microphthalmia blanc (mib).

We have examined the general and skeletal manifestations of osteopetrosis in a new, mild osteopetrotic mutation in the rat, microphalmia blanc (mib). Newborn mutant (mib) rats exhibit the typical skeletal deformities and sclerosis of osteopetrosis at birth, which are reduced significantly during the first postnatal month but don't disappear entirely up to 8 months later. Osteoclast numbers, staining for TRAP and TraATPase, and bone resorption are reduced in mutants during the first 2 postnatal weeks but improve by 1 month. In mutants, serum concentrations of calcium and phosphorus are normal, but 1,25(OH)2 D levels are higher at 1 week than those in normal littermates. Neonatally, mutants exhibit extramedullary hemopoiesis in the spleen. These results are interpreted to mean that the transient perinatal skeletal sclerosis in mib rats is caused by reduced production and function of osteoclasts in this period. The recent description of transient, perinatal osteopetrosis in a child suggests that analyses of the early differences between mild and severe animal mutations might distinguish those children with osteopetrosis who need treatment from those who do not.

Aging↗
PubMed2005

Relapsing diabetes can result from moderately activating mutations in KCNJ11.

Neonatal diabetes can either remit and hence be transient or else may be permanent. These two phenotypes were considered to be genetically distinct. Abnormalities of 6q24 are the commonest cause of transient neonatal diabetes (TNDM). Mutations in KCNJ11, which encodes Kir6.2, the pore-forming subunit of the ATP-sensitive potassium channel (K(ATP)), are the commonest cause of permanent neonatal diabetes (PNDM). In addition to diabetes, some KCNJ11 mutations also result in marked developmental delay and epilepsy. These mutations are more severe on functional characterization. We investigated whether mutations in KCNJ11 could also give rise to TNDM. We identified the three novel heterozygous mutations (G53S, G53R, I182V) in three of 11 probands with clinically defined TNDM, who did not have chromosome 6q24 abnormalities. The mutations co-segregated with diabetes within families and were not found in 100 controls. All probands had insulin-treated diabetes diagnosed in the first 4 months and went into remission by 7-14 months. Functional characterization of the TNDM associated mutations was performed by expressing the mutated Kir6.2 with SUR1 in Xenopus laevis oocytes. All three heterozygous mutations resulted in a reduction in the sensitivity to ATP when compared with wild-type (IC(50) approximately 30 versus approximately 7 microM, P-value for is all <0.01); however, this was less profoundly reduced than with the PNDM associated mutations. In conclusion, mutations in KCNJ11 are the first genetic cause for remitting as well as permanent diabetes. This suggests that a fixed ion channel abnormality can result in a fluctuating glycaemic phenotype. The multiple phenotypes associated with activating KCNJ11 mutations may reflect their severity in vitro.

ATP-Binding Cassette Transporters↗
PubMed2003

Bacterial multicellularity as a possible source of antibiotic resistance.

Knowledge about survival of micro-organisms in stressful situations not only influences the evolutionary theory in a fundamental way, but bears an extraordinary importance in finding a global solution to a very concrete urgent problem of mankind, namely bacterial resistance to antibiotics. Recent in vitro experiments demonstrate that the adaptive mutation process involving transient hypermutators could be one of the most important mechanisms whereby bacterial cells achieve the antibiotic resistance. An effective response of the mutation rates to specific selective conditions and an increasing number of conclusive evidence that bacterial cells are indeed communicative and co-operative organisms lead us to a hypothesis that the emergence of the antibiotic resistant mutants through the so-called adaptive mutation is deeply connected with the multicellular organisation of bacterial cells.

Anti-Bacterial Agents↗
PubMed1994

Dental abnormalities in the osteopetrotic rat mutation microphthalmia blanc.

Dental manifestations of the mild, transient osteopetrosis in the rat mutation microphthalmia blanc (mib) were examined. Eruption of all teeth was delayed in mib rats compared to normal littermates. The delays ranged from 5 days for incisors to 3 and 2 days for the first and second molars. Normal rats had straight incisors in the sagittal plane that exhibited signs of wear, but in mib littermates the incisors were maloccluded, distorted, and showed no signs of wear. Radiographic and histological examination of the dentition of 1- and 4-week-old rats revealed that the apical end of incisors in mib rats failed to extend posteriorly to the third molar region as in normal siblings, but ended at the first molar. Histological examination of longitudinal sections of mandibles through the incisors of neonatal normal and mib rats showed that in 1-day-old mutants the incisor was closely surrounded by alveolar bone to which it was ankylosed. The incisor body in mib rats was also malformed, with an indented apical end. This ankylosis was temporary, being resolved by 3 days. These findings show that neonatal reductions in bone resorption cause incisor defects and delay the eruption of all teeth in mib rats. The malocclusion and distortion of incisors of mib rats are likely caused by temporary ankylosis of incisor matrices to alveolar bone. Taken together, these findings illustrate the concept that bone resorption is an essential and rate-limiting element of tooth eruption.

Animals↗
PubMed2004

Persistent NKH with transient or absent symptoms and a homozygous GLDC mutation.

Three of four nonketotic hyperglycinemia patients homozygous for a novel GLDC mutation (A802V) were treated by assisted respiration and/or sodium benzoate with or without ketamine and had transient neonatal or absent symptoms and normal developmental outcome, despite persisting biochemical evidence of nonketotic hyperglycinemia. This exceptional outcome may be related to the high residual activity of the mutant protein (32% of wild type) and therapeutic intervention during a critical period of heightened brain exposure and sensitivity to glycine.

Adult↗
PubMed2006

Amino acid residues Gln4020 and Lys4021 of the ryanodine receptor type 1 are required for activation by 4-chloro-m-cresol.

The ryanodine receptor type 1 (RyR1) and type 2 (RyR2), but not type 3 (RyR3), are efficiently activated by 4-chloro-m-cresol (4-CmC). We previously showed that a 173-amino acid segment of RyR1 (residues 4007-4180) is required for channel activation by 4-CmC (Fessenden, J. D., Perez, C. F., Goth, S., Pessah, I. N., and Allen, P. D. (2003) J. Biol. Chem. 278, 28727-28735). In the present study, we used site-directed mutagenesis to identify individual amino acid(s) within this region that mediate 4-CmC activation. In RyR1, substitution of 11 amino acids conserved between RyR1 and RyR2, but divergent in RyR3, with their RyR3 counterparts reduced 4-CmC sensitivity to the same degree as substitution of the entire 173-amino acid segment. Further analysis of various RyR1 mutants containing successively smaller numbers of these mutations identified 2 amino acid residues (Gln(4020) and Lys(4021)) that, when mutated to their RyR3 counterparts (Leu(3873) and Gln(3874)), abolished 4-CmC activation of RyR1. Mutation of either of these residues alone did not abolish 4-CmC sensitivity, although Q4020L partially reduced 4-CmC-induced Ca(2+) transients. In addition, mutation of the corresponding residues in RyR3 to their RyR1 counterparts (L3873Q/Q3874K) imparted 4-CmC sensitivity to RyR3. Recordings of single RyR1 channels indicated that 4-CmC applied to either the luminal or cytoplasmic side activated the channel with equal potency. Secondary structure modeling in the vicinity of the Gln(4020)-Lys(4021) dipeptide suggests that the region contains a surface-exposed region adjacent to a hydrophobic segment, indicating that both hydrophilic and hydrophobic regions of RyR1 are necessary for 4-CmC binding to the channel and/or to translate allosteric 4-CmC binding into channel activation.

Allosteric Site↗
PubMed1998

Growth restriction of dengue virus type 2 by site-specific mutagenesis of virus-encoded glycoproteins.

The three flavivirus glycoproteins prM, E and NS1 are formed by post-translational cleavage and are glycosylated by the addition of N-linked glycans. NS1 may form homodimers, whereas E may form homodimers, homotrimers or heterodimers (prM-E). Modification of these processes by mutagenesis of the proteins has the potential to generate viruses that are restricted in growth and are possible vaccine candidates. Using an SV40-based expression system, we previously analysed dimerization and secretion of the NS1 protein of dengue virus type 2 (DEN-2) with mutations in the conserved Cys residues, or within hydrophilic or hydrophobic regions, or at glycosylation sites. In this study, mutations which reduce cleavage at the DEN-2 prM/E signalase cleavage site are described. On the basis of earlier and current results with transient expression, six mutations which reduced NS1 dimerization and two mutations which inhibited prM/E cleavage were analysed individually for their effects on virus growth using a genomic length cDNA clone. Two viruses were obtained that showed reduced growth in cell culture and attenuation of neurovirulence when inoculated into 3-day-old mice. One of these viruses encoded NS1 that lacked the second glycosylation site, the other encoded a Ser --> Ile change at the -3 position of the prM/E cleavage site. A third virus encoding a mutation in NS1 within a hydrophilic region grew as well as the parental virus. No virus was detected for the remaining five mutations.

Animals↗