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PubMed2002

Functional analysis of a carboxyl-terminal phosphorylation mutant of the bovine papillomavirus E1 protein.

The papillomavirus E1 protein is essential for viral DNA replication, and phosphorylation of E1 appears to regulate protein function and DNA replication. Serine 584 of bovine papillomavirus E1 is in a conserved motif resembling a CK2 consensus site, and is phosphorylated by CK2 in vitro. Mutation of serine 584 to alanine eliminates replication of the viral genome in transient replication assays. Wild-type and mutant E1 proteins were expressed from recombinant baculoviruses and used to assess biochemical functions of the amino acid 584 substitution. Helicase enzyme activity, E1 binding to the viral E2 protein and to cellular DNA polymerase alpha-primase were all unaffected in the mutant protein. Binding of E1 to viral replication origin DNA sequences was reduced in the mutant, but not eliminated. The carboxyl-terminal region of the protein appears to play a role in regulating E1 function, and adds to a complex picture emerging for papillomavirus DNA replication control.

Amino Acid Substitution↗
PubMed1993

Glucocorticoid receptor binding site in the mouse alpha-amylase 2 gene mediates response to the hormone.

Amylase gene expression has been shown to be positively regulated by glucocorticoids. Previous reports have suggested that this effect is indirect. We have addressed this question in a mouse exocrine pancreas cell line, 266-6, in which basal level of expression of amylase mRNA is low but inducible by glucocorticoids. In these cells the effect of glucocorticoids is not inhibited by cycloheximide at early time points. Reporter plasmids containing 224 base pairs of mouse amylase 5'-flanking DNA are positively regulated by glucocorticoids in gene transfer experiments. Glucocorticoid receptor purified from rat liver binds to the amylase promoter from position -56 to -33 and at the start of transcription. Site-directed mutation at the upstream position (-47 to -42) eliminates response to glucocorticoids in transient gene transfer experiments. Thus, glucocorticoid regulation of the mouse amylase gene is a direct effect and is mediated via a receptor binding site in the promoter region of the gene. Inhibition of the hormone response by cycloheximide at later time points after induction suggests the additional requirement for a short-lived factor. The DNA binding domain of the glucocorticoid receptor binds to a single site in the amylase promoter as a monomer, suggesting that both receptor binding sites as well as an additional short-lived factor are required to obtain induction.

Animals↗
PubMed2001

Emergence of a distinct pattern of viral mutations in chimpanzees infected with a homogeneous inoculum of hepatitis C virus.

BACKGROUND & AIMS: Prospective, long-term study of viral evolution and immunologic responses in chimpanzees infected with a homogeneous hepatitis C virus (HCV) population is crucial in understanding the pathogenesis of HCV-host interactions. METHODS: A molecular clone was constructed of HCV genotype 1b and RNA transcribed from this clone inoculated intrahepatically into chimpanzee X0142. Serum was taken from X0142 at week 2 and inoculated intravenously into a second chimpanzee (X0234). Detailed virologic, serologic, and immunologic analyses of these 2 chimpanzees were performed. RESULTS: Both chimpanzees developed persistent viremia, with titers of 10(3) to 10(5) genomes/mL, for 80 weeks (X0142) and 55 weeks (X0234) of follow-up. A late antibody response against the nonstructural proteins and a weak, transient T-helper proliferative response were detected in both animals. In X0142, 25 mutations emerged in the virus population by week 78 and 15 in X0234 by week 35. A relatively large proportion of mutations affecting protein sequences appeared in the NS5A gene (33% in X0142 and X0234 combined), and 5 mutations were common to both chimpanzees. CONCLUSIONS: In this long-term study of the molecular evolution of HCV genotype 1b from a cloned source, the appearance of a distinct pattern of mutations is suggestive of an adaptive response of HCV in vivo. In addition, a limited virus-specific immunity may contribute to HCV persistence.

Alanine Transaminase↗
PubMed1995

Arg506Gln factor V mutation (factor V Leiden) in patients with ischaemic cerebrovascular disease and survivors of myocardial infarction.

The point mutation Arg506- > Gln of factor V was recently shown to be an important and relatively common genetic cause of venous thromboembolism. Using a DNA technique based on polymerase chain reaction, we surveyed the blood samples of 236 patients with ischaemic stroke or a transient ischaemic attack, 122 survivors of myocardial infarction and 137 control subjects for the presence of this mutation. Although the frequency of the factor V mutation in patients with arterial disease (4.5%) was not significantly different from that in healthy blood donors (2.9%), a carrier status for this mutant gene was associated with symptoms of migraine and relatively mild angiographic abnormalities among patients with cerebrovascular disease. A more extensive study addressing the occurrence and significance of the mutant factor V mutation in patients with vasospastic cerebrovascular diseases seems to be warranted.

Adult↗
PubMed1997

Two missense mutations of H type alpha(1,2)fucosyltransferase gene (FUT1) responsible for para-Bombay phenotype.

BACKGROUND AND OBJECTIVES: Rare individuals (Bombay and para-Bombay phenotypes) fail to express the A, B and H antigens on erythrocyte membranes because of a lack in the H gene (FUT1)-encoded alpha(1,2)fucosyltransferase activity. In this study, we have found a para-Bombay individual (Bmh) who expressed B and H antigens in saliva but not on red blood cells. The FUT1 alleles of this person contained two single base changes (T460C and G1042A) in the coding region relative to the wild type allele. These substitutions may result in changes in two amino acid residues (Y154H and E348K). MATERIALS AND METHODS: Since the T460C and G1042A mutations destroy endonuclease RsaI and AvaI sites, respectively, we tested for these mutations using PCR-RFLP. RESULTS: Our findings indicated that this para-Bombay person was homozygous for the T460C and G1042A mutations, and that neither of these mutations was found in 136 randomly selected Japanese individuals. The measurement of the alpha(1,2)fucosyltransferase activity after transient expression of the FUT1 alleles in COS-7 cells indicated that the H-deficient allele-encoded enzyme had no detectable activity. Moreover, transfection by chimera FUT1 allele contains only the T460C mutation, or only the G1042A mutation, and yielded 1.0 or 9.3%, respectively, of the activities compared to transfection by the wild type allele. CONCLUSIONS: These results suggest that the two mutations in combination are responsible for the inactivation of the FUT1-encoded enzyme activity.

ABO Blood-Group System↗
PubMed2002

Functional cloning of BRF1, a regulator of ARE-dependent mRNA turnover.

To identify regulators of AU-rich element (ARE)-dependent mRNA turnover we have followed a genetic approach using a mutagenized cell line (slowC) that fails to degrade cytokine mRNA. Accordingly, a GFP reporter construct whose mRNA is under control of the ARE from interleukin-3 gives an increased fluorescence signal in slowC. Here we describe rescue of slowC by a retroviral cDNA library. Flow cytometry allowed us to isolate revertants with reconstituted rapid mRNA decay. The cDNA was identified as butyrate response factor-1 (BRF1), encoding a zinc finger protein homologous to tristetraprolin. Mutant slowC carries frame-shift mutations in both BRF1 alleles, whereas slowB with intermediate decay kinetics is heterozygous. By use of small interfering (si)RNA, independent evidence for an active role of BRF1 in mRNA degradation was obtained. In transiently transfected NIH 3T3 cells, BRF1 accelerated mRNA decay and antagonized the stabilizing effect of PI3-kinase, while mutation of the zinc fingers abolished both function and ARE-binding activity. This approach, which identified BRF1 as an essential regulator of ARE-dependent mRNA decay, should also be applicable to other cis-elements of mRNA turnover.

3' Untranslated Regions↗
PubMed1998

Activity of a ritonavir plus saquinavir-containing regimen in patients with virologic evidence of indinavir or ritonavir failure.

OBJECTIVE: To evaluate the virologic activity of a ritonavir plus saquinavir-containing regimen in patients who have failed an indinavir or ritonavir-containing regimen. DESIGN: Patients were identified through a retrospective study evaluating the incidence of indinavir or ritonavir failure in our clinic. PATIENTS: Eighteen patients failing indinavir or ritonavir therapy and who switched to a ritonavir-saquinavir-containing regimen were evaluated. Indinavir or ritonavir failure was defined as a plasma viral load > 1500 copies/ml (branched DNA) after 16 weeks of continuous therapy. INTERVENTIONS: All patients switched to ritonavir (400 mg twice daily) plus saquinavir (400 mg twice daily) and received concurrent therapy with two nucleoside reverse transcriptase inhibitors (NRTI). Twelve of the 18 patients modified their NRTI regimen at the time ritonavir-saquinavir was initiated. OUTCOME MEASURES: Plasma viral load was monitored using a branched DNA assay. Genotypic analysis was performed using a point mutation differential hybridization technique, and was confirmed with direct sequencing. RESULTS: Fourteen out of 18 patients completed at least 24 weeks of therapy; the remaining four patients discontinued therapy after week 12 due to a lack of virologic response or intolerance. Plasma viral load decreased a median 1.4 log10 after 4 weeks of treatment with ritonavir-saquinavir. Only four patients had a greater than 0.5 log10 decrease in viral load after 24 weeks of therapy. In eight out of 10 patients evaluated, the V82A mutation was present at the time of the switch to ritonavir-saquinavir. Viral rebound on ritonavir-saquinavir was associated with the emergence of mutations at amino acids 46, 48, 54 and 90. CONCLUSION: The combination of ritonavir, saquinavir and two NRTI resulted in a moderate but transient suppression of viral replication in patients who have failed indinavir or ritonavir therapy. Failure of ritonavir-saquinavir may be associated with the emergence of mutations associated with resistance to ritonavir/saquinavir monotherapy, particularly the L90M mutation.

Anti-HIV Agents↗
PubMed1993

Mutations in the conserved loop of human U5 snRNA generate use of novel cryptic 5' splice sites in vivo.

We have analyzed base pairing interactions between the U5 snRNA and 5' exon sequences during pre-mRNA splicing in a mammalian in vivo system. We constructed synthetic U5 genes with mutations that alter four bases (C3, U4, U5 and U6) within the invariant 9 nt U5 sequence GCCUUUUAC; transient transfection of HeLa cells with these U5 sequences cloned into a U1 expression vector yielded high levels of the mutant snRNAs. To test their function, we cotransfected a rabbit beta-globin gene containing one of two mutations (G1-->A or T2-->A) in the essential GT dinucleotide at the 5' end of the second intron. Certain U5 loop mutants activated novel 5' splice sites only in mutant rabbit beta-globin transcripts. One novel site surprisingly resides in the first exon; its use is invariably coupled to utilization of a particular cryptic 5' splice site in the second exon. All of the newly activated cryptic 5' splice sites exhibit complementarity with the mutant U5 loop in the exon 1-5 nt upstream of the cryptic site, extending previous results in yeast. However, the register of the potential pairing is not identical at the various novel cryptic 5' splice sites, indicating that the interaction between the U5 loop and the 5' exon may be more flexible than previously believed.

Animals↗
PubMed2006

Impact of the mutation A21G (Flemish variant) on Alzheimer's beta-amyloid dimers by molecular dynamics simulations.

Soluble oligomers of the amyloid beta-protein (Abeta) are linked to Alzheimer's disease. Irrespective of the nature of the nucleus before fibril growth, dimers are essential species in Abeta assembly, but their transient character has precluded, thus far, high-resolution structure determination. We have investigated the effects of the point mutation A21G on Abeta dimers by performing high temperature all-atom molecular dynamics simulations of Abeta(40), Abeta(42), and their Flemish variants (A21G) starting from their fibrillar conformations. Abeta dimers are found in equilibrium between various topologies, and the absence of common structural features shared by the four species makes problematic the design of a unique inhibitor for blocking dimers. We also show that the impact of the point mutation A21G on Abeta structure and dynamics varies from Abeta(40) to Abeta(42). Finally, we provide a possible structural explanation for the reduced aggregation rate of Abeta fibrils containing the Flemish disease-causing mutation.

Amyloid beta-Peptides↗
PubMed2000

Sensory rhodopsin II from the haloalkaliphilic natronobacterium pharaonis: light-activated proton transfer reactions.

In the present work the light-activated proton transfer reactions of sensory rhodopsin II from Natronobacterium pharaonis (pSRII) and those of the channel-mutants D75N-pSRII and F86D-pSRII are investigated using flash photolysis and black lipid membrane (BLM) techniques. Whereas the photocycle of the F86D-pSRII mutant is quite similar to that of the wild-type protein, the photocycle of D75N-pSRII consists of only two intermediates. The addition of external proton donors such as azide, or in the case of F86D-pSRII, imidazole, accelerates the reprotonation of the Schiff base, but not the turnover. The electrical measurements prove that pSRII and F86D-pSRII can function as outwardly directed proton pumps, whereas the mutation in the extracellular channel (D75N-pSRII) leads to an inwardly directed transient current. The almost negligible size of the photostationary current is explained by the long-lasting photocycle of about a second. Although the M decay, but not the photocycle turnover, of pSRII and F86D-pSRII is accelerated by the addition of azide, the photostationary current is considerably increased. It is discussed that in a two-photon process a late intermediate (N- and/or O-like species) is photoconverted back to the original resting state; thereby the long photocycle is cut short, giving rise to the large increase of the photostationary current. The results presented in this work indicate that the function to generate ion gradients across membranes is a general property of archaeal rhodopsins.

Archaeal Proteins↗
PubMed2003

Mutations in the Ca2+/H+ transporter CAX1 increase CBF/DREB1 expression and the cold-acclimation response in Arabidopsis.

Transient increases in cytosolic free calcium concentration ([Ca2+]cyt) are essential for plant responses to a variety of environmental stimuli, including low temperature. Subsequent reestablishment of [Ca2+]cyt to resting levels by Ca2+ pumps and antiporters is required for the correct transduction of the signal [corrected]. C-repeat binding factor/dehydration responsive element binding factor 1 (Ca2+/H+) antiporters is required for the correct transduction of the signal. We have isolated a cDNA from Arabidopsis that corresponds to a new cold-inducible gene, rare cold inducible4 (RCI4), which was identical to calcium exchanger 1 (CAX1), a gene that encodes a vacuolar Ca2+/H+ antiporter involved in the regulation of intracellular Ca2+ levels. The expression of CAX1 was induced in response to low temperature through an abscisic acid-independent pathway. To determine the function of CAX1 in Arabidopsis stress tolerance, we identified two T-DNA insertion mutants, cax1-3 and cax1-4, that display reduced tonoplast Ca2+/H+ antiport activity. The mutants showed no significant differences with respect to the wild type when analyzed for dehydration, high-salt, chilling, or constitutive freezing tolerance. However, they exhibited increased freezing tolerance after cold acclimation, demonstrating that CAX1 plays an important role in this adaptive response. This phenotype correlates with the enhanced expression of CBF/DREB1 genes and their corresponding targets in response to low temperature. Our results indicate that CAX1 ensures the accurate development of the cold-acclimation response in Arabidopsis by controlling the induction of CBF/DREB1 and downstream genes.

Abscisic Acid↗
PubMed2002

Ca2+ activation of RyR1 is not necessary for the initiation of skeletal-type excitation-contraction coupling.

Although an elevation in myoplasmic Ca2+ can activate the skeletal muscle ryanodine receptor (RyR1), the function of this Ca2+ activation is unclear because extracellular Ca2+ influx is unnecessary for skeletal-type EC coupling. To determine whether Ca2+ activation of RyR1 is necessary for the initiation of skeletal-type EC coupling, we examined the behavior of RyR1 with glutamate 4032 mutated to alanine (E4032A-RyR1) because this mutation had been shown to dramatically reduce activation by Ca2+. Proc. Natl. Acad. Sci. USA. 98:2865-2870). Analysis after reconstitution into planar lipid bilayers revealed that E4032A-RyR1 was negligibly activated by 100 microM Ca2+ (P(o) too low to be measured). Even in the presence of both 2 mM caffeine and 2 mM ATP, P(o) remained low for E4032A-RyR1 (ranging from <0.0001 in 100 microM free Ca2+ to 0.005 in 2 mM free Ca2+). Thus, the E4032A mutation caused a nearly complete suppression of activation of RyR1 by Ca2+. Depolarization of E4032A-RyR1-expressing myotubes elicited L-type Ca2+ currents of approximately normal size and myoplasmic Ca2+ transients that were skeletal-type, but about fivefold smaller than those for wild-type RyR1. The reduced amplitude of the Ca2+ transient is consistent either with the possibility that Ca2+ activation amplifies Ca2+ release during EC coupling, or that the E4032A mutation generally inhibits activation of RyR1. In either case, Ca2+ activation of RyR1 does not appear to be necessary for the initiation of Ca2+ release during EC coupling in skeletal muscle.

Adenosine Triphosphate↗
PubMed2004

Mutations conferring drug resistance affect eukaryotic expression of HIV type 1 reverse transcriptase.

Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.

Animals↗
PubMed2002

A mutation in GJB3 is associated with recessive erythrokeratodermia variabilis (EKV) and leads to defective trafficking of the connexin 31 protein.

Erythrokeratodermia variabilis (EKV) is a skin disorder characterized by variable (transient) erythemas and fixed keratosis. The disorder maps to chromosome 1p34-35, a location that contains the GJB3 gene encoding the gap junction protein connexin 31. Until now, only heterozygote mutations in the form of dominant inheritance have been described in this gene associated with EKV. We report here a homozygote mutation in the connexin 31 gene, found in a family that shows recessive inheritance of the disorder, thus providing the first molecular support for a recessive variant of EKV. The entire GJB3 coding sequence was scanned for mutations by sequencing. We detected a T-->C transition at position 101 of the coding sequence, which replaces a leucine with a proline at residue 34 of the protein (L34P). Evolutionary analysis shows that this mutation is located at a highly conserved region of connexin in the first putative transmembrane helix (TMH). In transfected keratinocytes, L34P connexin 31 had a cytoplasmic distribution, suggesting that the mutant form of this protein will not form normal gap junctions between adjacent cells. The change of leucine to proline is likely to alter the structure of the first TMH of connexin by inducing a kink, thus influencing connexon structure and function.

Amino Acid Sequence↗
PubMed1994

Cell type specific patterns of mRNA splicing in hepatoma cells transfected with the mutated albumin minigene of Nagase analbuminemic rats.

We constructed an SV40-derived expression vector containing a mutated albumin minigene of Nagase analbuminemic rats (NAR), and introduced it into cultured cells. Transient expression of the minigene mRNA was determined by RT-PCR. Three kinds of aberrant mRNAs were expressed by the non-hepatic cells COS-1, and the undifferentiated human hepatoma cells HLE, transfected with the minigene. Their predominant mRNA lacked exon H (delta H), while mRNAs lacking exons H and I, or exons G and H were less abundant. This pattern of the mRNAs was similar to that of albumin mRNAs in the liver of old NAR. In contrast, a differentiated type of human hepatoma cell line, HepG2, expressed only delta H mRNA, like young NAR. These findings indicate that the expression system of the mutated minigene in cultured hepatoma cells is useful for understanding two-exon-skipping of albumin pre-mRNA of NAR.

Alternative Splicing↗
PubMed2005

Transient structural ordering of the RNA-binding domain of carnation mottle virus p7 movement protein modulates nucleic acid binding.

Plant viral movement proteins bind to RNA and participate in the intra- and intercellular movement of the RNAs from plant viruses. However, the role and magnitude of the conformational changes associated with the formation of RNA-protein complexes are not yet defined. Here we describe studies on the relevance of a preexisting nascent alpha-helix at the C terminus of the RNA-binding domain of p7, a movement protein from carnation mottle virus, to RNA binding. Synthetic peptide analogues and single amino acid mutation at the RNA-binding domain of recombinant p7 protein were used to correlate the transient structural order in aqueous solution with RNA-binding potential.

Amino Acid Sequence↗
PubMed1996

[Cloning of the holocarboxylase synthetase cDNA and identification of mutations prevalent in Japanese HCS-deficient patients].

Holocarboxylase synthetase (HCS) plays an essential role in biotin utilization in cells and its deficiency causes biotin-responsive multiple carboxylase deficiency in humans. We have cloned the human HCS cDNA, which maps to chromosome 21q22.1. Two mutations in the HCS genes of Japanese patients with HCS deficiency have been identified: a transition from T to C which causes an amino acid substitution of proline for leucine at position 237 (L237P) and A single guanine base deletion (delta G1067) followed by premature termination. Transient expression in cultured fibroblasts from a patient after site-directed mutagenesis showed that the L237P mutation was responsible for decreased HCS activity. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the prevalence of the mutations--L237P and delta G1067--was 50% and 30%, respectively, among Japanese patients with HCS deficiency.

Amino Acid Sequence↗
PubMed2003

Genetic heterogeneity in erythrokeratodermia variabilis: novel mutations in the connexin gene GJB4 (Cx30.3) and genotype-phenotype correlations.

Erythrokeratodermia variabilis is an autosomal dominant genodermatosis characterized by persistent plaque-like or generalized hyperkeratosis and transient red patches of variable size, shape, and location. The disorder maps to a cluster of connexin genes on chromosome 1p34-p35.1 and, in a subset of families, results from mutations in the gene GJB3 encoding the gap junction protein connexin-31 (Cx31). A recent report suggested the involvement of another connexin gene (GJB4) in the etiology of erythrokeratodermia variabilis. In this study, we sequenced the coding region of GJB4 in 13 unrelated erythrokeratodermia variabilis families without detectable mutations in GJB3. Mutation analysis revealed six distinct missense mutations in five families and a sporadic case of erythrokeratodermia variabilis, all of which were not found in controls. Mutation G12D, identified in an extended Dutch family, lies in the predicted amino-terminus and may interfere with the flexibility of this domain, connexin selectivity, or gating polarity of gap junction channels. Other mutations (R22H, T85P, F137L, F189Y) were located in the transmembrane domains of Cx30.3, and are predicted to hinder regulation of voltage gating or alter the kinetics of channel closure. Affected individuals of two unrelated families harbored point mutations leading to amino acid substitution F137L, which was also reported in GJB3, yet the extent and severity of hyperkeratosis was milder compared to the corresponding mutation in GJB3. Two mutations (T85P, F137L) were associated with the occurrence of rapidly changing erythematous patches with prominent, circinate, or gyrate borders in affected children but not in adults, supporting the notion that this feature is specific to Cx30.3 defects. Nevertheless, we observed highly variable intrafamilial phenotypes, suggesting the strong influence of modifying genetic and epigenetic factors. In addition to pathogenic mutations, we identified several missense mutations and a 4 bp deletion within the GJB4 coding region, which might represent either inconsequential polymorphisms or recessive mutations. In conclusion, our results demonstrate genetic heterogeneity in erythrokeratodermia variabilis, and emphasize that intercellular communication mediated by both Cx31 and Cx30.3 is crucial for epidermal differentiation.

Connexins↗