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PubMed1998

Optimized detection of DNA point mutations by double gradient denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis displays the highest detection rate among mutation scanning methods. In classical denaturing gradient gel electrophoresis the denaturant gradient range and migration times vary for every amplicon to be scanned, greatly affecting the routine application of the method. As an alternative, we developed double gradient denaturing gradient gel electrophoresis where a gradient of pore size is superimposed over the denaturing one, allowing maintenance of the zone-sharpening effect even over prolonged time runs, and adoption of identical run time conditions for all fragments analyzed. Here double gradient denaturing gradient gel electrophoresis has been applied to the analysis of a number of point mutations and polymorphisms located in several exons of three different genes, the cystic fibrosis transmembrane conductance regulator, the beta-globin and the p53 genes.

Cystic Fibrosis Transmembrane Conductance Regulato↗
PubMed1997

[Hot-point mutations of viral C gene during hepatitis B virus infection].

To investigate the relationship of the HBV C gene mutations with the disease progress, the hotpoint mutations of pre- C stop28 and C region L97 were examined with restrictio-fragment-length-polymorphism (RFLP) technique in 91 hepatitis patients with diverse clinical features and HBe status. Both mutations were almost not seen in acute hepatitis B and chronic asymptomatic virus carriers; rarely in chronic persistent hepatitis and frequently in chronic active hepatitis (CAH) and active liver cirrhosis (ALC), accounting for 80% and 78% in CAH and ALC respectively. The pre-C mutant was mixed with wild strain in 11 of 31 patients who were HBeAg-positive CAH and ALC; on the other hand, the wild strain also coexisted with variant in anti-HBe-positive cases. So did the L97 in both HBeAg- and anti- HBe-positive cases. Possibly, both the mutant and the wild strain are usually in a relatively growth and decline status. So, the hot-point mutations of HBV C gene were closely related with the disease activity.

Adult↗
PubMed1994

A spontaneous point mutation in the human T-cell leukemia virus type 1 pX gene leads to expression of a novel doubly spliced pX-mRNA that encodes a 25-kD, amino-terminal deleted rex protein.

Primary RNA transcripts of the human T-cell leukemia virus type 1 (HTLV-1) are processed into mature mRNA by a complex series of splicing events. In this paper, we report the finding of a novel doubly spliced pX mRNA in two out of eight HTLV-1-infected cell lines and in one out of 13 peripheral blood mononuclear cells from HTLV-1-infected individuals. The second splicing for this novel pX mRNA is different from that for the known doubly spliced pX mRNA. A novel acceptor site in this splicing was generated by a single point mutation (G to A) at nucleotide 7,337 of the pX gene. This mRNA contained a complete open reading frame that encodes an amino-terminal truncated p27rex protein with 189 amino acids. A new 25-kD protein was detected in the cell lines expressing the novel pX mRNA by an antibody against the carboxy-terminal peptide of p27rex and was termed p25rex. Although the function of p25rex is not clear, we clarified that p25rex is a cytoplasmic phosphoprotein and its function is different from the transcriptional regulator function of p27rex. The possibility that the mutated virus is replicable only in cells coinfected with the wild type HTLV-1 may explain why the incidence of the mutants observed here is low.

Amino Acid Sequence↗
PubMed2000

A point mutation in the transmembrane domain of the hemagglutinin of influenza virus stabilizes a hemifusion intermediate that can transit to fusion.

A hemagglutinin (HA) of influenza virus having a single semiconserved Gly residue within the transmembrane domain mutated to Leu (G520L) was expressed on cells; these cells were bound to red blood cells. By decreasing pH at 23 degrees C rather than 37 degrees C, an intermediate with properties expected of hemifusion just as the membranes are about to transit to full fusion was captured. As evidence: 1) increasing temperature to 37 degrees C at neutral pH allowed fusion to proceed; 2) after achieving the intermediate, the two membranes did not separate from each other after proteolytic cleavage of G520L because cells treated with proteinase K could not fuse upon temperature increase but could fuse upon the addition of chlorpromazine; and 3) at the point of the intermediate, adding exogenous lipids known to promote or inhibit the creation of hemifusion did not significantly alter the lipid dye spread that occurred upon increasing temperature, implying that at the intermediate, contacting membrane leaflets had already merged. A stable intermediate of hemifusion that could transit to fusion was also generated for wild-type HA, but pH had to be reduced at the significantly lower temperature of 4 degrees C. The fusion pores generated by G520L did not enlarge, whereas those induced by wild-type HA did. The finding that a state of transitional hemifusion can be readily obtained via a point mutation without the need for unusually low temperature supports the hypothesis that hemifusion occurs before pore formation.

Animals↗
PubMed2002

Paraneoplastic erythrocytosis associated with an inactivating point mutation of the von Hippel-Lindau gene in a renal cell carcinoma.

The von Hippel-Lindau (VHL) tumor suppressor gene targets hypoxia-inducible transcription factors (HIFs) for proteasomal degradation. Erythrocytosis due to inappropriate production of erythropoietin (EPO), one of the HIF target genes, is a classic albeit rare finding in patients with renal cancer. We report the clinical to molecular analysis in a patient in whom a thrombotic myocardial infarction was the first manifestation of a clear cell renal carcinoma associated with an elevated serum EPO level (109 U/L) and erythrocytosis (hemoglobin 200 g/L [20 g/dL]). The tumor strongly expressed EPO messenger RNA and the 2 regulatory subunits HIF-1alpha and HIF-2alpha. Sequence analysis of tumor tissue identified a point mutation of the VHL gene (nucleotide 701 T > C) with a predicted amino acid exchange (Leu163Pro). This structural change, although located at distance to the HIF-binding region, was found to inhibit binding of HIF-1alpha to VHL, thus leading to accumulation of HIF, which drives EPO production.

Carcinoma, Renal Cell↗
PubMed1993

Propeptide processing during factor IX biosynthesis. Effect of point mutations adjacent to the propeptide cleavage site.

Factor IX is synthesized in a precursor form with a propeptide that contains the gamma-carboxylation recognition site, an element which directs the post-translational gamma-carboxylation of adjacent glutamic acid residues. After protein synthesis, the propeptide is cleaved to yield the mature Factor IX. To study propeptide processing, anti-proFactor IX antibodies were prepared using a synthetic peptide based upon the sequence of the Factor IX propeptide. Immunoaffinity-purified anti-proFactor IX antibodies were reactive with Factor IX Cambridge, a mutant form of Factor IX containing the propeptide, but were not reactive with Factor IX. These antibodies were used to examine the proteolytic processing of forms of Factor IX containing point mutations at P6, P3, P2, P1, P1', P2', and P3' adjacent to the propeptide cleavage site in order to determine the requirement of each of these amino acids for propeptide cleavage. Furthermore, the hierarchy of different pairs of basic residues at positions P1 and P2 was analyzed. The mutated cDNA constructs were expressed in Chinese hamster ovary cells. Propeptide processing was examined using intrinsically labeled Factor IX immunoprecipitated with either anti-pro-Factor IX antibodies or anti-Factor IX:total antibodies, and the Factor IX species were separated by SDS-gel electrophoresis. Under the expression conditions employed, the propeptide of wild type Factor IX was almost completely removed, whereas Factor IX mutated to threonine at P1 was not cleaved. The percentage of propeptide cleaved varied with the amino acid sequences of residues P2 and P1, respectively: Lys-Arg (93%), Arg-Arg (66%), Thr-Arg (33%), Arg-Lys (19%), Lys-Lys (10%), and Lys-Thr (< 1%). Apart from alterations of basic amino acids at P1 and P2, nonconservative mutations at P6 and P3 decreased propeptide cleavage, whereas conservative mutations at P3, P1', P2', or P3' resulted in cleavage efficiencies approximately equal to that for wild type Factor IX. These results indicate that the preference of paired basic residues at P1 and P2 is similar to other endopeptidases active toward proteins secreted through the constitutive pathway and that the propeptide residues NH2-terminal to these paired basic residues are important in defining enzyme-substrate binding.

Amino Acid Sequence↗
PubMed1994

Point mutations in the chloroplast 16s rRNA gene confer streptomycin resistance in Nicotiana plumbaginifolia.

In a previous paper we reported the isolation of streptomycin-resistant mutants from Nicotiana plumbaginifolia and presented evidence for chloroplast control of the resistance trait. To understand the molecular basis of the resistance in these mutants, we sequenced three regions in the chloroplast 16s rRNA gene, which correspond to the 5' terminus, the 530 loop, and the 900 stem/loop of Escherichia coli 16s rRNA, and compared them with the sequences of the wild-type. Our results show that: (1) nine mutants have a C to T change at position 912, (2) one mutant (SR1021) has a G to A change at position 885, (3) one mutant has a C to T change at position 526, based on E. coli numbering; and (4) three mutants do not have any change in the regions analyzed. The point mutation detected in SR1021 has not been reported previously. In E. coli 16s rRNA, position 885 is protected from chemical probing by ribosomal protein S12 and is closely juxtaposed with the streptomycin-binding region (positions 912-915) in the predicted secondary structure. It is likely that the G to A transition at this position is a novel mutation for streptomycin resistance.

Amino Acid Sequence↗
PubMed1998

Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity.

Evidence of the functional significance of two naturally occurring mutations at nt 40 or 41 in the Sp1 motif in the promoter proximal segment of the upstream regulatory region (URR) of human papillomavirus (HPV) type 18 is presented. In electrophoretic mobility shift assays, Sp1 protein bound more efficiently to the Sp1 mutant motifs than to the prototype; while in both HeLa and HT3 cells, luciferase activity controlled by the mutant URRs was upregulated 2- and 3-fold, or 4- and 6-fold, in comparison with the prototype URR or HeLa cell-derived URR respectively. The HeLa URR represents a more appropriate baseline for promoter activity, containing a series of point mutations representative of most HPV-18 cancer isolates, including one in the Yin Yang 1 (YY1) site at the P105 promoter. The effect of the Sp1 mutations was found to be largely maintained in the context of the HeLa URR containing the prototype YY1 site.

Alanine↗
PubMed1993

Benzodiazepine-induced motor impairment linked to point mutation in cerebellar GABAA receptor.

The selectively outbred alcohol-non-tolerant (ANT) rat line is highly susceptible to impairment of postural reflexes by benzodiazepine agonists such as diazepam. ANT cerebella are generally devoid of diazepam-insensitive high-affinity binding of the benzodiazepine [3H]Ro15-4513, whereas in non-selected strains such binding marks a granule-cell-specific GABAA (gamma-aminobutyric acid) receptor containing the alpha 6 subunit. A critical determinant for diazepam insensitivity of this 'wild-type' cerebellar GABAA receptor is an arginine residue in alpha 6 position 100, where other alpha subunits carry a histidine. Here we report that the alpha 6 gene of ANT rats is expressed at wild-type levels but carries a point mutation generating an arginine-to-glutamine substitution at position 100. In consequence, alpha 6(Q100)beta 2 gamma 2 receptors show diazepam-mediated potentiation of GABA-activated currents and diazepam-sensitive binding of [3H]Ro15-4513. Our results suggest that cerebellar motor control may be a distinct behavioural correlate of the alpha 6-subunit-containing GABAA receptor subtype.

Affinity Labels↗
PubMed1999

Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase.

The alymphoplasia (aly) mutation of mouse is autosomal recessive and characterized by the systemic absence of lymph nodes (LN) and Peyer's patches (PP) and disorganized splenic and thymic structures with immunodeficiency. Although recent reports have shown that the interaction between lymphotoxin (LT) and the LT beta-receptor (Ltbeta r, encoded by Ltbr) provides a critical signal for LN genesis in mice, the aly locus on chromosome 11 is distinct from those for LT and its receptor. We found that the aly allele carries a point mutation causing an amino acid substitution in the carboxy-terminal interaction domain of Nf-kappa b-inducing kinase (Nik, encoded by the gene Nik). Transgenic complementation with wild-type Nik restored the normal structures of LN, PP, spleen and thymus, and the normal immune response in aly/aly mice. In addition, the aly mutation in a kinase domain-truncated Nik abolished its dominant-negative effect on Nf-kappa b activation induced by an excess of Ltbeta r. Our observations agree with previous reports that Ltbeta r-deficient mice showed defects in LN genesis and that Nik is a common mediator of Nf-kappa b activation by the tumour necrosis factor (TNF) receptor family. Nik is able to interact with members of the TRAF family (Traf1, 2, 3, 5 and 6), suggesting it acts downstream of TRAF-associating receptor signalling pathways, including Tnfr, Cd40, Cd30 and Ltbeta r. The phenotypes of aly/aly mice are more severe than those of Ltbr-/- mice, however, indicating involvement of Nik in signal transduction mediated by other receptors.

Amino Acid Sequence↗
PubMed2005

Detection of DNA point mutation by atom transfer radical polymerization.

We report here a new DNA detection method in which polymer growth in atom transfer radical polymerization (ATRP) is used as a means to amplify detection signals. In this method, DNA hybridization and ligation reactions led to the attachment of ATRP initiators on a solid surface where specific DNA sequences were located. These initiators subsequently triggered the growth of poly(hydroxyethyl methacrylate) (PHEMA) at the end of immobilized DNA molecules and formed polymer brushes. The formation of PHEMA altered substrate opacity, rendering the corresponding spots readily distinguishable to the naked eye. A second ATRP reaction to form branched polymers on the surface drastically improved the visibility of DNA hybridization and significantly shortened the detection time. The resulting polymer film was characterized using infrared spectroscopy, ellipsometry, contact angle measurements, and atomic force microscopy. Direct visualization of 1 fmol of target DNA molecules of interest was demonstrated. A proof-of-principle experiment to detect DNA point mutation was conducted. The perfectly matched DNA targets were distinctively differentiated from those with mutations. The demonstrated capability to detect DNA mutation with direct visualization laid the groundwork for the future development of detector-free testing kits in single-nucleotide polymorphism screenings.

DNA↗
PubMed1994

Preferential beta-hexosaminidase (Hex) A (alpha beta) formation in the absence of beta-Hex B (beta beta) due to heterozygous point mutations present in beta-Hex beta-chain alleles of a motor neuron disease patient.

Deficiency of the lysosomal enzyme beta-hexosaminidase B (beta-Hex B) (a homodimer, beta beta), caused by a defect in the HEX B gene encoding the beta-chain, is usually accompanied by an absence of beta-Hex A (a heterodimer, alpha beta), thereby causing Sandhoff disease. However, we have earlier demonstrated the presence of partial beta-Hex A (30-50% of normal) even in the absence of beta-Hex B in an adult with motor neuron disease. The patient is a compound heterozygote with normal beta-chain message and one HEX B point mutation originating from each asymptomatic parent. Since the non-expression of beta-Hex B was post-transcriptional, we transfected COS-7 cells to understand the effect of each mutation on beta-Hex B activity. Transfection of the A1367-->C mutant (maternal allele) construct produced no overexpressed beta-Hex B, indicating that the encoded Tyr456-->Ser beta-chain was non-functional. Chou-Fasman analysis predicted that the Tyr456-->Ser mutation would cause a dramatic change in beta-chain folding (which often inhibits formation of functional dimers). This explains the complete lack of beta-Hex B in the transfectants and a partial deficiency of beta-Hex A and B (50% of normal) in the patient's mother. Since immunoprecipitated beta-Hex A (alpha beta) protein from patient fibroblasts showed the presence of mature beta-chains even though there was no beta-Hex B (beta beta) protein, the mutant beta-chain inherited from the father (who has normal beta-Hex A and B) must undergo preferential association with the normal alpha-chains in the patient, thus producing only beta-Hex A. Transient expression of the A619-->G mutant (paternal allele) construct produced beta-Hex B activity comparable to the wild type (approximately 10-20-fold over mock-transfected) whereas stable expression produced normal message but no beta-Hex B activity (wild type beta-Hex B expression: only 2-fold over mock-transfected). The lack of increased beta-Hex B after stable expression of the Ile207-->Val beta-chains at a lower copy number indicates the absence of self-association at low concentrations of Ile207-->Val beta-chain. In the patient who also has a non-functional Tyr456-->Ser allele, the effective concentration of beta-chains is reduced to 50% of normal and the remaining Ile207-->Val beta-chains fail to self-associate but can still dimerize with the abundant normal alpha-chains thus producing partial beta-Hex A and no beta-Hex B.

Adult↗
PubMed1994

Nonradiometric ELISA-based quantitation and validation of polymerase chain reaction-amplified DNA, including detection of point mutations, without allele-specific amplification, or ligation.

We describe two simple novel procedures, one direct and the other involving hybridization, for the enzyme-linked immunosorbent assay (ELISA)-based detection, quantitation, and validation of polymerase chain reaction (PCR)-amplified DNA. Both procedures are applicable to any PCR reaction, and do not require specially synthesized or enzyme-tagged oligonucleotides. We obtained accurate quantitation of PCR-amplified human cc10kDa cDNA with a sensitivity of about 0.6 fmoles. This cDNA was also used to detect single-base insertions, deletions, and substitutions specifically. Additionally, we could readily distinguish zinc-finger Y chromosome-specific genomic sequences in mixtures of male and female cells and normal and mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene sequences in crude fibroblast lysates. To our knowledge, this is the first report of point mutation detection in solution by ELISA without allele-specific amplification or ligation. These novel procedures have vast potential for basic and clinical applications, including gene expression studies, rapid screening of genetic diseases, detection of oncogene and anti-oncogene mutations, and identification of pathogens (e.g., HIV-1) in clinical specimens.

Base Sequence↗
PubMed2002

A novel cis AB allele derived from a B allele through a single point mutation.

BACKGROUND: The very rare cis AB phenotype, first described in 1964, corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B substances. Until now, gene sequences of only two cis AB alleles were partially characterized. One involved the A1*02 allele with a single nonsynonymous substitution at codon 268, whereas the second arose from a single nonsynonymous substitution at codon 266 in exon 7 of a B1*01 allele. STUDY DESIGN AND METHODS: A cis AB phenotype was identified in a French family. The serologic characteristics of this phenotype were determined. The cis AB allele was characterized from exon 6 to exon 7 by cloning and sequencing. RESULTS: The cis AB.tlse(*)01 allele is identical to B(1*)01 except for a single point mutation at nucleotide position 700, where a T replaces a C, implying a change of amino acid 234 (the B(1*)01 proline being replaced by a serine). CONCLUSION: The cis AB.tlse(*)01 allele clearly differs from all previously reported ABO, including the two previous cis AB described.

ABO Blood-Group System↗
PubMed1996

Significance of two point mutations present in each HEXB allele of patients with adult GM2 gangliosidosis (Sandhoff disease) homozygosity for the Ile207-->Val substitution is not associated with a clinical or biochemical phenotype.

The molecular defects in the HEXB gene encoding the common beta-subunit of lysosomal beta-hexosaminidase A (beta-Hex A, alpha beta) and beta-Hex B (beta beta) were investigated in a Portuguese family affected with late onset Sandhoff disease (GM2-gangliosidosis variant 0). This family comprised two unaffected daughters and three affected sibs who developed at about age 17 cerebellar ataxia and mental deficiency. Their parents were consanguineous and clinically asymptomatic. There was no detectable beta-Hex B activity and a profound reduction in the activity of beta-Hex A in the leukocytes and transformed lymphoid cell lines from the affected sibs. The expected intermediate values were observed in the parents as well as in one daughter and her children. Western analysis revealed the presence of reduced, but detectable amounts of mature beta-chain protein in cell lysates from the probands and intermediate levels in the parents. Nucleotide sequencing of amplified, reverse-transcribed beta-chain mRNA demonstrated the presence of two single point mutations: an A619 to G transition in exon 5 (Ile207-->Val), and a G1514 to A transition in exon 13 (Arg505-->Gln). Both of these two mutations have been previously linked to the adult form of Sandhoff disease in compound heterozygote patients. All three affected sibs were found to be homoallelic for both mutations. Interestingly, while the mother was heterozygous for each mutation, the father was homozygote for the A619-->G substitution and heterozygote for the G1514-->A transition. Since the father is homozygote for the A619-->G mutation but expresses a biochemical phenotype consistent with a carrier of Sandhoff disease and is clinically asymptomatic, this substitution is likely a neutral mutation. We confirmed this hypothesis by finding this transition present in 4 of 30 alleles from normal individuals. We conclude that homozygosity for the G1514-->A mutation is exclusively responsible for the adult form of Sandhoff disease in this family, and that the A619-->G substitution is not a deleterious mutation but rather a common HEXB polymorphism.

Adult↗
PubMed2001

The molecular basis of an avian plumage polymorphism in the wild: a melanocortin-1-receptor point mutation is perfectly associated with the melanic plumage morph of the bananaquit, Coereba flaveola.

BACKGROUND: Evolution depends on natural selection acting on phenotypic variation, but the genes responsible for phenotypic variation in natural populations of vertebrates are rarely known. The molecular genetic basis for plumage color variation has not been described in any wild bird. Bananaquits (Coereba flaveola) are small passerine birds that occur as two main plumage variants, a widespread yellow morph with dark back and yellow breast and a virtually all black melanic morph. A candidate gene for this color difference is the melanocortin-1 receptor (MC1R), a key regulator of melanin synthesis in feather melanocytes. RESULTS: We sequenced the MC1R gene from four Caribbean populations of the bananaquit; two populations of the yellow morph and two populations containing both the yellow morph and the melanic morph. A point mutation resulting in the replacement of glutamate with lysine was present in at least one allele of the MC1R gene in all melanic birds and was absent in all yellow morph birds. This substitution probably causes the color variation, as the same substitution is responsible for melanism in domestic chickens and mice. The evolutionary relationships among the MC1R haplotypes show that the melanic alleles on Grenada and St. Vincent had a single origin. The low prevalence of nonsynonymous substitutions among yellow haplotypes suggests that they have been under stabilizing selection, whereas strong selective constraint on melanic haplotypes is absent. CONCLUSIONS: We conclude that a mutation in the MC1R is responsible for the plumage polymorphism in a wild bird population and that the melanic MC1R alleles in Grenada and St. Vincent bananaquit populations have a single evolutionary origin from a yellow allele.

Animals↗
PubMed2003

A novel point mutation in the hormone binding domain of the androgen receptor associated with partial and minimal androgen insensitivity syndrome.

Mutations in the coding sequence of the androgen receptor (AR) gene result in a wide range of androgen insensitivity syndromes (AIS). We report an extended family in which at least five male individuals in different generations suffer from partial AIS. The index patient presented at birth with ambiguous genitalia; the karyotype was 46,XY and subsequent sex assignment male. Elevated stimulated testosterone (T) and normal baseline gonadotropins were found. Family history revealed four additional adult males affected with various abnormalities of their external genitalia. Molecular analysis of the coding sequence of the AR gene revealed in all a novel point mutation in exon 6, changing threonine to isoleucine at codon position 800 in the hormone-binding domain. We conclude that phenotypic variations in mild AR defects are striking and can remain undetected even until late in life.

Adult↗
PubMed1995

Induction of cell death by myristylated death domain of p55 TNF receptor is not abolished by Iprcg-like point mutation in death domain.

We transiently expressed the intracellular domains of p55 TNF receptor (TNFR1) as either a cytosolic- or a membrane-associated form and examined their effects on the endogenous receptor-mediated gene expression as well as on cell viability. We found that gene expression as measured by luciferase activity under NF-kappa B-controlling elements was blocked by all forms of the intracellular domain of TNFR1. The blockade of reporter gene expression was due to the cell death induced by the intracellular domain of TNFR1 per se. The killing mechanism of the intracellular domain peptides appeared to be apoptotic. Interestingly, myristylated form of the intracellular domain, consisting of mainly death domain showed the most potent cell-killing activity. Moreover, this myristylated death domain could still induce cell death even if lprcg-like point mutation (Leu351 to Ala), which has been reported to abrogate TNF-induced cytotoxicity, was introduced. This result suggests that the myristylated death domain activates an additional death signaling pathway which is not involved in TNF-induced cell death.

Apoptosis↗