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PubMed1985

Point mutations in the 3' minor domain of 16S rRNA of E.coli.

Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing.

Base Sequence↗
PubMed1989

Direct detection of point mutations by mismatch analysis: application to haemophilia B.

Rapid detection of point mutations in genomic DNA has been achieved by chemical mismatch analysis of heteroduplexes formed between amplified wild-type and target sequences in the human factor IX gene. Amplification and mismatch detection (AMD) analysis of DNA from relatives of haemophilia B patients permitted carrier diagnosis by direct identification of the presence or absence of the mutation in all cases, thus eliminating the need for the informative segregation of polymorphic markers. This extends diagnostic capability to virtually all haemophilia B families. AMD analysis permits detection of all sequence variations in genomic DNA and is therefore applicable to direct diagnosis of X-linked and autosomal diseases and for identification of new polymorphisms for genetic mapping.

Amino Acid Sequence↗
PubMed1991

Enhanced stability of subtilisin by three point mutations.

This study was undertaken to characterize the effect of three point mutations made on aprA-subtilisin on the stability of the protein to both heat- and detergent-induced denaturation. Asparagine residues at positions 109 and 218 were replaced with serine residues to prevent the possible cyclization between these asparagines and the adjacent glycine residues and hence to increase the long-term stability. The effect of these substitutions on conformational stability was examined by thermal denaturation. At high calcium concentrations, the Ser109-substituted analog showed a 3 degrees C higher transition temperature than that of aprA-subtilisin, while the Ser218 substituted analog had a 4 degrees C higher transition temperature. The analog with both changes had a 7 degrees C higher transition temperature than that of the original aprA-subtilisin, indicating that the contributions of the individual mutations were additive. The analog with both mutations also exhibited increased stability in the presence of sodium dodecyl sulfate (SDS) when compared to aprA-subtilisin. In addition to the above two mutations, the asparagine at position 76, located in the high affinity Ca(2+) binding loop of subtilisin, was changed to aspartic acid. The effect of this mutation on the thermal stability of the protein was examined at different calcium concentrations. The analog with all three mutations exhibited little dependence on calcium concentration below 1 mM levels, while the proteins without the mutation at asparagine-76 displayed a strong dependence of melting temperature on Ca(2+) concentration in this range. At much higher calcium concentrations, the analog with three mutations showed an increase in stability similar to that observed with aprA-subtilisin. The analog with three mutations also exhibited greater stability to SDS-induced denaturation than both aprA-subtilisin and the Ser109- and Ser218-substituted analogs. The activation energy barrier for loss of structure in 1% SDS for the analog with all three mutations was increased over that for aprA-subtilisin by 16 kcal/ml. These results suggest that the mutation of asparagine-76 to aspartic acid increases the affinity of the primary Ca(2+) binding site.

Circular Dichroism↗
PubMed1986

Somatic cell variants of H-2Kb: a point mutation in the first extracellular domain results in altered immune recognition.

A cell-surface-associated variant H-2K product was expressed by an Abelson virus-induced pre-B-cell line after chemical mutagenesis with ethyl methane sulfonate. The variant cell line (R8.313) was previously demonstrated to have altered allodeterminants in Kb as demonstrated by both Kb-specific monoclonal antibody binding and alloreactive cytotoxic T lymphocyte (CTL) cytolysis. The mutant H-2Kb gene from R8.313 was cloned and characterized in detail. DNA sequence analysis of the region of the gene corresponding to the three extracellular domains identified a single point mutation resulting in a leucine-to-phenylalanine substitution at amino acid residue 82. The site of mutation within the alpha 1 domain was confirmed by oligonucleotide hybridization analysis. Mouse L-cell fibroblasts transfected with the mutant gene were recognized with the same monoclonal antibody binding and CTL lytic pattern as the R8.313 cell line, confirming that the altered phenotype of the mutant cell line was due to a point mutation in the H-2Kb gene. These data further extend the hypothesis that the region of amino acid residues 70-90 in the alpha 1 domain is important in the formation of both antibody and CTL-defined recognition structures on major histocompatibility complex class I molecules.

Animals↗
PubMed1998

Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae.

Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated. Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345). The latter mutation clearly affects the 3' end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation. The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA. These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation.

Base Sequence↗
PubMed1979

Possible point mutation sites in LSc, 2ab poliovirus RNA and a protein covalently linked to the 5'-terminus.

Poliovirus type I, vaccine strain (LSc, 2ab), which is a temperature- and actinomycin D-sensitive mutant derived from type I Mahoney strain, was grown in HeLa cells in the presence of 32P and a low concentration of actinomycin D. Seven and a half h p.i., genome 32P-RNA was recovered from the purified virion. Analysis of RNase TI digests of the RNA by two-dimensional gel electrophoresis revealed that three possible point mutation sites exist in the large and unique oligonucleotides in the fingerprint. Neither a capping structure nor a nucleotide such as pppNp, ppNp or pNp, was detected by ion exchange column chromatography at pH 5.0 after digestion of virion RNA with RNase T2. Instead, a 32P-labelled compound, which could be digested with Pronase or proteinase K, was eluted at the void volume of the column. Proteinase K digests of the 32P-labelled compound contained pUp or pU as a single labelled c"mpound, when genome RNA was digested with RNase T2 or nuclease P1, respectively, before digestion with the proteinase. Our data locate possible point mutation sites on the genome of a mutant strain (LSc, 2ab) of type I poliovirus and show that a protein (VPg) is covalently bound to the 5'-terminus of RNA. The protein (VPg) of LSc, 2ab strain migrates faster than capsid protein VP4 (mol. wt. 7000 to 8000) in a polyacrylamide gel and is thus similar to the VPg of the wild-type virus.

Genes, Viral↗
PubMed2001

Detection of DNA point mutations and mRNA expression levels by rolling circle amplification in individual cells.

Rolling circle amplification has been useful for detecting point mutations in isolated nucleic acids, but its application in cytological preparations has been problematic. By pretreating cells with a combination of restriction enzymes and exonucleases, we demonstrate that rolling circle amplification in situ can detect gene copy number and single base mutations in fixed cells with efficiencies up to 90%. It can also detect and quantify transcribed RNA in individual cells, making it a versatile tool for cell-based assays.

Base Sequence↗
PubMed1998

Structural and dynamic effects of point mutations in the recognition helix of the glucocorticoid receptor DNA-binding domain.

We have studied the wild type and two variants of the glucocorticoid receptor DNA-binding domain (GRDBD): in one variant the three residues (the 'P-box' in GRDBD) that are essential for the discrimination between GREH and EREH are mutated to those in the estrogen receptor DBD (GRDBDega) and the other variant is a point mutation of one P-box residue, Ser459Gly (GRDBDggv). Molecular dynamics simulations (0.5-0.7 ns) have been performed on the GRDBDs, free in solution as well as in complex with the half-site response elements of the glucocorticoid (GREH) and estrogen (EREH) receptors. The residues which are central when forming the protein dimer interface in GRE-(GRDBD)2 (the 'D-box') were found to have different conformations in the different GRDBD-DNA complexes. This is consistent with experimental results showing that the cooperativity of dimeric GRDBD binding to DNA strongly depends on both the response element and the P-box residues. In our simulations the structures of GREH-GRDBDgsv (i.e. wild-type) and GREH-GRDBDggv were more similar to each other than to the respective GRDBDs bound to EREH. This is due to a thymine methyl group which is present in the major groove of the GREH and prevents the first zinc coordinating subdomain in GRDBD to approach GREH, but which is absent in EREH. Thus, EREH-GRDBD is able to respond more to the Ser459Gly mutation than GREH-GRDBD.

Amino Acid Sequence↗
PubMed2006

CUPSAT: prediction of protein stability upon point mutations.

CUPSAT (Cologne University Protein Stability Analysis Tool) is a web tool to analyse and predict protein stability changes upon point mutations (single amino acid mutations). This program uses structural environment specific atom potentials and torsion angle potentials to predict DeltaDeltaG, the difference in free energy of unfolding between wild-type and mutant proteins. It requires the protein structure in Protein Data Bank format and the location of the residue to be mutated. The output consists information about mutation site, its structural features (solvent accessibility, secondary structure and torsion angles), and comprehensive information about changes in protein stability for 19 possible substitutions of a specific amino acid mutation. Additionally, it also analyses the ability of the mutated amino acids to adapt the observed torsion angles. Results were tested on 1538 mutations from thermal denaturation and 1603 mutations from chemical denaturation experiments. Several validation tests (split-sample, jack-knife and k-fold) were carried out to ensure the reliability, accuracy and transferability of the prediction method that gives >80% prediction accuracy for most of these validation tests. Thus, the program serves as a valuable tool for the analysis of protein design and stability. The tool is accessible from the link http://cupsat.uni-koeln.de.

Databases, Protein↗
PubMed2004

Cells resistant to HTI-286 do not overexpress P-glycoprotein but have reduced drug accumulation and a point mutation in alpha-tubulin.

HTI-286, a synthetic analogue of hemiasterlin, depolymerizes microtubules and is proposed to bind at the Vinca peptide site in tubulin. It has excellent in vivo antitumor activity in human xenograft models, including tumors that express P-glycoprotein, and is in phase II clinical evaluation. To identify potential mechanisms of resistance induced by HTI-286, KB-3-1 epidermoid carcinoma cells were exposed to increasing drug concentrations. When maintained in 4.0 nmol/L HTI-286, cells had 12-fold resistance to HTI-286. Cross-resistance was observed to other Vinca peptide-binding agents, including hemiasterlin A, dolastatin-10, and vinblastine (7- to 28-fold), and DNA-damaging drugs, including Adriamycin and mitoxantrone (16- to 57-fold), but minimal resistance was seen to taxanes, epothilones, or colchicine (1- to 4-fold). Resistance to HTI-286 was retained when KB-HTI-resistant cells were grown in athymic mice. Accumulation of [(3)H]HTI-286 was lower in cells selected in intermediate (2.5 nmol/L) and high (4.0 nmol/L) concentrations of HTI-286 compared with parental cells, whereas accumulation of [(14)C]paclitaxel was unchanged. Sodium azide treatment partially reversed low HTI-286 accumulation, suggesting involvement of an ATP-dependent drug pump. KB-HTI-resistant cells did not overexpress P-glycoprotein, breast cancer resistance protein (BCRP/ABCG2/MXR), MRP1, or MRP3. No mutations were found in the major beta-tubulin isoform. However, 4.0 nmol/L HTI-286-selected cells had a point mutation in alpha-tubulin that substitutes Ser for Ala(12) near the nonexchangeable GTP-binding site of alpha-tubulin. KB-HTI-resistant cells removed from drug became less resistant to HTI-286, no longer had low HTI-286 accumulation, and retained the Ala(12) mutation. These data suggest that HTI-286 resistance may be partially mediated by mutation of alpha-tubulin and by an ATP-binding cassette drug pump distinct from P-glycoprotein, ABCG2, MRP1, or MRP3.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1985

Posttranslational cleavage of proinsulin is blocked by a point mutation in familial hyperproinsulinemia.

Familial hyperproinsulinemia is characterized by the accumulation of proinsulin-like material (PLM) in the plasma of affected patients. This disorder is inherited in an autosomal dominant fashion. The accumulation of PLM is thought to be due to the impaired conversion of proinsulin to insulin. Although PLM has been suggested to have an amino acid substitution, it has been impossible to locate and identify a substituted amino acid, due to the difficulty in isolating sufficient amounts of PLM from plasma samples. Therefore, we analyzed leukocyte DNA from one member of a proinsulinemic family, and we found a point mutation that changed guanine to adenine in the insulin gene. This transition implies that a substitution of histidine for arginine has occurred at amino acid position 65. Furthermore, it indicates that arginine at 65 is essential for the conversion of proinsulin to insulin. Our results suggest a novel mechanism by which disease can be incurred: a heritable disorder can result from a posttranslational processing abnormality caused by a point mutation.

Amino Acid Sequence↗
PubMed2003

Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans.

Restriction enzyme mediated insertional mutagenesis using a plasmid, pUCATPH, that confers hygromycin resistance, generated loss-of-pathogenicity mutants of Leptosphaeria maculans, the fungus that causes blackleg disease of Brassica napus. Of 516 L. maculans transformants analysed, 12 were pathogenicity mutants. When eight of these mutants were crossed to an isolate that attacks B. napus, cosegregation of pUCATPH sequences and loss of pathogenicity was not observed, suggesting that these mutations were not linked to plasmid sequences. In seven of eight crosses analysed, progeny with the hygromycin resistance gene were hygromycin-sensitive. Sequence analysis of an amplified fragment of pUCATPH in six clones derived from one 'silenced' progeny showed mutation of GC to AT on one DNA strand, reminiscent of repeat-induced point mutation (RIP) in Neurospora crassa. One loss-of-pathogenicity mutant had pUCATPH inserted in the promoter of a gene with an open reading frame of 529 amino acids that had no database match. Reintroduction of a wild-type copy of the gene to this mutant restored the ability to form lesions on cotyledons of B. napus.

Amino Acid Sequence↗
PubMed1998

A novel complex allele and two new point mutations in type 2 (acute neuronopathic) Gaucher disease.

Gaucher disease, the most prevalent inherited sphingolipidosis, is characterized by lipid laden histiocytes in the spleen, liver and bone marrow sinusoids of affected individuals. It results from deleterious mutations in the functional gene of glucocerebrosidase (acid beta-glucosidase, EC. 3.2.1.45) and is transmitted as an autosomal recessive trait. Three clinical forms of Gaucher disease have been described: Type 1 non-neuronopathic, type 2 acute neuronopathic, and type 3 subacute neuronopathic. In this report, we describe the identification and characterization of three novel mutations from two patients who died with type 2 Gaucher disease. Two heterozygous missense point mutations, one at cDNA nucleotide 238A (E41L) and the other at cDNA nucleotide 508T (R131C) were identified, both in the context of a cDNA nucleotide 1448C (L444P) mutation in the second allele. One of these L444P mutations was identified as a novel complex allele resulting from a crossover involving the glucocerebrosidase functional gene and pseudogene beginning between genomic nucleotides 5689 and 5723 and extending through the rest of the coding sequence. Based on the recent identification and sequence analysis of the metaxin gene and pseudogene contiguous with the glucocerebrosidase pseudogene and functional gene respectively, we have developed a PCR-based method for the analysis of the origin and extent of this recombination.

Adult↗
PubMed2001

Point mutations in alpha-subunit of human cardiac Na+ channels alter Na+ current kinetics.

Dietary polyunsaturated fatty acids (PUFAs) prevent ischemia-induced fatal cardiac arrhythmias in animals and probably in humans. This action results from inhibition of ion currents for Na+, Ca2+, and possibly other ions. To extend understanding of this protection we are seeking a possible binding site for the PUFAs on the alpha-subunit of the human cardiac Na+ channel, hH1alpha, transiently expressed in HEK293t cells. Three mutated single amino acid substitutions with lysine were made in the alpha-subunit at Domain 4-Segment 6 (D4-S6) for F1760, Y1767 and at D1-S6 for N406. These are in the putative sites of binding of local anesthetics and batrachotoxin, respectively. The mutants F1760K, Y1767K, and N406K, separately and to different extents, affected the current density, the steady-state inactivation potential, accelerated inactivation, delayed recovery from inactivation, and affected voltage-dependent block, but did not affect activation of the hH1alpha. It is essential to learn that single point mutations in D1-S6 and D4-S6 alone significantly modify the kinetics of human cardiac hH1alpha Na+ currents. The effects of PUFAs on these mutant channels will be the subject of subsequent reports.

Amino Acids↗
PubMed2002

[Different clinical phenotypes of siblings with familial amyotrophic lateral sclerosis showing Cys146Arg point mutation of superoxide dismutase 1 gene].

We reported different clinical phenotypes of siblings with familial amyotrophic lateral sclerosis (FALS) showing Cys146Arg point mutation of superoxide dismutase 1 (SOD1) gene. They showed differences in disease onset of age, progression and severity. The propositus, a 50-year old woman, had muscle wasting and weakness in right lower leg with dysesthesia at the onset, which showed slow progression without other neurological symptoms until 2 years after the onset when bulbar palsy appeared. She has been alive for 3.5 years after the onset. In contrast, a 61-year old man, elder brother of the propositus, had bulbar palsy at the onset, which showed rapid progression along with muscle weakness and wasting of upper extremities. Two years after the onset, he died due to respiratory failure. Detailed clinical features of FALS with Cys146Arg mutation of SOD1 have not been reported. Our cases suggest that FALS with the same SOD1 mutation could show variable clinical feature and course, and that some factors other than SOD1 mutation should be considered for the pathogenesis of FALS.

Amyotrophic Lateral Sclerosis↗
PubMed1990

Point mutation in codons 12 and 61 of the Ha-ras gene in rat urinary bladder carcinomas induced by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide.

Male F344 rats were fed N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) for up to 4 wk, then were given the basal diets (Prolab 3200 or AIN-76A) with or without 5% sodium saccharin for up to 100 wk. Eleven transitional cell carcinomas (TCCs), one undifferentiated carcinoma, and two sarcomas of the urinary bladder were examined for the expression of ras gene product, p21, by immunohistochemical staining and western blot analysis. Point mutation in codons 12 or 61 of the Ha-ras genes amplified by polymerase chain reaction was examined by a slot-blot screening procedure using allele-specific oligonucleotide probes. Immunohistochemical staining showed enhanced immunoreactivity with the antibody to ras p21 in seven TCCs and one undifferentiated carcinoma. Western blot analysis showed faster migration of the p21 band in 6 of 11 TCCs. Oligonucleotide hybridization revealed the point mutation in codon 12 of Ha-ras gene (GGA----GTA in 1 TCC) and in codon 61 (CAA----CGA in 5 TCCs and CAA----CTA in 1 TCC). Two mutations in codons 12 and 61 coexisted in one tumor, which were found to be present in different Ha-ras alleles. The incidence of Ha-ras gene mutations were similar in groups treated with (3 of 6) or without (3 of 8) sodium saccharin. These results suggest the involvement of activated Ha-ras gene in rat urinary bladder carcinogenesis induced by FANFT.

Animals↗
PubMed1998

p53 point mutations and thymidylate synthase messenger RNA levels in disseminated colorectal cancer: an analysis of response and survival.

Recent studies suggest that there may be a strong correlation between the p53 status of a tumor and a patient's response to chemotherapy. Therefore, we determined p53 status in 36 patients with disseminated colorectal cancer by cDNA sequencing and immunohistochemical staining, as well as by the gene expression level of thymidylate synthase (TS), the target enzyme of 5-fluorouracil (5-FU), by reverse transcription-PCR. Ten patients (28%) experienced a clinical response to 5-FU chemotherapy. Overall, TS expression and response to chemotherapy were associated: 9 of 18 (50%) patients with TS < or = 3.0 x 10(-3) responded, compared to 1 of 18 (6%) patients with TS > 3.0 x 10(-3) (P = 0.003). p53 mutations were found in 21 of 36 patients (58%) using cDNA cycle sequencing, and p53 protein overexpression was found in 20 of 32 patients (62%) using immunohistochemistry staining. Overall p53 status and response to chemotherapy were associated: 5 of 10 (50%) patients with wild-type p53 or negative p53 staining experienced a response, but only 5 of 26 (19%) patients with mutant p53 or p53 overexpression responded. TS expression, but not expression of p53, was significantly associated with overall survival (P = 0.002). Patients with wild-type p53 had significantly lower TS levels compared to patients with mutated p53 (P = 0.044). In this study, we also present data linking specific p53 point mutations to TS expression levels and resistance to 5-FU. Although the number of patients is relatively small, these results identify p53 status and TS gene expression as associated with response in disseminated colorectal cancer; independent studies are needed to confirm these findings and to provide information leading to a better understanding of the role of 5-FU-based chemotherapy in the treatment of colorectal cancer.

Adult↗
PubMed1999

Characterization of Neurospora crassa mutants isolated following repeat-induced point mutation of the beta subunit of fatty acid synthase.

Neurospora crassa cel-2 mutants were isolated following repeat-induced point mutation using part of the gene encoding beta-fatty acid synthase. These mutants are phenotypically less leaky than cel-1, which has a defective alpha-fatty acid synthase. The cel-2 mutant had a strict fatty acid (16:0) requirement for growth, and synthesized less fatty acid de novo than cel-1. Unlike cel-1, cel-2 has impaired fertility, and homozygous crosses are infertile, suggesting a low but strict requirement for fatty acid synthesis during sexual development. Like cel-1, cel-2 synthesized unusually high levels of the polyunsaturate 18:3(Delta9,12,15), and elongated 18:2(Delta9,12 )and 18:3(Delta9,12,15 )to 20:2(Delta11,14) and 20:3(Delta11,14,17), respectively. These fatty acids are not synthesized by wild-type, except following treatment with cerulenin (a fatty acid synthase inhibitor), demonstrating that inhibition of fatty acid biosynthesis results in a relative increase in both fatty acid desaturation and elongation activity.

Alleles↗