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PubMed1992

Determination of point mutational spectra of benzo[a]pyrene-diol epoxide in human cells.

The primary goal of our research consists of developing means sufficiently sensitive to allow assessment of human exposure to environmental carcinogens. We describe here a new approach for analyzing point mutational spectra and a test for its validity and precision using cultured human cells exposed to high doses of environmental carcinogens. The approach in its present form includes a) treatment of independent large cultures of human cells with a carcinogen, b) selection of mutant cells en masse by 6-thioguanine resistance, c) amplification of sequences of interest directly from 6TGR cells using high-fidelity polymerase chain reaction, and d) separation of mutant sequences from nonmutant sequences using denaturing gradient gel electrophoresis. We report use of this protocol to observe induced mutational spectra in exon 3 of the hprt gene in cultured human cells by benzo[a]pyrene-diol epoxide (BPDE), an active form of the widely distributed environmental carcinogen benzo[a]pyrene. BPDE induced predominantly G to T transversions within this target sequence. The variation of the frequency of the mutations among independent cultures is consistent with the interpretation that each of them corresponds to a hotspot.

Benzopyrenes↗
PubMed1987

The embryonic expression of the Notch locus of Drosophila melanogaster and the implications of point mutations in the extracellular EGF-like domain of the predicted protein.

The Notch locus of Drosophila melanogaster is one of a small number of zygotically acting 'neurogenic' genes necessary for the correct segregation of neural from epidermal lineages during embryogenesis. The predicted gene product is implicated in a cell interaction mechanism required to achieve this ectodermal differentiation. We have examined wild-type Notch expression by in situ hybridization and find it to be expressed in more cells than we would have predicted given a sole function in regulating neurogenesis. We conclude from these data that Notch plays a more general role in development. In order to assess the dependence of Notch expression on other neurogenic gene function we have hybridized Notch probes to Enhancer of split mutants which are known to interfere with expression of Notch phenotypes. We intimate that the nature of interaction between these genes is not at the level of transcription. Instead, the DNA sequence of split, which is a missense mutation in the EGF-like extracellular domain of the Notch protein, suggests a direct biochemical interaction between Notch and E(spl) proteins. The similar site of a second point mutation, AxE2, implies that protein interactions also occur between Notch proteins. Finally we discuss the general implications of our findings with a view to the models and mechanisms of Notch action in regulating individual cellular interactions during development.

Animals↗
PubMed1992

Characterization of unprocessed insulin proreceptors in COS 7 cells transfected with cDNA with Arg735----Ser735 point mutation at the cleavage site.

We previously reported on patients with severe insulin resistance due to unprocessed insulin proreceptors. A structural change of the cleavage site from Arg-Lys-Arg-Arg to Arg-Lys-Arg-Ser due to G----T point mutation appeared to be the cause for failure to process the proreceptors. To determine whether the mutation of insulin proreceptors at the cleavage site was responsible for unprocessed insulin receptors and to elucidate the structural and binding characteristics of the proreceptors, we transfected cDNA with the mutation in COS 7 cells and examined the expressed insulin receptors. At 72 hours after transfection, insulin binding increased to the maximum in cells transfected with either normal or mutated cDNA, and insulin binding was 40 and 14 times higher than that of nontransfected cells, respectively. The declining rate of insulin binding after reaching the maximum was delayed in cells transfected with mutated cDNA. Affinity cross-linking and surface-labeling studies showed a 135-kilodalton (kD), normal alpha-subunit in the cells transfected with normal cDNA and a 210-kD proreceptor in the mutant cells. The proreceptors were cleaved by trypsin to yield normal-sized alpha- and beta-subunits. The sensitivity to trypsin was similar to that demonstrated in patients' cells, and the most effective concentration for the cleavage was 0.025%. Autophosphorylation resulted in decreased 32P incorporation into proreceptors of cells transfected with mutated cDNA at both basal and insulin-stimulated states, without a change in insulin sensitivity. Competitive binding studies with insulin, proinsulin, and miniproinsulin showed that the proreceptors had a lower relative affinity for proinsulin, but this characteristic disappeared after trypsin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1991

Higher frequency of point mutations in the c-K-ras 2 gene in human colorectal adenomas with severe atypia than in carcinomas.

Human colorectal carcinomas may be induced from adenomas or they may occur de novo. To examine which is the main pathway, we analyzed point mutations at codon 12 in the c-K-ras 2 gene in 73 colorectal carcinomas, 13 metastatic tumors, 72 adenomas and 30 normal tissues. The c-K-ras 2 codon 12 mutation frequency was 0/30 in normal tissues, 0/17 in adenomas with mild atypia, 3/37 (8.1%) in adenomas with moderate atypia, 15/18 (83.3%) in adenomas with severe atypia, 19/73 (26.0%) in primary carcinomas and 3/13 (23.1%) in metastatic tumors. The mutation frequency in adenomas with severe atypia was much higher than that in carcinomas. These results indicate that many colorectal carcinomas may not be induced through adenomas with severe atypia.

Adenoma↗
PubMed1986

Tallysomycin-induced mitotic aneuploidy and point mutations in Aspergillus nidulans.

Tallysomycin is an antibiotic compound structurally related to bleomycin, and like bleomycins and phleomycins also shows antitumour activity. We have investigated the genetic activity of tallysomycin in the ascomycete Aspergillus nidulans for malsegregation of chromosomes at mitosis and for point mutations. We found that the antibiotic at very low concentrations from 0.025 to 0.2 micrograms/ml had an inhibitory effect on colonial growth of up to 50% and it increased the number of mitotic malsegregants from 311% to 607% over the control value. Tallysomycin at concentrations between 2 and 16 micrograms/ml (which inhibited the germination of conidia from 30 to 90%) also increased the number of methionine suppressor revertants from 59 to 368 per 10(6) conidia. With regard to the mechanism for induced mitotic segregation it was shown that this may involve non-disjunction of chromosomes which through the intermediate formation of unstable aneuploids resulted in formation of stable haploid and diploid segregants.

Aneuploidy↗
PubMed1979

Point mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells. I. Application to genetic toxicological testing.

We have systematically evaluated the mouse lymphoma TK+/- leads to TK-/- mutagenesis assay to determine if this somatic-cell test system would be a useful addition to the routine screening battery already used in our laboratory for the detection of chemical mutagens. During these investigations we observed that, with certain modifications of the basic assay, mutagenicity data could be obtained in as little as 9 days once the relative cytotoxic properties of the test substance were known. By improving the culturing conditions, we were able to reduce the serum requirements by as much as 50--75% without appreciably altering either cell viability or the recovery of chemically-induced mutants. Phenotypic stability of test-derived trifluorothymidine resistant (TFTR) mutants was confirmed by demonstrating cross-resistance to bromodeoxyuridine and concomitant sensitivity to methotrexate (THMG) in TFTR cells grown for 20 generations under non-selective conditions. While reduced growth rates resulting from temporary cell-division delay in treated cells is probably not a contributing factor to the observed mutation frequencies, only TFTR colonies which formed large distinct colonies in the presence of trifluorothymidine were clearly phenotypically stable mutants when spontaneous mutants were isolated and verified. When a non-mutagen, a weak mutagen, and a well-established mutagen were compared at equitoxic doses under these modified conditions, clear quantitative differences were seen in the respective mutation frequencies induced by these 3 types of agents. With these technical modifications, we feel this assay is both reliable and amenable to the screening of diverse chemical compounds for point-mutational activity in a mammalian cell.

Animals↗
PubMed1988

Effect of point mutations on the in-vitro pore properties of maltoporin, a protein of Escherichia coli outer membrane.

Maltoporin (LamB protein), a protein of Escherichia coli outer membrane forms ionic channels with a selectivity for maltose and maltodextrins (Dargent et al., 1987). The effect of different point mutations on maltoporin pore properties was investigated in vitro with planar bilayers. The mutations belong to three classes in terms of selective maltose transport in vivo: class A (substitution at positions 259 and 382) does not affect maltose transport, class B (position 163 and 245) decreases maltose transport down to 20 to 30%, and class C (position 18) almost completely abolishes selective maltose transport. This in-vitro study reveals that class A does not affect the pore properties in contrast to class B substitutions. The class B maltoporins are still able to form channels but display some specific features and altered specificity for maltose and maltodextrins. The substitution (Gly18----Val) alters trimer stability and impedes pore function (class C mutant). Thus, there is a good correlation between the specific transport properties of the mutated maltoporins in vivo and their behavior in vitro. These data, in combination with the asymmetric orientation of the protein within the bilayer and topological considerations, indicate that residues 245 and 163 do not belong to the selectivity filter. Mutations at these sites cause hindrance at the mouth of the pore on the outer domain of maltoporin.

Amino Acid Sequence↗
PubMed1993

Double strand conformation polymorphism (DSCP) detects two point mutations at codon 280 (AAC-->ATC) and at codon 431 (TAC-->AAC) of the blood coagulation factor VIII gene.

Hemophilia A is a hereditary, X-linked, bleeding disorder that is caused by a defect in the factor VIII gene. Here, we report two novel point mutations in the factor VIII gene that result in an aberrant electrophoretic mobility of double strand PCR fragments (double strand conformation polymorphism, DSCP). In exon 9 a TAC-->AAC mutation at codon 431, replacing Tyr by Asn, was observed in a family (A211) with moderately severe hemophilia A. A family with mild hemophilia A revealed an A-->T mutation in codon 280 (exon 7) that results in the replacement of Asn by Ile. One of these two mutations was not detected in an analysis based on single strand conformation polymorphisms (SSCP). At present we have no explanation for the effect of the nucleotide changes on the electrophoretic mobility of double strand DNA. Although DSCP is not able to detect all mutations the combination of DSCP analysis with SSCP analysis increases the sensitivity in a screening for factor VIII mutations.

Base Sequence↗
PubMed1991

Novel point mutations leading to type 1 antithrombin deficiency and thrombosis.

Direct sequencing of antithrombin III (AT) gene fragments specifically amplified by the polymerase chain reaction was utilized to identify the molecular basis of type 1 AT deficiency in two unrelated kindreds, both with thrombotic disease. Two novel point mutations were identified, deletion of a T from the second position of codon 81 in one propositus and insertion of a G in codon 424 in the second kindred. The AT 81(-T) frameshift mutation leads to a premature stop signal in codon 89, while the AT 424(+G) allele has a premature stop only one codon short of the normal gene. The latter mutation changes the eight carboxy terminal residues of AT, including 429Cys, and increases the proportion of polar amino acids in this region. We suggest that altered folding of the mutant protein may explain the AT deficiency.

Adult↗
PubMed1989

Point mutations in the regulatory region of the ilvGMEDA operon of Escherichia coli K-12.

The ilvGMEDA operon of Escherichia coli K-12 is preceded by a regulatory region containing a promoter, a leader, and an attenuator. This region has been extensively characterized biochemically. In this note point mutations of the regulatory region are reported. The effect of these mutations on expression from the ilv regulatory region supports the previous biochemical analysis.

Amino Acid Sequence↗
PubMed1992

Infrequent point mutations in codons 12 and 61 of ras oncogenes in human hepatocellular carcinomas.

DNA from human hepatocellular carcinomas (HCC) were analysed for the presence of mutations in codons 12 and 61 of the K-ras, H-ras and N-ras genes. The relevant ras sequences were amplified in vitro using the polymerase chain reaction and point mutations detected by selective hybridisation using mutation-specific synthetic oligonucleotides. In one of the 19 HCCs a mutation in codon 61 of the K-ras gene was detected, whilst in 3/19 HCCs a mutation was found in codon 61 of the N-ras gene. The mutations were all heterozygous A-T transversions and were found in HCCs arising in patients with underlying cirrhosis. In two of these patients where the corresponding normal tissue was available only the wild-type ras gene was detected, indicating that oncogenic activation of the ras gene was a consequence of somatic mutation. In another patient the same mutation in codon 61 of the N-ras gene was found in cirrhotic liver tissue and in all four patients the same mutation was also detected in formalin-fixed, paraffin-embedded liver biopsy HCC tissue obtained at diagnosis. These results indicate that mutational activation of the ras genes at codon 61 is an infrequent but possibly early event in the development of HCC in Britain.

Aged↗
PubMed1992

High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II.

The plasmids pUC18 and pUC19 are pBR322 derivatives that replicate at a copy number several fold higher than the parent during growth of Escherichia coli at 37 degrees C. We show here that the high copy number of pUC plasmids results from a single point mutation in the replication primer, RNA II, and that the phenotypic effects of this mutation can be suppressed by the Rom (RNA one modulator)/Rop protein or by lowering the growth temperature to 30 degrees C. The mutation's effects are enhanced by cell growth at 42 degrees C, at which copy number is further increased. During normal cell growth, the pUC mutation does not affect the length or function of RNA I, the antisense repressor of plasmid DNA replication, but may, as computer analysis suggests, alter the secondary structure of pUC RNA II. We suggest that the pUC mutation impedes interactions between the repressor and the primer by producing a temperature-dependent alteration of the RNA II conformation. The Rom/Rop protein may either promote normal folding of the mutated RNA II or, alternatively, may enable the interaction of sub-optimally folded RNA II with the repressor.

Bacterial Proteins↗
PubMed1991

Genotyping of the aldehyde dehydrogenase 2 (ALDH2) gene using the polymerase chain reaction: evidence for single point mutation in the ALDH2 gene of ALDH2-deficiency.

About half of all Japanese lack the activity of aldehyde dehydrogenase 2 (ALDH2), and suffer a flush after alcohol intake due to the marked elevation of blood acetaldehyde concentration. The cause of ALDH2 deficiency is thought to be a single point mutation in codon 487 of the ALDH2 gene. However, this mutant ALDH2 gene has not yet been cloned and sequenced. We amplified and cloned the exon 12 of the ALDH2 gene using polymerase chain reaction (PCR), and revealed that normal GAA coding glutamic acid is replaced for AAA coding lysine in codon 487 of the mutant ALDH2 gene. Based on this finding, we performed the genotyping of the ALDH2 gene using PCR and allele-specific oligonucleotide probes. The genotypes of 13 subjects with ALDH2-active phenotype were all homozygous for the normal ALDH2 gene (ALDH2(1)), while in 9 subjects with ALDH2-deficient phenotype 2 subjects were homozygous for the mutant ALDH2 gene (ALDH2(2)) and the other 7 subjects were heterozygous for both genes, indicating that the mutant ALDH2 gene is dominant. In 20 normal control subjects, the prevalence of ALDH2(1)/ALDH2(1), ALDH2(1)/ALDH2(2) and ALDH2(2)/ALDH2(2) was 45%, 45% and 10% respectively. On the other hand, in 36 alcoholic liver disease patients, the prevalence of the genotypes was 83%, 17% and 0%. These results confirmed the previous observation that the incidence of ALDH2 deficiency is much lower in alcoholic liver disease patients than in the general population, and suggested that most of the ALDH2 deficient patients with alcoholic liver disease are heterozygous for the normal and mutant ALDH2 genes.

Aldehyde Dehydrogenase↗
PubMed1992

Point mutations in the dystrophin gene.

Defining the range of mutations in genes that cause human disease is essential to determine the mechanisms of genetic variation and the function of gene domains and to perform precise carrier and prenatal diagnosis. The mutations in one-third of Duchenne muscular dystrophy patients remain unknown as they do not involve gross rearrangements of the dystrophin gene. The size and complexity of the gene have prohibited the systematic definition of point mutations. We have developed a method for the identification of these mutations by nested amplification, chemical mismatch detection, and sequencing of reverse transcripts of trace amounts of dystrophin mRNA from peripheral blood lymphocytes. Analysis of the entire coding region (11 kilobases) in seven patients has resulted in detection of a sequence change in each case that is clearly sufficient to cause the disease. All mutations should cause premature translational termination, and the resulting phenotypes are thus equivalent to those caused by frameshifting deletions. The results support a particular functional importance for the C-terminal region of dystrophin. Application of this approach to mutation detection will extend direct carrier and prenatal diagnosis to virtually every affected family.

Adolescent↗
PubMed1990

The Escherichia coli K-12 metJ193 allele contains a point mutation which alters the hydrophobic pocket responsible for in vitro binding of S-adenosylmethionine: effects on cell growth and induction of met regulon expression.

The metJ193 allele encodes one of two identified temperature-sensitive Escherichia coli K-12 met repressors. The nucleotide sequence of the metJ193 allele was determined. The point mutation was a T to A transversion at base 170 of the metJ193 open reading frame and resulted in the substitution of leucine by glutamine at the 56th amino acid residue of the MetJ193 protein. The mutational lesion altered the hydrophobic pocket responsible for in vitro binding of the corepressor S-adenosylmethionine by wild-type MetJ. MetJ193 protein formed at the permissive temperature (28 degrees C) allowed slow derepression of met regulon expression when cultures were shifted to the nonpermissive temperature (34 degrees C). When 28 degrees C cultures of strains bearing two metJ193 alleles were transferred from methionine-containing medium to minimal medium, derepression of met regulon expression did not occur quickly enough to avoid a lag in growth due to the methionine deprivation. The inability of the MetJ193 protein to easily accomplish transition between apo- and active-repressor conformations was also demonstrated by using a maxicell system to study expression of a plasmid-borne copy of the E. coli metF transcription unit. These results confirm the importance of the leucine 56 residue for the structure and function in vivo of the wild-type MetJ protein.

Alleles↗
PubMed1991

Point mutation in the human dystrophin gene: identification through western blot analysis.

Using antibodies directed against the amino-terminus of dystrophin, we identified a truncated protein in a Duchenne muscular dystrophy patient. Antibodies directed against the carboxy-terminus failed to identify any cross-reactive material, a result consistent with premature termination of dystrophin translation. The estimated molecular mass of 126 kDa predicted the approximate location of the mutation in the mRNA and in the gene. Sequencing of cloned PCR products from patient muscle cDNA revealed a nonsense mutation, which was confirmed by direct sequencing of amplified patient genomic DNA. The mutation, a G to T transversion, at position 3714 changes a glutamic acid codon to an Amber stop codon. Translation of mRNA containing this mutation would be expected to result in a truncated protein with a molecular mass of 133 kDa, in close agreement with the 126 kDa estimated by Western blot analysis. This is the first reported case of a point mutation in this very large human gene.

Adolescent↗
PubMed1990

The site of an immune-selected point mutation in the transmembrane protein of human immunodeficiency virus type 1 does not constitute the neutralization epitope.

We previously reported the in vitro generation of a neutralization-resistant variant of the molecularly cloned isolate of human immunodeficiency virus type 1 (HIV-1), HXB2D. The molecular basis for the resistance was shown to be a point mutation in the env gene, causing the substitution of threonine for alanine at position 582 of gp41. Here, we show the variant to be resistant to syncytium inhibition as well as to neutralization by the immune-selecting serum. Moreover, 30% of HIV-positive human sera able to neutralize the parental virus have significantly decreased ability to neutralize the variant. As the A-to-T substitution thus has general relevance to the interaction of HIV-1 with the host immune system, we investigated further the biologic and immunologic bases for the altered properties. Synthetic peptides corresponding to the 582 region failed to compete in infectivity, neutralization, or syncytium inhibition assays and did not elicit neutralizing antibodies. Furthermore, human antibodies, affinity purified on synthetic peptide resins, bound to gp41 and peptides from the 582 region but did not possess neutralizing antibody activity. Some viral constructs in which the AVERY sequence in the 582 region was altered by site-directed mutagenesis were not infectious, indicating that the primary structure in this region is crucial for viral infectivity. Constructs predicted to possess a local secondary structure similar to that of the variant nevertheless behaved like the parental virus and remained neutralization sensitive. These results suggest that the requirements for neutralization resistance in this region are very precise. Our results with synthetic peptides show that the 582 region does not by itself constitute a neutralization epitope. Moreover, the degree of flexibility in amino acid substitution which allows maintenance of neutralization sensitivity suggests that position 582 does not form part of a noncontiguous neutralization epitope. The basis for neutralization resistance of the immune-selected variant is more likely a conformational change altering a neutralization epitope at a distant site.

Amino Acid Sequence↗
PubMed1985

Identification of a point mutation in the adenosine deaminase gene responsible for immunodeficiency.

Deficiency of adenosine deaminase (ADA) is the cause of an autosomal recessive form of immunodeficiency. We sought to define, at a molecular level, the mutations responsible for ADA deficiency in the cell line GM-1715, derived from an immunodeficient patient. Full-length complementary DNA (cDNA) for ADA was synthesized and cloned from the cell line. Sequence analysis of the clones revealed a point mutation in codon 101 (CGG to CAG) that predicts an amino acid change from arginine to glutamine. Southern blot analysis, based on silent polymorphisms in the cDNA sequence, indicated that only one of the defective alleles of the GM-1715 line had been sequenced. The mutation that was identified appears to be responsible for the loss of function in this allele, since the predicted primary structure of the enzyme is otherwise entirely normal.

Adenosine Deaminase↗