Distribution and difference of APOBEC-induced mutations in the TpCpW context of HBV DNA between HCC and non-HCC

Hepatitis B virus (HBV) DNA is vulnerable to editing by human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) cytidine deaminases. However, the distribution of APOBEC-induced mutations on HBV DNA is not well characterized. To this end, we obtained the HBV DNA sequence of HBV-infected individuals with and without hepatocellular carcinoma (HCC and non-HCC groups, respectively) from NCBI database and calculated the r_apo values of APOBEC-induced TpCpW→TpKpW mutation prevalence in HBV DNA. The results showed that the APOBEC-induced mutations were mainly distributed in the minus strand of non-HCC-derived HBV DNA (r_apo = 2.04), while the mutation on the plus-strand was weaker (r_apo = 0.99). There were high APOBEC-induced mutation regions in the minus strand of HBV DNA 1 to 1000 nucleotides (nts) region and in the plus-strand of HBV DNA 1000 to 1500 nts region; the mutations in the 1 to 1000 nts region were mainly TpCpW→TpTpW mutation types (total T/G: 111/18) and a number of these were missense mutations (missense/synonymous: 35/94 in P gene, 17/15 in S gene, and 5/10 in X gene). The difference between minus to plus-strand r_apo of HCC-derived HBV DNA (1.96) was greater than that of the non-HCC group (1.05). The minus-strand r_apo of HCC-derived HBV DNA regions 1000 to1500nts and 1500 to 2000 nts (r_apo = 4.2 and 4.2) was also higher than that of the same regions of non-HCC-derived HBV DNA (r_apo = 1.2 and 1.1). Finally, the ratio of minus to plus-strand r_apo was used to distinguish HCC-derived HBV DNA from non-HCC-derived HBV DNA. This study unraveled the distribution characteristics of APOBEC-induced mutations on double strands of HBV DNA from HCC and non-HCC samples. Our findings would help understand the mechanism of APOBECs on HBV DNA and may provide important insights for the screening of HCC.     

RAD58 (XRS4)—A new gene in the RAD52 epistasis group

The RAD58 (XRS4) gene of Saccharomyces cerevisiae has been previously identified as a DNA repair gene. In this communication, we show that RAD58 also encodes an essential meiotic function. The spore inviability of rad58 strains is not rescued by a spo13 mutation. The rad50 mutation suppresses spore inviability of a spo13 rad58 strain suggesting that RAD58 acts after RAD50 in meiotic recombination. The rad58-4 mutation does not prevent mitotic recombination events. Haploid rad58 cells fail to carry out G₂-repair of gamma-induced lesions, whereas rad58/rad58 diploids are able to perform some diploid-specific repair of these lesions.     

Isolation and molecular analysis of the gene for cytochrome c1 from Kluyveromyces lactis

By ethyl methanesulphonate mutagenesis of the yeast Kluyveromyces lactis we have isolated five nuclear mutants that were unable to grow on non-fermentable carbon sources. The mutations were found to belong to three complementation groups. After functional complementation of the mutation in one of these mutants we have cloned the structural gene for cytochrome c ₁, named KlCYT1. This gene has been assigned to chromosome VI and its nucleotide sequence exhibited 74.3% identity to the homologous gene of S. cerevisiae. Received: 6 February 1996 / 2 April 1996     

Increased diuron resistance in the joint expression of mutations located at the DIU2, DIU3 and DIU4 loci of Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, diuron blocks the respiratory pathway at the level of the bc₁ complex. Two mitochondrially inherited loci, DIU1 and DIU2, located in the cytochrome b gene, and two nuclearly inherited loci, DIU3 and DIU4, have previously been identified. The present work genetically characterizes two double mutants. One mutant, Diu-217, carries two nuclearly inherited mutations, diu3-217a and diu-217b; the second mutant, Diu-783, carries the previously described nuclear mutation diu3-783 and a mitochondrial mutation diu2-783. Each mutation, independent of its location, exhibits a weak diuron resistance. The joint expression of two or three mutations leads to a cumulative or a cooperative enhanced diuron-resistant phenotype.     

Cloning and characterization of KlCOX18, a gene required for activity of cytochrome oxidase in Kluyveromyces lactis

We describe the isolation and initial characterization of KlCOX18, a gene that is essential for the assembly of a functional cytochrome oxidase in the yeast Kluyveromyces lactis. Cells carrying a recessive nuclear mutation in this gene are respiratory deficient and contain reduced levels of cytochromes a and a ₃ . The KlCOX18 gene has been cloned by complementation of the respective nuclear mutation, sequenced, and disrupted. KlCOX18 is located on chromosome II and contains an open reading frame of 939 base pairs. The corresponding protein exhibits 70.4% similarity to the Cox18p of Saccharomyces cerevisiae. It contains three possible membrane-spanning domains and a putative amino-terminal mitochondrial import sequence. The strain carrying a null mutation in KlCOX18 does not grow on non-fermentable carbon sources and is deficient in both cytochrome c oxidase and respiratory activity. It is proposed that KlCox18p, like its S. cerevisiae counterpart, provides an important function at a later step of the cytochrome oxidase assembly pathway. Received: 25 March / 3 July 1997     

Mutations Affecting the Delayed Rectifier Potassium Current in Drosophila

Mutations and pharmacological agents have been used to resolve and analyze several K⁺ currents in Drosophila. Mutations that affect channels carrying the voltage-activated I_A and the Ca²⁺-activated I_CF have helped greatly in analyzing the structure, function and regulation of these channels. We now report mutations that selectively affect the delayed rectifier current, I_R. Flies mutagenized with ethylmethanesulfonate were screened for temperature-induced paralysis. Paralytic mutants identified in the screen were examined for K⁺ currents in the larval body-wall muscles. The z66 mutant larvae showed a significant reduction in I_K. The mutation did not affect other K⁺ currents (I_A, I_CF, or I_CS) or the Ca²⁺ channel current in the muscles. Another mutation, z4, which showed reduced I_K, failed to complement z66. Genetic analysis localized the gene disrupted by z66 and z4 to the left arm of chromosome 3, in the 63A1–63B6 region on polytene chromosomes. The z66 and the z4 mutations, which lie in the Shab K⁺ channel gene, provide an opportunity to undertake analysis of the functioning of these channels and to study the role of these channels in membrane excitability.     

The nude mutation results in impaired primary antibody repertoire

We have studied the effect of the nude mutation and/or T lymphocytes on the development of V gene germ-line repertoire in neonatal athymic (nu/nu) and euthymic (+/nu) littermates. A total of 2.35 × 10⁶ and 1.47 × 10⁶ B lymphocyte clones from nu/nu and +/nu neonates, respectively, were examined for the expression of select V_H (J558, J606, S107, 36–60, 7183 and Q52) and V_x (1, 2, 8 and 9) gene families as well as V_H (J558, S107) + V_x (1, 9) associations. Data showed that the nude mutation, whether homozygous or heterozygous, significantly affects V_H and V_x gene expression as well as V_H and V_x pairings and, thus, provide evidence for a defective development of B cell repertoire in both athymic nude (nu/nu) and euthymic (+/nu) mice. In addition, an analysis of 3.34 × 10⁶ B lymphocyte clones from adult C57BL/6 mice showed non-stochastic association between V_HJ558 + V_x1 gene families that suggests restrictions on clonal population in order to maintain homeostasis in the immune system. Studies outlined here, therefore, describe an hitherto unknown defect in the development of B lymphocyte repertoire as a result of the nude mutation which is independent of thymic dysgenesis.     

SLC40A1 Q248H allele frequencies and associated SLC40A1 haplotypes in three West African population samples

Background: Ferroportin is a transmembrane protein responsible for iron export from enterocytes and macrophages. Mutation c.744G → T (Q248H), located in exon 6 of the ferroportin gene SLC40A1, is found as a polymorphism in populations of African origin. This mutation has been extensively analysed in African-Americans, but poorly studied in native African populations.

Aim: To increase information about Q248H mutation frequency in native sub-Saharan populations examining three West African populations.

Subjects and methods: Samples from S. Tomé e Príncipe (n = 115), Angola (n = 156) and Republic of Guinea (n = 170) were analysed for Q248H mutation and for two polymorphisms, IVS1( ? 24)G → C and microsatellite (CGG)_n, using standard molecular methodology.

Results: The estimated frequencies of Q248H allele were 2.2% in S. Tomé e Príncipe, 3.5% in Angola and 4.1% in Republic of Guinea. Analysis of polymorphisms IVS1( ? 24)G → C and (CGG)_n showed mutation allele c.744T to be strongly associated with haplotype IVS1( ? 24)G/(CGG)₇.

Conclusions: This study confirmed the presence of Q248H mutation at polymorphic frequencies in three native sub-Saharan populations. Analysis of two additional markers in the same gene support a single origin of the mutant allele c.744T in the haplotype background IVS1( ? 24)G/(CGG)₇.     

Systematic screening for mutations in the promoter and the coding region of the 5-HT1A gene

In the present study we sought to identify genetic variation in the 5-HT_1A receptor gene which through alteration of protein function or level of expression might contribute to the genetic predisposition to neuropsychiatric diseases. Genomic DNA samples from 159 unrelated subjects (including 45 schizophrenic, 46 bipolar affective, and 43 patients with Tourette's syndrome, as well as 25 healthy controls) were investigated by single-strand conformation analysis. Overlapping PCR (polymerase chain reaction) fragments covered the whole coding sequence as well as the 5′ untranslated region of the 5-HT_1A gene. The region upstream to the coding sequence we investigated contains a functional promoter. We found two rare nucleotide sequence variants. Both mutations are located in the coding region of the gene: a coding mutation (A→G) in nucleotide position 82 which leads to an amino acid exchange (Ile→Val) in position 28 of the receptor protein and a silent mutation (C→T) in nucleotide position 549. The occurrence of the Ile-28-Val substitution was studied in an extended sample of patients (n = 352) and controls (n = 210) but was found in similar frequencies in all groups. Thus, this mutation is unlikely to play a significant role in the genetic predisposition to the diseases investigated. In conclusion, our study does not provide evidence that the 5-HT_1A gene plays either a major or a minor role in the genetic predisposition to schizophrenia, bipolar affective disorder, or Tourette's syndrome. © 1995 Wiley-Liss, Inc.     

The ogd1 and kgd1 mutants lacking 2-oxoglutarate dehydrogenase activity in yeast are allelic and can be differentiated by the cloned amber suppressor

The activity of mitochondrial 2-oxoglutarate dehydrogenase in S. cerevisiae can be impaired either by the ogd1 or the kgd1 mutation. The OGD1 gene and two suppressor genes were isolated by complementation of the ogd1 mutant. The complementation of the kdg1 mutant by the OGD1 gene, an allelism test, and meiotic mapping, revealed that the ogd1 and kgd1 mutations are allelic. The two mutations were differentiated by the cloned suppressor gene which was able to partially complement ogd1, but not kgd1. The molecular analysis of the suppressor gene revealed its identity with the natural tRNA _CAG ^Gln gene found in the upstream region of URA10.     

The role of the outer membrane protein gene ybjX in the pathogenicity of avian pathogenic Escherichia coli

Avian pathogenic Escherichia coli (APEC) are important pathogens that cause serious economic losses to the poultry industry worldwide. The PhoP/Q system is essential for APEC pathogenicity, and its mutation leads to a decrease in ybjX gene expression, but whether ybjX regulated by PhoP/Q plays a role in pathogenicity remains unclear. The role of ybjX in APEC pathogenicity was investigated using a ybjX mutant strain constructed from APEC strain AE17 by the Red recombination method. The mutant strain displayed significantly reduced adherence to and invasion of chicken embryo fibroblast cell line (DF-1) cells. Moreover, N-phenyl-1-naphthylamine (NPN) assays revealed an increase in outer membrane permeabilization for the ybjX mutant, suggesting ybjX affected susceptibility to avian beta-defensin through this mechanism. The ybjX mutation increased susceptibility to avian beta-defensin 9 (AvBD9), and in vivo LD₅₀ assays revealed reduced bacterial virulence in a chicken model. Taken together, these results indicated that mutation of ybjX may attenuate APEC pathogenicity, not only by affecting bacterial adhesion and invasion, but also via reducing resistance to avian beta-defensin by increasing outer membrane permeabilization of bacteria.     

Infrequent mutations of the TP53 gene and no amplification of the MDM2 gene in hepatoblastomas

We have investigated the mutation of the TP53 gene in hepatoblastomas (HBLs) by using polymerase chain reaction-single strand conformation polymorphism and direct sequencing in 38 HBL tumor samples and in two HBL cell lines. We detected the TP53 gene mutation in an anaplastic hepatoblastoma cell line, but no aberration of the TP53 gene (exons 5–9) was found in tumor samples and in the other HBL cell line. The mutation of the cell line was a missense mutation from GAC (asparagine) to CAC (histidine) at codon 281, which was different from the G-to-T transversion of codon 249 that is frequently found in adult hepatocellular carcinomas (HCCs). In addition, we performed Southern blot analysis of the MDM2 gene, but we did not find MDM2 gene amplification in 19 cases tested. Our results suggest that, in contrast to the findings in HCCs in adults, TP53 gene aberrations are not involved in the development or progression of HBLs in children. Genes Chromosom Cancer 15:187–190 (1996). © 1996 Wiley-Liss, Inc.     

Mitochondrial ATP synthase subunit 9 is not required for viability of the petite-negative yeast Kluyveromyces lactis

Specific mutations in nuclear MGI genes encoding the α, β and γ subunits of the mitochondrial inner membrane F₁-ATPase complex allow mitochondrial DNA (mtDNA) to be lost from K. lactis. In the absence of a mutation in any of these three nuclear genes, loss of mtDNA is lethal. These results imply that mtDNA encodes a gene that is essential. Likely candidates for such an essential role are the ATP6, 8 and 9 genes coding for proteins of the ATP synthase-F₀ component. The present study removes ATP9 from contention as a vital mitochondrial gene because in a respiratory deficient mutant, Gly^– 3.9, lacking a nuclear mgi mutation, we have found that a rearrangement in mtDNA has deleted 22 amino acids from the carboxy terminus of the 75 amino-acid subunit-9 protein. Rearrangement in mtDNA has occurred by recombination at a 23-bp repeated sequence in the introns of the ATP9 and large ribosomal RNA (LSU) subunit genes. These two introns, of 394 (ATP9) and 410 (LSU) nucleotides, both belong to group 1. Received: 5 December 1996 / 6 March 1997     

Novel ALG8 mutations expand the clinical spectrum of congenital disorder of glycosylation type Ih

Congenital disorders of glycosylation (CDG) are an expanding group of inherited disorders caused by defects in the N- or O-Glycosylation of proteins and lipids. Several CDG subtypes have been described so far, including CDG type Ih which is caused by a deficiency of the dolichyl-P-Glc:Glc₁Man₉GlcNAc₂-PP-dolichyl α1,3-glucosyltransferase (hALG8). The defect leads to an accumulation of Dol-PP-GlcNAc₂Man₉ and Dol-PP-GlcNAc₂Man₉Glc₁ in the endoplasmic reticulum of patients’ fibroblasts that can be detected by analyzing the lipid-linked oligosaccharyl intermediates. Five patients with CDG-Ih have been described so far. The clinical presentation of four of these patients was severe with death in early infancy. In this report, we describe two mildly affected siblings with CDG-Ih caused by two novel mutations.While one mutation (c.1434delC) causes a frame shift resulting in a premature termination codon (p.485X), the point mutation of the other allele (c.845C>T, p.A282V) causes an amino acid replacement in a highly conserved region of the hALG8 gene. The two siblings show similar symptoms, including pseudo-gynecomastia, epicanthus, muscular hypotonia, mental retardation and ataxia, expanding the genetic and clinical spectrum of CDG-Ih.     

Genotype–phenotype correlations analysis of mutations in the phenylalanine hydroxylase (PAH) gene

The aims of our research were to define the genotype–phenotype correlations of mutations in the phenylalanine hydroxylase (PAH) gene that cause phenylketonuria (PKU) among the Israeli population. The mutation spectrum of the PAH gene in PKU patients in Israel is described, along with a discussion on genotype–phenotype correlations. By using polymerase chain reaction/denaturing high-performance liquid chromatography (PCR/dHPLC) and DNA sequencing, we screened all exons of the PAH gene in 180 unrelated patients with four different PKU phenotypes [classic PKU, moderate PKU, mild PKU, and mild hyperphenylalaninemia (MHP)]. In 63.2% of patient genotypes, the metabolic phenotype could be predicted, though evidence is also found for both phenotypic inconsistencies among subjects with more than one type of mutation in the PAH gene. Data analysis revealed that about 25% of patients could participate in the future in (6R)-l-erythro-5, 6, 7, 8-tetrahydrobiopterin (BH₄) treatment trials according to their mutation genotypes. This study enables us to construct a national database in Israel that will serve as a valuable tool for genetic counseling and a prognostic evaluation of future cases of PKU.     

A novel mutation in the flavin-containing monooxygenase 3 gene (FMO3) of a Norwegian family causes trimethylaminuria

Loss-of-function mutations in the flavin-containing monooxygenase 3 gene (FMO3) cause the inherited disorder trimethylaminuria (TMAuria), or fish-odour syndrome. Here we describe the identification in a family from northern Norway of a novel causative mutation of TMAuria. A female child within the family presented with a TMAuria-like phenotype. The child and her mother were found to be heterozygous for a novel mutation (R238Q) in exon 6 of FMO3. The child’s father lacked this mutation, but was heterozygous for a double polymorphic variant, E158K/E308G, which was not present in the child. During a consultation with her doctor the mother mentioned an uncle whom she remembered as having a strong body odour. This discussion led to genetic counselling of the uncle and analysis of his DNA showed him to be homozygous for the R238Q mutation. Analysis of the mutant FMO3 expressed in bacteria revealed that the R238Q mutation abolished catalytic activity of the enzyme and is thus a causative mutation for TMAuria. The specificity constant (k_cat/K_M) of the K158/G308 variant was 43% of that of ancestral FMO3. Because the child is heterozygous for the R238Q mutation and no other mutation known to cause TMAuria was detected in her DNA she is predicted to suffer from transient childhood TMAuria, whereas her great-uncle has primary TMAuria.     

Chromium sensitive mutants of the yeast Saccharomyces cerevisiae

Summary Ten mutants of Saccharomyces cerevisiae sensitive to CrO₃ were obtained and genetically analyzed. All of them were recessive. Segregation of the sensitivity after crossing them with a wild type strain indicated that the sensitivity of some mutants was determined by single mutations and that of others was caused by multiple mutations. Among 6 mutations found in mutants having single mutations responsible for the sensitivity, 6 loci were distinguished by complementation. One of them was identified as LYS7, the gene previously known as the structure gene for homocitric dehydrase, in terms of co-segregation and co-reversion of chromium sensitivity and lysine dependency which did not complement an authentic lys7 mutation.     

The development and prevalidation of an in vitro mutagenicity assay based on MutaMouse primary hepatocytes,Part I: Isolation,structural, genetic,and biochemical characterization

As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in an in vitro gene mutation assay due to their metabolic competence, their “normal” phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in an in vitro gene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PH in vitro gene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct-acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration-dependent increases in mutant frequency (MF) ranging from 2.6- to 14.4-fold over the control. None of the four nonmutagens, including two misleading, or “false,” positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3-nitrobenzanthrone [3-NBA], benzo[a]pyrene [BaP], and aflatoxin B₁ [AFB1]) to least (i.e., N-ethyl-N-nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in an in vitro gene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348–360, 2019. © 2019 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.     

Modulation of the H2O2-induced SOS response in escherichia coli PQ300 by amino acids,metal chelators,antioxidants, and scavengers of reactive oxygen species

The SOS chromotest is a simple colorimetric genotoxicity assay that monitors DNA repair by measuring the induction of the gene sfiA in Escherichia coli K-12. E. coli PQ300, a diagnostic SOS tester strain for the detection of oxidative genotoxins, carries a mutation in a key gene for antioxidative defense, _oxyR. This mutation renders PQ300 more sensitive to oxidative genotoxins, particularly to H₂O₂. We found that induction of the SOS response by H₂O₂ in E. coli PQ300 is dependent on the composition of the incubation medium; a substantially reduced response was obtained in minimal phosphate buffered saline (PBS) as opposed to complex Luria broth (LB) medium. Supplementation of PBS with histidine or cysteine stimulated H₂O₂-induced SOS induction to levels exceeding those found in LB medium. Low concentrations of glutathione (20–70 μM) also enhanced the H₂O_2-induced SOS response in E. coli PQ300, whereas higher concentrations (>150 μM) were protective. Preincubation of tester cells with the chelators o-phenanthroline, 2,2-dipyridyl, and ethylenediaminetetraacetic acid (EDTA) protected cells from the effects of H₂O₂, although EDTA was only partially effective. Pretreatment of PQ300 with the antioxidant ascorbic acid or the hydroxyl radical scavenger dimethyl sulfoxide also diminished the SOS response, whereas mannitol and glucose were ineffective. The results show that the net effect of H₂O_2-induced DNA damage is influenced by the balance of oxidative and antioxidative factors and, furthermore, can be modulated by constituents of the extracellular milieu. © 1993 Wiley-Liss, Inc.     

Genetic dissection of dopamine and serotonin synthesis in the nervous system of Drosophila melanogaster

Catecholamine- and serotonin-containing neurons were studied in pale, a Drosophila third-chromosome recessive lethal mutant. Using histofluorescent and immunocytochemical techniques, we show that this mutation only alters catecholamine levels in the CNS. Both the presence of catecholamine-neurons and the expression of serotonin are not affected by the mutation. Furthermore, we show that normal characteristics of catecholamine neurons, such as the presence of the enzyme DOPA decarboxylase and the selective uptake properties are normal in pale mutants. We suggest that pale is either the tyrosine hydroxylase structural gene, or a gene controlling tyrosine hydroxylase activity in Drosophila. The similar genetic location of the putative tyrosine hydroxylase gene and the mutation pale supports the former suggestion.