Third Base Of Codon Research Articles

Comparative genomic analysis of thermophilic fungi reveals convergent evolutionary adaptations and gene losses

Thermophily is a trait scattered across the fungal tree of life, with its highest prevalence within three fungal families (Chaetomiaceae, Thermoascaceae, and Trichocomaceae), as well as some members of the phylum Mucoromycota. We examined 37 thermophilic and thermotolerant species and 42 mesophilic species for this study and identified thermophily as the ancestral state of all three prominent families of thermophilic fungi. Thermophilic fungal genomes were found to encode various thermostable enzymes, including carbohydrate-active enzymes such as endoxylanases, which are useful for many industrial applications. At the same time, the overall gene counts, especially in gene families responsible for microbial defense such as secondary metabolism, are reduced in thermophiles compared to mesophiles. We also found a reduction in the core genome size of thermophiles in both the Chaetomiaceae family and the Eurotiomycetes class. The Gene Ontology terms lost in thermophilic fungi include primary metabolism, transporters, UV response, and O-methyltransferases. Comparative genomics analysis also revealed higher GC content in the third base of codons (GC3) and a lower effective number of codons in fungal thermophiles than in both thermotolerant and mesophilic fungi. Furthermore, using the Support Vector Machine classifier, we identified several Pfam domains capable of discriminating between genomes of thermophiles and mesophiles with 94% accuracy. Using AlphaFold2 to predict protein structures of endoxylanases (GH10), we built a similarity network based on the structures. We found that the number of disulfide bonds appears important for protein structure, and the network clusters based on protein structures correlate with the optimal activity temperature. Thus, comparative genomics offers new insights into the biology, adaptation, and evolutionary history of thermophilic fungi while providing a parts list for bioengineering applications.

Association between rat decompression sickness resistance, transthyretin single nucleotide polymorphism, and expression: A pilot study.

Decompression sickness (DCS) is a systemic syndrome that can occur after an environmental pressure reduction. Previously, we showed that the plasmatic tetrameric form of transthyretin (TTR) nearly disappeared in rats suffering DCS but not in asymptomatic ones. In this pilot study, we assessed whether the resistance to DCS could be associated with polymorphism of the gene of TTR. For this study, Sanger sequencing was performed on purified PCR products from the liver of 14-week-old male and female standard and DCS-resistant rats (n = 5 per group). Hepatic TTR mRNA expression was assessed by RT-qPCR in 18-19 week-old male and female standard and resistant rats (n = 6 per group). There is a synonymous single nucleotide polymorphism (SNP) on the third base of codon 46 (c.138 C > T). The thymine allele was present in 90% and 100% of males and females standard, respectively. However, this allele is present in only 30% of DCS-resistant males and females (p = 0.0002301). In the liver, there is a significant effect of the resistance to DCS (p = 0.043) and sex (p = 0.047) on TTR expression. Levels of TTR mRNA were lower in DCS-resistant animals. To conclude, DCS resistance might be associated with a SNP and a lower expression of TTR.

Codon usage bias in the chloroplast genomes of Cymbidium species in Guizhou, China

Cymbidium plants play a vital role in biodiversity conservation, evolutionary research, and biological protection. Understanding codon usage bias (CUB) in Cymbidium plants is crucial for unraveling the mechanism of species evolution and gene expression. In this study, we systematically analyzed the CUB and identified sources of variance in chloroplast genomes of 12 Cymbidium species to explore the main factors influencing CUB. Using CodonW 1.4.2 software and CUSP program, we screened 52 coding DNA sequences (CDS) within the complete chloroplast genome sequences of the twelve Cymbidium species. Subsequently, neutral, ENC-plot, and PR2-plot mapping analyses were performed to explore the relationships between code usage and genetic elements. The results revealed a consistent bias for guanine-cytosine (GC) bases at the first and second positions (GC1 > GC2 > GC3) of chloroplast genome codons in all 12 Cymbidium species. Moreover, the third base of codons exhibited a predilection for adenine/thymine (A/T) or uracil (U). The effective number of codons (ENC) ranged from 48.6 % to 49.49 %, with an average of 49.07, indicating a weak CUB. Additionally, codon adaptation index (CAI) ranged from 0.167 to 0.171, further supporting the weak CUB. The mapping analyses consistently highlighted natural selection as the primary factor influencing CUB in the 12 orchid chloroplast genomes while acknowledging the impact of other factors, such as mutations. Through optical codon analysis, we identified the top ten preferred codons (UUA, AUU, UCU, CCU, ACU, GCU, AAA, CGA, AGU, and GGU), all of which ended with A or U. These findings provide valuable insights for future studies involving codon modification of foreign genes and the development of high-expression transgenic techniques in Cymbidium plants.

Diversity of genomic adaptations to the post-fire environment in Pezizales fungi points to crosstalk between charcoal tolerance and sexual development.

Wildfires drastically impact the soil environment, altering the soil organic matter, forming pyrolyzed compounds, and markedly reducing the diversity of microorganisms. Pyrophilous fungi, especially the species from the orders Pezizales and Agaricales, are fire-responsive fungal colonizers of post-fire soil that have historically been found fruiting on burned soil and thus may encode mechanisms of processing these compounds in their genomes. Pyrophilous fungi are diverse. In this work, we explored this diversity and sequenced six new genomes of pyrophilous Pezizales fungi isolated after the 2013 Rim Fire near Yosemite Park in California, USA: Pyronema domesticum, Pyronema omphalodes, Tricharina praecox, Geopyxis carbonaria, Morchella snyderi, and Peziza echinospora. A comparative genomics analysis revealed the enrichment of gene families involved in responses to stress and the degradation of pyrolyzed organic matter. In addition, we found that both protein sequence lengths and G + C content in the third base of codons (GC3) in pyrophilous fungi fall between those in mesophilic/nonpyrophilous and thermophilic fungi. A comparative transcriptome analysis of P. domesticum under two conditions - growing on charcoal, and during sexual development - identified modules of genes that are co-expressed in the charcoal and light-induced sexual development conditions. In addition, environmental sensors such as transcription factors STE12, LreA, LreB, VosA, and EsdC were upregulated in the charcoal condition. Taken together, these results highlight genomic adaptations of pyrophilous fungi and indicate a potential connection between charcoal tolerance and fruiting body formation in P. domesticum.

The novel Ideal Symmetry Genetic Code table – Common purine-pyrimidine symmetry net for all RNA and DNA species

Ever since Nirenberg’s discovery in 1961 in which codons code individual amino acids, numerous scientists searched for symmetries within the genetic code. The standard genetic code (SGC) table is an alphabetic artificial construct based on the U-C-A-G ordering of nucleotides without natural symmetries. Up to the present, complete symmetry in the genetic code has not been found, leaving doubt as to whether the symmetrical nature as the protector of order even exists. Our novel Ideal Symmetry Genetic Code (ISyGC) table reflects a unique fundamental physicochemical purine-pyrimidine symmetry net for all more than thirty known variations of nuclear and mitochondrial genetic codes. The nuclear genetic code for RNA and DNA viruses also contains the same purine-pyrimidine symmetry net. We show that the ISyGC table leads to automatic transformation into a DNA sequence akin to the 5′3 codon and 3′5 anticodon patterns. As a result of purine-pyrimidine symmetries between codons in the ISyGC table, algorithms of the first two bases as well of the third base of codons show how tRNA cognate anticodons can recognize synonymous codons during mRNA decoding. We show that the ISyGC purine-pyrimidine net with its physicochemical properties represents an evolutionary common “frozen accident” at the onset of each genetic code creation and RNA to DNA evolution. As such, during all of evolution the unique fundamental purine-pyrimidine symmetry net of all genetic codes remains unchangeable. In this way, evolution is a road paved with symmetries.

Genomic Evidence for Simultaneous Optimization of Transcription and Translation through Codon Variants in the pmoCAB Operon of Type Ia Methanotrophs.

Understanding the interplay between genotype and phenotype is a fundamental goal of functional genomics. Methane oxidation is a microbial phenotype with global-scale significance as part of the carbon biogeochemical cycle and a sink for greenhouse gas. Microorganisms that oxidize methane (methanotrophs) are taxonomically diverse and widespread around the globe. In methanotrophic bacteria, enzymes in the methane oxidation metabolic module (KEGG module M00174, conversion of methane to formaldehyde) are encoded in four operons (pmoCAB, mmoXYZBCD, mxaFI, and xoxF). Recent reports have suggested that methanotrophs in Proteobacteria acquired methane monooxygenases through horizontal gene transfer. Here, we used a genomic meta-analysis to infer the transcriptional and translational advantages of coding sequences from the methane oxidation metabolic modules of different types of methanotrophs. By analyzing isolate and metagenome-assembled genomes from phylogenetically and geographically diverse sources, we detected an anomalous nucleotide composition bias in the coding sequences of particulate methane monooxygenase genes (pmoCAB) from type Ia methanotrophs. We found that this nucleotide bias increases the level of codon bias by decreasing the GC content in the third base of codons, a strategy that contrasts with that of other coding sequences in the module. Further codon usage analyses uncovered that codon variants of the type Ia pmoCAB coding sequences deviate from the genomic signature to match ribosomal protein-coding sequences. Subsequently, computation of transcription and translation metrics revealed that the pmoCAB coding sequences of type Ia methanotrophs optimize the usage of codon variants to maximize translation efficiency and accuracy, while minimizing the synthesis cost of transcripts and proteins.IMPORTANCE Microbial methane oxidation plays a fundamental role in the biogeochemical cycle of Earth's system. Recent reports have provided evidence for the acquisition of methane monooxygenases by horizontal gene transfer in methane-oxidizing bacteria from different environments, but how evolution has shaped the coding sequences to execute methanotrophy efficiently remains unexplored. In this work, we provide genomic evidence that among the different types of methanotrophs, type Ia methanotrophs possess a unique coding sequence of the pmoCAB operon that is under positive selection for optimal resource allocation and efficient synthesis of transcripts and proteins. This adaptive trait possibly enables type Ia methanotrophs to respond robustly to fluctuating methane availability and explains their global prevalence.

Cloning and Genomic Organization of a Rhamnogalacturonase Gene from Locally Isolated Strain of Aspergillus niger.

The rhg gene encoding a rhamnogalacturonase was isolated from the novel strain A1 of Aspergillus niger. It consists of an ORF of 1.505kb encoding a putative protein of 446 amino acids with a predicted molecular mass of 47kDa, belonging to the family 28 of glycosyl hydrolases. The nature and position of amino acids comprising the active site as well as the three-dimensional structure were well conserved between the A. niger CTM10548 and fungal rhamnogalacturonases. The coding region of the rhg gene is interrupted by three short introns of 56 (introns 1 and 3) and 52 (intron 2) bp in length. The comparison of the peptide sequence with A. niger rhg sequences revealed that the A1 rhg should be an endo-rhamnogalacturonases, more homologous to rhg A than rhg B A. niger known enzymes. The comparison of rhg nucleotide sequence from A. niger A1 with rhg A from A. niger shows several base changes. Most of these changes (59%) are located at the third base of codons suggesting maintaining the same enzyme function. We used the rhamnogalacturonase A from Aspergillus aculeatus as a template to build a structural model of rhg A1 that adopted a right-handed parallel β-helix.

Conformational Preferences of Modified Nucleoside N(4)-Acetylcytidine, ac4C Occur at “Wobble” 34th Position in the Anticodon Loop of tRNA

Conformational preferences of modified nucleoside, N(4)-acetylcytidine, ac(4)C have been investigated using quantum chemical semi-empirical RM1 method. Automated geometry optimization using PM3 method along with ab initio methods HF SCF (6-31G**), and density functional theory (DFT; B3LYP/6-31G**) have also been made to compare the salient features. The most stable conformation of N(4)-acetyl group of ac(4)C prefers "proximal" orientation. This conformation is stabilized by intramolecular hydrogen bonding between O(7)···HC(5), O(2)···HC2', and O4'···HC(6). The "proximal" conformation of N(4)-acetyl group has also been observed in another conformational study of anticodon loop of E. coli elongator tRNA(Met). The solvent accessible surface area (SASA) calculations revealed the role of ac(4)C in anticodon loop. The explicit molecular dynamics simulation study also shows the "proximal" orientation of N(4)-acetyl group. The predicted "proximal" conformation would allow ac(4)C to interact with third base of codon AUG/AUA whereas the 'distal' orientation of N(4)-acetyl cytidine side-chain prevents such interactions. Single point energy calculation studies of various models of anticodon-codon bases revealed that the models ac(4)C(34)(Proximal):G3, and ac(4)C(34)(Proximal):A3 are energetically more stable as compared to models ac(4)C(34)(Distal):G3, and ac(4)C(34)(Distal):A3, respectively. MEPs calculations showed the unique potential tunnels between the hydrogen bond donor-acceptor atoms of ac(4)C(34)(Proximal):G3/A3 base pairs suggesting role of ac(4)C in recognition of third letter of codons AUG/AUA. The "distal" conformation of ac(4)C might prevent misreading of AUA codon. Hence, this study could be useful to understand the role of ac(4)C in the tertiary structure folding of tRNA as well as in the proper recognition of codons during protein biosynthesis process.

Aflatoxins, Hepatitis and Hepatocellular Carcinoma: A Special Focus on Turkey’s Current Status

Aflatoxins, Hepatitis and Hepatocellular Carcinoma: A Special Focus on Turkey’s Current Status Mycotoxins are food contaminants produced as secondary metabolites by different kinds of fungi. The major groups of mycotoxins are aflatoxins (AFs), ochratoxins, patulin and fusarium toxins. Aflatoxin B1 (AFB1) is classified as carcinogenic to humans (Group I) by The International Agency for Research on Cancer. The principle target organ of AFB1 is liver. AFB1 causes hotspot mutation of p53 gene at third base of codon 249, and takes the form of G…>T transversions. Besides, both hepatits B virus (HBV) and hepatits C virus (HCV) infections were suggested to be responsible HCC. HCC is the most frequent primary liver cancer and the most severe complication of chronic liver disease. The annual number of new cases worldwide represents more than 5% of human cancers and is the third leading cause of cancer-related deaths. One or more causative agents can be identified in most HCC cases. AFB1 exposure with viral hepatitis is suggested to lead to HCC. This review will focus on AFB1, viral hepatitis, their synergistic and additive effects and the measures that should be taken to prevent HCC stemming from AFB1 exposure with viral hepatitis.

Aflatoxins metabolism, effects on epigenetic mechanisms and their role in carcinogenesis

Chronic consumption of aflatoxin-contaminated foods is a global problem in both developing and developed countries especially where there is poor regulation of their levels in foods. In the body, aflatoxins (AFBs) mainly AFB1 are biotransformed to various metabolites especially the active AFB1-exo-8,9-epoxide (AFBO). The AFB, AFBO and other metabolites interact with various biomolecules in the body including nucleic acids such as DNA and RNA and the various metabolic pathways such as protein synthesis, glycolytic pathway and electron transport chain involved in ATP production in body cells. The AFB interacts with DNA to form AFB-DNA adducts causing DNA breakages. The AFB and its metabolites induce the up regulation of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) through gene expression that regulates the metabolizing enzymes such as CYP450 involved in Phase I and Phase II metabolism of xenobiotics. AFB activates these nuclear receptors to produce the metabolizing enzymes. The AFB1 is metabolized in the body by cytochrome P450 (CYP450) enzyme isoforms such as CYP1A2, CYP1A2, CYP3A4/ CYP3A5, and CYP3A7 in fetus, glutathione S-transferase, aflatoxin B1-aldehyde reductase leading to reactive metabolites, some of which can be used as aflatoxin exposure biomarkers. These enzymes are involved in the Phase I and Phase II metabolic reactions of aflatoxins. The CYP1A2 is the principal metabolizer of aflatoxin at low concentrations while the reverse is true for CYP3A4. The accumulation of AFB and its metabolites in the body especially the AFB1-exo-8,9-epoxide depletes the glutathione (GSH) due to the formation of high amounts of epoxides and other reactive oxygen species (ROS). The AFB, AFB1-exo-8,9-epoxide and other metabolites also affect the epigenetic mechanisms including the DNA methylation, histone modifications, maturation of miRNAs as well as the daily formation of single nucleotide polymorphism (SNP) where AFB exposure may facilitate the process and induces G:C to T:A transversions at the third base in codon 249 of TP53 causing p53 mutations reported in hepatocellular carcinoma (HCC). The changes in epigenetic mechanisms lead to either epigenetic inactivation or epigenetic derepression and all these affect the gene expression, cellular differentiation and growth. AFB also through epigenetic mechanisms promotes tumorigenesis, angiogenesis, invasion and metastasis in hepatocellular carcinoma. However, the formation of the small amounts of AFB1 from AFB2 is suspected to cause the carcinogenicity of AFB2 in humans and animals. Chronic aflatoxins exposure leads to formation of reactive AFBO metabolites in the body that could activate and de-activates the various epigenetic mechanisms leading to development of various cancers.

The complete mitochondrial genome of two recently derived species of the fish genus Nannoperca (Perciformes, Percichthyidae)

Here we report the complete sequence of mitochondrial genomes for two sister taxa of freshwater teleosts, the recently derived Yarra pigmy perch Nannoperca obscura and the southern pigmy perch Nannoperca australis. These represent the first complete mitochondrial genomes for Percichthyidae (Perciformes), a family mostly distributed in Australia. The de novo genome assembly of 316,430 pyrosequencing reads from 454 libraries has produced the entire mitochondria for N. obscura and a nearly complete version for N. australis. The mtDNA genome from the latter was completed through the design of one primer set and standard Sanger sequencing for genome finishing, followed by the hybrid assembly of reads with MIRA software using N. obscura sequence as reference genome. The complete mitogenomes of N. obscura and N. australis are 16,496 and 16,494 bp in size, respectively. Both genomes contain 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a control region. Several characteristics of mitochondria typically found in teleost fishes were detected, such as: (i) most genes found in the heavy strand, with the exception of ND6 and eight tRNA genes; (ii) avoidance of G as the third base of codons; (iii) presence of gene overlapping; (iv) percentage of bases usage. We found only eight indels and 197 nucleotide substitutions between these Nannoperca mitogenomes, consistent with a previous hypothesis of recent speciation. The data reported here provide a resource for comparative analysis of recent evolution of mitochondrial genomes.

Molecular Signatures of Environmental Mutagens in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the 5th most common cancers, with 80% of the cases occurring in low resource countries. Its etiology is dominated by complex interplay between chronic infection by hepatitis virus B or C (HBV, HCV), metabolic diseases and exposure to environmental carcinogens. In areas of high incidence of HCC, the most common risk factors are chronic HB carriage and exposure to a mycotoxin, aflatoxin B1 (AFB1), which contaminates many staples and causes mutations at the third base of codon 249 in the TP53 tumor suppressor gene in hepatocytes (R249S mutation). In this review, we summarize studies using a very sensitive and quantitative detection method, short-oligonucleotide mass analysis, to measure R249S in cell-free DNA isolated from the plasma of asymptomatic subjects and patients with chronic liver disease and/or HCC. These studies have identified that high levels of R249S were strongly associated with HBV-related HCC. Low to intermediate levels of R249S, in contrast, were detectable in asymptomatic subjects exposed to AFB1, with seasonal variations informative of the complex interactions between mutagenesis by AFB1 and chronic infection by HBV. Overall, we suggest that formation of R249S occurs in response to AFB1 exposure, well ahead of cancer development, thus generating large populations of cells at high risk for neoplastic transformation. In addition, R249S mutations may inactivate pro-apoptotic activities of p53 and contribute to rendering hepatocytes resistant to liver cell destruction by chronic inflammation, thus limiting chronic liver disease symptoms.

Two New α-Thalassemia Point Mutations that are Undetectable by Biochemical Techniques

We report two new point mutations causing α-thalassemia (α-thal) that could not be characterized by conventional biochemical studies. The first mutation is a single base substitution at codon 123 of the α1-globin gene [α123(H6)Ala→Pro, GCC>CCC (α1)] and leads to the substitution of a proline residue in the H helix. The resulting unstable hemoglobin (Hb) variant has been named Hb Voreppe. The second is a frameshift of the α2 gene due to a deletion (−C), either of the third base of codon 112 or of the first base of codon 113, that causes a premature stop codon at position 132.

TP53 mutations and hepatocellular carcinoma: insights into the etiology and pathogenesis of liver cancer

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide and the major risk factors include chronic infections with the hepatitis B (HBV) or C (HCV) virus, and exposure to dietary aflatoxin B(1) (AFB(1)) or alcohol consumption. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC, for example, somatic mutations in the p53 tumor suppressor gene (TP53) and the activation of the WNT signal transduction pathway. AFB(1) frequently induces G:C to T:A transversions at the third base in codon 249 of TP53 and cooperates with HBV in causing p53 mutations in HCC. The detection of TP53 mutant DNA in plasma is a biomarker of both AFB(1) exposure and HCC risk. Chronic infection with HBV and HCV viruses, and oxyradical disorders including hemochromatosis, also generate reactive oxygen/nitrogen species that can both damage DNA and mutate cancer-related genes such as TP53. Certain mutant p53 proteins may exhibit a 'gain of oncogenic function'. The p53 biological network is a key responder to this oxidative and nitrosative stress. Depending on the extent of the DNA damage, p53 regulates the transcription of protective antioxidant genes and with extensive DNA damage, transactivates pro-oxidant genes that contribute to apoptosis. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC and the integrated HBx is frequently mutated. Mutant HBx proteins still retain their ability to bind to p53, and attenuate DNA repair and p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology of TP53 mutations during the molecular pathogenesis of HCC.

Degeneracy in the genetic code and its symmetries by base substitutions

Degeneracy in the genetic code is known to minimise the deleterious effects of the most frequent base substitutions: transitions at the third base of codons are generally synonymous substitutions. Transversions that alter degeneracy were reported by Rumer. Here the other transversions are shown to leave invariant degeneracy when applied to the first base of codons. As a summary, degeneracy is considered with respect to all three types of base substitutions, the transitions and the two types of transversions. The symmetries of degeneracy by base substitutions are independent of the representation of the genetic code and discussed with respect to the quasi-universality of the genetic code. To cite this article: J.-L. Jestin, C. R. Biologies 329 (2006).

A polymorphism at codon 31 of gene p21 is not associated with primary open angle glaucoma in Caucasians

BackgroundPrimary open angle glaucoma (POAG) is considered to be a neurodegenerative optic neuropathy, in which cell death occurs by apoptosis. p21, is an important protective component of the apoptotic pathway, regulating cellular arrest in the presence of DNA damage. An unstable or altered p21 protein could modify the cellular response to genomic injury and abolish the effect of p21. A previous study on a Chinese cohort suggested that the p21 codon 31 polymorphism may alter the state of apoptosis in glaucomatous optic neuropathy, failing to protect the ganglion cells. The aim of this study was to test the hypothesis that a p21 codon 31 polymorphism is associated with POAG on a Caucasian cohort.Methods140 POAG patients and a control group of 73 healthy individuals were included in the study. All the subjects were of Caucasian origin. Genomic DNA was amplified by polymerase chain reaction, followed by enzymatic restriction fragment length polymorphism technique (PCR-RFLP). Patients and controls were genotyped for a single nucleotide polymorphism (C/A transversion) in the third base of codon 31 of p21, which leads to a serine (Ser)/arginine (Arg) substitution.ResultsThe distribution of the genotypes in the POAG patients showed 128 (91.4%) Ser homozygotes, 10 (7.1%) Ser/Arg heterozygotes and 2 (1.5%) Arg homozygotes. In the control cohort, there were 61 (83.6%) Ser homozygotes and 12 (16.4%) Ser/Arg heterozygotes. No Arg homozygotes were present amongst the control group. Both the allelic and genotypic frequencies of the Ser or Arg residues at codon 31 were not significantly different between POAG patients and controls (Fisher's exact test, P = 0.20 for alleles and P = 0.0561 for genotypes).ConclusionThis study suggests that the p21 codon 31 polymorphism does not contribute to the risk of POAG in the Caucasian population.

Mutational spectrum and genotoxicity of the major lipid peroxidation product, trans-4-hydroxy-2-nonenal, induced DNA adducts in nucleotide excision repair-proficient and -deficient human cells.

trans-4-Hydroxy-2-nonenal (4-HNE), a major product of lipid peroxidation, is able to interact with DNA to form 6-(1-hydroxyhexanyl)-8-hydroxy-1,N(2)-propano-2'-deoxyguanosine (4-HNE-dG) adducts, but its genotoxicity and mutagenicity remain elusive. It has been reported that 4-HNE treatment in human cells induces a high frequency of G.C to T.A mutations at the third base of codon 249 (AGG*) of the p53 gene, a mutational hot spot in human cancers, particularly in hepatocellular carcinoma. This G.C to T.A transversion at codon 249, however, has been thought to be caused by etheno-DNA adducts induced by the endogenous metabolite of 4-HNE, 2,3-epoxy-4-hydroxynonanal. We have recently found that 4-HNE preferentially forms 4-HNE-dG adducts at the GAGG*C/A sequence in the p53 gene including codon 249 (GAGG*C). Our finding supports the possibility that G.C to T.A mutations at codon 249 may be induced by 4-HNE-dG adducts. To investigate this possibility, we determined the mutational spectrum induced by 4-HNE-dG adducts in the supF gene of shuttle vector pSP189 replicated in human cells. We have found that 4-HNE-dG adducts are mutagenic and genotoxic in human cells, and that G.C to T.A transversions are the most prevalent mutations induced by 4-HNE-dG adducts. Furthermore, 4-HNE-dG adducts induce a significantly higher level of genotoxicity and mutagenicity in nucleotide excision repair (NER)-deficient human and Escherichia coli cells than in NER-proficient cells, indicating that NER is a major pathway for repairing 4-HNE-dG adducts in both human and E. coli cells. Together, these results suggest that 4-HNE-dG adducts may contribute greatly to the G.C to T.A mutation at codon 249 of the p53 gene, and may play an important role in carcinogenesis.

Molecular characterization of 5'-deoxy-5'-methylthioadenosine phosphorylase-deficient mutant clones of murine lymphoma cell line R1.1.

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP), the polyamine and purine metabolic enzyme, is ubiquitously expressed in normal human tissues and cells, but is deficient in a variety of human and murine malignant cell lines. MTAP-deficient mutants were previously selected from murine lymphoma cell line R1.1 that contains abundant MTAP activity, to analyze the metabolic consequences of MTAP deficiency. Two mutants, one with partial deficiency (F clone) and the other with complete deficiency (H5 clone), were found to have the MTAP protein by immunoblotting. However, the molecular mechanism of enzyme deficiency in these mutants has not been established. In this study, we cloned the mouse cDNA and analyzed MTAP mRNA in the two mutants by reverse transcription-polymerase chain reaction (RT-PCR) followed by direct sequencing. Both mutants have a single nucleotide substitution at the third base of codon 223, which results in a change of cysteine to tyrosine (C223Y). The MTAP mRNA level determined by RT-PCR was significantly lower in the mutant with complete deficiency than in wild-type (WT) R1.1 cells, whereas the mRNA level in the mutant with partial deficiency was comparable to that in WT R1.1 cells. C223Y mutation may cause conformational alteration of the methylthioribose-binding site and decrease the substrate binding. Thus, C223Y may account for the partial deficiency in the F clone, but the complete deficiency with a remarkably low MTAP mRNA level in the H5 clone may be a result of transcriptional abnormality in addition to C223Y mutation.

Novel mutations of neurofibromatosis type 1 gene in small cell lung cancers.

Mutations in the GTPase-activating protein-related domain (GRD) of the von Recklinghausen neurofibromatosis type 1 (NF1) gene have been reported in several tumors that were not previously associated with NF1. We analyzed 37 cases of lung cancer, including 9 cases of small cell carcinoma, to detect mutations in this domain. DNA was extracted from the tumors, and single-strand conformation polymorphism and direct sequencing were used to detect mutations. Three cases of small cell carcinoma had mutations in NF1-GRD. There were two incidences of an A --> G transition at the second base of codon 1415 resulting in Glu --> Gly substitution, and one incidence of an A --> G transition at the third base of codon 1411 (Pro) without amino acid substitution. All three patients had both hilar and mediastinal lymphatic metastasis. A mutation in this region could imply a poor prognosis.

TP53 and liver carcinogenesis.

Primary hepatocellular carcinoma (HCC) is one of the most common malignancies and has the fourth highest mortality rate worldwide. The major risk factors, including chronic infections with the hepatitis B or C virus, are exposure to dietary aflatoxin B1(AFB1), vinyl chloride, or alcohol consumption. Southern China and sub-Saharan Africa have the highest dietary AFB1 exposure, making it and hepatitis B virus (HBV) the major causes of cancer mortality in these geographic areas. Recent studies have discovered genetic and epigenetic changes involved in the molecular pathogenesis of HCC, including somatic mutations in the p53 tumor suppressor gene (TP53). AFB1 induces typical G:C to T:A transversions at the third base in codon 249 of p53. Chronic active hepatitis B and C (HCV) infection, and further inflammatory and oxyradical disorders including Wilson disease (WD) or hemochromatosis, generate reactive oxygen/nitrogen species that can damage DNA and mutate the p53 gene. The X gene of HBV (HBx) is the most common open reading frame integrated into the host genome in HCC. The integrated HBx is frequently mutated and has a diminished ability to function as a transcriptional cotransactivator and to activate the NF-kappa B pathway. However, the mutant HBx proteins still retain their ability to bind to and abrogate p53-mediated apoptosis. In summary, both viruses and chemicals are implicated in the etiology and molecular pathogenesis of HCC. The resultant molecular changes in the ras and Wnt signal-transduction pathways, and the p53 and Rb tumor suppressor pathways significantly contribute to liver carcinogenesis