DNA Base Substitutions Research Articles

MELAS-Derived Neurons Functionally Improve by Mitochondrial Transfer from Highly Purified Mesenchymal Stem Cells (REC).

Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episode (MELAS) syndrome, caused by a single base substitution in mitochondrial DNA (m.3243A>G), is one of the most common maternally inherited mitochondrial diseases accompanied by neuronal damage due to defects in the oxidative phosphorylation system. There is no established treatment. Our previous study reported a superior restoration of mitochondrial function and bioenergetics in mitochondria-deficient cells using highly purified mesenchymal stem cells (RECs). However, whether such exogenous mitochondrial donation occurs in mitochondrial disease models and whether it plays a role in the recovery of pathological neuronal functions is unknown. Here, utilizing induced pluripotent stem cells (iPSC), we differentiated neurons with impaired mitochondrial function from patients with MELAS. MELAS neurons and RECs/mesenchymal stem cells (MSCs) were cultured under contact or non-contact conditions. Both RECs and MSCs can donate mitochondria to MELAS neurons, but RECs are more excellent than MSCs for mitochondrial transfer in both systems. In addition, REC-mediated mitochondrial transfer significantly restored mitochondrial function, including mitochondrial membrane potential, ATP/ROS production, intracellular calcium storage, and oxygen consumption rate. Moreover, mitochondrial function was maintained for at least three weeks. Thus, REC-donated exogenous mitochondria might offer a potential therapeutic strategy for treating neurological dysfunction in MELAS.

DCas9-Mediated PCR-Free Detection of Oncogenic Mutation by Nonequilibrium Nanoelectrokinetic Selective Preconcentration.

Cutting-edge nanoelectrokinetic technology in this work provides a breakthrough for the present clinical demands of molecular diagnosis to detect a trace amount of oncogenic mutation of DNA in a short time without an erroneous PCR procedure. In this work, we combined the sequence-specific labeling scheme of CRISPR/dCas9 and ion concentration polarization (ICP) mechanism to separately preconcentrate target DNA molecules for rapid detection. Using the mobility shift caused by dCas9's specific binding to the mutant, the mutated DNA and normal DNA were distinguished in the microchip. Based on this technique, we successfully demonstrated the dCas9-mediated 1-min detection of single base substitution (SBS) in EGFR DNA, a carcinogenesis indicator. Moreover, the presence/absence of target DNA was identified at a glance like a commercial pregnancy test kit (two lines for positive and one line for negative) by the distinct preconcentration mechanisms of ICP, even at the 0.1% concentration of the target mutant.

An integrative in-silico analysis discloses a novel molecular subset of colorectal cancer possibly eligible for immune checkpoint immunotherapy

BackgroundHistorically, the molecular classification of colorectal cancer (CRC) was based on the global genomic status, which identified microsatellite instability in mismatch repair (MMR) deficient CRC, and chromosomal instability in MMR proficient CRC. With the introduction of immune checkpoint inhibitors, the microsatellite and chromosomal instability classification regained momentum as the microsatellite instability condition predicted sensitivity to immune checkpoint inhibitors, possibly due to both high tumor mutation burden (TMB) and high levels of infiltrating lymphocytes. Conversely, proficient MMR CRC are mostly resistant to immunotherapy. To better understand the relationship between the microsatellite and chromosomal instability classification, and eventually discover additional CRC subgroups relevant for therapeutic decisions, we developed a computational pipeline that include molecular integrative analysis of genomic, epigenomic and transcriptomic data.ResultsThe first step of the pipeline was based on unsupervised hierarchical clustering analysis of copy number variations (CNVs) versus hypermutation status that identified a first CRC cluster with few CNVs enriched in Hypermutated and microsatellite instability samples, a second CRC cluster with a high number of CNVs mostly including non-HM and microsatellite stable samples, and a third cluster (7.8% of the entire dataset) with low CNVs and low TMB, which shared clinical-pathological features with Hypermutated CRCs and thus defined Hypermutated-like CRCs. The mutational features, DNA methylation profile and base substitution fingerprints of these tumors revealed that Hypermutated-like patients are molecularly distinct from Hypermutated and non-Hypermutated tumors and are likely to develop and progress through different genetic events. Transcriptomic analysis highlighted further differences amongst the three groups and revealed an inflamed tumor microenvironment and modulation Immune Checkpoint Genes in Hypermutated-like CRCs.ConclusionTherefore, our work highlights Hypermutated-like tumors as a distinct and previously unidentified CRC subgroup possibly responsive to immune checkpoint inhibitors. If further validated, these findings can lead to expanding the fraction of patients eligible to immunotherapy.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p= 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

Programmable C:G to G:C genome editing with CRISPR-Cas9-directed base excision repair proteins

Many genetic diseases are caused by single-nucleotide polymorphisms. Base editors can correct these mutations at single-nucleotide resolution, but until recently, only allowed for transition edits, addressing four out of twelve possible DNA base substitutions. Here, we develop a class of C:G to G:C Base Editors to create single-base genomic transversions in human cells. Our C:G to G:C Base Editors consist of a nickase-Cas9 fused to a cytidine deaminase and base excision repair proteins. Characterization of >30 base editor candidates reveal that they predominantly perform C:G to G:C editing (up to 90% purity), with rAPOBEC-nCas9-rXRCC1 being the most efficient (mean 15.4% and up to 37% without selection). C:G to G:C Base Editors target cytidine in WCW, ACC or GCT sequence contexts and within a precise three-nucleotide window of the target protospacer. We further target genes linked to dyslipidemia, hypertrophic cardiomyopathy, and deafness, showing the therapeutic potential of these base editors in interrogating and correcting human genetic diseases.

Two Power Curves Yielding the Energy of a Lifetime in Evo-SETI Theory

This paper presents a new mathematical model for the ENERGY that a living being needs in order to live its whole life between birth and death. This also applies to a civilization made up by many living beings. The model is based on a LOGELL POWER CURVE, which is a curve in time made up by a lognormal probability density between birth and peak, followed by an ellipse between peak and death (LOGELL means LOGnormal plus ELLipse). We derive analytic equations yielding the ENERGY in terms of three free parameters only: the time of birth b, the time of the power peak p, and the time of death, d. The author’s previously published papers about his so-called Evo-SETI Theory (Evo-SETI stands for Evolution and SETI) cover the biological evolution over the last 3.5 billion years described as an increase in the number of living species from one (RNA) to the current (say) 50 million. Past mass extinctions make this evolution become a stochastic process having an exponential mean value, called Geometric Brownian Motion (GBM). In those papers, a lifetime, rather than a logell, was a b-lognormal, i.e., a lognormal starting at instant b (birth) and descending straight to death at its descending inflexion point. Our mathematical discovery of the Peak-Locus Theorem showed that the GBM exponential is the geometric locus of all the peaks of the b-lognormals. Since b-lognormals are probability densities, the area under each of them always equals 1 (normalization condition); going from left to right on the time axis, the b-lognormals become more and more “peaky,” so they last less and less in time. This “level of civilization” is what physicists call (Shannon) ENTROPY of information, meaning that the higher Species have higher information content than the lower Species. This author also proved mathematically that for all GBMs, the (Shannon) Entropy of the b-lognormals grows LINEARLY in time. The Molecular Clock, well known to geneticists since 1962, shows that the DNA base-substitutions occur LINEARLY in time since they are neutral with respect to Darwinian selection. The conclusion is that the Molecular Clock and the LINEAR increase of EvoEntropy in time are just the same thing! In other words, we derived the Molecular Clock mathematically as a part of our Evo-SETI Theory. Finally, this linearly growing entropy is just the new EvoSETI SCALE to measure the evolution of life on Exoplanets (measured in bits). In conclusion, our invention of the logell power curve, described in this paper, provides a new mathematical tool for our Evo-SETI mathematical description of Life, History and SETI.

DNA Damage and Chromatin Conformation Changes Confer Nonhost Resistance: A Hypothesis Based on Effects of Anti-cancer Agents on Plant Defense Responses.

Over the last decades, medical research has utilized DNA altering procedures in cancer treatments with the objective of killing cells or suppressing cell proliferation. Simultaneous research related to enhancing disease resistance in plants reported that alterations in DNA can enhance defense responses. These two opposite perspectives have in common their effects on the center for gene transcription, the nuclear chromatin. A review of selected research from both anticancer- and plant defense-related research provides examples of some specific DNA altering actions: DNA helical distortion, DNA intercalation, DNA base substitution, DNA single cleavage by DNases, DNA alkylation/methylation, and DNA binding/exclusion. The actions of the pertinent agents are compared, and their proposed modes of action are described in this study. Many of the DNA specific agents affecting resistance responses in plants, e.g., the model system using pea endocarp tissue, are indeed anticancer agents. The tumor cell death or growth suppression in cancer cells following high level treatments may be accompanied with chromatin distortions. Likewise, in plants, DNA-specific agents activate enhanced expression of many genes including defense genes, probably due to the chromatin alterations resulting from the agents. Here, we propose a hypothesis that DNA damage and chromatin structural changes are central mechanisms in initiating defense gene transcription during the nonhost resistance response in plants.

Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid β peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

Energy of Extra-Terrestrial Civilizations according to Evo-SETI Theory

Energy of Extra-Terrestrial Civilizations according to Evo-SETI Theory

Evo-SETI SCALE to measure Life on Exoplanets

Darwinian Evolution over the last 3.5 billion years was an increase in the number of living species from 1 (RNA?) to the current 50 million. This increasing trend in time looks like being exponential, but one may not assume an exactly exponential curve since many species went extinct in the past, even in mass extinctions. Thus, the simple exponential curve must be replaced by a stochastic process having an exponential mean value. Borrowing from financial mathematics (“Black–Scholes models”), this “exponential” stochastic process is called Geometric Brownian Motion (GBM), and its probability density function (pdf) is a lognormal (not a Gaussian) (Proof: see ref. Maccone [3], Chapter 30, and ref. Maccone [4]). Lognormal also is the pdf of the statistical number of communicating ExtraTerrestrial (ET) civilizations in the Galaxy at a certain fixed time, like a snapshot: this result was found in 2008 by this author as his solution to the Statistical Drake Equation of SETI (Proof: see ref. Maccone [1]). Thus, the GBM of Darwinian Evolution may also be regarded as the extension in time of the Statistical Drake equation (Proof: see ref. Maccone [4]). But the key step ahead made by this author in his Evo-SETI (Evolution and SETI) mathematical model was to realize that LIFE also is just a b-lognormal in time: every living organism (a cell, a human, a civilization, even an ET civilization) is born at a certain time b (“birth”), grows up to a peak p (with an ascending inflexion point in between, a for adolescence), then declines from p to s (senility, i.e. descending inflexion point) and finally declines linearly and dies at a final instant d (death). In other words, the infinite tail of the b-lognormal was cut away and replaced by just a straight line between s and d, leading to simple mathematical formulae (“History Formulae”) allowing one to find this “finite b-lognormal” when the three instants b, s, and d are assigned.Next the crucial Peak-Locus Theorem comes. It means that the GBM exponential may be regarded as the geometric locus of all the peaks of a one-parameter (i.e. the peak time p) family of b-lognormals. Since b-lognormals are pdf-s, the area under each of them always equals 1 (normalization condition) and so, going from left to right on the time axis, the b-lognormals become more and more “peaky”, and so they last less and less in time. This is precisely what happened in human history: civilizations that lasted millennia (like Ancient Greece and Rome) lasted just centuries (like the Italian Renaissance and Portuguese, Spanish, French, British and USA Empires) but they were more and more advanced in the “level of civilization”. This “level of civilization” is what physicists call ENTROPY.Also, in refs. Maccone [3] and [4], this author proved that, for all GBMs, the (Shannon) Entropy of the b-lognormals in his Peak-Locus Theorem grows LINEARLY in time. The Molecular Clock, well known to geneticists since 50 years, shows that the DNA base-substitutions occur LINEARLY in time since they are neutral with respect to Darwinian selection. In simple words: DNA evolved by obeying the laws of quantum physics only (microscopic laws) and not by obeying assumed “Darwinian selection laws” (macroscopic laws). This is Kimura׳s neutral theory of molecular evolution. The conclusion is that the Molecular Clock and the b-lognormal Entropy are the same thing.At last, we reach the new, original result justifying the publication of this paper. On exoplanets, molecular evolution is proceeding at about the same rate as it did proceed on Earth: rather independently of the physical conditions of the exoplanet, if the DNA had the possibility to evolve in water initially. Thus, Evo-Entropy, i.e. the (Shannon) Entropy of the generic b-lognormal of the Peak-Locus Theorem, provides the Evo-SETI SCALE to measure the evolution of life on exoplanets.

Major Histocompatibility complex-DMB allelic diversity in old and new world nonhuman primates: Intraspecies pattern of evolution

Nineteen different new MHC-DMB complete cDNA sequences have been obtained in thirteen different individuals belonging to the following primate species/families: Hylobates lar, Papio hamadryas, Macaca mulatta, Macaca fascicularis, Cercopithecus aethiops and Saguinus oedipus. Exonic allelism has been recorded all along the DM molecule domains and analyses of the critical residues in the conformation of the MHC-DR peptide-binding site were done; it was found an evolutionary pressure over the putative peptide-binding region of the DMB molecule that favours synonymous changes. These results are in contrast with the ones found in the MHC class I and class II genes, where non-synonymous DNA base substitutions are favoured The immunoreceptor inhibition motif Tyr230-X231-X232-Leu233 (ITIM) is invariantly present in all extant studied primates since 40 million years ago. It confirms the important function for this molecule, directing DR molecules towards the endosomal/ lysosomal class II compartment and sending inhibitory signals to cells in order to stop synthesis of unnecessary MHC-DR molecules. Some Macaca individuals DMB molecules (appear on Earth more than ten million years ago) dobear both short (without ITIM) and long cytoplasmic tails (with ITIM), similarly to what has been found in human individuals. These differences may have important functional implications. Other molecules, like NK-cell receptors and Fc receptors, bear this type of tyrosine-based inhibitory motifs in order to switch off specific cell functions. MHC-DMB variations occurring within each species suggest that their polymorphism may have an intraspecific evolution, since alleles of the same species cluster together, as it occurs in other MHC related genes (Bf, C4d). Other MHC class I and class II molecules, phylogenetic trees show a trans-species pattern of evolution. Finally, a cluster grouping human and gorilla DMB cDNA sequences is obtained using a dendrogram (for the MHC genes, i.e.: C4d trees); this is in contrast to others' results that obtain a human/chimpanzee cluster using different DNA sequences.Keywords: MHC (Major Histocompatibility Complex), MHC-DMB, HLA-DMB, primates, evolution, ITIM, MHC Class II metabolism, gibbon, macaque, New World Monkeys.

Murine melanomas accelerated by a single UVR exposure carry photoproduct footprints but lack UV signature C>T mutations in critical genes.

Ultraviolet radiation (UVR) exposure increases malignant melanoma (MM) risk, but in the context of acute, not cumulative exposure. C>T and CC>TT changes make up the overwhelming majority of single base substitutions (SBS) in MM DNA, as both precursor melanocytes and melanocytic lesions have incurred incidental exposures to sunlight. To study the mutagenic mechanisms by which acute sunburn accelerates MM, we sequenced the exomes of spontaneous and neonatal UVB-induced Cdk4-R24C::Tyr-NRASQ61K mouse MMs. UVR-induced MMs carried more SBSs than spontaneous MMs, but the levels of genomic instability, reflected by translocations and copy number changes, were not different. C>T/G>A was the most common SBS in spontaneous and UVR-induced MMs, only modestly increased in the latter. However, they tended to occur at the motif A/GpCpG (reflecting C>T transition due to spontaneous deamination of cytosine at CpG) in spontaneous MMs, and T/CpCpC/T (reflecting the effects of pyrimidine dimers on either side of the mutated C) in UVR-induced MMs. Unlike MMs associated with repetitive exposures, we observed no CC>TT changes. In addition, we also found UVR 'footprints' at T>A/A>Ts (at NpTpT) and T>C/A>G (at CpTpC). These footprints are also present in MMs from a chronic UVR mouse model, and in some human MMs, suggesting that they may be minor UVR signature changes. We found few significantly somatically mutated genes (~6 per spontaneous and 15 per UVR-induced melanoma) in addition to the Cdk4 and NRAS mutations already present. Trp53 was the most convincing recurrently mutated gene; however, in the UVR-induced MMs no Trp53 mutations were at C>T/G>A, suggesting that it was probably mutated during tumour progression, not directly induced by UVR photoproducts. The very low load of recurrent mutations convincingly induced by classical UVB-induced dimer photoproducts may support a role for cell extrinsic mechanisms, such as photoimmunosuppression and inflammation in driving MM after acute UVB exposure.

One-Dimensional Multichromophor Arrays Based on DNA: From Self-Assembly to Light-Harvesting.

Light-harvesting complexes collect light energy and deliver it by a cascade of energy and electron transfer processes to the reaction center where charge separation leads to storage as chemical energy. The design of artificial light-harvesting assemblies faces enormous challenges because several antenna chromophores need to be kept in close proximity but self-quenching needs to be avoided. Double stranded DNA as a supramolecular scaffold plays a promising role due to its characteristic structural properties. Automated DNA synthesis allows incorporation of artificial chromophore-modified building blocks, and sequence design allows precise control of the distances and orientations between the chromophores. The helical twist between the chromophores, which is induced by the DNA framework, controls energy and electron transfer and thereby reduces the self-quenching that is typically observed in chromophore aggregates. This Account summarizes covalently multichromophore-modified DNA and describes how such multichromophore arrays were achieved by Watson-Crick-specific and DNA-templated self-assembly. The covalent DNA systems were prepared by incorporation of chromophores as DNA base substitutions (either as C-nucleosides or with acyclic linkers as substitutes for the 2'-deoxyribofuranoside) and as DNA base modifications. Studies with DNA base substitutions revealed that distances but more importantly relative orientations of the chromophores govern the energy transfer efficiencies and thereby the light-harvesting properties. With DNA base substitutions, duplex stabilization was faced and could be overcome, for instance, by zipper-like placement of the chromophores in both strands. For both principal structural approaches, DNA-based light-harvesting antenna could be realized. The major disadvantages, however, for covalent multichromophore DNA conjugates are the poor yields of synthesis and the solubility issues for oligonucleotides with more than 5-10 chromophore modifications in a row. A logical alternative approach is to leave out the phosphodiester bridges between the chromophores and let chromophore-nucleoside conjugates self-assemble specifically along single stranded DNA as template. The self-organization of chromophores along the DNA template based on canonical base pairing would be advantageous because sequence selective base pairing could provide a structural basis for programmed complexity within the chromophore assembly. The self-assembly is governed by two interactions. The chromophore-nucleoside conjugates as guest molecules are recognized via hydrogen bonds to the corresponding counter bases in the single stranded DNA template. Moreover, the π-π interactions between the stacked chromophores stabilize these self-assembled constructs with increasing length. Longer DNA templates are more attractive for self-assembled antenna. The helicity in the stack of porphyrins as guest molecules assembled on the DNA template can be switched by environmental changes, such as pH variations. DNA-templated stacks of ethynyl pyrene and nile red exhibit left-handed chirality, which stands in contrast to similar covalent multichromophore-DNA conjugates with enforced right-handed helicity. With ethynyl nile red, it is possible to occupy every available binding site on the templates. Mixed assemblies of ethynyl pyrene and nile red show energy transfer and thereby provide a proof-of-principle that simple light-harvesting antennae can be obtained in a noncovalent and self-assembled fashion. With respect to the next important step, chemical storage of the absorbed light energy, future research has to focus on the coupling of sophisticated DNA-based light-harvesting antenna to reaction centers.

Evo-SETI Entropy identifies with Molecular Clock

Darwinian evolution over the last 3.5 billion years was an increase in the number of living species from 1 (RNA?) to the current 50 million. This increasing trend in time looks like being exponential, but one may not assume an exactly exponential curve since many species went extinct in the past, even in mass extinctions. Thus, the simple exponential curve must be replaced by a stochastic process having an exponential mean value. Borrowing from financial mathematics (“Black-Sholes models”), this “exponential” stochastic process is called Geometric Brownian Motion (GBM), and its probability density function (pdf) is a lognormal (not a Gaussian) (Proof: see Ref. [3], Chapter 30, and Ref. [4]). Lognormal also is the pdf of the statistical number of communicating ExtraTerrestrial (ET) civilizations in the Galaxy at a certain fixed time, like a snapshot: this result was found in 2008 by this author as his solution to the Statistical Drake Equation of SETI (Proof: see Ref. [1]). Thus, the GBM of Darwinian evolution may also be regarded as the extension in time of the Statistical Drake equation (Proof: see Ref. [4]). But the key step ahead made by this author in his Evo-SETI (Evolution and SETI) mathematical model was to realize that LIFE also is just a b-lognormal in time: every living organism (a cell, a human, a civilization, even an ET civilization) is born at a certain time b (“birth”), grows up to a peak p (with an ascending inflexion point in between, a for adolescence), then declines from p to s (senility, i.e. descending inflexion point) and finally declines linearly and dies at a final instant d (death). In other words, the infinite tail of the b-lognormal was cut away and replaced by just a straight line between s and d, leading to simple mathematical formulae (“History Formulae”) allowing one to find this “finite b-lognormal” when the three instants b, s, and d are assigned.Next the crucial Peak-Locus Theorem comes. It means that the GBM exponential may be regarded as the geometric locus of all the peaks of a one-parameter (i.e. the peak time p) family of b-lognormals. Since b-lognormals are pdf-s, the area under each of them always equals 1 (normalization condition) and so, going from left to right on the time axis, the b-lognormals become more and more “peaky”, and so they last less and less in time. This is precisely what happened in Human History: civilizations that lasted millennia (like Ancient Greece and Rome) lasted just centuries (like the Italian Renaissance and Portuguese, Spanish, French, British and USA Empires) but they were more and more advanced in the “level of civilization”. This “level of civilization” is what physicists call ENTROPY. In Refs. [3] and [4], this author proved that, for all GBMs, the (Shannon) Entropy of the b-lognormals in his Peak-Locus Theorem grows LINEARLY in time.At last, we reach the new, original result justifying the publication of this paper. The Molecular Clock, well known to geneticists since 50 years, shows that the DNA base-substitutions occur LINEARLY in time since they are neutral with respect to Darwinian selection. In simple words: DNA evolved by obeying the laws of quantum physics only (microscopic laws) and not by obeying assumed “Darwinian selection laws” (macroscopic laws). This is Kimura׳s neutral theory of molecular evolution.The conclusion of this paper is that the Molecular Clock and the b-lognormal Entropy are the same thing.And, on exoplanets, molecular evolution is proceeding at about the same rate as it did proceed on Earth: rather independently of the physical conditions of the exoplanet, if the DNA had the possibility to evolve in water initially.

APOBEC3B can impair genomic stability by inducing base substitutions in genomic DNA in human cells

Human APOBEC3 proteins play pivotal roles in intracellular defense against viral infection by catalyzing deamination of cytidine residues, leading to base substitutions in viral DNA. Activation-induced cytidine deaminase (AID), another member of the APOBEC family, is capable of editing immunoglobulin (Ig) and non-Ig genes, and aberrant expression of AID leads to tumorigenesis. However, it remains unclear whether APOBEC3 (A3) proteins affect stability of human genome. Here we demonstrate that both A3A and A3B can induce base substitutions into human genome as AID can. A3B is highly expressed in several lymphoma cells and somatic mutations occur in some oncogenes of the cells highly expressing A3B. Furthermore, transfection of A3B gene into lymphoma cells induces base substitutions in cMYC gene. These data suggest that aberrant expression of A3B can evoke genomic instability by inducing base substitutions into human genome, which might lead to tumorigenesis in human cells.

Evolutionary patterns of carbohydrate transport and metabolism in Halomonas boliviensis as derived from its genome sequence: influences on polyester production

BackgroundHalomonas boliviensis is a halophilic bacterium that is included in the γ-Proteobacteria sub-group, and is able to assimilate different types of carbohydrates. H. boliviensis is also able to produce poly(3-hydroxybutyrate) (PHB) in high yields using glucose as the carbon precursor. Accumulation of PHB by microorganisms is induced by excess of intracellular NADH.The genome sequences and organization in microorganisms should be the result of evolution and adaptation influenced by mutation, gene duplication, horizontal gen transfer (HGT) and recombination. Furthermore, the nearly neutral theory of evolution sustains that genetic modification of DNA could be neutral or selected, albeit most mutations should be at the border between neutrality and selection, i.e. slightly deleterious base substitutions in DNA are followed by a slightly advantageous substitutions.ResultsThis article reports the genome sequence of H. boliviensis. The chromosome size of H. boliviensis was 4 119 979 bp, and contained 3 863 genes. A total of 160 genes of H. boliviensis were related to carbohydrate transport and metabolism, and were organized as: 70 genes for metabolism of carbohydrates; 47 genes for ABC transport systems and 43 genes for TRAP-type C4-dicarboxylate transport systems. Protein sequences of H. boliviensis related to carbohydrate transport and metabolism were selected from clusters of orthologous proteins (COGs). Similar proteins derived from the genome sequences of other 41 archaea and 59 bacteria were used as reference. We found that most of the 160 genes in H. boliviensis, c.a. 44%, were obtained from other bacteria by horizontal gene transfer, while 13% of the genes were acquired from haloarchaea and thermophilic archaea, only 34% of the genes evolved among Proteobacteria and the remaining genes encoded proteins that did not cluster with any of the proteins obtained from the reference strains. Furthermore, the diversity of the enzymes derived from these genes led to polymorphism in glycolysis and gluconeogenesis. We found further that an optimum ratio of glucose and sucrose in the culture medium of H. boliviensis favored cell growth and PHB production.ConclusionsResults obtained in this article depict that most genetic modifications and enzyme polymorphism in the genome of H. boliviensis were mainly influenced by HGT rather than nearly neutral mutations. Molecular adaptation and evolution experienced by H. boliviensis were also a response to environmental conditions such as the type and amount of carbohydrates in its ecological niche. Consequently, the genome evolution of H. boliviensis showed to be strongly influenced by the type of microorganisms, genetic interaction among microbial species and its environment. Such trend should also be experienced by other prokaryotes. A system for PHB production by H. boliviensis that takes into account the evolutionary adaptation of this bacterium to the assimilation of combinations of carbohydrates suggests the feasibility of a bioprocess economically viable and environmentally friendly.

Synthesis of a Benzophenone C-Nucleoside as Potential Triplet Energy and Charge Donor in Nucleic Acids

A synthetic route to the C-nucleoside that bears benzophenone as a DNA base substitution directly at the anomeric center of the 2′-deoxyribofuranoside was worked out. Furthermore, the α-anomer of this artificial nucleoside was converted synthetically into the corresponding DNA building block and incorporated into two representative oligonucleotides by automated phosphoramidite chemistry. The chromophore-modified DNA was characterized by methods of optical spectroscopy. The absorption band at ∼350 nm can be used for selective excitation of the benzophenone chromophore outside the nucleic acid absorption range, which makes the benzophenone nucleoside potentially useful for photochemical and photobiological applications.

Power of the likelihood ratio test for models of DNA base substitution

The goal of this work is to study the properties of the likelihood ratio (LR) tests comparing base substitution models. These are the most widely used hypothesis tests. With mild regularity conditions, we show that the asymptotic distribution of the LR statistic test, under the alternative hypothesis, is a non-central chi-square distribution. The asymptotic normal distribution of the LR test is proved when the sequence length S goes to infinity. We also propose a consistent estimator for the non-centrality parameter D. Through asymptotic theory and based on this consistent estimator for D, we propose a low computational cost estimator for the power of the LR test. The methodology is applied to 17 different gene sequences of the ECP–EDN family in primates.

Genotoxicity of polar and apolar extracts obtained from Qualea multiflora and Qualea grandiflora

Ethnopharmacological relevanceThe species Qualea grandiflora and Qualea multiflora, which belong to the Vochysiaceae family, are common in the Brazilian savannah (Cerrado biome), and the local inhabitants use these species to treat external ulcers and gastric diseases and as an anti-inflammatory agent. Studies have demonstrated that these plants contain compounds that exhibit pharmacological activities; however, the risks associated with their consumption are not known. Material and methodsIn the present study, the mutagenicity of polar and apolar extracts from Qualea grandiflora and Qualea multiflora were assessed by employing the Ames assay with and without metabolic activation. Additionally, phytochemical analyses (HPLC–ESI-IT-MS, HPLC–UV-PDA and GC–IT-MS) were performed to identify the chemical constituents present in these species, including the evaluation of physico-chemical properties, such as polarity or apolarity of the organic compounds, which are related to each fraction obtained. These studies provide important information regarding the biochemical behaviour of these compounds. ResultsAll extracts exhibited mutagenicity, inducing frameshift mutations and base substitutions in DNA. Phytochemical analysis identified terpenes, ellagic acid derivatives and phytosteroids. ConclusionsThe mutagenicity observed might be due to the presence of pentacyclic triterpenes and polyphenols, which are able to generate reactive oxygen species (ROS) and result in the potential to cause DNA damage. The genetic risk identified in this present work shows that special attention should be considered for the use of compounds obtained from these plant species in medicinal treatments. Further studies must be conducted to identify safe therapeutic doses.

High-Throughput Sequencing of Core STR Loci for Forensic Genetic Investigations using the Roche Genome Sequencer FLX Platform

The analysis and profiling of short tandem repeat (STR) loci is routinely used in forensic genetics. Current methods to investigate STR loci, including PCR-based standard fragment analyses and capillary electrophoresis, only provide amplicon lengths that are used to estimate the number of STR repeat units. These methods do not allow for the full resolution of STR base composition that sequencing approaches could provide. Here we present an STR profiling method based on the use of the Roche Genome Sequencer (GS) FLX to simultaneously sequence multiple core STR loci. Using this method in combination with a bioinformatic tool designed specifically to analyze sequence lengths and frequencies, we found that GS FLX STR sequence data are comparable to conventional capillary electrophoresis-based STR typing. Furthermore, we found DNA base substitutions and repeat sequence variations that would not have been identified using conventional STR typing.