Unstable, non-mutational expression of resistance to the thymidine analogue, trifluorothymidine in CHO cells.

In Saccharomyces cerevisiae, methyl methanesulphonate and diepoxybutane produced efficiently lethal, as well as mutagenic, damage in nuclear DNA. However, in the same conditions, these agents did not induce cytoplasmic petite mutations and poorly induced point mutations (resistance to erythromycin and chloramphenicol) in mitochondrial DNA. Possible reasons for these differences are discussed.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae. I. Ultraviolet radiation.

UV efficiently induces mutations in mitDNA , conferring resistance to erythromycin. Mitochondrial chloramphenicol-resistant mutants are probably also induced by UV, but almost 90% of mutants with such phenotype are non-mitochondrial; therefore it is possible to estimate accurately the frequences of the induced presumptive mitochondrial capr mutations.     

Mutagenic DNA repair in Escherichia coli. VII. Constitutive and inducible manifestations.

Incubation of E. coli WP2 in the presence of chloramphenicol (CAP) for 90 min before and 60 min after γ-irradiation had no effect on the induction of Trp⁺ mutations. Bacteria that had been treated with CAP for 90 min prior to UV irradiation showed normal or near normal yields of induced mutations to streptomycin or colicin E2 resistance. Most of these mutations lost their photoreversibility (indicating “fixation”) during continued incubation with CAP for a further 60 min after irradiation, during which time neither protein nor DNA synthesis was detectable. It is suggested that CAP-sensitive protein synthesis is not required for mutagenic (error-prone) repair of lesions in pre-existing DNA, arguing against an inducible component in this repair.In contrast the frequency of UV-induced mutations to Trp⁺ (largely at suppressor loci) was drastically reduced by CAP pretreatment, confirming the need for an active replication fork for UV-mutagenesis at these loci. It is known from the work of others that CAP given after UV abolishes mutagenesis at these loci. We conclude that CAP-sensitive protein synthesis (consistent with a requirement for an inducible function) is necessary for mutagenic repair only in newly-replicated DNA (presumably at daughter strand gaps) and not in pre-existing DNA. The data are consistent with but do not prove the hypothesis that CAP-sensitive and insensitive modes of mutagenesis reflect minor differences in the operation of a single basic mutagenic repair system.     

Nitrous acid mutagenesis of duplex DNA as a three-component system.

Purified native Hemophilus influenzae DNA is relatively insusceptible to nitrous acid (NA) mutagenesis in vitro, but is readily mutated following denaturation. NA mutagenicity for duplex DNA is significantly increased in the presence of various alcohols, glycols, phenols or primary amines. Phenol-extracted DNA contains dissociable contaminants of low molecular weight that enhance NA mutagenesis. Enhancement of NA mutagenesis by phenol and by spermine is due to the formation of unstable molecular species. We propose that reactive organic nitroso compounds are formed which then serve as delivery vehicles to promote mutagenicity of native DNA, perhaps via transnitrosation reactions. Similar reactions probably occur in vivo to promote NA-induced base substitution (but not frameshift) mutations in Salmonella typhimurium and in Escherichia coli. The possible significance of these observations to carcinogenesis is discussed.     

The Fundamental Theorem of Neutral Evolution: Rates of Substitution and Mutation Should Factor in Premeiotic Clusters

Mutations do not always arise as single events. Many new mutations actually occur in the cell lineage before germ cell formation or meiosis and are therefore replicated premeiotically. The increased likelihood of substitutions caused by these clusters of new mutant alleles can change the fundamental theorem of neutral evolution.     

Mitochondrial mutagenesis in Saccharomyces cerevisiae V. Ethyl methanesulfonate

Mitochondrial genomes are more sensitive to the lethal action of EMS than are nuclear genomes of S. cerevisiae. EMS induces efficiently only some types of mutation in nuclear genomes of yeast, and probably the same is true for induction of mutations non-lethal to the mitochondrial genomes.     

An investigation of noradrenaline uptake and release by the CATH.a cell line

The cell bodies of ascending noradrenergic neurons in the brain are located predominantly in the locus coeruleus. An in vitro model of locus coeruleus neurons could prove to be a useful tool in the investigation of noradrenergic neural networks and their associated pathophysiologies. The CATH.a cell line demonstrates some of the properties expected of locus coeruleus neurons, and the present study investigated the neurotransmitter uptake and release properties of the CATH.a cells. It was surprising that the CATH.a cells failed to accumulate [3H]noradrenaline ([3H]NA), suggesting the lack of a functional NA transporter. RT-PCR supported this finding by demonstrating the absence of NA transporter mRNA. Treatment of CATH.a cells with various differentiating agents failed to increase the [3H]NA uptake. Endogenous NA release was studied using HPLC detection, which revealed a lack of depolarisation-induced increases in endogenous NA release. A human NA transporter-transfected CATH.a cell line was generated (termed RUNT), and a study of the [3H]NA uptake revealed that the RUNT cells displayed significant uptake that could be blocked by cocaine (10 microM). Furthermore, the uptake capacity could be dramatically increased by differentiation of the cells with dibutyryl cyclic AMP (1 mM) for 24 h. Using dibutyryl cyclic AMP-differentiated RUNT cells, high K+ concentrations (50 mM) significantly increased [3H]NA release above basal levels.     

Development of spontaneous mutations in somatic mamalian cells and DNA replication. II. Expression of gene mutations in cultured cells]

The dependence of spontaneous rate and phenotypic expression of 6-mercaptopurine resistance mutations of DNA replication synthesis was studied in cultured Chinese hamster cells. Spontaneous mutations arising with a constant rate per cell per time unit independently on DNA replication rate were shown to be expressed only in the course of subsequent cell divisions. The frequency of N-nitrosomethylurea induced mutations in cells with reduced and normal DNA replication rate is approximately the same. However, DNA replication synthesis is necessary for the phenotypic expression of both induced and spontaneous mutations. The causes of the phenotypic lag are discussed.     

Computational studies of H5N1 influenza virus resistance to oseltamivir

Influenza A (H5N1) virus is one of the world's greatest pandemic threats. Neuraminidase (NA) inhibitors, oseltamivir and zanamivir, prevent the spread of influenza, but drug‐resistant viruses have reduced their effectiveness. Resistance depends on the binding properties of NA‐drug complexes. Key residue mutations within the active site of NA glycoproteins diminish binding, thereby resulting in drug resistance. We performed molecular simulations and calculations to characterize the mechanisms of H5N1 influenza virus resistance to oseltamivir and predict potential drug‐resistant mutations. We examined two resistant NA mutations, H274Y and N294S, and one non‐drug‐resistant mutation, E119G. Six‐nanosecond unrestrained molecular dynamic simulations with explicit solvent were performed using NA‐oseltamivir complexes containing either NA wild‐type H5N1 virus or a variant. MM_PBSA techniques were then used to rank the binding free energies of these complexes. Detailed analyses indicated that conformational change of E276 in the Pocket 1 region of NA is a key source of drug resistance in the H274Y mutant but not in the N294S mutant.     

Kinetic and structural changes in HsmtPheRS,induced by pathogenic mutations in human FARS2

Mutations in the mitochondrial aminoacyl‐tRNA synthetases (mtaaRSs) can cause profound clinical presentations, and have manifested as diseases with very selective tissue specificity. To date most of the mtaaRS mutations could be phenotypically recognized, such that clinicians could identify the affected mtaaRS from the symptoms alone. Among the recently reported pathogenic variants are point mutations in FARS2 gene, encoding the human mitochondrial PheRS. Patient symptoms range from spastic paraplegia to fatal infantile Alpers encephalopathy. How clinical manifestations of these mutations relate to the changes in three‐dimensional structures and kinetic characteristics remains unclear, although impaired aminoacylation has been proposed as possible etiology of diseases. Here, we report four crystal structures of HsmtPheRS mutants, and extensive MD simulations for wild‐type and nine mutants to reveal the structural changes on dynamic trajectories of HsmtPheRS. Using steady‐state kinetic measurements of phenylalanine activation and tRNA^Phe aminoacylation, we gained insight into the structural and kinetic effects of mitochondrial disease‐related mutations in FARS2 gene.     

线粒体功能障碍与孤独症

孤独症谱系障碍（ASDs）患儿中约有5%伴有线粒体功能紊乱.线粒体功能紊乱会损害对能量高度依赖的生理进程,如神经发育和神经可塑性,从而导致孤独症.本文综述了孤独症个体中线粒体过量的活性氧（reactive oxygen species,ROS）产生及其抗氧化系统减弱、呼吸链复合物异常、线粒体基因突变及与线粒体功能相关的基因组DNA编码的蛋白质异常等方面的研究,旨在阐述线粒体系统多方面的紊乱在孤独症个体中均有所体现,希望能够对孤独症的发病机制和治疗提供帮助.     

应用高剂量/庇护所策略管理Bt作物抗性的三个基本假设：综述与展望

目前, 抗虫转基因作物的抗性管理方法主要是高剂量/庇护所策略。该策略的有效性取决于3个基本的假设条件：（1）抗虫转基因作物（Bt作物）表达出高剂量的杀虫蛋白, 该剂量使得靶标害虫对Bt杀虫蛋白的抗性表现型为功能性完全隐性或近于完全隐性, 进而使得Bt作物可以杀死几乎所有的抗性杂合个体和所有的敏感性个体;（2）靶标害虫种群的Bt抗性基因起始频率处于很低的水平;（3）源自转基因作物田和非转基因作物田(庇护所)的成虫在田间随机混合并交配。这3个假设必须同时满足, 缺一不可。本文就这3个假设的理论基础和经验研究的进展进行了综合论述, 并着重讨论了随机交配假设的最新研究进展以及今后的研究方向和方法。     

Inhibition of viral group-1 and group-2 neuraminidases by oseltamivir: A comparative structural analysis by the ScrewFit algorithm

The viral surface glycoprotein neuraminidase (NA) allows the influenza virus penetration and the egress of virions. NAs are classified as A, B, and C. Type-A NAs from influenza virus are subdivided into two phylogenetically distinct families, group-1 and group-2. NA inhibition by oseltamivir represents a therapeutic approach against the avian influenza virus H5N1. Here, structural bases for oseltamivir recognition by group-1 NA1, NA8 and group-2 NA9 are highlighted by the ScrewFit algorithm for quantitative structure comparison. Oseltamivir binding to NA1 and NA8 affects the geometry of Glu119 and of regions Arg130-Ser160, Val240-Gly260, and Asp330-Glu382, leading to multiple NA conformations. Additionally, although NA1 and NA9 share almost the same oseltamivir-bound final conformation, they show some relevant differences as suggested by the ScrewFit algorithm. These results indicate that the design of new NA inhibitors should take into account these family-specific effects induced on the whole structure of NAs.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Further sequence analysis of the phage lambda receptor site. Possible implications for the organization of the lamB protein in Escherichia coli K12

We present the DNA sequence alterations due to seven lamB missense mutations yielding resistance to phages lambda and K10. They reveal five different amino acid positions in the LamB protein. Three positions (245, 247 and 249) define a new region required for phage adsorption. The two other positions (148 and 152) belong to a region where mutations to phage resistance has already been detected. These two regions are hydrophilic and could belong to turns of the protein located at the surface of the cell. All the missense mutational alterations to phage resistance sequenced in the LamB protein correspond to 10 sites located in four different segments of the polypeptide chain. We discuss their location in terms of the notion of phage receptor site and of a working model for the organization of this protein in the outer membrane of Escherichia coli.     

Analysis of oseltamivir resistance substitutions in influenza virus glycoprotein neuraminidase using a lentivirus-based surrogate assay system

Influenza A virus poses a great threat to global health, and oseltamivir (trade marked as Tamiflu), which targets influenza surface glycoprotein neuraminidase (NA), is used clinically as a major anti-influenza treatment. However, certain substitutions in NA can render an influenza virus resistant to this drug. In this study, using a lentiviral pseudotyping system, which alleviates the safety concerns of studying highly pathogenic influenza viruses such as avian influenza H5N1, that utilizes influenza surface glycoproteins (hemagglutinin or HA, and NA) and an HIV-core combined with a luciferase reporter gene as a surrogate assay, we first assessed the functionality of NA by measuring pseudovirion release in the absence or presence of oseltamivir. We demonstrated that oseltamivir displays a dose-dependent inhibition on NA activity. In contrast, a mutant NA (H274Y) is more resistant to oseltamivir treatment. In addition, the effects of several previously reported substitution NA mutants were examined as well. Our results demonstrate that this lentivirus-based pseudotyping system provides a quick, safe, and effective way to assess resistance to neuraminidase inhibitors. And we believe that as new mutations appear in influenza isolates, their impact on the effectiveness of current and future anti-NA can be quickly and reliably evaluated by this assay.     

Use of transposon-promoted deletions in DNA sequence analysis

The usefulness of the dideoxy method for DNA sequencing can be greatly extended by the use of transposon-generated deletions. These deletions have the unique property of extending from a fixed nucleotide at the transposon terminus to various sites outside it. A plasmid (pAA3.7) carrying Tn9, which allows positive selection of such deletions as galactose-resistant colonies of Escherichia coli, is described. A cloned gene can thus be subdivided into a series of overlapping sequences, all of which are fused to a common sequence at the transposon terminus. Restriction fragments carrying the segments fused by deletions are cloned in M13, and sequenced using a primer complementary to the Tn9 terminus. Complete nucleotide sequence of the gene is assembled from sequence overlaps found in deletions with end-points approximately 350 base-pairs apart. The method is rapid, requires minimal in vitro manipulation, and is free from redundant information normally produced in shotgun sequencing.     

Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3,N⁶-etheno-ATP. phosphate/carbamoyl-d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme.We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3,N⁶-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose.Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies.Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation.Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London &; Schmidt (1972). We suggest, however, a modification of the London &; Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.     

Pseudovirus-based neuraminidase inhibition assays reveal potential H5N1 drug-resistant mutations

The use of antiviral drugs such as influenza neuraminidase (NA) inhibitors is a critical strategy to prevent and control flu pandemic, but this strategy faces the challenge of emerging drug-resistant strains. F or a highly pathogenic avian influenza (HPAI) H5N1 virus, biosafety restrictions have significantly limited the efforts to monitor its drug responses and mechanisms involved. In this study, a rapid and biosafe assay based on NA pseudovirus was developed to study the resistance of HPAI H5N1 virus to NA inhibitor drugs. The H5N1 NA pseudovirus was comprehensively tested using oseltamivir-sensitive strains and their resistant mutants. Results were consistent with those in previous studies, in which live H5N1 viruses were used. Several oseltamivir-resistant mutations reported in human H1N1 were also identifi ed to cause decreased oseltamivir sensitivity in H5N1 NA by using the H5N1 NA pseudovirus. Thus, H5N1 NA pseudoviruses could be used to monitor HPAI H5N1 drug resistance rapidly and safely.