Guanine Residues Research Articles

Pyrrolo[2,1-c][1,4]benzodiazepine as a Scaffold for the Design and Synthesis of Anti- Tumour Drugs

Compounds that bind in the minor groove of DNA have found use in the experimental treatment of cancer and certain infectious diseases. Furthermore, agents which target and can recognize discrete sequences of DNA have the potential to offer selective therapies by modulating the activity of specific transcription factors or genes. For this reason, a number of sequence-selective DNA binding agents have been evaluated with a range of affinities and recognition fidelities. In this respect, the pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are of interest as they bind to guanine residues in the minor groove with a preference for Pu-G-Pu sequences. A dramatic increase in cytotoxicity and sequence selectivity has been achieved by linking two PBD units to form PBD dimers as cross-linking agents on opposite DNA strands (e.g., interstrand cross-links). SJG-136 is currently undergoing Phase I evaluation in both the United States (through the NCI) and United Kingdom (through Cancer Research United Kingdom). This review will focus on design, synthesis and structure activity relationship studies of pyrrolobenzodiazepines as anticancer therapeutics reported since 2003.

8-Oxoguanine DNA glycosylase (Ogg1) causes a transcriptional inactivation of damaged DNA in the absence of functional Cockayne syndrome B (Csb) protein

We have analysed the effect of oxidative guanine lesions on the expression of a transfected reporter gene in mouse embryonic fibroblasts deficient in Cockayne syndrome B protein (Csb) and/or the 8-oxoguanine DNA glycosylase (Ogg1). We used a highly sensitive flow cytometry-based approach and quantitative real-time PCR to measure the changes in gene expression caused by the presence of oxidised guanine residues generated by photosensitisation in the vector DNA. In wild-type cells, small numbers (one or three) of oxidised guanines did not affect gene expression at short times after transfections, whereas progressive reduction of the transgene expression was observed at later time points. Although Ogg1 has a major contribution to the repair of oxidised guanine bases, its absence did not have a strong effect on the gene expression. In contrast, the lack of functional Csb protein caused a pronounced inactivation of the damaged reporter gene. Most strikingly, an additional Ogg1 deficiency significantly attenuated this effect. The results indicate that the processing of oxidative guanine modifications by Ogg1 can mediate host cell inactivation rather than reactivation of the damaged genes and that this effect is strongly enhanced in the absence of Csb.

FAS Promoter Polymorphism: Outcome of Childhood Acute Myeloid Leukemia. A Children's Oncology Group Report

FAS is a cell surface receptor involved in apoptotic signal transmission. Deregulation of this pathway results in down-regulation of apoptosis and subsequent persistence of a malignant clone. A single nucleotide polymorphism resulting in guanine-to-adenine transition in the FAS promoter region (position -1377) is thought to reduce stimulatory protein 1 transcription factor binding and decrease FAS expression. Previous work has shown increased risk of developing acute myeloid leukemia (AML) in adult patients with a variant allele at this site. The same authors have shown that the presence of an adenine residue rather than a guanine residue at -1,377 bp significantly attenuates transcription factor stimulatory protein 1 binding and may contribute to a reduction in FAS expression and ultimately to the enrichment of apoptosis-resistant clones in AML. We hypothesized that FAS genotype by altering susceptibility to apoptosis might affect outcome of childhood AML therapy. Four hundred forty-four children treated for de novo AML on a uniform protocol were genotyped for FAS 1377. There were no significant differences in overall survival, event-free survival, treatment-related mortality, or relapse rate between patients with FAS 1377GG genotype versus 1377GA/1377AA genotypes. FAS 1377 genotype does not alter outcome of de novo AML in children.

Influence of decreasing solvent polarity (1,4-dioxane/water mixtures) on the stability and structure of complexes formed by copper(II), 2,2′-bipyridine or 1,10-phenanthroline and guanosine 5′-diphosphate: evaluation of isomeric equilibria†‡

The stability constants of the 1 : 1 complexes formed between Cu(Arm)2+, where Arm = 2,2′-bipyridine or 1,10-phenanthroline, and guanosine 5′-diphosphate (GDP)3− or its monoprotonated form H(GDP)2− were determined by potentiometric pH titrations in water and in water containing 30 or 50% (v/v) 1,4-dioxane (25°C; I = 0.1 M, NaNO3). The stability of the binary Cu(GDP)− complex is enhanced due to macrochelate formation of the diphosphate-coordinated Cu2+ with N7 of the guanine residue as previously shown. In Cu(Arm)(GDP)− the N7 is released from Cu2+ and the stability enhancement of more than one log unit in aqueous solution is clearly attributable to intramolecular stack formation between the aromatic rings of Arm and the guanine moiety. Indeed, stacked isomers occur to more than 90% in equilibrium with open unstacked forms. Surprisingly, the same formation degrees of the stacks are observed for Cu(Arm)(dGMP) complexes, where dGMP2− = 2′-deoxyguanosine 5′-monophosphate, despite the fact that the overall stability of the latter species is by about 2.7 log units lower. In 1,4-dioxane–water mixtures stack formation is drastically reduced, probably due to hydrophobic solvation of the aromatic rings by the ethylene bridges of 1,4-dioxane. The relevance of these results regarding biological systems is indicated. †This study is dedicated to Professor Dr Alfredo Mederos on the occasion of his retirement from the University of La Laguna (Spain) with the very best wishes for all of his future endeavors. ‡This is part 70 of the series Ternary Complexes in Solution; for parts 69 and 68 see 14 and 15, respectively.

Interaction of Dinuclear Ruthenium(II) Supramolecular Cylinders with DNA: Sequence‐Specific Binding, Unwinding, and Photocleavage

Metallosupramolecular chemistry was used to design a new class of synthetic agents, namely, tetracationic supramolecular cylinders, that bind strongly and noncovalently in the major groove of DNA. To gain additional information on interactions of the cylinders with DNA we explored DNA unwinding and sequence-specific binding properties, as well as DNA photonuclease activity of ruthenium(II) metallosupramolecular cylinder [Ru(2)L(3)](4+), where L is a bis-pyridylimine ligand. We found that [Ru(2)L(3)](4+) unwinds negatively supercoiled plasmid DNA and exhibits binding preference to regular alternating purine-pyrimidine sequences in a similar way to the [Fe(2)L(3)](4+) analogue. Photocleavage studies showed that, unlike [Fe(2)L(3)](4+), [Ru(2)L(3)](4+) induces single-strand breaks on irradiation by visible and UVA light and cleaves DNA mainly at guanine residues contained preferentially in regularly alternating purine-pyrimidine nucleotides. As [Ru(2)L(3)](4+) binds and cleaves DNA in a sequence-dependent manner, it may provide a useful tool for basic and applied biology, such as for controlled manipulation of the genome.

Abstract B45: Prenatal exposure to polycyclic aromatic hydrocarbons (PAH), PAH-DNA adducts and genomic DNA methylation in cord blood

Abstract B45 Polycyclic aromatic hydrocarbons (PAHs) are widespread organic carcinogenic pollutants. When metabolized in vivo, PAHs form reactive diol epoxides that bind to DNA at guanine residues forming adducts. PAH exposure has also been associated with alterations in genomic cytosine methylation. Both PAH-DNA adducts and altered DNA methylation have been associated with increased cancer risk. We sought to explore the relationship between prenatal PAH exposure, global DNA methylation and PAH-DNA adducts in a New York City birth cohort. Cord blood was collected from deliveries of non-smoking, African Americans and Dominicans, ages 18-35, residing in N. Manhattan and S. Bronx. Personal air monitors measured PAH during their 8th month of pregnancy. We measured benzo[a]pyrene (B[a]P)-DNA adducts (which serve as a proxy for PAH-DNA adducts) in cord blood using high-performance liquid chromatography-fluorescence. We measured genomic DNA methylation in cord blood leukocytes using the MethylampTM global DNA methylation quantification kit. Demographic and epidemiologic risk factors were collected prospectively, including maternal age, ethnicity, parity, and prenatal tobacco smoke exposure, and child’s gender We found that prenatal PAH exposure was associated global DNA hypomethylation such that average methylation among those in the highest quartile of prenatal PAH exposure was 1.17 ng/100 ng total DNA as compared 1.83 ng/100 ng total DNA in the lowest quartile (p < 0.01). Conversely, those with detectable PAH-DNA adducts had higher levels of DNA methylation (1.43 ng/100 ng total DNA) as compared to those with non-detectable adducts (1.16 ng/100 ng total DNA) (p = 0.02). Using multiple variable linear regression, PAH exposure was independently associated with hypomethylation (average difference in methylation among those in the highest vs. lowest quartile of exposure = -0.42, 95% CI: -0.75, -0.09) and the presence of DNA adducts was associated with hypermethyaltion (average difference in methylation among those with detectable vs. non-detectable adducts = 0.28, 95% CI: 0.05, 0.49). Adjusting for potential confounders did not significantly alter these independent associations, indicating that the lowest level of global DNA methylation was found among those with non-detectable adducts and high PAH exposure, and the highest level of DNA methylation was found among those with detectable adducts and low PAH exposure. While the mechanism by which prenatal PAH exposure results in DNA adducts is well understood, the way exposure may impact methylation is not known. These data suggest that in the presence of DNA adducts, PAH exposure is associated with a reduction in methylation, but when adducts are non-detectable, the same PAH exposure is associated with an even larger methylation reduction. It is possible that the presence of adducts may be partially preventing PAH compounds (or metabolites) from accessing the DNA, leading to less of a change in methylation. It is also possible that the shift in global methylation from PAH exposure may prevent adduct formation. These speculations as well as the associations observed in this analysis require confirmation in other settings/populations. Because methylation changes are potentially reversible, it is possible that if these associations are confirmed, methylation may serve as a target for intervention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B45.

Absence of MGMT promoter methylation in endometrial cancer

Objective O 6-methylguanine-DNA methyltransferase (MGMT) acts to repair DNA damaged by alkylation of guanine residues. MGMT promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes [Ogino S, Meyerhardt JA, Kawasaki T, et al. CpG island methylation, response to combination chemotherapy, and patient survival in advanced microsatellite stable colorectal carcinoma. Virchows Arch 2007;450:529–37; Rodriguez MJ, Acha A, Ruesga MT, Rodriguez C, Rivera JM, Aguirre JM. Loss of expression of DNA repair enzyme MGMT in oral leukoplakia and early oral squamous cell carcinoma. A prognostic tool? Cancer Lett 2007;245:263–8; Ishii T, Murakami J, Notohara K, et al. Oesophageal squamous cell carcinoma may develop within a background of accumulating DNA methylation in normal and dysplastic mucosa. Gut 2007;56:13–9]. The loss of MGMT activity and promoter methylation is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas [Weaver KD, Grossman SA, Herman JG. Methylated tumor-specific DNA as a plasma biomarker in patients with glioma. Cancer Invest 2006;24:35–40; Esteller M, Garcia-Foncillas J, Andion E, et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N Engl J Med 2000;343:1350–4; Hegi ME, Diserens AC, Gorlia T, et al. MGMT gene silencing and benefit from temozolomide in glioblastoma. N Engl J Med 2005;352:997–1003]. We sought to determine if MGMT promoter methylation plays a role in endometrial cancer. Methods One hundred and twenty primary endometrial cancers were analyzed for MGMT promoter methylation by combined bisulfite restriction analysis (COBRA). The cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. Twenty-one endometrioid and mixed endometrioid ovarian cancers were also analyzed. A subset of the primary tumors was analyzed for MGMT expression by immunohistochemistry. Results No MGMT promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. One of the 21 endometrioid ovarian cancers showed methylation. Immunohistochemistry revealed moderate to high level expression of MGMT in the primary endometrial tumors. Conclusion MGMT promoter methylation is an infrequent event in endometrial cancer. MGMT expression and the ability to repair damaged alkylguanine residues could in part explain the limited response of endometrial tumors to alkylating chemotherapy.

A Novel Conformationally Constrained Parallel G Quadruplex

Guanine-rich DNA sequences are known to form highly ordered structures called G quadruplexes. These structures play an important role in many relevant biological processes, such as telomere stabilization, oncogene activation, and the regulation of the immunoglobulin switch region. The G-quadruplex motif is based on the association of planar G quartets of four guanine residues that are held together by eight Hoogsteen-type hydrogen bonds (Figure 1A). The G-quadruplex motif requires monovalent cations, such as Na and K , for stabilization. A wide variety of topologies can be adopted depending on the number of strands involved in the structure, the strand direction, as well as variations in loop size and sequence (Figure 1). The structure of parallel-stranded as well as antiparallel-stranded quadruplexes have been extensively studied by using different methods, such as NMR spectroscopy, X-ray diffraction and circular dichroism, but the exact conformation present in vivo is still under discussion. The design of small molecules that can bind to G quadruplexes has thus received attention because these nucleic acid motifs represent valuable pharmaceutical targets. For this purpose, a large number of small molecules has been evaluated for their binding with these particular DNA structures. However, as mentioned, the G quadruplex can adopt different topologies that can confuse the study of recognition phenomena. The design of a system that is able to mimic a well-defined conformation of G quadruplex is thus of great interest to precisely study the molecular interactions that can occur with small organic molecules. In 1985, Mutter proposed the TASP concept (template-assembled synthetic proteins) for the design of folded proteins. These pioneering works described the use of a cyclodecapeptide that allows the preparation of artificial proteins with a predetermined three-dimensional structure. Despite a large number of examples that use this template, to our knowledge, it has not been applied for the design of a specific folded structure of nucleic acid. With this in mind, we investigated the use of a peptidic scaffold as a topological template that directs the intramolecular assembly of covalently attached oligonucleotides into a single characteristic folding topology of G quadruplex. We anticipated that the scaffold should permit the preorganization of the DNA strands and the stabilization of the quadruplex structure. We report herein the synthesis and characterization of the novel water-soluble peptidic scaffold–oligonucleotide conjugate 1 that mimics the parallel-stranded conformation of G quadruplex (Scheme 1). We demonstrate that the use of the scaffold allows the precise control of the conformation of the quadruplex and dramatically increases the stability of the motif—all the more so as the formation of the quadruplex motif is possible even without the addition of any monovalent cations, such as K . We also show that mimic 1 can be used for surface functionalization, and this permits the study of the molecular interaction with G-quadruplex ligands by using surface plasmon resonance (SPR). The scaffold used for the synthesis of mimic 1 is a cyclic decapeptide with two independently functionalizable faces, which are due to the orientation of the lysine side-chains. On one side, the four oligonucleotides derived from the human telomeric sequence d(TTAGGGT) were anchored by using oxime bond formation, and a biotin residue was incorporated on the other side for attachment to streptavidin-immobilized surfaces. Earlier work from our laboratory has demonstrated that the oxime coupling strategy allows the efficient preparation of peptide–oligonucleotide conjugates. Figure 1. G-quartet motif and possible folded structures of the G quadruplex. A) G quartet; B) intermolecular parallel form; C) intramolecular parallel form; D) intramolecular antiparallel form.

DNA-Binding and Physical Studies of Pt(4′-NR2-trpy)CN+ Systems (trpy = 2,2′:6′,2′′-Terpyridine)

This paper focuses on DNA-binding interactions exhibited by Pt(dma-T)CN(+), where dma-T denotes 4'-dimethylamino-2,2':6',2''-terpyridine, and includes complementary studies of the corresponding pyrr-T complex, where pyrr-T denotes 4'-(N-pyrrolidinyl)-2,2':6',2''-terpyridine. The chromophores are useful for understanding the interesting and rather intricate DNA-binding interactions exhibited by these and related systems. One reason is that the terpyridine ligands employed provide intense visible absorption and enhanced photoluminescence signals. Incorporating cyanide as a coligand further aids analysis by suppressing covalent binding. Physical methods utilized include X-ray crystallography for structures of the individual inorganic complexes. Viscometry as well as spectral studies of the absorbance, emission, and circular dichroism (CD) yield information about interactions with a variety of DNA hosts. Although there is no sign of covalent binding under the conditions used, most hosts exhibit two phases of uptake. Under conditions of high loading (low base-pair-to-platinum ratios), the dma-T complex preferentially binds externally and aggregates on the surface of the host, except for the comparatively rigid host [poly(dG-dC)]2. Characteristic signs of the aggregated form include a bisignate CD signal in the charge-transfer region of the spectrum and strongly bathochromically shifted emission. When excess DNA is present, however, the complex shifts to intercalative binding, preferentially next to G[triple bond]C base pairs if available. Once the complex internalizes into DNA it becomes virtually immune to quenching by O2 or solvent, and the emission lifetime extends to 11 micros when [poly(dI-dC)]2 is the host. On the other hand, the host itself becomes a potent quenching agent when G[triple bond]C base pairs are present because of the reducing strength of guanine residues.

Cytotoxicity, Cellular Uptake, and DNA Interactions of New Monodentate Ruthenium(II) Complexes Containing Terphenyl Arenes

We have compared the cancer cell cytotoxicity, cell uptake, and DNA binding properties of the isomeric terphenyl complexes [(eta(6)-arene)Ru(en)Cl](+), where the arene is ortho- (2), meta- (3), or para-terphenyl (1) (o-, m-, or p-terp). Complex 1, the X-ray crystal structure of which confirms that it has the classical "piano-stool" geometry, has a similar potency to cisplatin but is not cross-resistant and has a much higher activity than 2 or 3. The extent of Ru uptake into A2780 or A2780cis cells does not correlate with potency. Complex 1 binds to DNA rapidly and quantitatively, preferentially to guanine residues, and causes significant DNA unwinding. Circular and linear dichroism, competitive binding experiments with ethidium bromide, DNA melting, and surface-enhanced Raman spectroscopic data are consistent with combined intercalative and monofunctional (coordination) binding mode of complex 1. This unusual DNA binding mode may therefore make a major contribution to the high potency of complex 1.

The Human Cytomegalovirus UL44 C Clamp Wraps around DNA

Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the "circle." The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a "hybrid" of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both.

Oxidatively Generated Damage to the Guanine Moiety of DNA: Mechanistic Aspects and Formation in Cells

Nuclear DNA and other molecules in living systems are continuously exposed to endogenously generated oxygen species. Such species range from the unreactive superoxide radical (O2*-)the precursor of hydrogen peroxide (H2O2)to the highly reactive hydroxyl radical (*OH). Exogenous chemical and physical agents, such as ionizing radiation and the UVA component of solar light, can also oxidatively damage both the bases and the 2-deoxyribose moieties of cellular DNA. Over the last two decades, researchers have made major progress in understanding the oxidation degradation pathways of DNA that are most likely to occur from either oxidative metabolism or exposure to various exogenous agents. In the first part of this Account, we describe the mechanistic features of one-electron oxidation reactions of the guanine base in isolated DNA and related model compounds. These reactions illustrate the complexity of the various degradation pathways involved. Then, we briefly survey the analytical methods that can detect low amounts of oxidized bases and nucleosides in cells as they are formed. Recent data on the formation of oxidized guanine residues in cellular DNA following exposure to UVA light, ionizing radiation, and high-intensity UV pulses are also provided. We discuss these chemical reactions in the context of *OH radical, singlet oxygen, and two-quantum photoionization processes.

Electrochemical Study on DNA Damage Based on the Direct Oxidation of 8‐Hydroxydeoxyguanosine at an Electrochemically Modified Glassy Carbon Electrode

AbstractThe study of DNA damage induced by Fenton reaction (Fe2+/H2O2) in vitro was performed based on the direct electrochemical oxidation of 8‐hydroxydeoxyguanosine (8‐OH‐dG), the biomarker of DNA oxidative damage, at an electrochemically modified glassy carbon electrode (GCE). The effects of antioxidants, such as ascorbic acid, and hydroxyl‐radical scavenger (mannitol) on the DNA damage were also investigated. 8‐OH‐dG, the oxidation product of guanine residues in DNA, has shown significantly oxidative peak on the electrochemically modified GCE. The oxidative peak current of 8‐OH‐dG was linear with the damaged DNA concentration in the range of 10–200 mg/L. The experimental results demonstrate that ascorbic acid has ambivalent effect on DNA oxidative stress. It can promote DNA oxidative damage when ascorbic acid concentration is below 1.5 mM and protect DNA from damage in the range of 1.5–2.5 mM. As a hydroxyl‐radical scavenger, mannitol inhibits significantly DNA oxidative damage. The influence of Fe2+, as reactant, and EDTA as iron chelator in the system were also studied. The proposed electrochemical method can be used for the estimation of DNA oxidative damage from new point of view.

Electrochemical Study of the Interaction Mechanism of Proflavine (PF) with DNA Using Carbon Paste (CPE) and Hanging Mercury Drop (HMDE) Electrode

Proflavine binds with DNA in a complicated manner. This work involves the electrochemical study of this interaction using differential pulse voltammetry at a carbon paste electrode (CPE) and alternating current voltammetry at a hanging mercury drop electrode (HMDE). At the CPE the peak current intensity at 1.0 V (corresponding to the oxidation of the guanine residues) decreased by increasing the concentration of proflavine. At the HMDE, a decrease in the current intensity of the DNA peak at − 1.2 V (corresponding to segmental desorption) was also observed by increasing the concentration of proflavine. These results confirmed, electrochemically, that proflavine intercalates within the DNA double helix and changes its conformation.

Structure-activity relationships in the acronycine and benzo[b]acronycine series: Role of the pyran ring

The pyranoacridone alkaloid acronycine (1), originally isolated from Acronychia baueri Schott (Rutaceae), has shown interesting properties against solid tumors. Significant improvements in terms of solubility and potency were obtained with derivatives modified in the pyran ring, such as diesters of cis-1,2-dihydroxy-1,2-dihydroacronycine (2) and cis-1,2-dihydroxy-1,2-dihydrobenzo[b]acronycine (3). Their mechanism of action implies alkylation of the 2-amino group of DNA guanine residues by the carbocation resulting from the elimination of the ester leaving group at position 1 of the drug.

Detection of Gamma Radiation-Induced Oxidation in Animal and Bacterial DNA

Gamma radiation-induced oxidative DNA damage, in terms of production of 8-hydroxydeoxyguanine (8-OHdG), was studied in the DNA samples from Chinese hamster ovary cell line (CHO-K1), bovine, chicken, Escherichia coli and Deinococcus radiodurans. A radiation dose response with respect to the formation of the 8-OHdG residues from the DNA molecules was observed by employing enzyme-linked immunosorbent assay (ELISA) detection system using monoclonal antibody. At high radiation doses (>5 kGy), the formation or detection of the 8-OHdG residues from bacterial DNA was declined. It was apparent that the oxidative DNA damage was dependent on the base composition of the DNA molecule. However, an inverse relation was observed between the dose limit and nitrogenous base composition especially the guanine residue. Nonetheless, DNA base composition might to be considered in cases of the radiation-induced oxidation of food or other DNA samples. Keywords: Gamma radiation-induced oxidation, 8-Hydroxydeoxyguanine (8-OHdG), ELISA, CHO-K1, Bovine, Chicken, Escherichia coli, Deinococcus radiodurans DOI: http://dx.doi.org/10.3329/bjm.v23i2.877 Bangladesh J Microbiol, Volume 23, Number 2, December 2006, pp 130-132

Establishment of reverse genetics system of betanodavirus for the efficient recovery of infectious particles

Betanodaviruses, a member of the family Nodaviridae, have small positive-stranded bipartite RNA genomes and are the causal agent of viral nervous necrosis in marine-farmed fish. To facilitate the study of betanodavirus, infectious cDNA clones of its two genomic RNAs were generated. The full-length cDNA of the new Redspotted grouper nervous necrosis virus strain (SG2001Nag) RNA1 and RNA2 were co-transcribed by T7 RNA polymerase in baby hamster kidney cells expressing T7 RNA polymerase. The transcription of precise viral RNAs from cDNAs neither lead to viral protein synthesis nor the production of infectious particles. However, the additional two guanine residues following T7 promoter increased the transcription of viral RNAs from cDNAs, and 1.0 × 10 6 TCID 50/ml of infectious particles was collected from the transfected cells. The ability to reproduce the entire life cycle of betanodavirus from cDNA clones by this reverse genetics system would therefore facilitate a further analysis of the mechanism of betanodavirus RNA replication, structure, and assembly. These findings may thus help in the future development of a betanodavirus vaccine.

DNA Interactions of Monofunctional Organometallic Osmium(II) Antitumor Complexes in Cell-Free Media

This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops × M. saxatilis and zebrafish Danio rerio

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

Incorporation of Oxidized Guanine Nucleoside 5′-Triphosphates in DNA with DNA Polymerases and Preparation of Single-Lesion Carrying DNA

We investigated the incorporation of oxidatively modified guanine residues in DNA using three DNA polymerases, Escherichia coli Kf exo+, Kf exo-, and Taq DNA polymerase. We prepared nucleoside 5'-triphosphates with modified bases (dN (ox)TP) including imidazolone associated with oxazolone (dIzTP/dZTP), dehydroguanidinohydantoin (dOGhTP), and oxaluric acid (dOxaTP). We showed that the single-nucleotide incorporation of these dN (ox)TP at the 3'-end of a primer DNA strand was possible opposite C or G for dIzTP/dZTP, opposite C for dOGhTP using the Klenow fragment, and opposite C for dOxaTP using Taq. The efficiency of these misincorporations was compared to that of the nucleoside 5'-triphosphate modified with the mutagenic guanine lesion 8-oxo-G opposite A or C as well as to that of the natural dNTPs. The reaction was found not competitive. However, the ability of Kf exo- to further copy the whole template DNA strand from the primer carrying one modified residue at the 3'-end proved to be easy and rapid. The two-step polymerization process consisting of the single-nucleotide extension followed by the full extension of a primer afforded a method for the preparation of tailored double-stranded DNA oligonucleotides carrying a single modified base at a precise site on any sequence. This very rapid method allowed the incorporation of unique residues in DNA that were not available before due to their unstable character.