High-resolution Polyacrylamide Gel Electrophoresis Research Articles

In Vitro Chemical Mapping of G-Quadruplex DNA Structures by Bis-3-Chloropiperidines.

G-quadruplexes (G4s) are biologically relevant, non-canonical DNA structures that play an important role in gene expression and diseases, representing significant therapeutic targets. Accessible methods are required for the in vitro characterization of DNA within potential G-quadruplex-forming sequences (PQSs). B-CePs are a class of alkylating agents that have proven to be useful chemical probes for investigation of the higher-order structure of nucleic acids. This paper describes a new chemical mapping assay exploiting the specific reactivity of B-CePs with the N7 of guanines, followed by direct strand cleavage at the alkylated Gs. Namely, to distinguish G4 folds from unfolded DNA forms, we use B-CeP 1 to probe the thrombin-binding aptamer (TBA), a 15-mer DNA able to assume the G4 arrangement. Reaction of B-CeP-responding guanines with B-CeP 1 yields products that can be resolved by high-resolution polyacrylamide gel electrophoresis (PAGE) at a single-nucleotide level by locating individual alkylation adducts and DNA strand cleavage at the alkylated guanines. Mapping using B-CePs is a simple and powerful tool for the in vitro characterization of G-quadruplex-forming DNA sequences, enabling the precise location of guanines involved in the formation of G-tetrads.

Particle Size Distribution of Plasma Lipoproteins in Donkeys from Death Valley Compared to a Sampling of Horses.

Simple SummaryDonkeys, like horses, belong to the Equidae family. Though they share some physical attributes, much of their physiology is quite different from horses. Both horses and donkeys can develop clinical issues associated with obesity; insulin dysregulation; and altered lipid metabolism, often termed equine metabolic syndrome or donkey metabolic syndrome, respectively. Unlike large breed horses, however, donkeys, ponies, and miniature horses are more susceptible to developing dyslipidemias. This study aimed to use a novel Lipoprint® assay to compare the lipid profiles of apparently healthy donkeys and large-breed horses to establish a baseline for future work that may lead to the use of this test methodology for the evaluation and monitoring of equids with endocrine disorders.The clinical evaluation of lipid metabolism in equids is often limited to the measurement of total cholesterol and triglyceride concentrations. This provides a limited picture of metabolic state and general health, given the continuous exchange of lipid species between various lipoproteins. Major lipoprotein classes in equids include high-density lipoprotein (HDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL), and chylomicrons (CM). Unlike large breed horses, donkeys are highly susceptible to hepatic lipidosis. Currently, serum triglyceride concentrations serve as a surrogate marker of hepatic lipid exportation. Both VLDL, indicative of hepatic exportation, and its metabolic end-product, LDL, are rich in triglycerides, and contribute to this value. Diagnostic assays that distinguish VLDL from LDL could be useful in better recognizing the hepatic pathology in donkeys. The compositional differences of lipoproteins across species limit the use of commercially available assays developed for the measurement of human lipoproteins in domestic animals. In this study, we evaluated a high-resolution polyacrylamide gel electrophoresis method (Lipoprint®) for separating major lipoprotein classes and sub-fractionating LDL and HDL based on particle size in a large group of donkeys, and compared the pattern to a representative set of horses. Donkeys proved an HDL-rich species, with HDL accounting for the bulk of all lipoproteins (average 78.45%, SD 6.6%, range 92.2–55%). VLDL accounted for a large portion of the total (average 21.6%, SD 6.6%, range 37.1–7.8%), with minimal amounts of LDL detected. The horses tested had higher proportions of VLDL as compared to donkeys (31.7% and 21.6%, respectively p = 0.00008). The later finding draws into question the purported relationship between VLDL, high triglycerides, and hepatic lipidosis, given the incidence of the disease in donkeys is far higher than in horses.

Advanced Gel Electrophoresis Techniques Reveal Heterogeneity of Humic Acids Based on Molecular Weight Distributions of Kinetically Inert Cu2+-Humate Complexes.

Humic acids (HAs) play important roles for the fate of metal ions in the environment. Most chemical speciation models involving HAs assume heterogeneous metal ion binding. However, these models also assume that the binding affinities of metal ions with HAs are the same regardless of the molecular weight (MW) ranges of the HAs involved. Here, we develop new polyacrylamide gel electrophoresis (PAGE) techniques to investigate the MW distributions of HAs with strongly complexed Cu2+ ions. By combining contaminant metal-free and high-resolution PAGE for HAs, this work was able to provide accurate MW distributions for the complexed metal ions. The MW distribution of Cu2+ binding ability per quantity of HA indicates that strong metal-binding moieties in HAs are heterogeneous in terms of MW. Coupling of the PAGE techniques with UV-vis and excitation-emission matrix (EEM) spectrometry-parallel factor analysis (PARAFAC) methods revealed new insights into kinetically inert interactions between HAs and Cu2+ ions. By this method, we found that the protein-like fluorescence components in the high- and low-MW regions cooperatively responded through Cu2+ binding. Thus, the advanced gel electrophoresis techniques developed herein are able to shed new light on the heterogeneity of metal binding affinities of HAs in terms of MW.

Use of in-gel peroxidase assay for cytochrome c to visualize mitochondrial complexes III and IV.

ABSTRACTThe in-gel activity assay (IGA) is a powerful technique that uses enzymatic activity and compares intensities of detected bands in mitochondrial respiratory chain supercomplexes, and it is applicable to eukaryotic organisms. However, no IGA has been established for complex III because of the difficulty of access by ubiquinol, a substrate for complex III. Herein, we demonstrate that cytochrome c (Cyt c) showed peroxidase activity on IGA as a component of complexes III and IV. We used pre-incubation with sodium dodecyl sulfate (SDS) before IGA to loosen complexes in the gel after high-resolution clear native polyacrylamide gel electrophoresis (hrCN-PAGE), a refinement of blue native PAGE. The signals of IGA based on peroxidase activity were obtained using enhanced chemiluminescence solution. Then, the gel was directly used in western blotting or hrCN/SDS two-dimensional PAGE. Our findings indicate that IGA for Cyt c reflected the indirect activity of complexes III and IV.

Correlation Between High-Density Lipoprotein and Monocyte Subsets in Patients with Stable Coronary Heart Disease.

BackgroundHigh-density lipoprotein (HDL) consists of heterogeneous particles with a variety of structures and functions. Its role in atherosclerosis has been gradually recognized. Studies have shown dysfunction of small HDL in patients with coronary artery disease (CAD). Monocytes play an important role in atherosclerosis, which can be divided into 3 subgroups based on the expression of surface markers CD14 and CD16. This study aimed to investigate the association between HDL and monocyte subsets in CAD patients.Material/MethodsA total of 90 patients with stable CAD were selected in this study. Monocytes were divided into classical monocytes (CM, CD14++CD16−), intermediate monocytes (IM, CD14++CD16+), and non-classical monocytes (NCM, CD14+CD16++). HDL components in serum were determined by high-resolution polyacrylamide gel electrophoresis (detected by Quantimetrix HDL Lipoprint system, referring to HDL subfractions analysis: A new laboratory diagnostic assay for patients with cardiovascular diseases and dyslipoproteinemia).ResultsSerum level of small HDL was positively correlated with circulating proinflammatory NCM (r=0.30; p=0.004), negatively correlated with CM, and not correlated with IM. We also found that disease severity was not associated with diabetes mellitus, glycosylated hemoglobin, hypertension, smoking history, or statin dosage.ConclusionsOur study confirmed that small HDL level is associated with an increase in NCM and a decrease in CM, suggesting the proinflammatory relationship between small HDL and intrinsic immune function during the progression of stable CAD.

Dispersion quality of amine functionalized multiwall carbon nanotubes plays critical roles in polymerase chain reaction enhancement

Impact of dispersion quality of NH2-MWCNTs (13–18 nm in diameter with a length between 1 and 12 µm, >99 % purity) in the amplification efficiency of a random DNA oligonucleotide library (96 bp) was investigated. Amplification yield in the presence of non-filtered NH2-MWCNT dispersion, filtered NH2-MWCNT dispersion and surface-attached NH2-MWCNTs was explored, and physical interactions between NH2-MWCNTs and major PCR reagents including DNA template, wild type Taq DNA polymerase enzyme and primers were determined using high resolution polyacrylamide gel electrophoresis, dynamic light scattering, UV–Vis-NIR spectroscopy and scanning electron microscopy techniques. The results revealed that presence of NH2-MWCNT dispersion which was sonicated, centrifuged and filtered, enhanced the total PCR efficiency up to 70 % while the presence of NH2-MWCNT only centrifuged after sonication, inhibited the reaction significantly at similar concentrations. Furthermore, the NH2-MWCNTs coupled covalently onto magnetic microspheres, contributed for the specificity enhancement whilst decreasing the amplification efficiency by 30 % at the maximum concentration, which suggests a removable enhancement system for sensitive applications. On the other hand, the relative hydrodynamic size distribution measurements displayed a clear difference between the filtered NH2 and non-filtered NH2-MWCNT water dispersions, which justifies the inhibition of the amplification by the non-filtered NH2-MWCNTs containing big agglomerates and bundles. Finally, we demonstrated that major PCR components adsorb onto the NH2-MWCNTs with diverse affinities, and maintain their functions after adsorption, which provides a good framework to further develop tunable NH2-MWCNT-carriers to be utilized in various nanobiotechnology and material science applications.

Detection of a Mixed Infection in a Culture-Negative Brain Abscess by Broad-Spectrum Bacterial 16S rRNA Gene PCR

We describe the identification of two bacterial pathogens from a culture-negative brain abscess by the use of broad-spectrum 16S rRNA gene PCR. Simultaneous detection of Fusobacterium nucleatum and Porphyromonas endodontalis was possible due to a 24-bp length difference of their partially amplified 16S rRNA genes, which allowed separation by high-resolution polyacrylamide gel electrophoresis.

Sequence-specific alkylation of DNA by pyrrole-imiclazole polyamides through cooperative interaction

We designed and synthesized an alkylating N-methylpyrrole (Py) -N-methylimidazole (Im) polyamide and l-(chloromethyl)-5-hydroxy-l,2-dihydro-3H benz[e]indole (seco-CBI) conjugate 1. DNA alkylating activities of conjugate 1 were evaluated by high-resolution denaturing polyacrylamide gel electrophoresis with DNA fragment containing human telomere repeat sequence. These results revealed that simultaneous treatment of the DNA fragment with conjugate 1 and Distamycin A (Dist) alkylate at the 5(')-GGTTAGGGTTA-3' sequence effectively due to cooperative interaction. Moreover, this combination has achieved 11-base-pair (bp) recognition. Thus, it is suggested that the utilization of one alkylating agent possessing long Py-Im polyamide moiety and short Py-Im polyamide partner can be an expedient way to attain the extension and precision of the recognition of DNA sequence.

Late-Stage Polyribitol Phosphate Wall Teichoic Acid Biosynthesis in Staphylococcus aureus

Wall teichoic acids are cell wall polymers that maintain the integrity of the cellular envelope and contribute to the virulence of Staphylococcus aureus. Despite the central role of wall teichoic acid in S. aureus virulence, details concerning the biosynthetic pathway of the predominant wall teichoic acid polymer are lacking, and workers have relied on a presumed similarity to the putative polyribitol phosphate wall teichoic acid pathway in Bacillus subtilis. Using high-resolution polyacrylamide gel electrophoresis for analysis of wall teichoic acid extracted from gene deletion mutants, a revised assembly pathway for the late-stage ribitol phosphate-utilizing enzymes is proposed. Complementation studies show that a putative ribitol phosphate polymerase, TarL, catalyzes both the addition of the priming ribitol phosphate onto the linkage unit and the subsequent polymerization of the polyribitol chain. It is known that the putative ribitol primase, TarK, is also a bifunctional enzyme that catalyzes both ribitol phosphate priming and polymerization. TarK directs the synthesis of a second, electrophoretically distinct polyribitol-containing teichoic acid that we designate K-WTA. The biosynthesis of K-WTA in S. aureus strain NCTC8325 is repressed by the accessory gene regulator (agr) system. The demonstration of regulated wall teichoic acid biosynthesis has implications for cell envelope remodeling in relation to S. aureus adhesion and pathogenesis.

Synthesis and biological properties of pyrrole-imidazole polyamide conjugates

We have developed a series of conjugates between pyrrole (Py)-imidazole (Im) polyamides and 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H benz[e]indole (seco-CBI) with an indole linker. High resolution polyacrylamide gel electrophoresis revealed that these conjugates alkylated DNA at predetermined sequences. Then, we demonstrated that conjugates 1 and 2 have DNA alkylation activities at double stranded human telomere sequence and potent cytotoxicities in cancer cell lines. In addition, we showed that conjugate 3 alkylates DNA with ten-base-pair recognition sequence in the presence of partner polyamide 4, which suggested alkylation through 3-4 heterodimer formation.

Association of Pro12Ala polymorphism in the peroxisome proliferator–activated receptor γ2 gene with small dense low-density lipoprotein in the general population

The Pro12Ala polymorphism of the peroxisome proliferator–activated receptor γ2 (PPAR γ2) gene has been reported to predict a lower risk for developing type 2 diabetes mellitus. However, its effect on the lipid profile has been disputable. Among low-density lipoproteins, small dense low-density lipoprotein (sdLDL) particles have been linked to a greater risk for coronary artery disease. The purpose of this study was to investigate the genetic effect of the Pro12Ala polymorphism in the PPAR γ2 gene on the presence of sdLDL in the general Japanese population. In 379 subjects (aged 54 ± 13 years), body mass index, percentage of body fat, blood pressure, and biochemical profiles were measured. Pro12Ala polymorphism was genotyped by polymerase chain reaction–restriction fragment length polymorphism. The area of sdLDL subfractions (sdLDL4-7) was analyzed by high-resolution polyacrylamide gel electrophoresis. The frequency of the Ala12 allele in PPAR γ2 was 0.04. There was no difference in total cholesterol, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol levels between genotypes. However, subjects with the X/Ala genotype (Pro/Ala + Ala/Ala) had significantly higher serum triglyceride levels ( P = .001) and a larger area of sdLDL4-7 ( P = .002) than those with the Pro/Pro genotype. Multiple regression analysis revealed that the Ala12 allele was a significant variable contributing to the variance in the increased area of sdLDL4-7 ( P = .040). In conclusion, the Pro12Ala polymorphism in the PPAR γ2 gene was positively associated with an enlarged area of sdLDL4-7. This polymorphism may play a role in the genetic predisposition to increases in sdLDL4-7.

Oxidation of Guanine in Double-Stranded DNA by [Ru(bpy)2dppz]Cl2 in Cationic Reverse Micelles

DNA oxidation has been investigated in the medium of cationic reverse micelles (RMs). The oxidative chemistry is photochemically initiated using the DNA intercalator bis(bipyridine)dipyridophenazine ruthenium(II) chloride ([Ru(bpy)2dppz]Cl2) bound to duplex DNA in the RMs. High-resolution polyacrylamide gel electrophoresis (PAGE) is used to reveal and quantify guanine (G) oxidation products, including 8-oxo-7,8-dihydroguanine (8OG). In buffer solution, the addition of the oxidative quenchers potassium ferricyanide or pentaamminechlorocobalt(III) dichloride leads to an increase in the amount of piperidine-labile G oxidation products generated via one-electron oxidation. In RMs, however, the yield of oxidatively generated damage is attenuated. With or without ferricyanide quencher in the RMs, the yield of oxidatively generated products is approximately the same. Inclusion of the cationic quencher [CoCl(NH3)5]2+ in the RMs increases the amount of oxidation products generated but not to the extent that it does in buffer solution. Under anaerobic conditions, all of the samples in RMs, with or without added oxidative quenchers, show decreased levels of piperidine-labile oxidation products, suggesting that the primary oxidant in RMs is singlet oxygen. G oxidation is enhanced in D2O and deuterated heptane and is diminished in the presence of sodium azide in RMs, also supporting 1O2 as the main G oxidant in RMs. Isotopic labeling experiments show that the oxygen atom in 8OG produced in RMs is not from water. The observed change in the G oxidation mechanism from a one-electron process in buffer to mostly 1O2 in RMs illustrates the importance of both DNA structure and DNA environment on the chemistry of G oxidation.

Molecular design of DNA alkylating pyrrole-imidazole polyamides with longer recognition sequence

The sequence-specificity, and DNA alkylating activity of the conjugate 1, which consists of N-methylpyrrole (Py)-N-methylimidazole (Im) polyamides, 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) with indole linker, were investigated in the absence or presence of partner Py-Im polyamide 2. High-resolution denaturing polyacrylamide gel electrophoresis showed that the specificity of DNA alkylation by 1 modulated in the presence of partner 2. We found that sequence-specific DNA alkylation by 1 and 2 with 10 base pair (bp) match recognition sequence through heterodimer formation. This result indicates one possibility of DNA alkylation with longer recognition sequence by different two molecules.

DNA Oxidation in Anionic Reverse Micelles: Ruthenium-Mediated Damage at Guanine in Single- and Double-Stranded DNA

One-electron guanine oxidation in DNA has been investigated in anionic reverse micelles (RMs). A photochemical method for generating Ru3+ from the ruthenium polypyridyl complex tris(2-2'-bipyridine)ruthenium(II) chloride ([Ru(bpy)3]Cl2) is combined with high-resolution polyacrylamide gel electrophoresis (PAGE) to quantify piperidine-labile guanine oxidation products. As characterized by emission spectroscopy of Ru(bpy)3(2+), the addition of DNA to RMs containing Ru(bpy)3(2+) does not perturb the environment of Ru(bpy)3(2+). The steady-state quenching efficiency of Ru(bpy)3(2+) with K3[Fe(CN)6] in buffer solution is approximately 2-fold higher than that observed in RMs. Consistent with the difference in quenching efficiency in the two media, a 1.5-fold higher yield of piperidine-labile damage products as monitored by PAGE is observed for duplex oligonucleotide in buffer vs RMs. In contrast, a 13-fold difference in the yield of PAGE-detected G oxidation products is observed when single-stranded DNA is the substrate. Circular dichroism spectra showed that single-stranded DNA undergoes a structural change in anionic RMs. This structural change is potentially due to cation-mediated adsorption of the DNA phosphates on the anionic headgroups of the RMs, leading to protection of the guanine from oxidatively generated damage.

Development of a PCR‐based diagnostic test detecting a nt230(del4) MDR1 mutation in dogs: verification in a moxidectin‐sensitive Australian Shepherd

A subpopulation of dogs of the Collie and Australian Shepherd breeds show increased sensitivity to central nervous actions of ivermectin, doramectin, loperamide, and probably several other drugs. The molecular background for this greater sensitivity is a nonsense mutation in the MDR1 efflux pump, which is part of the functional blood-brain barrier and normally limits drug penetration into the brain. This report describes a rapid PCR-based method for detection of this nt230(del4) MDR1 mutation using a small amount of genomic DNA from blood cells. Thereby, homozygous intact, homozygous mutated, and heterozygous mutated MDR1 genotypes can be clearly differentiated by high resolution polyacrylamide gel electrophoresis. Using this diagnostic test two Collies and one Australian Shepherd were screened for the nt230(del4) MDR1 mutation. The Collies had no history of altered drug sensitivity and showed homozygous intact and heterozygous mutated MDR1 alleles, respectively. However, the Australian Shepherd developed clear signs of neurotoxicity including ataxia, crawling, acoustic and tactile hyperexcitability, and miosis after a single dose of moxidectin (400 microg/kg). For this dog two mutated MDR1 alleles were detected. This report describes for the first time moxidectin neurotoxicosis in a dog with a homozygous MDR1 mutation.

Osteocalcin fragment in bone matrix enhances osteoclast maturation at a late stage of osteoclast differentiation

Although 14-day-old mouse embryonic calvarial cells cultured in plastic culture dishes in the presence of 1alpha,25-dihydroxyvitamin D3[1alpha,25-(OH)2D3] for 7 days could barely resorb bone slices, the same calvarial cells cultured with an ethylenediaminetetraacetic acid (EDTA) extract from bovine bone powder under the same conditions stimulated pit formation on bone slices in a dose-dependent manner. Therefore, the present study was conducted to purify and characterize this osteoclast maturation-inducing factor(s) from the bone matrix. The protein having osteoclast maturation-inducing activity in the EDTA extract was purified by gel filtration over Superdex 75 preparation grade and chromatography on hydroxyapatite, Mono Q, and C8 reversed-phase HPLC by monitoring the ability of the eluted fractions to elicit pit formation on bone slices. The molecular weight of the purified protein estimated by high-resolution polyacrylamide gel electrophoresis was 5.7 kDa and 6.8 kDa in the respective absence and presence of 2-mercaptoethanol. The sequence of the 30-amino-acid purified protein corresponded to the 7th to 36th residues of bovine osteocalcin. The osteocalcin fragment, missing the initial 6 residues at the N-terminal region, exhibited higher osteoclast maturation-inducing ability than bovine intact osteocalcin on a per weight basis. The osteocalcin fragment had no effect on the expression of receptor activator of nuclear factor (NF)-kappaB ligand (RANKL) and osteoprotegerin (OPG) genes in calvarial cells, nor did it enhance the bone-resorbing activity of mature osteoclasts. When the osteocalcin fragment was added to late-stage (days 4-7) or to early-stage (days 0-3) cultures of calvarial cells pretreated with 1alpha,25-(OH)2D3, its stimulatory effect was observed in the late-stage cultures rather than in the early-stage ones. In addition, the osteocalcin fragment directly enhanced the formation of osteoclasts with bone-resorbing ability from Mac-1+ c-Fms+ cells in the presence of macrophage colony-stimulating factor (MCSF) and RANKL. These results suggest that the osteocalcin fragment in bone matrix is involved in osteoclast maturation, especially at a late stage of osteoclast differentiation.

LDL particle size and lipid composition are risk factors for microalbuminuria in normotensive and normocholesterolemic patients with type 2 diabetes

The study was to evaluate the influence of particle size and lipid composition of low-density lipoprotein (LDL) on urinary albumin excretion and oxidative susceptibility of LDL, and to define association between LDL particle size and α-tocopherol content in LDL from normotensive and normocholesterolemic patients with type 2 diabetes. Twenty-three patients with type 2 diabetes (13 males, 10 females) were studied, and none of these patients had hypertension, hypercholesterolemia and overt proteinuria. The baseline body mass index of all patients was less than 28 kg/m 2. All patients were hospitalized in Hirosaki University Hospital and took dietary therapy whose total intake was restricted to less than 30 kcal/kg of ideal body weight for 3 weeks. Their plasma glucose levels were controlled within fasting plasma glucose <140 mg/dl and 2-h postprandial plasma glucose <200 mg/dl. LDL particle size was evaluated by using high-resolution polyacrylamide gel electrophoresis (Lipoprint LDL™ System) and expressed by Rf value. LDL was incubated with 0.25 μM CuSO 4 for 20 h, and the degree of LDL oxidation was determined by malondialdehide analysis. Twenty-four-hour urinary C-peptide excretion and plasma triglyceride concentration in patients with microalbuminuria were significantly higher than those in normoalbuminuric patients. Rf values in microalbuminuric patients were significantly greater than those in normoalbuminuric patients. There were significantly inverse correlations between Rf value and α-tocopherol content in LDL, and between Rf value and LDL-free cholesterol/LDL-total cholesterol. Thiobarbituarte-reactive substance level in LDL had a tendency to correlate with Rf value and significantly inverse correlation to α-tocopherol content in LDL. In type 2 diabetics without hypertension, hypercholesterolemia and obvious obesity, smaller LDL particle size, accompanied by mild hyperinsulinemia and mild hypertriglyceridemia seems to be one of the important factors responsible for microalbuminuria. In addition, the present study suggests that the decrease of α-tocopherol content in small LDL particle is associated with oxidative susceptibility to Cu 2+-induced oxidation.

A BAC-based physical map of the chicken genome.

A genome-wide physical map constructed with bacterial artificial chromosomes (BACs) is an essential component in linking phenotypic traits to the responsible genetic variation in the genomes of plants and animals. We have constructed a physical map of the chicken genome from 57,091 BACs (7.9-fold haploid genome coverage) by restriction fingerprint analysis using high-resolution polyacrylamide gel electrophoresis. The physical map consists of 2331 overlapping BAC contigs and is estimated to span 1510 Mb in physical length. BAC contigs were verified manually and by screening the BACs with 367 DNA markers. A total of 361 of the contigs have been anchored to the existing chicken genetic map. This map represents the first genome-wide, BAC-based physical map of the chicken genome. It provides a powerful platform for many areas of chicken genomics, including targeted marker development, fine mapping of genes and QTL alleles, positional cloning, analysis of avian genome organization and evolution, chicken-mammalian comparative genomics, and large-scale genome sequencing.

Mechanistic aspects of CoII(HAPP)(TFA)2 in DNA bulge-specific recognition.

A novel octahedral complex CoII(HAPP)(TFA)2 [hexaazaphenantholine-cyclophane (HAPP), trifluoroacetate (TFA)] is a DNA bulge-specific probe with single-strand DNA cleavage activity in the presence of H2O2. This complex exhibits low affinity towards double-stranded DNA and low reactivity toward single-stranded DNA. Metal-HAPP complexes with different coordination number and ring size were synthesized and their selectivity and reactivity for DNA bulges were compared. The DNA sequence at the bulge site influences the intensity of cleavage at the bulge and the flanking sites after piperidine treatment. Cleavage specificity of CoII(HAPP)(TFA)2 was characterized extensively using scavenger reagents to quench the cleavage reaction and high-resolution polyacrylamide gel electrophoresis. In addition, 3'-phosphoglycolate cleavage products were trapped and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. These data were used to deduce that the DNA cleavage pathway for CoIIHAPP2+ in the presence of H2O2 involves 4'-H abstraction of the deoxyribose moiety.

Inhibition of transcription at a coding sequence by alkylating polyamide.

Transcription from DNA sequence-specifically alkylated by a hairpin polyamide (ImPyPy-gamma-ImPyLDu86, 1) was investigated. High-resolution polyacrylamide gel electrophoresis demonstrated that conjugate 1 alkylated a 993-bp DNA fragment, in accordance with the Py-Im recognition rule, predominantly at the one match site on the GFP-encoding strand and at four sites (I'-IV') on the noncoding strand. Alkylation of DNA inhibited the formation of full-length mRNA and caused the transcription of truncated mRNA. Polyacrylamide gel electrophoresis demonstrated that the length of the truncated mRNA was consistent with the alkylated site on the coding strand. Complete inhibition of full-length mRNA synthesis was observed in the presence of 50 nM 1. In clear contrast, the hydrolyzed derivative of 1, designated 2, produced no truncated mRNA, nor did it significantly retard transcription: >80% transcription of full-length mRNA was observed at 50 nM. These results clearly indicate that inhibition of transcription can be achieved with alkylating Py-Im polyamide even in the coding regions of genes.