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At least 1,099 records · Page 61Linked to original sources
PubMed1983

Mapping restriction sites on large DNAs by electron microscopy.

We have developed a novel technique to map restriction sites on large duplex DNAs by electron microscopy. In this method, the sample DNA is first cut with a restriction enzyme. The resulting fragments are briefly digested with Escherichia coli exonuclease III, and treated with wheat germ RNA polymerase II to fill-in with RNA the resulting gaps. These small RNAs, complementary to sequences immediately adjacent to either side of the restriction site, are isolated from the DNA template and R-looped to the full-length DNA. When this material is prepared by the formamide-cytochrome spreading technique, small bubbles are visible wherever there is a restriction site on the DNA. Improved methods of mapping are outlined.

Bacteriophage lambda↗
PubMed1986

Presence of random single-strand gaps in mycobacteriophage I3 DNA.

The genomic double-stranded DNA of mycobacteriophage I3, when denatured with alkali, heat, formamide or dimethylsulfoxide, breaks down to heterogeneous-sized single-strand (ss) fragments smaller than the expected intact unit genome length suggesting the presence of random ss interruptions on both the strands. The occurrence of the interruptions at random is also demonstrated by two-dimensional gel electrophoresis of the restriction fragments of I3 DNA. These interruptions have no adverse effect on the phage infectivity or DNA transfectivity. Studies with nuclease BAL 31 and end-labeling analysis confirm the presence of random interruptions. Detailed analysis using T4 DNA ligase, nuclease S1 and DNA polymerase I Klenow fragment revealed that the interruptions are in the form of small gaps rather than single phosphodiester bond breaks. The average length of the gap is about 10 nucleotides long and there are 13 to 14 such gaps per DNA molecule.

DNA, Single-Stranded↗
PubMed1990

Detection of bovine viral diarrhea virus in serum from cattle by dot blot hybridization assay.

A dot blot hybridization assay was developed for use as a rapid screening test to detect bovine viral diarrhea virus (BVDV) in serum from infected cattle. A 1.1. kilobase cDNA, prepared from the BVDV genome, was molecularly cloned and used in this study. Insert cDNA was removed from the pUC9 plasmid vector by Pst-I restriction endonuclease digestion and purified from plasmid DNA by agarose electrophoresis and electroelution. The hybridization probe was prepared by nick translation in the presence of gamma dCT32P and labelled to a specific activity of 2 x 10(8) cpm/micrograms of DNA. Specificity was determined by dot blot hybridization of infected cell culture supernate from nine different BVDV strains. The probe hybridized equally with all strains of BVDV tested, which included four cytopathic and five noncytopathic strains of BVDV. Serum was collected from veal calves with respiratory tract disease, unthriftiness, anorexia, and/or poor conditions. Serum samples were treated with nonidet P40 detergent and denatured with formaldehyde and heat prior to application on 1.2 micron nylon membrane filters using a vacuum dot blot apparatus. Hybridization was done under relatively stringent conditions (50% formamide at 42 degrees C). A total of 141 serum samples from different calves were tested and of these samples, 55 (39%) were positive by dot blot hybridization for BVDV RNA. Eight calves (33%) out of 24, tested 3 to 4 weeks later, remained positive for BVDV RNA.

Animals↗
PubMed1990

Hybridization probes for the detection and differentiation of two serotypes of porcine rotavirus.

A dot hybridization assay was developed to detect and differentiate two serotypes of porcine rotavirus by hybridization of specific probes to rotavirus RNA dotted onto nylon membranes. The assay was conducted using radiolabeled probes prepared from cDNA clones of OSU (serotype 5) and Gottfried (serotype 4) rotavirus gene 9 segments. The probes were prepared by excision of full length cDNA copies of gene 9 inserts from recombinant plasmids followed by nick translation and 32P-dCTP labeling. Rotavirus RNA samples used in the assay were prepared from tissue culture, and intestinal contents from gnotobiotic and conventional pigs. Optimal conditions for hybridization consisted of 52 degrees C, 50% formamide, and 5 x SSC. Hybridization studies with the OSU and Gottfried gene 9 probes have shown them to be specific with only low levels of cross-reactivity with heterologous rotavirus preparations. Sensitivity studies have demonstrated the ability of the probes to detect at least 2 ng of rotavirus RNA. Further development and refinement of this technique may provide a useful tool for the detection and differentiation of porcine rotavirus serotypes.

Animals↗
PubMed1988

Cell growth dynamics in long-term bladder carcinogenesis.

A biologically based probabilistic model of the carcinogenic process has been developed based on a two-stage theory of carcinogenesis. The model has been validated utilizing experimental urinary bladder carcinogenesis studies in the rat, with an emphasis on quantification of cell dynamics. Critical parameters tracked through this process include mitotic rates, cell loss and birth rates, and irreversible cellular transitions from normal to initiated to transformed states. Analyses demonstrate the sensitivity of tumor incidence to the timing and magnitude of changes to these cellular variables. Modeling has been applied to genotoxic compounds, such as N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide, and non-genotoxic compounds, such as sodium saccharin. For the latter compounds, complex administration regimens have been studied, including two-generation experiments, initiation-promotion experiments, and sodium saccharin administration following ulceration and regenerative hyperplasia. Modeling indicates that the effects of such compounds can be explained entirely on the basis of cytotoxicity and consequent hyperplasia. Quantitative modeling based on biological processes has the potential for direct application to carcinogenic risk assessment.

Cell Division↗
PubMed1995

Poly(ethyleneoxide) for high-resolution and high-speed separation of DNA by capillary electrophoresis.

Capillary electrophoresis (CE) with polyacrylamide gels has already been demonstrated to allow single-base resolution of single-stranded DNA. However, linear polyacrylamide is not an ideal matrix because of a high viscosity and difficulties in preparing the polymer with well defined pore sizes. Alternatively, poly(ethyleneoxide) (PEO) with a large range of molecular masses from 300,000 to 8,000,000 is available commercially. In addition, it is easy to prepare homogeneous solutions to provide highly reproducible separation performance with sufficient resolution. Single-base resolution of double-stranded DNA between 123 and 124 base pairs can be achieved by the use of homogeneous matrices prepared from PEO (2.5% M(r) 8,000,000), and even better resolution is achieved by using mixed polymer matrices. With further work, it should be possible to change the fractions and the total amounts of polymers to achieve even higher resolution for different samples with different size ranges of fragments. Another advantage of mixed polymer matrices is that relatively high resolution can be obtained while maintaining a relatively low viscosity compared to linear polyacrylamide with identical contents of formamide and urea, which makes it easier to fill these matrices into small capillaries.

DNA↗
PubMed1990

Metabolism of aromatic and heterocyclic amine bladder carcinogens: bioanalytical considerations.

Aromatic and heterocyclic amines are environmental chemicals which can cause bladder cancer in man. Because these chemicals cause carcinomas at a site distal to their portals of entry, metabolic processes are involved in initiation of their carcinogenic effects. N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT) and its deformylated analogue, ANFT, were used as model compounds to assess metabolism. Electrochemical properties of ANFT made liquid chromatography with electrochemical detection a specific and sensitive method for analysis. Peroxidatic metabolism of ANFT by prostaglandin H synthase (PHS) in the presence of N-acetylcysteine resulted in the formation of 2-amino-4-(5-nitro-2-furyl)-5-(N-acetylcystein-S-yl)thiazole (ANFT-MA). This thioether product has an oxidation potential significantly lower than ANFT. Rat urinary excretion of ANFT-MA was significantly decreased with peroxidase inhibitors, 6-n-propyl-2-thiouracil and methimazole. Inhibitors did not alter excretion of ANFT or prostaglandin E2, a PHS product of arachidonic acid metabolism. 1H and 13C-NMR were selected to explore potential structural differences between ANFT and FANFT which might explain preferential PHS metabolism of ANFT. Evidence for a "zwitterion" configuration for ANFT but not FANFT was observed. ANFT in the "zwitterion" configuration would be a better reducing co-substrate. Chemical synthesis and GC-MS fragmentation patterns identified 3-(2,3-dihydro-1-methyl-2-pyrrolyl)pyridine as a peroxidatic product of nicotine metabolism. This peroxidatic product was found in urine from a cigarette smoker in an amount approximately 6% that observed for continine. Thus, a potential rôle for peroxidative metabolism was demonstrated in man.

Animals↗
PubMedDate not supplied

Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA.

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47 degrees C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.

Avian Leukosis Virus↗
PubMed1988

Novel method of detecting single base substitutions in RNA molecules by differential melting behavior in solution.

We have developed a quick and reliable way of detecting point mutations in RNA molecules. This method involves melting RNA-RNA heteroduplexes of varying lengths in a series of tubes containing a stepwise salt or formamide gradient, followed by polyacrylamide gel electrophoresis to distinguish between single- and double-stranded RNA molecules. The manipulations required are technically simple, and the method is sensitive enough to detect destabilization of the highest melting domain of a dsRNA duplex by a single base mismatch. When this method is used in parallel with denaturing gradient gel electrophoresis, which detects point mutations in low-melting domains of duplexes, it should now be possible to rapidly screen for mutations located throughout the length of any RNA molecule whose wild-type sequence is known.

Base Composition↗
PubMed1988

Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus.

cDNA, synthesized on bovine coronavirus (BCV) genomic RNA templates, could be used to detect very small quantities (i.e. 1 pg) of viral RNA by hybridization with either radioisotopic-labelled or biotinylated recombinant plasmids. Virus was optimally attached to nitrocellulose membranes when spotted in 1 x SSC, whereas 20 x SSC was superior for viral RNA. Denaturation and RNA fixation of both RNA, still encapsidated in virus particles and isolated genomic RNA, was achieved by baking of the blots in vacuum. Virus detection in the supernatant of infected HRT-18 cells was feasible, but improved significantly after proteinase K treatment. No homology was observed between virus cDNA with either plasmid DNA or nucleic acid isolated from non-infected HRT-18 cells. Hybridization with radioisotopic-labelled probes in higher formamide concentrations (up to 60%) increased the detection signals, possibly by reducing reassociation of the probe. Significant detection amplification (30-50 times) was achieved in the case of biotinylated probes by stimulation of hyperpolymer formation on already hybridized target sequences, by additional hybridization with biotinylated pUC-19. A detection amplification was also obtained when hybridization was done with two probes (pBC-52 and pBC-247), containing non-overlapping viral sequences. Although the detectability was surpassed by biotinylated probes, sensitivity was superior in radioisotopic virus detection.

Animals↗
PubMed1988

Development and application of RNA probes for the study of picornaviruses.

Single-stranded RNA probes were developed from cloned cDNA fragments derived from four picornaviruses; poliovirus type 1, coxsackievirus B3, ECHOvirus 9 and Theiler's murine encephalomyelitis virus. In comparative testing with a nick-translated cDNA probe, the RNA probe was found to be 10-100-fold more sensitive. Hybridization conditions were optimized for RNA probes to picornavirus targets. Longer hybridization times gave an improved signal, as did a temperature of 50 degrees C. Formamide concentration had little effect on signal strength. A crude transcription mix performed as well or better as a probe than did more purified preparations of RNA. Finally, a combination of the three probes derived from human picornaviruses detected all 16 serotypes of human enteroviruses tested. Used individually, the four probes detected different spectra of animal and human picornaviruses, shedding more light on the interrelationships among these pathogens.

DNA↗
PubMed1989

Use of alkaline northern blot hybridization for the identification of genetic relatedness of the fourth gene of rotaviruses.

A recently developed alkaline northern blot hybridization assay (Li, J. K. K., Parker, B. & Kowalik, T., Analytical Biochemistry 163, 210-18, 1987) was used to assess the genetic relatedness of the fourth gene of human rotavirus strains recovered from children with diarrhea and from asymptomatic neonates. Genomic double stranded (ds) RNAs of the rotavirus strains were separated by polyacrylamide gel electrophoresis and were blotted to nylon membranes (Gene Screen Plus or Zeta Probe membranes). The blotted RNAs were then probed with 32P-labelled single-stranded (ss) RNA probes prepared by in vitro transcription from single-shelled particles of the different strains. When analysed under a condition of high stringency (52 degrees C, 2.5 x SSC, 50% formamide) that allowed up to 21% of nucleotide mismatch, a high degree of the fourth gene homology was observed among strains recovered from asymptomatic neonates (asymptomatic rotaviruses) or among strains recovered from infants and children with diarrhea (symptomatic rotaviruses), while the homology of the fourth gene between the asymptomatic and symptomatic strains was considerably lower. It is of particular interest that the fourth gene of the AU-1 and AU228 strains recovered from children with diarrhea failed to hybridize to the corresponding gene of either asymptomatic or symptomatic rotavirus strains but showed a high degree of homology with the fourth gene of a feline rotavirus recovered from an apparently healthy cat. These data indicate that a new group of the fourth gene is present among symptomatic rotaviruses and that the fourth gene of this group is genetically related to the corresponding gene of a feline rotavirus.

Animals↗
PubMed1991

Comparison of solution hybridization efficiencies using alkaline phosphatase-labelled and 32P-labelled oligodeoxynucleotide probes.

The hybridization efficiencies of oligonucleotide probes directly labelled with alkaline phosphatase and probes labelled with 32P were compared by quantitating the enzyme activity or radioactivity associated with hybridization targets over time. The targets tested included both synthetic oligonucleotides (53 bases in length) and single-stranded and double-stranded cloned M13 DNA (7350 bases long). Hybrid molecules were separated from unhybridized probes using size exclusion FPLC. This system allowed quantitative analysis of the time course and efficiency of hybridization for both probes and targets in complex hybridization media containing protein blocking agents, formamide, and carrier DNA. Similar maximum hybridization efficiencies were attained for probes labelled with either radioactivity or alkaline phosphatase as a marker. The reaction rate constant for oligonucleotide hybridization to long M13 targets was 3.6 x 10(5) mol-1 s-1 for a probe labelled with alkaline phosphatase, and 5.8 x 10(5) mol-1 s-1 for the same probe labelled with 32P.

Alkaline Phosphatase↗
PubMed1995

A 7Li and 19F NMR relaxation study of LiCF3SO3 in plasticised solid polyether electrolytes.

7Li and 19F NMR relaxation time (T1, T2, T1 rho) measurements have been used to probe the dynamics of LiCF3SO3 dissolved in an amorphous co-polymer poly(ethylene oxide-co-propylene oxide), and in particular the influence of the plasticising agents propylene carbonate and dimethyl formamide. The changes in relaxation behaviour of 19F and 7Li with increasing plasticiser concentration are very different, as is the effect of each plasticiser. These differences can be explained qualitatively in terms of the interaction between the plasticiser and the ions. Preliminary 7Li T1 rho measurements reveal two components at low temperatures.

Dimethylformamide↗
PubMed1994

Image analysis quantification of the Miles assay.

The Miles assay for vascular permeability has high intra- and interassay coefficients of variation (CVs). Quantification, usually by dye extraction and spectrophotometry, is time consuming. In this study, quantification by this means was compared with image analysis using the Olympus CUE-2 Image Analyzer (version 4). The test substance was recombinant human vascular permeability factor (rhVPF). The quantification process took approximately 10 min with image analysis. Formamide extraction and spectrophotometry required 1 hr of preparation, 4-6 days of incubation, and 1 hr for filtration and spectrophotometry. Between assay CVs ranged from 0 to 30% for spectrophotometry, but were all < 10% for image analysis. The sensitivity (2SD above the negative control mean) of the image analysis approach was 64 +/- 25 ng/mL, whereas for spectrophotometry it was 65 +/- 29 ng/mL. Interanimal CVs for rhVPF at 200 and 1000 ng/mL were 15% and 26% when assessed by spectrophotometry and 7% and 22%, respectively, by image analysis. The R2 value for the correlation of image analysis with spectrophotometry was 81.4%. Test substances injected close to the spine evoked a greater permeability response than those injected laterally: at 200 ng/mL p = 0.005, at 1000 ng/mL p = 0.1 (unpaired t tests).

Animals↗
PubMed2025

Methods for purification and characterization of nicked tRNAs.

While tRNA-derived fragments (tDRs) play important roles in gene expression regulation, it is technically challenging to distinguish bona fide tDRs from nicked tRNAs. This is because analytical techniques used to study RNA, such as northern blot, RT-qPCR or sequencing involve the use of denaturing reagents (e.g., phenol, formamide, urea) or physical procedures (e.g., heat) that convert nicked tRNAs into tRNA halves or other tDRs. In this chapter, we describe a protocol that enables the purification of nicked tRNAs under non-denaturing conditions that preserve their 3D structure. Purified nicked tRNAs can then be either enzymatically repaired into almost full-length tRNAs, or chromatographically separated from single-stranded tDRs before detection. These protocols will allow researchers to distinguish between structurally distinct but sequence identical tDRs and nicked tRNAs, disentangling their biological functions.

RNA, Transfer↗
PubMed2004

Proton NMR study of the heme environment in bacterial quinol oxidases.

The heme environment and ligand binding properties of two relatively large membrane proteins containing multiple paramagnetic metal centers, cytochrome bo3 and bd quinol oxidases, have been studied by high field proton nuclear magnetic resonance (NMR) spectroscopy. The oxidized bo3 enzyme displays well-resolved hyperfine-shifted 1H NMR resonance assignable to the low-spin heme b center. The observed spectral changes induced by addition of cyanide to the protein were attributed to the structural perturbations on the low-spin heme (heme b) center by cyanide ligation to the nearby high-spin heme (heme o) of the protein. The oxidized hd oxidase shows extremely broad signals in the spectral region where protons near high-spin heme centers resonate. Addition of cyanide to the oxidized bd enzyme induced no detectable perturbations on the observed hyperfine signals, indicating the insensitive nature of this heme center toward cyanide. The proton signals near the low-spin heme b558 center are only observed in the presence of 20% formamide, consistent with a critical role of viscosity in detecting NMR signals of large membrane proteins. The reduced bd protein also displays hyperfine-shifted 1H NMR signals, indicating that the high-spin heme centers (hemes b595 and d) remain high-spin upon chemical reduction. The results presented here demonstrate that structural changes of one metal center can significantly influence the structural properties of other nearby metal center(s) in large membrane paramagnetic metalloproteins.

Cyanides↗
PubMed2006

Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase.

This study demonstrates the immobilization of carbohydrate containing turnip peroxidase on an inexpensive bioaffinity adsorbent, Concanavalin A-cellulose support. The bioaffinity support was prepared simply by incubating cellulose powder with jack bean extract at 4 degrees C. Cellulose powder adsorbed 30 mg concanavalin A/g of the matrix. Concanavalin A adsorbed cellulose has been employed for the simultaneous purification and immobilization of glycoenzymes directly from ammonium sulphate fractionated proteins of turnip. The obtained bioaffinity support was quite effective in high yield immobilization of peroxidase from turnip and it retained 672 U/g. Turnip peroxidase immobilized on concanavalin A-cellulose support retained 80% of the initial activity. Immobilized turnip peroxidase preparation was quite resistant against the denaturation mediated by pH, heat, urea, guanidinium-HCl, Surf Excel, cetyltrimethylammonium bromide and water-miscible organic solvents; dimethyl formamide, dioxane and n-propanol. Low concentration of detergents like Surf Excel and cetyltrimethylammonium bromide enhanced the activity of soluble and immobilized turnip peroxidase.

Brassica rapa↗