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At least 1,063 records · Page 59Linked to original sources
PubMed1977

Two adenovirus mRNAs have a common 5' terminal leader sequence encoded at least 10 kb upstream from their main coding regions.

The messenger RNAs encoding two late adenovirus serotype 2 (Ad2) proteins, fiber and 100K, were purified by hybridization to restriction endonuclease fragments of Ad2 DNA followed by electrophoresis on polyacrylamide gels containing 98% formamide. The 5' terminal oligonucleotides generated by RNAase T1 digestion of the messengers were selected by dihydroxyboryl-cellulose chromatography. Both mRNAs gave an identical 5'-undecanucleotide with the general structure 7mG5'ppp5'AmC(m)U(C4,U3)G. This undecanucleotide could be removed by mild RNAase treatment from the mRNA after hybridization to DNA fragments containing the main coding sequence of the messenger. In contrast, a small region defined by Bal I-E (14.7-21) protects this undecanucleotide from RNase. A second region contained within both Hind III-B (17-31.5) and Hpa I-F (25.5-27.9), although unable to protect the undecanucleotide, hybridizes to both fiber and 100K mRNAs and protects a similar sequence of 100-150 nucleotides. These observations suggest that both mRNAs contain a long common sequence, complementary to at least two different sites on the Ad2 genome remote from the start of these two genes. The implications of these findings are discussed, and a general mechanism is presented for the biosynthesis of mRNAs from larger precursor molecules, based on intramolecular ligation.

Adenoviruses, Human↗
PubMed1977

Discrete sizes of replication intermediates in Drosophila cells.

The size of DNA replication intermediates from Drosophila cells pulse-labeled with 3H-thymidine for 30-120 sec was determined by electrophoresis in formamide-polyacrylamide gels. Replication intermediates were formed in three discrete size classes, with median lengths of 61, 125 and 240 nucleotides. Replication intermediates in the 125 nucleotide size class occurred most frequently. Two of the three size classes may contain discrete species of replication intermediates about 90-400 nucleotides long. The data also suggested that some larger replication intermediates accumulate in pulse-labeled cells. We concluded that 61 nucleotide molecules give rise to 125 and 240 nucleotide molecules, which then form high molecular weight DNA. Mechanisms for forming these replication intermediates are discussed.

Cell Line↗
PubMed1977

Messenger RNA for myosin polypeptides: isolation from single myogenic cell cultures.

Messenger RNA which stimilates the synthesis of myosin heavy chain in a reticulocyte lysate has been isolated from single myogenic cell cultures. Specific myosin polypeptides have been identified by immunoprecipitation with an antibody made to purified adult chicken skeletal muscle myosin. This mRNA binds to oligo(dT)-cellulose, and an active fraction from sucrose gradients migrates as 26S on formamide-polyacrylamide gels. The relative amount of this RNA increases dramatically at the time of terminal differentiation.

Cell Differentiation↗
PubMed1978

The transcription map of mouse mitochondrial DNA.

Nine transcripts complementary to mouse L cell mitochondrial DNA have been detected, sized and mapped to restriction fragments using the method of Berk and Sharp (1977). RNA isolated from L cell mitochondria was hybridized to 32P-labeled, cloned L cell mitochondrial DNA restriction fragments in 70% formamide under conditions 5 degrees C above the melting temperature of the DNA-DNA duplex, but approximately 15 degrees C below the melting temperature of the RNA-DNA duplex. The heteroduplexed material was then treated with the single-strand-specific nuclease S1, whick cleaves the single-stranded DNA not protected by RNA-DNA duplex formation into oligonucleotides and leaves intact 32P-labeled, single-stranded DNA replicas complementary to the transcripts. The single-stranded DNA replicas were then resolved and sized by alkaline agarose gel electrophoresis. Hybridization to strand-separated, 32P-labeled L cell mitochondria DNA restriction fragments under the same conditions showed that all nine transcripts hybridized exclusively to the heavy strand (H strand) of restriction fragments isolated as the dense strand from alkaline CsCl gradients, indicating that all stable transcripts 300 bases or longer detected by this technique originate from genes on the H strand. The two most abundant transcripts homologous to mitochondrial DNA map adjacent to the origin of replication. This result is consistent with map positions assigned to the large and small mitochondrial ribosomal RNAs isolated from Xenopus laevis and HeLa cells. Six of the other seven transcripts map continuously in approximately 40% of the genome. Only one transcript of 950 bases maps in the first quadrant of the genome as defined by the origin and direction of mitochondrial DNA replication, and it does not lie within the D loop region. The genetic function of the remaining 75% of this region of the genome is yet to be determined.

DNA Replication↗
PubMed1979

Length-independent separation of DNA restriction fragments in two-dimensional gel electrophoresis.

When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60 degrees C in a concentration gradient of a urea-formamide mixture, each Eco RI fragment of lambda or E. coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment has been retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a two-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the Eco RI digest of E. coli DNA. Corresponding fragments from an isogenic lambda lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.

Coliphages↗
PubMed1980

Some yeast mitochondrial RNAs are circular.

11S and 18S fractions of yeast mitochondrial RNAs, isolated by electrophoresis through agarose gels, have been found by electron microscopy to contain approximately 50% circular molecules. Circles in the 11S fraction have a contour length of 0.36 +/- 0.02 micron, which is approximately equal to the length of the majority of linear molecules also present. Circles in the 18S fraction have an average length of 0.78 +/- 0.11 micron. The size distribution is broader than for the 11S fraction, and we cannot exclude the possibility that more than one size class may be present. The 11S circular RNA forms circular R loops and RNA-DNA hybrids with DNA fragments of the oxi 3 region of mtDNA, which contains the structural gene for subunit 1 of cytochrome oxidase. As judged from the electron micrographs, the complete RNA participates in hybrid formation and the sequences coding for it appear to be continuous. Both 11S and 18S circles withstand treatment with DNAase and pronase. They are not eliminated by treatment with 1 M glyoxal in 50% formamide for 1 hr at 50 degrees C. We conclude that they are covalently closed. The function of the circular RNAs is unknown. They may be active as mRNAs, storage forms, or arise in a cut-and-splice process which generates mRNAs from longer transcripts.

Mitochondria↗
PubMed1988

Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.

In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells. We conclude that both 70Z/3 and HeLa cells contain apparently cytosolic NF-kappa B in a form with no evident DNA-binding activity, and phorbol esters both release the inhibition of binding and cause a translocation to the nucleus.

Cell Line↗
PubMed1988

Homology mapping of T-DNA regions on three Agrobacterium rhizogenes Ri plasmids by electron microscope heteroduplex studies.

Recombinant plasmids carrying segments of the Agrobacterium rhizogenes T-DNA regions of the three Ri plasmids 1855 (TL-DNA only), 8196, and 2659 were used for establishing homology maps by electron microscope examination of heteroduplexes. Plasmid DNA was linearized by digestion with suitable restriction endonucleases in order to generate large T-DNA segments. Heteroduplexes were prepared in 50% formamide and spread under standard conditions. Measurements of double and single strands allowed the drawing of homology maps. The three T-DNAs share mainly two homologous sequences of respectively about 2.5 and 1.5 kb, bracketing a largely nonhomologous central part which is about 5.5 kb long. The T-DNAs from pRi1855 and pRi2659 appear to be more related to each other than to that of pRi8196. With reference to the published nucleotide sequence of the TL-DNA of pRiA4 (probably identical to that of pRi1855), ORFs 8 and 14 seem to be the most conserved sequences of the three T-DNAs. The significance of these conserved sequences is unclear since the genetic loci involved in rhizogenicity of agropine strains identified previously are located in nonhomologous regions.

Base Sequence↗
PubMed1981

A 13C-NMR study of non-planar distortions in amides.

The technique of 13C-NMR spectroscopy of oriented systems to problems of biological importance has been suggested and used to investigate non-planar distortions in substituted amides--models for peptides. The studies in conjunction with the proton magnetic resonance data on 15N-[13C]methyl[13C]formamide oriented in a nematic solvent provide all the direct dipolar couplings between the interacting nuclei in the system. When the 13C- and the 1H-NMR experiments are performed under non-identical conditions, 22 different direct dipolar couplings are obtained. It is demonstrated that they can be used to determine unambiguously non-planar distortions around the nitrogen atom together with other geometrical data and the molecular order.

Amides↗
PubMed1985

Isolating and sequencing the predominant 5'-ends of a specific mRNA in cells. I. Purification by filter hybridization.

Procedures are considered for purification of a specific procaryotic RNA by successive hybridizations to DNA immobilized to nitrocellulose with special consideration of problems associated with subsequent end-labeling in the T4 polynucleotide kinase reaction. (1) Inhibitors of the kinase can be associated with the plasmid but were removed by electrophoresis of the DNA fragment through polyacrylamide. (2) Residual soluble acrylamide, contaminating the DNA and preventing its efficient retention to nitrocellulose, could be removed by DE52 chromatography. (3) Short denatured DNA required high salt (0.9 M) to bind to nitrocellulose but reannealed quickly at those salt concentrations unless applied at less than or equal to 0.3 micrograms/ml at 4 degrees C with a flow rate of 1 ml/min. (4) The kinetics of the hybrid reaction were a function of DNA length, concentration, and temperature. (5) Formamide was a more effective denaturing agent to remove hybrid RNA from the filter than either 12 M urea or 8 M guanidine-HCl, but caused significant release of DNA from the nitrocellulose as well as another potent inhibitor of the kinase reaction. The release of DNA and other kinase inhibitors was greatly reduced by eluting in boiling water.

Base Sequence↗
PubMed1996

Optimization of Fast-FISH for alpha-satellite DNA probes.

It has been shown for several highly repetitive DNA probes that the newly introduced Fast-FISH (fast-fluorescence in situ hybridization) technique is well suited for quantitative microscopy. The advantage of omitting denaturing chemical agents (e.g., formamide) in the hybridization buffer results in a short hybridization time and a considerable reduction of the number of subsequent washing steps. Choosing the appropriate hybridization temperature and time allows to clearly discriminate major and minor binding sites by quantitative fluorescence microscopy. To further optimize the procedure with reference to reproducibility, a fully programmable thermal-cycler was applied for thermal de- and renaturation. Here, the optimized renaturation conditions for two commercially available alpha-satellite probes (specific for chromosomes 1 and X) are described. For the Boehringer chromosome-1-specific DNA probe, two highly fluorescent binding sites were obtained for 72 degrees C hybridization temperature and 60 min hybridization time. For the Oncor chromosome-X-specific DNA probe, the optimal conditions were found at 74 degrees C and 60 min hybridization time. In both cases the major binding sites were clearly discriminated from only a few weakly fluorescent minor binding sites on metaphase spreads as well as in interphase cell nuclei.

DNA Probes↗
PubMed1983

Evans blue fluorescence: quantitative and morphological evaluation of vascular permeability in animal tissues.

The dye Evans blue was used to monitor vascular protein leakage. Fluorometric measurement of Evans blue in formamide extracts of rat tracheal tissue was performed after induction of protein leakage by electrical vagus nerve stimulation and compared with the widely used colorimetric detection. The fluorescence method was approximately 100 X more sensitive than the colorimetric method. Furthermore, Evans blue fluorescence (excitation at 620 nm, emission at 680 nm) was used for microscopic investigation of cryostat sections of tracheal tissue. Extravasated Evans blue after electrical nerve stimulation was mainly found in the subepithelial layer of the trachea obviously bound to tissue constituents. It is suggested that Evans blue fluorescence can be applied for quantification of protein leakage with high sensitivity which opens the possibility of measuring this reaction in very small regions with high accuracy, as well as for tissue localization of protein leakage at the microscopic level.

Animals↗
PubMed1995

A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.

This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.

Animals↗
PubMed1980

Enhancement of sister-chromatid exchange in Chinese hamster ovary cells by nitrofurans.

The nitrofurans, N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT), N-[4-(5-nitro-2-furyl)-2-thiazolyl]acetamide (NFTA), 2-amino-4-(5-nitro-2-furyl)thiazole (ANFT), 2-methyl-4-(5-nitro-2-furyl)thiazole (MNFT), 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2), nitrofurantoin, 5-nitro-2-furfurylidene diacetate and 5-nitro-2-furamidoxime were assayed for the enhancement of sister-chromatid exchange (SCE) in Chinese hamster ovary (CHO) cells and for the mutagenic activity in Salmonella typhimurium strain TA100. All of the nitrofurans significantly increased the frequency of SCE in CHO cells, and they were mutagenic for Salmonella. The increases in the frequencies of SCE were well correlated with the mutagenic activities in Salmonella.

Animals↗
PubMed1987

Effects of chemical combinations on the induction of aneuploidy in Saccharomyces cerevisiae.

Nocodazole, ethyl acetate, acetone and methyl ethyl ketone all are known to induce aneuploidy. Treatment of yeast strain D61.M with mixtures containing ineffective low levels of nocodazole and ineffective low levels of these solvents was highly effective in inducing aneuploidy. Ineffective low levels of nocodazole mixed with ineffective low levels of methyl 2-benzimidazolecarbamate also gave elevated frequencies of aneuploidy. Dimethyl formamide, a solvent that does not induce aneuploidy, mixed with low levels of nocodazole gave no increase in aneuploidy frequency above those levels seen in controls.

Acetates↗
PubMed1985

Inhibitory effect of organic solvents on the mutagenicity of N-nitrosodialkylamines in Salmonella.

The influence of organic solvents on the mutagenicity of 11 N-nitrosamines was examined in Salmonella typhimurium TA100 using the Ames's liquid incubation assay in the presence of rat-liver S9. The mutagenic activities of N-nitrosodimethylamine, N-nitrosodiethylamine, 6 oxidative derivatives of N-nitrosopropylamine and N-nitroso-2,6-dimethylmorpholine were considerably decreased by addition of dimethyl sulfoxide, dimethyl formamide, acetone, 95% ethanol or acetonitrile, which are recommended for use as solvents in the assay by Ames's group, to the incubation mixture. The mutagenic activities of N-nitrosodipropylamine and N-nitrosodibutylamine, which are barely soluble in water, were also suppressed by increasing concentrations of dimethyl sulfoxide. These organic solvents did not appear to exert their influence by desmutagenic and antimutagenic actions. In contrast, the recoveries of unmetabolized carcinogens from preincubation mixtures and from agar plates were significantly higher in the presence of organic solvents than in their absence. The results indicate that the inhibitory effect is a result of interference with the process of metabolic activation by liver S9.

Animals↗
PubMed1980

In vitro 32P-labelling of viroid RNA for hybridization studies.

A method is described for the in vitro labelling of viroid RNA for use in hybridization studies. The citrus exocortis viroid (approximately 350 nucleotides) is degraded by hot formamide hydrolysis to fragments ranging from small oligonucleotides to near full lengths, and subsequently labelled to high specific activity by enzymatically attaching 32P to the 5'-end of each molecule. The cleavage step leaves 5' hydroxyl groups which allows the polynucleotide kinase to directly label the RNA fragments without prior enzymatic dephosphorylation. The method is simple, requires no special equipment, and provides a radioactive RNA probe sufficient for most types of hybridization studies.

Isotope Labeling↗
PubMed1986

Detection of citrus exocortis viroid in crude extracts by dot-blot hybridization: conditions for reducing spurious hybridization results and for enhancing the sensitivity of the technique.

Dot-blot assays to detect citrus exocortis viroid (CEV), in clarified sap and unfractionated total nucleic acid preparations of CEV-infected Gynura aurantiaca and chrysanthemum, were impaired by the non-specific binding of the radioactive probe shown by the healthy controls. This non-specific background was considerably reduced by the addition to the hybridization mixture, of the fraction of nucleic acids from healthy plants which are insoluble in 2 M LiCl (containing mainly the large ribosomal RNAs). Sample denaturation with formaldehyde was found to provide a high increase of hybridization, when compared with samples either denatured with formamide or directly spotted. Nitrocellulose was observed to be a better solid support than charge-modified nylon, in terms of the sensitivity of viroid detection by spot hybridization.

Collodion↗