Hot Formamide Research Articles

Improved procedure for the measurement of telomere length in whole cells by PNA probe and flow cytometry

Peptide nucleic acid (PNA) probes hybridize to denatured telomeric sequences in cells permeabilized in hot formamide. In reported protocols, the hybridization was conducted in solutions with high formamide concentrations to avoid the DNA renaturation that can hamper binding of the oligo-PNA probe to specific sequences. We postulated that telomeric DNA, confined in the nuclear microvolume, is not able to properly renature after hot formamide denaturation. Therefore, to improve hybridization conditions between the probe and the target sequences, it might be possible to add probe to sample after the complete removal of formamide. After telomeric DNA denaturation in hot formamide solution and several washes to remove the ionic solvent, cells were hybridized overnight at room temperature with human telomere-specific PNA probe conjugated with Cy5 fluorochrome, Cy5-OO-(CCCTAA)(3) . After stringency washes and staining with ethidium bromide, the cells were analysed by flow cytometry and by using a confocal microscope. Using three continuous cell lines, different in DNA content and telomere length, and resting human peripheral blood T and B lymphocytes, we demonstrated that the oligo-PNA probe hybridized to telomeric sequences after complete removal of formamide and that in the preserved nucleus, telomeric sequence denaturation is irreversible. According to our experience, oligo-PNA binding results is efficient, specific and proportional to telomere length. These, our original findings, can form the technological basis of actual in situ hybridization on preserved whole cells.

Studies on Reaction of Hydroxycoumarin Compounds with Formamide.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Denaturing RNA electrophoresis in TAE agarose gels

Current methods of analytical RNA electrophoresis are based on the utilization of either complicated laboratory instrumentation or toxic, carcinogenic, or expensive chemicals. We suggest here the use of classical Tris–acetate–ethylenediamine tetraacetic acid (TAE) agarose gels combined with prior denaturation of RNA samples in hot formamide for the electrophoretic separation of RNA species. We present a brief comparison of the proposed TAE/formamide method with the most common 3-( N-morpholino)propanesulfonic acid/formaldehyde agarose gel protocol and show that both methods produce comparable results for size determination of RNA molecules and subsequent Northern blotting of gels. In addition to purified RNA samples, the robustness of the TAE/formamide protocol is demonstrated by its suitability for the analysis of RNA quality in crude yeast cell lysates containing large amounts of proteins, DNA, and other contaminating molecules. We therefore propose the TAE/formamide agarose electrophoresis as a rapid, simple, and cheaper alternative to current methods of RNA electrophoresis. Additionally, another benefit is the reduced exposure of laboratory personnel to hazardous chemicals.

Studies on reaction of hydroxycoumarin compounds with formamide

3-Hydroxycoumarin, on reaction with hot formamide, produced 3-formylaminocoumarin (2) which underwent deformylation on fusion with p-toluenesulphonic acid monohydrate to produce very pure variety of 3-aminocoumarin. 4-Hydroxycoumarin and 4-hydroxyhenzo[b]thiopyran-2-one (7) on similar reaction produced benzo[I]pyrano[4,3-d]oxazol-4-one (6) and benzo[1]thiopyrano[4,3-d]oxazol-4-one (8) respectively, under effectively non-oxidising reaction condition.

One-Lane Chemical Sequencing of 3′-Fluorescent-Labeled DNA

A method for DNA sequencing that combines limited chemical degradation of 3′-fluorescent-labeled DNA with densitometric analysis using an automatic sequencer is presented. The DNA sequence is determined in a single electrophoretic lane by monitoring the intensities of bands representing products of cleavage at the four bases obtained by solvolysis in hot aqueous piperidine (10%) followed by treatment with hot formamide. An application of the method for the detection of point mutations is reported.

Biochemical and immunochemical properties of the cell surface of Renibacterium salmoninarum

The biochemical composition of the cell envelope of Renibacterium salmoninarum was investigated in a total of 13 strains isolated from different salmonid fish species at various geographical locations of the United States, Canada, and Europe. A marked similarity with the type strain R. salmoninarum ATCC 33209 was found both in the peptidoglycan and the cell wall polysaccharide. The primary structure of the peptidoglycan was found to be consistent with lysine in the third position of the peptide subunit, a glycyl-alanine interpeptide bridge between lysine and D-alanine of adjacent peptide subunits, and a D-alanine amide substituent at the alpha-carboxyl group of D-glutamic acid in position 2 of the peptide subunit. The cell wall polysaccharide contained galactose as the major sugar component which was accompanied by rhamnose, N-acetylglucosamine, and N-acetylfucosamine. The polysaccharide amounted to more than 60% of the dry weight of the cell walls. It was found to be covalently linked to the peptidoglycan and was released by hot formamide treatment. On gel filtration chromatography the extracted polysaccharide behaved like a homogeneous polymeric compound. The purified cell wall polysaccharide showed antigenic activity with antiserum obtained by immunization of rabbits with heat-inactivated trypsinized cells of R. salmoninarum. Immunoblotting experiments with nontrypsinized cell walls and antisera raised against R. salmoninarum cells revealed that antigenic proteins were attached to the cell walls.

Triazines and related products. Part 28. Conversion of 3-aryl-1-(2-cyanophenyl)triazenes into 3-arylquinazolin-4(3H)-ones with formamide

The thermolysis of 4-anilino-1,2,3-benzotriazines and their precursor 3-aryl-1-(2-cyanophenyl)-triazenes in hot formamide to give 3-arylquinazolin-4(3H)-ones in high yield is described. The reaction cannot be extended to the preparation of 2-alkyl-3-arylquinazolinones. In hot acetamide–diglyme 4-(4-nitroanilino)-1,2,3-benzotriazine (5c) gives a high yield of the 3-substituted 4-(4-nitrophenylimino)-3,4-dihydro-1,2,3-benzotriazine (11c).

Improved streptococcal grouping antisera containing polyethylene glycol.

Antisera to streptococcal groups A through G containing 4% polyethylene glycol 6000 were prepared and evaluated. Seventy strains of homologous and heterologous beta- and non-beta-hemolytic streptococci were included in the evaluation. Homologous reactions were determined against extracts prepared by four extraction methods: hot hydrochloric acid (Lancefield) extraction, autoclave extraction, hot formamide extraction, and nitrous acid extraction. Enhancement of the precipitin reaction in the presence of polyethylene glycol 6000 permitted a fourfold dilution of all antisera for use in the capillary precipitin test. At this dilution, the potency of the antisera exceeded requirements established for these reagents.

Simplified coagglutination test for serological grouping of beta-hemolytic streptococci.

A new coagglutination kit consisting of a plastic slide with dried antibody-coated staphylococci was evaluated for the grouping of beta-hemolytic streptococci of groups A, B, C, and G. The test was compared with the classical precipitation test, using hot formamide antigen extracts with 224 strains of groups A, B, C, and G streptococci. An agreement of 100% was found between the new coagglutination and the classical precipitation procedures. No false-positive results were obtained with group F and D streptococci; however, group L streptococci reacted with the group A reagent. The test procedure could be shortened by using suspensions of colonies from overnight cultures on blood agar plates in small volumes of Todd-Hewitt broth without further incubation. All 74 strains of groups A, B, C, and G were correctly identified from suspensions in Todd-Hewitt broth; however, suspending the colonies in 0.9% saline or phosphate-buffered saline resulted in lower sensitivities.

A short synthesis of 5‐methylhistamine

AbstractMethoxybromination of 4,5‐dihydro‐2‐methylfuran (4), followed by treatment of the resulting bromoketal 5 with hot formamide, gave 4‐(2‐hydroxyethyl)‐5‐methyl‐1H‐imidazole (3) in 25% yield. This method provides easy access to the selective H2‐agonist 4‐methylhistamine (1) via the chloromethyl intermediate 2.

Characterization of a Regular Array in the Wall of Lactobacillus buchneri and its Reattachment to the Other Wall Components

SUMMARY: A regular array exhibiting hexagonal periodicity with a centre-to-centre spacing of about 6 nm was found on the wall surface of freeze-etched Lactobacillus buchneri. The regular array was composed of a subunit protein with a molecular weight of about 55000 as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Subunits isolated from the wall by extraction with guanidine hydrochloride reassembled into the original regular array after dialysis against distilled water. The subunits could reattach to wall fragments from which most of the teichoic acid had been extracted with cold trichloroacetic acid, but not to wall fragments from which most of the neutral polysaccharide had been removed by treatment with hot formamide. Heterologous reattachment of the subunits took place on to Lactobacillus casei subsp. casei wall fragments which had been partially hydrolysed under mild acid conditions. These observations suggest that the subunit protein binds to a neutral polysaccharide moiety in the underlying wall layer but not to peptidoglycan or teichoic acid.

Reassembly of the regularly arranged subunits in the cell wall os Lactobacillus brevis and their reattachment to cell walls.

The reassembly of tetragonally arranged subunits in the cell wall of Lactobacillus brevis and the reattachment of the subunits to cell wall fragments were investigated by electron microscopy. The subunits dissociated from the cell wall with guanidine hydrochloride (GHCl) reassembled into the same regular array as seen in native cell wall after dialysis against neutral buffer even in the absence of specific cations. The subunits could also reattach to the cell wall fragments from which they had been removed by treatment with GHCl, sodium dodecyl sulfate or cold trichloroacetic acid but not to those treated with hot formamide. Heterologous reattachment of the subunits occurred on cell wall fragments obtained from L. fermentum but not on those from L plantarum or L. casei subsp. casei. On the basis of these observations and chemical analyses of the cell wall fragments, the subunits of L. brevis appeared to be bound by hydrogen bonds to a neutral polysaccharide moiety in the cell wall but not to peptidoglycan or teichoic acid.

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 (~350 nucleotides) is degraded by hot formamide hydrolysis to fragments ranging from small oligonucleotides to near full lengths, and subsequently labelled to high specific activities 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.

Detection of candida antigenemia by counterimmunoelectrophoresis in patients with invasive candidiasis.

Because of the difficulty in diagnosis of invasive candidiasis, an assay that makes use of counterimmunoelectrophoresis (CIE) was developed to detect candida antigen. A cell-wall polysaccharide of Candida was extracted with hot formamide, ethanol precipitation, and gel filtration. Rabbit antiserum was tested by CIE against sera from 20 healthy individuals, 15 patients with mucosal candidiasis, and 48 compromised patients with cultures positive for Candida. Antigen was detected in sera from 13 patients by CIE; eight of these patients were eventually proven to have invasive disease. None of the antigen-negative patients for whom autopsy was performed had evidence of invasive candidiasis. Healthy individuals and patients with superficial mucosal candidiasis were also antigen-negative. The detection of candida antigenemia by CIE is specific for invasive disease and serves as a guide in initiation and follow through of antifungal therapy.

Antigens of Streptococcus mutans: cellular localization of the serotype-specific polysaccharide of strain AHT and release during exponential growth.

The serotype-specific polysaccharide of Streptococcus mutans AHT (serotype a) was shown to be loosely associated with the cell surface of this organism. The antigen was extracted from whole cells by boiling in sodium acetate buffer, pH 4.0, for 10 min. The purified product was found to be a diheteroglycan of galactose and glucose (3.6:1, molar ratio). The antigen possessed serological characteristics similar to the a antigen previously extracted from purified cell walls with hot formamide. Its physicochemical structure was identical to the previously studied wall antigen. Electron micrographs, developed after immunocytological labeling of this antigen on whole cells, revealed it to compose a dense microcapsule surrounding the microbe. Analyses of spent culture fluids indicated that the antigen was released during exponential growth at a rate directly proportional to the increase in culture biomass. It is concluded that the serotype-specific antigen may be a prime immunogen due to its surface localization at both capsule and wall sites.

Chemical, immunochemical, and structural studies of the cross-reactive antigens of Streptococcus mutans AHT and B13.

Two antigenic polysaccharides were extracted from cell walls of the cross-reactive strains Streptococcus mutans AHT (a) and S. mutans B13 (d). The antigens extracted from walls by the hot formamide method, were purified by affinity chromatography on columns containing the galactose-specific lectin from the castor bean and were found to be diheteroglycans consisting of galactose and glucose. Antigenic specificities of both the serotype-specific and the cross-reactive sites on each polymer were studied: the AHT (a) antigen is determined by D-galactose linked 1 leads to 6 to adjacent sugar, the B13 (d) antigen is determined by D-glucose similarly linked to o its neighbor, and the cross-reactive (a--d) site present on both polymers consists of D-galactose linked 1 leads to 6 to a subterminal sugar moiety. Methylation analysis revealed structural similarities between the purified polysaccharides that may reflect the nature of the cross-reactive sites and differences that may reflect the natures of the specific haptenic regions. Based on these studies, a partial hypothetical structural model is proposed.

A different approach to RNA sequencing.

DURING the past few years techniques to simplify the systematic sequence determination of nucleic acids have become available; these involve the use of polyacrylamide gels from which the sequence may be directly and rapidly read off. The most recent of these, applicable to RNA fragments, rely on the base-specific nature of cleavage by T1 and U2 RNases1,2 and pancreatic RNase and RNase I from Physarum polycephalum2 by which a spectrum of 5′ terminally-labelled fragments may be produced and comigrated with a ‘ladder’ achieved by incubating the fragment with alkali or hot formamide. We describe here a method for the sequence determination of RNA fragments bearing a blocked 5′ terminus, in which the above enzymatic hydrolysis steps and their accompanying problems may be avoided.

Characterization of group H streptococcal temperate bacteriophage phi 227.

phi 227, a temperate phage from a group H streptococcus (Streptococcus sanguis), was propagated vegetatively in group H strain Wicky 4-EryR, and its characteristics were determined. A procedure dependent on multiplicity of infection, incubation time, and treatment of crude lysates with diatomaceous earth was found to optimize phage yield, resulting in titers of 1 X 10(10) to 2 X 10(10) PFU/ml. Without prior treatment with diatomaceous earth, subsequent purification procedures (methanol, ammonium sulfate, polyethylene glycol) gave recoveries of less than 1% of crude lysate titers. Adsorption of phi227 to host cells was relatively unaffected by the medium, but calcium (not substituted by magnesium) was required for formation of infectious centers. The phage receptor was present on purified cell walls, resisted trypsin and heat, and was removed ty hydrochloric acid, trichloracetic acid, and hot formamide: however, formamide-extracted material failed to inactivate phage, and the nature of the receptor is unknown. Single-step growth experiments showed a latent period of 39 min and a burst size of 100 PFU/infectious center; results were unaffected by omission of supplemental Ca2+, by supplementation with Mg2, addition of glucose, or changes of pH between 6.35 and 8.0; but increased temperature (40 to 43 degrees C) shortened the latent period and decreased the burst size. The latent period was prolonged in genetically competent host cells and in chemically defined medium; and in the latter, the burst size was smaller. Phage replication was sensitive to those metabolic inhibitors which inhibited the host streptococcus: these included rifampin, fluorodeoxyuridine, hydroxyurea, dihydrostreptomycin, and 6-P-hydroxyphenylazouracil. The data suggest that phi227 does not code for a rifampin-resistant RNA polymerase. However, in a rifampin-resistant host strain, phage replication and lysogen formation were both decreased suggesting that altered host core polymerase had less affinity for (some) promotors on the phi227 template. In transfection, a Ca2+-dependent stabilization step that was inhibited by Mg2+ was demonstrated; transformation was not affected by either Ca2+ or Mg2+, and the site and nature of the stabilization are unknown. More than one molecule of DNA was required for plaque formation. Biophysical characterization showed a type B phage of buoyant density (CsCl) 1.50, containing five proteins and 54.8% DNA. The duplex linear DNA had a molecular weight (calculated from contour length) of 23.2 X 10(6) and a guanine plus cytosine content (calculated from melting point) of 42.3 mol%. Similar characterizations of streptococcal phages, including biophysical data, have not been previously available.

Role of Candida albicans in Granulomatous Tissue Reactions. I. In Vitro Degradation of C. albicans and Immunospecificity of Split Products

Several mannan-containing fractions were obtained from whole cells, cell walls, and cytoplasm of Candida albicans by means of treatment with hot formamide or precipitation with (NH4)2SO4. The immunological and chemical characteristics of the fractions were compared with those of C. albicans mannan prepared by standard procedures. Antisera to C. albicans from rabbits immunized with whole cells of the organism were found to be primarily directed against the mannan content. With use of such antisera, mannan was localized in both the inner and outer layers of the cell wall, whereas the middle layer was found in all likelihood to represent the glucan polymer. Stepwise removal of mannan from whole cells or cell walls resulted in increasing loss of periodic acid-Schiff staining, immunofluorescence, and peroxidase reactivity. Thus, it appears that mannan is responsible for the ability of cell walls of C. albicans to be stained by periodic acid-Schiff or labeled with fluorescent antibody. The component of the pathogen most resistant to physical or chemical treatment was the glucan, which lacked all immunological reactivity.

The viroidlike structure and cellular location of anomalous RNA associated with the cadang-cadang disease.

Abstract The cadang-cadang associated RNA species, ccRNA-1 and ccRNA-2, were recovered from diseased coconut palm leaf using a range of procedures. These included varying the salt molarity, type of buffer, and reducing agent in the extraction medium, using either polyethylene glycol (PEG) or ammonium sulphate to precipitate the RNA-containing component from tissue extracts, and using three alternative procedures to extract nucleic acid from the PEG-precipitated material. The view that ccRNA-1 is uniquely associated with the disease was supported by showing that the similarly migrating RNA, which sometimes could be recovered from normal palms, differed structurally from ccRNA-1. Kinetics of digestion with ribonuclease A and the single-strand specific S1 endonuclease, and thermal denaturation studies, indicated that the ccRNA-1 molecule has both double- and single-stranded regions. Because disruption of base-pairing by melting or hot formamide treatment failed to affect the apparent molecular weight of ccRNA-1, it is concluded that ccRNA-1 has a looped or “hairpin” secondary structure, stabilized by base-pairing. Its buoyant density (1.62 g/cm3) falls between the expected values for a single-stranded and a double-stranded viral RNA. A comparative study with potato spindle tuber viroid (PSTV) showed that the S1 nuclease digestion kinetics of ccRNA-1 and PSTV were similar. In denaturing formamide polyacrylamide gels, the molecular weight of ccRNA-1 was below that of PSTV and was estimated to be 63,000–73,000. ccRNA-1 is soluble in 2 M LiCl, is able to penetrate 20% polyacrylamide gels, and its thermal denaturation profile responds to Mg2+ like that of citrus exocortis viroid RNA. The structure of ccRNA-1 therefore appears to resemble closely that of viroid RNA. Unlike the known viroids, ccRNA-1 is not sedimented from tissue extracts by low or high speed centrifugation, nor has its infectivity been demonstrated. Some variations in the ccRNA banding patterns in gels are described. The occasional appearance of duplex ccRNA-1 is attributed to the existence of two ccRNA-1 molecules which have the same number of base pairs, but which differ in having larger or smaller amounts of single-stranded RNA associated with them.