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PubMed1987

Molecular hybridization with RNA probes in concentrated solutions of guanidine thiocyanate.

Hybridizations with RNA probes were performed in 3-6 M guanidine thiocyanate concentrations where cells can be solubilized. At these concentrations the melting temperature of hybrids and the optimum temperature of hybrid formation are greatly reduced. The rate of hybridization in GuSCN at room temperature was greater than that in 50% formamide at 42 degrees C. Hybridizations were performed on DNA and RNA in cells dissolved in GuSCN without prior nucleic acid purification.

Animals↗

PubMed1987

Separation of DNA restriction fragments by ion-exchange chromatography on FPLC columns Mono P and Mono Q.

Separation of DNA restriction fragments by FPLC ion-exchange chromatography on Mono Q and Mono P columns was investigated. The columns were found to be particularly suitable for the separation of fragments up to 500-600 bp long. Larger fragments can also be separated although less effectively. We found the following practical working ranges for the parameters investigated: pH, 4 to 11; flow rate, 0.05 to 0.6 ml/min corresponding to separation times between 2 and 20 h. (better resolution is achieved at lower flow rates); gradient slope; between 0.5 mM eluting salt/ml buffer and over 5 mM/ml (better resolution is achieved at lower gradient slopes; eluting ionic strength was found to be independent of gradient slope); gradient composition, chloride salts of smaller monovalent cations eluted the DNA at lower ionic strengths but separations obtained were similar; additives, substances such as urea, formamide, and EDTA can be added without chromatographic effects; sample amount: amounts from 2.5 to 200 micrograms were applied, corresponding to single peak content of from 42 ng to 74 micrograms DNA. Yields were generally over 90% and the chromatographed DNA was fully accessible to restriction enzyme cleavage. Separations occurred predominantly according to DNA size, but AT-rich fragments were retarded in a predictable way.

Chromatography, High Pressure Liquid↗

PubMed1990

Synthesis of nucleic acid probes on membrane supports: a procedure for the removal of unincorporated precursors.

We have used DNA bound to small pieces of nylon membrane for the synthesis of radioactive probes. The DNA to be used for generating the probe(s) is first bound to nylon membranes and then introduced into the reaction mix. The labeling reaction takes place on the membrane and therefore allows easy removal of unincorporated precursors by simple washing for 1-2 min. The clean labeled probe is eluted from the membrane in formamide or in water and is ready for use. This DNA-membrane can be stored for reuse. Synthesis of probes on a solid support such as nylon membrane thus circumvents problems associated with chromatographic manipulations needed for the separation of labeled DNA from unicorporated precursors. Probes synthesized in this manner are as efficient in detecting nucleic acid sequences as those synthesized in solution.

Blotting, Northern↗

PubMed1990

Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA.

We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.

Azides↗

PubMed1988

Oxidation of cyanide to the cyanyl radical by peroxidase/H2O2 systems as determined by spin trapping.

The cyanyl radical was formed during the oxidation of potassium or sodium cyanide by horseradish peroxidase, lactoperoxidase, chloroperoxidase, NADH peroxidase, or methemoglobin in the presence of hydrogen peroxide. The spin adducts of the cyanyl radical with 5,5-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-alpha-phenylnitrone were quite stable at neutral pH. The identity of these spin adducts could be demonstrated using 13C-labeled cyanide and by comparison with the spin adducts of the formamide radical, a hydrolysis product of the cyanyl radical adduct. The enzymatic conversion of cyanide to cyanyl radical by peroxidases should be considered in addition to its well-known role as a metal ligand. Furthermore, since cyanide is used routinely as an inhibitor of peroxidases, some consideration should be given to the biochemical consequences of this formation of the cyanyl radical by the catalytic activity of these enzymes.

Cyanides↗

PubMed1992

Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi.

Previous studies have demonstrated that fungal pathogens of cyanogenic plants produce cyanide hydratase (CHT, EC 4.2.1.66), which converts HCN to formamide. Production of CHT in these fungi is thought to be a means of circumventing cyanide toxicity, and CHT is thus believed to be an important pathogenicity trait. In the present study, 13 species of fungi were assayed for CHT production, and all 7 species that were pathogens of sorghum, a cyanogenic plant, produced this enzyme. CHT was purified to apparent homogeneity from one of these sorghum pathogens, Gloeocercospora sorghi. The enzyme had a Km of 12 mM for KCN. Enzymatically functional CHT was obtained only as a large molecular entity of greater than 300 kDa. However, a polypeptide of approximately 45 kDa was identified as the only component of purified CHT detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 45-kDa polypeptide band could be resolved into three isozymes of pI 6.1, 6.3, and 6.5. Antibodies raised against the 45-kDa polypeptide inhibited the G. sorghi CHT activity and showed high specificity in Western blots to a polypeptide of approximately the same size. The evidence suggests that functional G. sorghi CHT is an aggregated protein that consists of 45-kDa polypeptides. A CHT with similar properties was also found in the fungus Colletotrichum graminicola, another pathogen of sorghum.

Chromatography, Ion Exchange↗

PubMed1985

Peptidoglycan from the potentially pathogenic oral bacterium Actinomyces viscosus is a B-cell mitogen.

Cell walls and peptidoglycan from Actinomyces viscosus, strain M-100 were compared for their ability to act as mitogens with spleen cells from germ-free Fischer rats. The cell walls were prepared from trypticase soy broth grown whole cells using a French press, followed by two consecutive washes with 0.1 M tris-HCl buffer, pH 8.0, 1 M NaCl and distilled water. Peptidoglycan was prepared from cell walls by three consecutive formamide extractions at 165 degrees C. On a dry-weight basis, the peptidoglycan was a significantly-better mitogen than cell walls, suggesting that the peptidoglycan is the major mitogenic component of A. viscosus cell walls. Mononuclear spleen cells were separated on a Nylon-wool column into a non-adherent subpopulation enriched for T lymphocytes and a weakly-adherent, plunger-removable subpopulation enriched for B lymphocytes. The non-adherent T-cell subpopulation responded strongly to the T-cell mitogen PHA, but was unresponsive to both the peptidoglycan and cell walls from A. viscosus. In contrast, the weakly-adherent enriched B-cell subpopulation was less responsive to PHA, but was strongly stimulated by A. viscosus peptidoglycan and cell walls. These results indicate that peptidoglycan and cell walls from A. viscosus are B-cell mitogens.

Actinomyces↗

PubMed1993

New method for the isolation of polyglycosylceramides from human erythrocyte membranes.

A new procedure was developed for the isolation of long-chain, highly polar glycosphingolipids from human erythrocytes. The membrane material left after extraction of membrane lipids with organic solvents was peracetylated in a mixture of formamide, pyridine and acetic anhydride, and the acetylated products were then extracted with chloroform. The material was fractionated and purified by means of Sephadex LH-20, Sephadex LH-60 and silica-gel chromatography. The final preparations were mixtures of highly polar glycosphingolipids containing from 7 to 31 sugar residues relative to sphingosine. GC-MS analysis of the sugar part of the isolated fractions showed the presence of branched polyglycosyl chains of N-acetyllactosaminyl type. Endo-beta-galactosidase (Bacteroides fragilis) liberated from the deacetylated material two glycosphingolipids, which were identified by fast atom bombardment-mass spectrometry as Hex-Cer and HexNAc-Hex-Hex-Cer with sphingosine and mainly 24 and 22 carbon fatty acids. Endoglycoceramidase (Rhodococcus) degraded polyglycosylceramides to free ceramides and free polysaccharides. The released sugars were fractionated by high-pH ion-exchange chromatography into fractions differing in sialic acid content. The procedure presented in this paper can be used for large and small scale preparations of complex glycosphingolipids. It proved to be especially suitable for screening for polyglycosylceramides in different tissues.

Carbohydrate Sequence↗

PubMed1975

Mitochondrial DNA of Tetrahymena pyriformis strain ST contains a long terminal duplication-inversion.

1. We have studied denatured Tetrahymena mtDNA by electron microscopy using the formamide technique. 2. After denaturation all DNA is single stranded, but within a few minutes single-stranded circles with a duplex tail are formed. 3. The duplex tail is 1.3 mum long, i.e. 8 percent of the length of native mtDNA, and it often contains a small single-stranded eye. 4. Digestion of the duplex DNA with exonuclease III of Escherichia coli abolishes its ability to form circles and duplex tails after denaturation. 5. Renaturation of denatured mtDNA leads to the formation of duplex circles with single-stranded section and/or duplex tails. In addition, a minority of duplex circles without apparent tails is formed, but these circles contain a small ambiguous section. 6. We conclude that this mtDNA contains a long terminal duplication-inversion, that could be involved in the replication of this linear mtDNA.

Animals↗

PubMed1975

Inhibitor of protein synthesis co-isolating with polyribosomal RNA.

Although low concentrations of total polyribosomal RNA from porcine parotid glands or rat pituitary cells in culture (GH3) isolated by standard dodecylsulphate/phenol, chloroform extraction techniques can direct the incorporation of radiolabeled amino acids into proteins using a cell-free protein-synthesizing system derived from wheat germ embryos, higher concentrations inhibit the translation of endogenous wheat germ mRNA, or added rabbit globin mRNA or polyribouridylic acid. This inhibitory activity is separated from poly(A)-rich RNA by oligo(dT)-cellulose chromatography. The inhibitory activity appears to reside in a heat-stable protein since it is inactivated by incubation with various proteases but not by DNAase I, pancreatic ribonuclease, alkaline hydrolysis, or treatment with formamide. Specificity of the inhibition is suggested since the inhibitory fraction prepared from GH3 cells also inhibits protein synthesis in a cell-free protein-synthesizing system derived from porcine parotid gland, while the inhibitory fraction prepared from porcine parotid gland has no inhibitory activity in this homologous system. Radioiodination and dodecylsulphate/polyacrylamide gel electrophoresis reveal several protein bands, the most prominent with an apparent molecular weight of 78 000.

Animals↗

PubMed1976

Molecular weight determinations on the alpha-globin and beta-globin mRNAs.

The globin mRNAs containing between 30 and 40 polyadenylate residues can be separated from thos mRNAs containing longer poly(A) regions by Millipore filter binding. The molecular weights of the alpha-and beta-globin mRNAs containing this size class of poly(A) have beed determined by lectrophoresis on 3.7% polyacrylamide gels in the presence of 99% formamide. Because the number of adenylic acid residues in these mRNAs is known, the number of non-poly(A) nucleotides can be accurately calculated. The molecular weight of the beta-globin mRNA is 235 000 +/- 28 000 (736 +/- 88 nucleotides) and that of the alpha-globin mRNA is 208 900 +/- 43 870 (653 +/- 78 nucleotides). By subtracting the number of nucleotides in the coding and poly(A) regions, the number of non-coding nucleotides in the beta-globin mRNA were calculated to be 261, 69 more than the 193 present in the alpha-globin mRNA. Comparison of size estimates of newly synthesized globin mRNAs containing longer average lengths of poly(A) shhowed that there is no comparable processin of the 5' termini of the alpha-and beta-globin mRNAs concomitant with the stepwise degradation of the poly(A) regions which occur as the mRNAs mature.

Animals↗

PubMed1976

Modified nucleosides and 5'-end groups in purified mouse immunoglobulin light chain mRNA and rabbit globin mRNA detected by borohydride labelling.

The borohydride reaction has been used to investigate modified nucleosides and end groups in purified immunoglobulin light chain mRNA and rabbit globin mRNA. 1. The light chain mRNA was isolated from the microsomal fraction of MOPC 41A mouse myelomas, which secrete kappa chains, by two cycles of oligo(dT) cellulose chromatography and glycerol gradient centrifugation. The 12 S mRNA was active in a Krebs II ascites cell-free system and appeared to be homogenous as judged by gradient centrifugation, polyacrylamide gel electrophoresis in 98% formamide and fingerprint analysis of 125I-labelled mRNA. 2. End group labelling of the light chain and globin mRNAs by oxidation with periodate and reduction with boro[3H]hydride showed that the RNAs have a 5'-terminal 7-methyl guanosine in 5'-pyrophosphate linkage with the next nucleoside. 3. To detect any modified residues in the interior of chains, nucleosides in complete digests of the mRNAs were converted by the borohydride reaction to 3H-labelled nucleoside trialcohols, which were fractionated by two dimensional chromatography (the Randerath technique). The light chain mRNA was found to contain N6-methyl adenosine (1 mole) but the rabbit globin mRNAs lacked this nucleoside. Deficiencies in this technique for analysis of minor constituents in large RNAs were noted.

Animals↗

PubMed1977

Ribonucleic acid from the higher plant Matthiola incana. Molecular weight measurements and DNA-RNA hybridisation studies.

The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.

Base Sequence↗

PubMed1977

Purification and properties of soybean leghemoglobin messenger RNA.

Poly(A)-containing leghemoglobin mRNA from soybean root nodules has been purified 84-fold, as judged by its ability to direct the in vitro synthesis of leghemoglobin in a wheat germ system. It has a poly(A) content of 8.6% and a molecular weight, estimated by formamide gel electrophoresis, of 260 000. mRNA with a molecular weight of around 143 000 would be sufficient to code for leghemoglobin. Thus, with respect to both its poly(A) content and its unexpectedly high molecular weight, leghemoglobin mRNA is similar to mRNAs isolated from animal tissues.

Hemeproteins↗

PubMed1978

Estrogen withdrawal in chick oviduct. Characterization of RNA synthesized in isolated nuclei using a mercurated precursor.

1. As a prerequisite for analyzing the effect of estrogen on transcription in chick oviduct, we describe suitable methods for the synthesis (under conditions restricting reinitiation), and isolation of RNA transcripts from oviduct nuclei in vitro, utilizing mercurated UTP (Hg-UTP) as an RNA precursor and chromatography on sulphydryl-Sepharose (SH-Sepharose) to recover mercurated RNA (Hg-RNA). The techniques described include treatment of Hg-RNA with p-hydroxymercuribenzoate, to improve the efficiency of binding to SH-Sepharose, and elution of Hg-RNA from SH-Sepharose after treatment with 60% formamide at 90 degrees C, to eliminate contamination by aggregated nucleic acid. 2. RNA synthesized by endogenous form B RNA polymerase (using either UTP or Hg-UTP as precursor) was recovered in nuclear lysates in the form of 30--85-S heterogeneous RNA . protein complexes, and after removal of protein, was 10--12 S in size. 3. The nature of RNA transcripts synthesized in vitro was examined by hybridization. More than 90% of the RNA was complementary to "unique" DNA sequences, and 50--60% of the hybridized RNA could be competed with homologous, steady-state nuclear RNA, indicating a significant degree with homologous, steady-state nuclear RNA, indicating a significant degree of homology between in vitro transcripts and in vivo RNA. The level of homology was similar whether RNA synthesis was performed in low salt, or in high salt in the presence of heparin. Possible reasons for only partial competition in these experiments are discussed. 4. Withdrawal of estrogen from chicks leads to a 50% reduction in endogenous RNA polymerase activities in nuclei within 48 h. Similar levels of competition with Hg-RNA transcripts for "unique" DNA were obtained using oviduct nuclear RNAs isolated before or after estrogen withdrawal, and even with liver nuclear RNA. Thus, in oviduct, those sequences present in primary transcripts, and analyzed under our experimental conditions, are present in different hormonal states and also in other chick tissues.

Amanitins↗

PubMed1978

The putative 15 S precursor of globin mRNA contains a poly (A) sequence.

[3H] Uridine or [3H] adenosine pulse-labelled nuclear RNA was isolated from chicken immature red blood cells and separated on denaturing formamide sucrose gradients. RNA of each gradient fraction was hybridized with unlabelled globin DNA complementary to mRNA (cDNA) and subsequently digested by RNAase A and RNAase T1. The experiments revealed two RNA species with globin coding sequences sedimenting 9 S and approx. 15 S, the latter probably representing a precursor of 9 S globin mRNA. A poly (A) sequence was demonstrated in this RNA by two different approaches. Nuclear RNA pulse-labelled with [3H] uridine was fractionated by chromatography on poly (U)-Sepharose. Part of the 15 S precursor was found in the poly(A)-containing RNA. In the second approach 15 S RNA pulse-labelled with [3H]adenosine was hybridized with globin cDNA, incubated with RNAase A and RNAase T1 and subjected to chromatography on hydroxyapatite. The hybrids were isolated and after separation of the strands degraded with DNAase I, RNAase A and RNAase T1. By this procedure poly(A) sequences of approximately 100 nucleotides could be isolated from the 15 S RNA with globin coding sequences. The poly(A) sequence was completely degraded by RNAase T2.

Animals↗

PubMed1978

Messenger RNA synthesis in synchronized Chinese hamster ovary cells.

Chinese hamster ovary cells were synchronized without inhibitors by mitotic selection and labelled in G1, S or G2 phase by incubation for 90 min with [3H]- OR [14C]uridine. Purified polyribosomes were extracted with phenol and the polyadenylated mRNA prepared by poly(U)-Sepharose chromatography. Poly-adenylated [3H]uridine-labelled mRNA from the G1 phase of the cell cycle was compared by exponential polyacrylamide gel electrophoresis in formamide with [14C] uridine-labelled polyadenylated nRNA from the S or G2 phase. The electrophoretic patterns obtained correspond to the size range expected for mRNA (7-28 S). No prominent differences were detected between mRNAs synthesized in different phases of the cell cycle. From these data we conclude that the major size classes of polyribosomal poly(A)-containing mRNA are synthesized in equal ratios throughout the cell cycle.

Cell Cycle↗

PubMed1979

Inserts within the mRNA sequence on the 15 S globin mRNA precursor from chicken.

[3H]Uridine pulse-labelled nuclear RNA of chicken immature red blood cells contains two RNA species with globin coding sequences sedimenting at 9 S and 15 S, the latter representing most probably the precursor to 9 S globin mRNA. Whether the globin mRNA sequence on the 15 S precursor is interrupted by interspersed sequences was investigated by hybridization of labelled nuclear 15 S RNA with an excess of unlabelled globin complementary DNA. After degradation of nonhybridized RNA by RNAase A the enzyme was removed by proteinase K and a subsequent phenol extraction. The complementary DNA hybrids were melted and the complementary DNA degraded by DNAase I. For the separation of complementary DNA protected RNA polyacrylamide gel electrophoresis in formamide was used. Three RNA fragments with approximately 320, 200 and 120 nucleotides were found in the case of the 15 S RNA, whereas only one 650-700 nucleotide fragment was detected when the same procedure was carried out with nuclear 9 S RNA. This means, that the chicken 15 S globin mRNA precursor contains at least two inserts within the mRNA sequence, which originate presumably by transcription of a gene with intervening sequences.

Animals↗