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PubMed1974

Localized molecular orbital description of nitrogen lone pairs.

The Boys localization procedure is applied to several molecules possessing nitrogen lone pairs. The Boys structures show a tendency for the N lone pairs to participate in bonding to nearby electron sinks. For example, the Boys structure for planar formamide has two equivalent tau-bond orbitals between the carbonyl carbon and nitrogen atoms. The change in this description with variation of geometry at nitrogen is treated, and analogous Edmiston-Ruedenberg localizations on formamide are included for comparison.

Journal Article↗
PubMed1975

Further evidence of a quantitative deficiency of chain-specific globin mRNA in the thalassemia syndromes.

Formamide gel electrophoresis separates the mRNA fraction from reticulocyte polyribosomes of adult humans into two major RNA species with migratory rates identical to those of the alpha- and beta-globin mRNAs of the rabbit. That these two RNAs of human origin are the globin mRNAs is further supported by the deficiency of the presumed beta mRNA in reticulocyte polyribosomes of fetuses and premature infants, whose cells make gamma chains in preference to beta chains. The globin mRNAs of reticulocyte polyribosomes from patients with hematological disorders were estimated by scanning the stained formamide gels. In contrast to individuals with either hemolytic anemia without hemoglobinopathy or sickle cell anemia who had beta mRNA to alpha mRNA ratios of approximately one, a patient with Hb S-beta-thalassemia had a ratio of beta mRNA to alpha mRNA of 0.75 while two subjects with homozygous beta-thalassemia had severe deficiencies of beta mRNA. Conversely, a patient with alpha-thalassemia (Hb H disease) had a ratio of beta mRNA to alpha mRNA on reticulocyte polyribosomes of 6. These data provide further evidence of a quantitative deficiency of chain-specific globin mRNA in patients with the thalassemia syndromes.

Adult↗
PubMed1989

Electron microscopy of supercoiled pEJ4 DNA containing homopurine.homopyrimidine sequences.

Supercoiled pEJ4 DNA (a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from the sea urchin P. miliaris histone gene spacer) was investigated by electron microscopy using three different spreading techniques i.e., formamide and aqueous variants of the Kleinschmidt technique and protein-free benzyldimethyl-alkyl ammonium chloride (BAC) technique at different pHs. If the specimens for electron microscopy were prepared at pH 5.6 and pH 4.0 (i.e., under conditions where the homopurine.homopyrimidine tract assumes an unusual conformation) a single thick "stem" or a "denaturation bubble" in a large number of DNA molecules were observed. No such changes were found in samples prepared at neutral pH and in linearized pEJ4 DNA prepared at pH 5.6. In specimens of a control supercoiled pUC19 DNA prepared at pH 5.6 and 4.0 practically no local changes were detected. The "denaturation bubbles" were observed by BAC techniques (probably due to secondary local DNA denaturation during the specimen preparation) while the more gentle formamide technique revealed only "stems". The "stems" were almost always positioned at the sites where the curvature of supercoiled DNA molecules occurred. The results are in agreement with presence of a protonated triplex H-form in homopurine.homopyrimidine tract bringing the first evidence of curvature or kinking of the DNA molecule connected with the occurrence of the H-form in supercoiled DNA.

Base Sequence↗
PubMed2001

Unusual DNA binding exhibited by synthetic distamycin analogues lacking the N-terminal amide unit under high salt conditions.

The binding of three analogues of the minor-groove binding antiviral antibiotic distamycin (Dst) with double-stranded (ds)-DNA were monitored using ds-DNA melting temperature (Tm) measurements, ethidium bromide (EtBr) displacement assay, footprinting analysis and induced circular dichroism (ICD). These compounds contained 3-5 N-methyl-pyrrole-carboxamide units and lacked the N-terminal formamide unit present in Dst. These experiments suggested that the present analogues did not compromise their AT-specificity despite the deletion of the N-terminal formamide unit. The binding affinities, however, were significantly affected. Interestingly, the analogue with three N-methyl-pyrrole-carboxamide units exhibited an initial decrease in ICD at > 40 mM salt concentrations. This was followed by a pronounced recovery of ICD at > 1.6 M salt concentrations, a phenomenon hitherto not observed with any other DNA binding molecules. The pentapyrrole analogue exhibited the highest binding affinity with CT-DNA under normal (40 mM) salt conditions. However, it suffered maximum relative dissociation under high salt conditions and did not exhibit any recovery in ICD at higher NaCl concentrations. The analogues possessing four and five pyrrole rings exhibited intense ICD signals with poly d(GC) in the ligand absorption region in the presence of 40 mM NaCl, unlike the one with three pyrrole rings. These ICD signals were however, highly susceptible to changes in ionic strength. Thus subtle modifications in the ligand molecular structure can have dramatic effect on their DNA binding properties.

Amides↗
PubMed1978

Free radicals in U.V.-irradiated aqueous solutions of substituted amides: an e.s.r. and spin-trapping study.

The radicals produced by reactions of hydroxyl radicals with alkyl substituted ureas and amides in aqueous solutions have been investigated. Hydroxyl radicals were produced by U.V. photolysis of H2O2 and the short-lived amide and urea radicals were spin-trapped by t-nitrosobutane and identified by e.s.r. For all N-alkyl derivatives of urea and acetamide, and for N,N-dimethyl propionamide and N,N-diethyl formamide, only radicals centred on N-alkyl groups were detected. Radicals situated only on alkyl groups attached to the carbonyl carbon were observed for dimethyl acetamide, trimethyl acetamide and butyramide. However, for N,N-dimethyl butyramide, N, N-diethyl butyramide, N-methyl propionamide and N, N-diethyl propionamide, free radicals were formed which were localized on the alkyl group attached to the amide carbon as well as those attached to nitrogen. The hydrogen atom bound to the carbonyl carbon was abstracted in N-ethyl formamide. Acyl radicals formed by C-N scission due to direct U.V. photolysis of N, N-dimethyl butyramide and N,N-dimethyl propionamide were also detected.

Acetamides↗
PubMed1962

Studies on the chemical structure of the streptococcal cell wall. II. The composition of group C cell walls and chemical basis for serologic specificity of the carbohydrate moiety.

The trypsinized cell walls of Group C streptococci contain two components, the group-specific carbohydrate and a mucopeptide polymer. Hot formamide extraction of Group C cell walls results in a soluble group-specific carbohydrate fraction and an insoluble mucopeptide residue. This mucopeptide, similar in composition to that of Groups A and A-variant streptococci, contains N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, lysine, and glycine. It is dissolved by the muralytic enzymes, including lysozyme, which does not attack the whole cell wall. Lysis of the cell wall by phage-associated lysin results in the release of soluble fragments composed of the elements of mucopeptide. Group C carbohydrate extracted with formamide is composed primarily of N-acetylgalactosamine and rhamnose. Serological studies suggest that the specificity of Group C carbohydrate is determined by the N-acetylgalactosamine.

Carbohydrates↗
PubMed1971

Tracer determinations of human red cell membrane permeability to small nonelectrolytes.

A flow system has been used to determine the permeability of human red cell membranes to four small nonelectrolytes labeled with (14)C. The permeability coefficients, omega, in units of mol dyne(-1) sec(-1) x 10(15), are: ethylene glycol, 6; urea, 13; formamide, 22; and methanol, 131. The values for urea and formamide are in good agreement with values obtained by Sha'afi, Gary-Bobo, and Solomon by the minimum method. The unusually high value for omega for methanol is ascribed to its solubility in the red cell membrane since its ether: water partition coefficient is 0.14, higher by more than an order of magnitude than the ether: water partition coefficient for water. The other three solutes are hydrophilic and are characterized by values of omega which behave consistently with those of other hydrophilic amides and ureas. The values of omega for the three hydrophilic solutes measured are also consistent with an equivalent pore radius of about 3.5 A in agreement with previous estimates made on the basis of other types of studies.

Amides↗
PubMed1994

Released chromatin: linearized DNA for high resolution fluorescence in situ hybridization.

Free DNA was prepared from routinely harvested and fixed cells for high resolution FISH mapping using either a sodium hydroxide/ethanol mixture or 70% formamide. Hybridization signals from cosmid probes appeared as extended lines. The average length of signals on DNA prepared with sodium hydroxide was significantly greater than with formamide. A set of overlapping cosmids from the HLA class II region was used to determine how precisely the actual overlap or gap between probes can be calculated from the measured overlap or gap between their signals. Lengths of the probe signals and their known kilobase lengths were used as an internal ruler. The mean values calculated from the measured length from 30 or more signals for each probe pair showed remarkable conformity with the known kilobase lengths of their overlaps and gaps. Immediately adjacent probes could also be ordered on the released DNA. These simple procedures dramatically increase the speed with which relationships between probes can be determined during contig construction.

Chromatin↗
PubMed1983

Heterology of mitochondrial DNA from mammals detected by electron microscopic heteroduplex analyses.

Heteroduplex analysis of mitochondrial DNA (mtDNA) from evolutionary closely related mammals (rat vs. mouse, man vs. monkey) are analyzed and compared to heteroduplex analysis of mt-DNA from more distantly related mammals (rat vs. man, rat vs. monkey, mouse vs. man, mouse vs. monkey and man vs. cow). Each analysis is transformed into a heteroduplex map and all maps are aligned to restriction enzyme maps and to genetic maps and where possible compared with the known sequence. We show that early evolutionary changes are seen mainly in URF2, URFA6L, URF6 and the D-loop region. The regions of rRNA, URF1, COI and COIII are generally very conserved regions but areas with some evolutionary activity can be localized. Heteroduplex analysis between distantly related species show much more heterology than do closely related species and the heteroduplex maps between all the distantly related species show a common pattern of heterology. Comparisons between the DNA sequence of mtDNA from man, cow and mouse and the equivalent heteroduplex maps show that base pair homologies higher than 73% are displayed as homologous regions. In the heteroduplex analysis of mtDNA's from more closely related species very few heterologies are displayed at 50% formamide but an increase in formamide concentration to 65-70% demonstrate also in these instances general heterologous regions.

Animals↗
PubMed1976

Size and structure of the genome of infectious pancreatic necrosis virus.

The genome of infectious pancreatic necrosis virus consists of two segments of dsRNA, in equimolar amounts, with molecular weights of 2.5 X 10(6) and 2.3 X 10(6) daltons, as determined by polyacrylamide gel electrophoresis and autoradiography. The viral RNA was resistant to ribonuclease, and in sucrose gradient it co-sedimented at 14S with RNase resistant RNA from virus infected cells. Upon denaturation in 98% formamide, the viral genome sedi-mented at 24S in formamide sucrose gradient and became sensitive to RNase. Denatured 24S viral RNA did revert to its undenatured 14S form upon recentrifugation in aquaeous sucrose gradient (0.1 M NaCL), but co-sedimented with the denatured large size class of reovirus 25S RNA. The same results were obtained if the native viral RNA was pre-treated with ribonuclease before denaturation, indicating the absence of exposed single strainded regions in the viral genome. Since infectious pancreatic necrosis virus contains only two dsRNA segments it does not belong to the family Reoviridae and may represent a new group of viruses.

Animals↗
PubMed1978

RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and mercury-labeled CTP.

Isolated nuclei of lactating mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25 degrees C. In presence of unmodified CTP, [3H]UMP incorporation into RNA increased linearly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essentially similar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibited by alpha-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A)+ and poly(A)- RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose affinity column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cell nuclei in vitro should now permit monitoring hormonal regulation of specific gene (casein) transcription in the mammary cells by molecular hybridization of the Hg-RNA with cDNA to casein mRNA.

Amanitins↗
PubMed1980

Different base per unit length ratios exist in single-stranded RNA and single-stranded DNA.

A significant difference was found to exist in the number of bases per unit length of single-stranded RNA as compared to single-stranded DNA when single-stranded RNA or DNA molecules of known nucleotide sequence were measured by electron microscopy using a cytochrome spreading technique. Using this technique, single-stranded RNA was found to have 17.5% more bases per unit of length than single-stranded DNA. These ratios were verified using four different denaturing conditions for the RNA: 80% formamide, 80% formamide plus glyoxal, 80% formamide/4M urea and 80% formamide/4M urea plus glyoxal. Molecules ranging in size from 1541 to 5386 nucleotides were examined and the number of bases per unit length was found to vary inversely with micrometer was consistent when RNA and DNA molecules of the same length were compared.

DNA, Single-Stranded↗
PubMed1984

Rat cytomegalovirus: studies on the viral genome and the proteins of virions and nucleocapsids.

The nucleocapsids (N-capsids) isolated from the nuclei of rat cytomegalovirus (RCMV)-infected rat embryo fibroblasts (REF) are composed of three major proteins: 142 X 10(3) (142K), 40K and 32K mol. wt. Nucleocapsids isolated from the cytoplasmic fraction (C-capsids) are composed of proteins found in N-capsids and five major and seven minor new protein species. Most of the proteins present in C-capsids are found in the extracellular enveloped virions, although the ratios vary. Proteins that are abundantly present, particularly in virions (mol. wt. 125K, 116K, 87K, 79K, 71K, 68K, 62K, 50K, 43K and 28K), are probably the major constituents of the viral envelope. The DNA recovered from extracellular virions was purified to homogeneity and by equilibrium centrifugation in CsCl one density class of 1.716 (+/- 0.001) g/ml was found. Contour length measurements showed one size class of a linear double-stranded DNA corresponding to an average mol. wt. of 144(+/-9) X 10(6) which is in good agreement with data obtained by restriction endonuclease analysis (REA), which yielded mol. wt. values of 132(+/-9) X 10(6) (HindIII), 138(+/-2) X 10(6) (EcoRI) and 137 X 10(6) (BglII). The REA patterns also revealed the presence of 0.25 M and 0.5 M fragments, which might indicate, in analogy with other cytomegalo- and herpesviruses, the existence of four different configurations of the RCMV genome. The infectivity of RCMV DNA was determined in subconfluent REF monolayers. A cytopathic effect characteristic of RCMV was observed 6 days post-transfection and up to 60 plaques/microgram DNA were obtained. Using DNA-DNA filter hybridization the degree of homology between the genomes of RCMV and murine or human CMV was examined. Under stringent conditions (50% formamide) values of 12(+/-2)% and 3(+/-1)% were found whereas under non-stringent conditions (20% formamide) values of 21(+/-2)% and 6 (+/-1)% were obtained, respectively.

Animals↗
PubMed1988

Utilization of histidine by Caulobacter crescentus.

Caulobacter crescentus has an inducible pathway which is responsible for the degradation of histidine. Induction of this pathway occurs in the presence of both glucose and ammonia. Growth yield experiments indicate that only two of the three available nitrogens are used for growth suggesting that formamide may be produced as a waste product. However, formamide was not detected in the culture fluid and formate was formed instead. These results suggest that histidine may be degraded in a novel pathway which results in the production of 1 mol each of ammonia, glutamate and formate per mol of histidine. The third nitrogen from histidine appears to be sequestered in some kind of secondary metabolite.

Gram-Negative Bacteria↗
PubMed1993

Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene.

The filamentous fungus Fusarium lateritium is cyanide tolerant, due, at least in part, to the induction by cyanide of the enzyme formamide hydrolyase (EC 4.2.1.66). This enzyme, more commonly known as cyanide hydratase, catalyses the hydration of cyanide to formamide. The enzyme was purified from F. lateritium and showed a subunit molecular mass of 43 kDa (as judged by SDS-PAGE), while the native protein appeared to form aggregates of up to 1217 kDa (as judged by gel-filtration and non-denaturing PAGE). mRNA samples from cultures grown with and without cyanide were in vitro translated and immunoprecipitated. This demonstrated that, in this species, the gene encoding the enzyme designated chy1, is cyanide inducible. Differential screening was used to isolate a cyanide hydratase cDNA clone which was subsequently used to obtain the corresponding genomic clone. A fragment of the cDNA clone encoding all but the first seven amino acids of the protein was expressed in E. coli using the expression vector pGEX-2T. Features of F. lateritium cyanide hydratase together with an analysis of the nucleotide sequence encoding this enzyme are presented.

Amino Acid Sequence↗
PubMed1997

An outer-membrane porin inducible by short-chain amides and urea in the methylotrophic bacterium Methylophilus methylotrophus.

The fmdA and fmdB genes encoding formamidase and a putative regulatory protein, respectively, from the methylotrophic bacterium Methylophilus methylotrophus were recloned with additional flanking DNA (pSW1). fmdC, encoding a weakly hydrophilic protein containing an N-terminal signal sequence, was identified upstream of fmdAB. The derived amino acid sequence of mature FmdC (M(r) 39204) showed that it was rich in beta-sheet and aromatic amino acids, and exhibited significant similarities to several outer-membrane porins from other bacteria. Cell fractionation studies showed that the protein was located in the outer membrane. Mature FmdC was purified and shown to consist of a single type of subunit (M(r) 40,000) with the predicted N-terminal amino acid sequence (GATISF-). SDS-PAGE and Western blotting of cells grown in continuous culture under various conditions showed that mature FmdC was induced by formamide, acetamide and urea, repressed by excess ammonia, and over-expressed during prolonged growth under formamide limitation. It is concluded that mature FmdC is a porin involved in the transport of short-chain amides and urea through the outer membrane of M. methylotrophus under conditions where these nitrogen sources are present at very low concentration.

Amides↗
PubMed1992

The Isolation of Actin from Pea Roots by DNase I Affinity Chromatography.

Native actin can be isolated from pea (Pisum sativum L.) roots by DNase I affinity chromatography, but the resulting yields and quality of actin are variable. By use of two assays for actin, a DNase I inhibition assay and a gel scanning assay, we identified several factors that increased actin yield. ATP is required for the actin in crude pea root extracts to bind to immobilized DNase I. Low amounts of ATP are hydrolyzed rapidly by an endogenous ATPase in the extract, and the actin then irreversibly loses the ability to bind to DNase I. High ATP concentrations (5-10 mm) or inhibition of the ATPase (with 10 mm pyrophosphate) are required for pea actin to retain DNase I binding ability. When adequate amounts of ATP are present, actin binding from the extract is further enhanced by basic pH, formamide, and soluble polyvinyl-pyrrolidone. Once actin is bound to the DNase I-agarose and washed free of extract, high ATP concentrations are not required to keep actin bound. Actin eluted from the DNase I-agarose with formamide retained its ability to polymerize into filaments with the addition of KCl and Mg(2+). The advantages and disadvantages of this procedure and its application to other plant materials are discussed.

Journal Article↗
PubMed1980

Reassociation Kinetics and Cytophotometric Characterization of Peanut (Arachis hypogaea L.) DNA.

The base composition of peanut (var. NC-17) DNA determined from thermal denaturation profiles showed an average guanine plus cystosine content of 34% which was in close approximation to 36% guanine plus cytosine calculated from the buoyant density. Buoyant density also indicated the absence of satellite DNA. The genome size, 2.0 x 10(9) base pairs, as determined by reassociation kinetics of the single copy DNA was close to the genome size determined by cytophotometry, 2.1 x 10(9) base pairs. Peanut DNA averaging 450 to 600 base pairs long, reassociated in phosphate buffer and fractionated by hydroxylapatite, indicated a DNA genome composition of 36% nonrepetitive or single copy DNA; reassociation in formamide and followed by optical methods indicated the repetitive DNA possesses highly repeated, intermediately repeated and rarely repeated components of DNA with DNA sequences repeated on the average about 38,000, 6,700, and 200 times each. Different criteria of reassociation in formamide revealed further subdivisions of these four separate components of DNA. The DNA of above mentioned NC-17 variety compared to Florigiant variety showed no differences in thermal denaturation profiles, buoyant density, or in genome size.

Journal Article↗