Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Formamides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources
PubMed1974

Glycerolphosphate-containing cell wall polysaccharides from Streptococcus sanguis.

Six glycerolphosphate-containing tetraheteroglycans, a, b-1, b-2, b-3, b-4, and b-5, have been purified from the formamide extracts of Streptococcus sanguis by alcohol and acetone precipitations, Sephadex G-75, and diethylaminoethyl-cellulose column chromatography. The polysaccharides were judged as at least 95% pure by analytical disc gel electrophoresis and immune double diffusion against rabbit antiserum. They were shown to be cell wall polysaccharides, since they formed a single band of identity in immune double diffusion with partially purified polysaccharide extracted from a purified cell wall preparation of S. sanguis. The polysaccharides were composed of l-rhamnose, d-glucose, and N-acetyl d-glucosamine in a similar molar ratio, but varied in their glycerol and phosphate contents. They exhibited four different mobilities in polyacrylamide disc gel electrophoresis at pH 8.9. When they were treated with formamide at 170 C for 20 min, the faster moving polysaccharide(s) yielded polysaccharides with mobilities corresponding to the other slower moving polysaccharides. These results indicate that the polysaccharides originated from the same cell wall polysaccharide and were produced as a result of breakage in the phosphodiester bonds during the formamide extraction procedure. A preliminary structural study shows that the terminal reducing sugar is l-rhamnose and that the glycerol moiety is probably linked to the polysaccharide through a phosphodiester bond.

Animals↗
PubMed2004

Enzymatic characterization of a prokaryotic urea carboxylase.

We identified the first prokaryotic urea carboxylase (UCA) from a member of the alpha subclass of the class Proteobacteria, Oleomonas sagaranensis. This enzyme (O. sagaranensis Uca) was composed of 1,171 amino acids, and its N-terminal region resembled the biotin carboxylase domains of various biotin-dependent carboxylases. The C-terminal region of the enzyme harbored the Met-Lys-Met motif found in biotin carboxyl carrier proteins. The primary structure of the enzyme was 45% identical to that of the urea carboxylase domain of urea amidolyase from Saccharomyces cerevisiae. O. sagaranensis Uca did not harbor the allophanate hydrolase domain found in the yeast enzyme, but a separate gene with structural similarity was found to be adjacent to the uca gene. Purified recombinant O. sagaranensis Uca displayed ATP-dependent carboxylase activity towards urea (V(max) = 21.2 micro mol mg(-1) min(-1)) but not towards acetyl coenzyme A (acetyl-CoA) and propionyl-CoA, indicating that the gene encoded a bona fide UCA and not an acetyl-CoA or propionyl-CoA carboxylase. The enzyme also exhibited high levels of activity towards acetamide and formamide. Kinetic parameters of the enzyme reaction were determined with ATP, urea, acetamide, and formamide. O. sagaranensis could grow on urea, acetamide, and formamide as sole nitrogen sources; moreover, ATP-dependent urea-degrading activity was found in cells grown with urea but not in cells grown with ammonia. The results suggest that the UCA of this organism may be involved in the assimilation of these compounds as nitrogen sources. Furthermore, orthologues of the O. sagaranensis uca gene were found to be widely distributed among Bacteria. This implies that there are two systems of urea degradation in Bacteria, a pathway catalyzed by the previously described ureases and the UCA-allophanate hydrolase pathway identified in this study.

Alphaproteobacteria↗
PubMed1990

Serotypic differentiation of group A rotaviruses with porcine rotavirus gene 9 probes.

The serotypic specificities of Gottfried and OSU porcine rotavirus gene 9 probes were investigated in a dot hybridization assay. The probes were reacted with homologous and heterologous serotypes of group A rotaviruses of human and animal origin. Hybridizations were conducted under relatively low-stringency (52 degrees C, no formamide, 5 x SSC) and high-stringency (52 degrees C, 50% formamide, formamide, 5 x SSC) conditions (1 x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Under conditions of relatively low stringency, the Gottfried and OSU gene 9 probes demonstrated broad cross-reactivity and were useful in the detection of homologous and heterologous serotypes of group A rotaviruses. Under conditions of relatively high stringency, the Gottfried and OSU gene 9 probes were serotype specific. The Gottfried gene 9 probe (serotype 4) hybridized with homologous Gottfried porcine rotavirus as well as the serotype 4 human rotaviruses ST3 and VA70. The OSU gene 9 probe (serotype 5) hybridized with homologous OSU porcine rotavirus and the serotype 5 equine rotavirus H1. Hybridization was not observed with the antigenically distinct group B and C porcine rotaviruses or with other porcine enteric viruses, including calicivirus and a coronavirus, transmissible gastroenteritis virus, regardless of stringency conditions. Analysis of 14 group A rotavirus-positive field samples resulted in the serotypic differentiation, collectively, of six serotype 4 or 5 porcine rotaviruses. No field samples reacted with both the Gottfried and OSU gene 9 probes.

Animals↗
PubMed1987

Use of polar aprotic solvents to release membranes from milk lipid globules.

In the search for alternatives to physical methods for release of membranes from milk lipid globules, aqueous solutions of the polar aprotic solvents dimethyl sulfoxide and dimethyl formamide were found to cause release of milk fat globule membrane. Membranes released with polar, aprotic solvents displayed some differences in polypeptide, enzymatic, and lipid composition in comparison with membranes released by churning. However, all polypeptide, enzymatic, and lipid constituents measured in membranes released by churning were also present in membranes released with dimethyl formamide and dimethyl sulfoxide. Antigenic and Concanavalin A binding activity of polypeptides was retained during exposure to dimethyl sulfoxide or dimethyl formamide. The solvent method was effective in releasing membrane from lipid globules of cow, rat, and guinea pig milks. This solvent method was rapid and minimized losses attendant with physical methods for release of milk fat globule membrane.

Animals↗
PubMed1984

DNA homology studies in Streptomyces using S1 nuclease.

The optimal reaction conditions for the determination of DNA-homology in Streptomyces species were established in the presence of formamide using S1 nuclease. The melting temperature of Streptomyces DNA was 90 degrees C in 0.42 M NaCl containing 20% formamide in which the denaturation was completed by boiling for 5 minutes. In the S1 reaction mixture consisting of 5 U of S1 nuclease, 0.168 M NaCl, 1 mM ZnSO4 and 8% formamide at pH 4.8, single-stranded DNA was hydrolyzed by more than 98%, while the hydrolysis of double-stranded DNA was less than 3%. From the analysis of homoduplex formation, the C0t1/2 was found at 20 hours, when a mixture of unlabeled DNA and index DNA was used at a ratio of 500:1.

Base Sequence↗
PubMed1975

Physical and chemical characterization of purified ovalbumin messenger RNA.

Preparative agarose gel electrophoresis under denaturing conditions has been successfully employed to purify large quantities of ovalbumin mRNA from hen oviducts. The mRNA thus prepared is physically homogeneous based on its migration as a single component on electrophoresis in both analytical acid-urea agarose gels and formamide-containing, neutral polyacrylaminde gels; it also sediments as a single peak in sucrose gradients containing 70% formamide. The mRNA is chemically free of ribosomal RNA contamination since its oligonucleotide fingerprint map after complete T1 ribonuclease digestion contains no detectable specific large oligonucleotide markers of ribosomal RNAs. It is also not contaminated by other biologically active messenger RNAs because, when it is added to the cell-free wheat germ translation system, the only protein product synthesized is ovalbumin as analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and specific immunoprecipitation. Ovalbumin mRNA has a nucleotide composition of 32.3% A, 21.0% G, 25.7% U, and 20.7% C [(A+U)/(G+C) equal 1.41]. The mRNA contains a heterogeneous poly(A) tract ranging from 20 to 140 residues with a number average chain length of 62 adenylate residues. The molecular weight of the sodium salt of the purified mRNA is approximately 650,000 +/- 63,000, corresponding to a chain length of 1890 +/- 180 nucleotides, as determined by electron microscopy under completely denaturing conditions. This value is in close agreement with the values obtained from: (a) sucrose gradient centrifugation in the presence of 70% formamide; (b) evaluation of poly(A) content in the mRNA and the number average chain length of its poly(A) tract; and (c) sedimentation velocity studies in the presence of 3% formaldehyde. When 125I-labeled ovalbumin mRNA is allowed to hybridize with a large excess of chick DNA, the observed kinetics of hybridization reveal no appreciable reaction between the mRNA and the repeated sequences of the chick DNA, although the mRNA appears to be approximately 600 nucleotides longer than necessary to code for ovalbumin. It thus appears that the entire ovalbumin mRNA is primarily transcribed from a unique sequence in the chick genome.

Adenine Nucleotides↗
PubMed1977

The pathogenesis of experimental bladder cancer.

The pathogenesis of signal morphological lesions of the urinary bladder induced in several species following administration of N-butyl-N-(4-hydroxybutyl)nitrosamine, bracken fern, or N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide is presented. Incidences of bladder neoplasia exceeding 80% were generated in the rat by each compound. Bladder neoplasia was induced in the following species by each substance: by N-butyl-N-(4-hydroxybutyl)nitrosamine in the mouse, hamster, guinea pig, and dog; by bracken fern in the guinea pig, mouse, and cow; and by N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide in mouse, hamster, and dog. The guinea pig appeared resistant to the bladder oncogenicity of N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide. Different species displayed a gradient of bladder neoplastic responsiveness. Hyperplasia was a consistent early lesion and was usually focal. Early hyperplastic lesions regressed following removal of the carcinogenic stimulus, but later lesions appeared to be irreversible. These animal systems appear useful in providing opportunities for investigations relevant to human bladder cancer.

Animals↗
PubMed1985

Mechanistic implications of the induction of thermotolerance in Chinese hamster cells by organic solvents.

Several organic solvents were shown to induce thermotolerance. These included: aliphatic alcohols (ethanol to octanol); local anesthetics; dimethyl sulfoxide; and dimethyl formamide. The minimum concentrations of the various alcohols required to induce tolerance were similar to the threshold doses for cytotoxicity. When only intramembrane concentrations were considered (by multiplying by the appropriate membrane:buffer partition coefficient), then the alcohols' threshold dose to induce tolerance was only mildly a function of carbon number. The efficiency increased almost linearly with a membrane dose from propanol to pentanol, but was slightly reduced for octanol. All alcohols induced tolerance rapidly (within a few hours after 30-min exposure), but the thermotolerance ratio decreased with increasing carbon number. Of the three anesthetics examined, lidocaine was a highly efficient inducer, procaine less so, and tetracaine did not induce tolerance even at a concentration mildly cytotoxic. Dimethyl sulfoxide and dimethyl formamide induced tolerance without cytotoxicity, but the kinetics of doing so was different from that seen with the other solvents. Only cells exposed to these water-soluble agents for 18 h or longer showed evidence of induced heat resistance. Our results indicate that membranes likely are the site for the initiation of solvent-induced cell killing and thermotolerance. The minimum dose of induction may be related to the degree of disorder induced by the individual alcohol, but the amount of tolerance induced is inversely related to this. The data on the effects of local anesthetics show that neither their common functional mode of inhibiting nerve transmission nor their similar level of cytotoxicity insures similar efficiency as tolerance inducers. Results with dimethyl sulfoxide and dimethyl formamide suggest that tolerance can be induced by more than one mechanism, or alternately, that a multiple-step mechanism can be activated at different points along this pathway. We also measured heat shock protein synthesis after several treatments; efficient inducers of thermotolerance also induced these proteins.

Alcohols↗
PubMed1978

Topography and numerical densities of intramembrane particles in chemical carcinogen-induced urinary bladder carcinomas in Fischer rats.

Integral membrane proteins are visualized as intramembrane particles (IMP) at the cleaved surfaces of freeze-fractured plasma membranes. Topographical distributions of the IMP of the urinary bladder epithelial cells membranes in normal Fischer rat bladder and noninvasive and invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide (FANFT)-induced bladder tumors are shown to be significantly different. Using several statistical methods that test IMP topography vis-a-vis the Poisson (random) hypothesis, it is demonstrated that IMP are mathematically randomly distributed in the large majority of plasma membranes of cells in normal rat bladder epithelium and in invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide tumors. In noninvasive rat bladder carcinomas, IMP are in a lattice-like arrangement in half of the tumor cells and randomly distributed in the remainder. IMP numerical densities are also altered in the course of neoplastic transformation. IMP are equally increased above control values in both noninvasive and invasive N-(4-(5-nitro-2-furyl)-2-thiazolyl)formamide tumors. Although transformation into noninvasive tumors is associated with increased numbers of IMP, there is no evidence that this parameter is specifically related to tumor biologic behavior in this model system.

Animals↗
PubMed1980

Effects of urine and continued exposure to carcinogen on progression of early neoplastic urinary bladder lesions.

Based on reports of regression of superficial bladder tumors after urinary diversion, a study was designed to measure the effects of urine and continued exposure to carcinogen on the incidence of progression of N-[4-(5-nitro-2-furyl)-2-thiazolyl]-formamide-induced early urinary bladder lesions to invasive tumor. After being fed 0.2% N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide diet for 14 weeks, one-half of the male Fischer rats had urinary diversion by ureterosigmoidostomy, and the remainder were sham operated. One-half of each of these two groups was continued on the N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide diet while the remaining animals were fed regular chow postoperatively. One-half of each of the four groups was sacrificed at 3 months, and the remainder were sacrificed at 6 months after ureterosigmoidostomy or sham-operation. The incidence and mean number of tumors as well as the incidence of invasive tumor were tabulated. The combined 3- and 6-month data indicate that excreted carcinogen in the urine influences progression of the preinvasive lesions more than urine alone or systemic carcinogen alone. However, urine alone had a significant effect (p < 0.025) on tumor incidence (8 of 19 sham-operated animals with tumor versus 1 of 18 diverted animals with tumor). Urine acts as a promoter in this experimental system. These findings may have clinical applications in the treatment of early transitional cell carcinoma.

Animals↗
PubMedDate not supplied

Non-enzymatic, low temperature fluorescence in situ hybridization of human chromosomes with a repetitive alpha-satellite probe.

In all DNA-DNA in situ hybridization (ISH) procedures described so far in the literature, the production of single-stranded target DNA sequences plays a decisive role. This can be achieved either by enzymatic treatment at physiological temperatures or by the separation of double-stranded DNA sequences. Denaturation by heat and chemical agents (e.g. formamide) is regarded as a prerequisite for the non-enzymatic ISH process. However, additional mechanisms of a non-enzymatic ISH procedure are conceivable which do not require high temperature treatment combined with formamide. Here, we report on a non-enzymatic, non-formamide, low temperature, fluorescence, in situ hybridization (FISH) procedure which allowed a microscopic visualization and quantitative fluorescence analysis of the binding sites of a repetitive DNA probe. Following only probe denaturation at 94 degrees C, hybridization was performed at 52 degrees C for 30 min, i.e. at nearly physiological temperatures. Moreover, increasing the hybridization time to 3 hours indicated that hybridization sites became also visible at 37 degrees C. Since the protocols are based on recently described Fast FISH developments, the technique will be called Low Temperature Fast-FISH (LTFF).

Cell Nucleus↗
PubMed1997

Transport of water in proximal kidney tubules from whole tubules to single channels: length and section of the selectivity filter of aquaporin-1.

Proximal straight tubule (PST) were dissected from rabbit kidneys, held with crimping pipettes in a chamber bathed in a buffered mannitol isosmotic solution (MBS, 295 mOsm/kg). Tubule cell volume changes with time (dV/Adt) after steps in MBS osmolality (delta Cs) were monitored on line with an inverted microscope, a TV camera and an image processor. Reflection coefficients sigma and osmotic permeability coefficients, Pos, for several solutes were measured using two methods. Method 1: sigma was calculated from the delta Csiso of impermeant and permeant solutes at which (dV/Adt)t-->0 = 0 (i.e., by a null point method). It is denoted as sigma 1. sigma 1 = 1.00 for mannitol (M), raffinose (R), sucrose (S), glycerol (G), acetamide (A) and urea (U). With formamide (F), sigma 1, Formamide = 0.62 +/- 0.05. These findings confirm our previous value of dp = 4.5 A for the diameter of the selectivity filter of the basolateral PST cell membrane water channel AQP1. Method 2: PST were exposed for 20 s to MBS made hyperosmotic by addition of a delta Cs of 35 mOsm/kg of R, S, M, G, A and U. Cells shrunk within 500 ms of t = 0 to their osmometric volume and remained shrunk for the 20 s of the osmotic challenge. Pos was measured from the shrinking curves. P(os) = 3000 +/- 25 microns/s with R, S, M, G, A and U. Method 2 also allowed to calculate sigma, denoted as sigma 2. sigma 2 = 1.00 for R, S, M, G, A and U. By contrast, the shrinking curve produced by a delta Cs of 35 mOsm/kg F was 1/5th to 1/6th slower and smaller (i.e., subosmometric) than that produced by a delta Cs of 35 mOsm/kg R, S, M, G, A and U. Furthermore, with F cells did not remain shrunk but recovered their original volume within 3 s. P(os) (measured with F) is denoted as P(os)*, P(os)* = 480 +/- 30 microns/s. sigma 2, Formamide = 0.16 +/- 0.01. Use of sigma 1, sigma 2 and P(os)* values in Hill's equations for the bimodal theory of osmosis leads to n = 2-9. Where n is the number of water molecules single filling within the channel selectivity filter, whose length must lie within 6 to 27 A, a value significantly lower than our previous value calculated from the P(os)/Pd* ratio.

Animals↗
PubMed1978

Quantum chemical investigations of charge-transfer interactions in relation to the electronic theory of cancer.

The results of ab initio 'supermolecule' calculations of the charge transfer between formamide and methylglyoxal, dimethylglyoxal and ethylglyoxal are compared for several different relative conformations of the constituent molecules. The extent and sign of the charge transfer is similar for all three molecules; the ketoaldehyde acts as an electron acceptor only for the stacked conformation. Similar calculations on alpha-hydroxytetronic acid as a model for ascorbic acid show that it can act as either an acceptor from formamide or a donor to glyoxal.

Animals↗
PubMed1990

Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling.

This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd-tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4-100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the non-specific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X-100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd-labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.

Bromodeoxyuridine↗
PubMed1992

Teratogenic assessment of four solvents using the Frog Embryo Teratogenesis Assay--Xenopus (FETAX).

The Frog Embryo Teratogenesis Assay--Xenopus (FETAX) was used to assess the teratogenic potential of four solvents. Embryos of the South African clawed frog, Xenopus laevis, were exposed for 96 h to ethanol, dimethyl sulfoxide (DMSO), formamide or glycerol formal. Exposure groups were maintained using a static renewal system in which the exposure media were changed at 24-h intervals. Survival was monitored at 24-h intervals. Length, as an indicator of growth effects, and developmental malformations were determined at the end of the assay (96 h). Using this information, the 96-h LC50, the 96-h EC50 (Malformation), and the no observable effect levels (NOELs) for mortality, malformation and length were determined for each solvent. The teratogenic index [TI = 96-h LC50/96-h EC50 (Malformation)] also was calculated for each of the solvents. DMSO appeared to be the least toxic or teratogenic solvent examined, with a pooled LC50 of 1.92%, a pooled EC50 (Malformation) of 1.57% and TI values of 1.20 and 1.24 in replicate trials. Formamide appeared to be the most toxic solvent, with a pooled LC50 of 1.04%. Data trends suggested that ethanol was the most teratogenic solvent tested, with a pooled EC50 (Malformation) of 1.04% and TI values of 1.42 and 1.50. The results obtained in the present work for ethanol and DMSO were compared to previously published FETAX results for these two solvents. The present results are in close agreement with these results from other laboratories, thus providing further evidence supporting the interlaboratory reproducibility of FETAX results.

Abnormalities, Drug-Induced↗
PubMed1987

Unresponsive, a behavioral mutant in Xenopus laevis: electrophysiological studies of the neuromuscular system.

Normal Xenopus laevis embryos begin movements at 1 day after fertilization. Embryos homozygous for the unresponsive mutation fail to move until 4 days after fertilization (just prior to feeding), after which they recover slowly. Electrophysiological studies were undertaken to determine the focus of this mutation. Formamide treatment of normal embryos was used to produce a phenocopy of the unresponsive condition, permitting direct comparisons between mutant and normal embryos. Intracellular recordings from muscle cells were obtained in formamide-treated and untreated preparations with both normal and unresponsive animals. Local electrical stimulation evoked either isolated endplate potentials and action potentials or after-discharges of these events in all preparations. A decrease in illumination also caused a burst of endplate potentials and action potentials. Therefore, the electrophysiology of the neuromuscular junction in unresponsive appears qualitatively normal; the effect of the mutation on the motor system is probably distal to the neuromuscular junction, either at or subsequent to excitation-contraction coupling.

Animals↗
PubMed1995

Chaotropic solvents increase the critical micellar concentrations of detergents.

Monomer detergent concentrations of Triton X-100, Chaps (3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate), and sodium dodecyl sulfate in guanidine hydrochloride, formamide, and urea solutions were measured by an ultrafiltration procedure. This simple and rapid procedure effectively separated the monomer forms of these detergents from their respective micelle forms. Critical micellar concentrations of these detergents in water measured by this procedure agreed well with previously reported values. Both ionic and nonionic chaotropic agents, e.g., guanidine hydrochloride, formamide, and urea, are demonstrated to significantly shift the equilibrium between the monomer and the micellar form of various detergents toward the direction of monomer in a concentration-dependent manner. Thus, monomer/micelle ratio of detergents in solution can be manipulated over a wide range by the concomitant use of chaotropic solvents. This has direct applications in experiments involving destruction of biomembranes and solubilization of hydrophobic molecules in aqueous solutions.

Detergents↗
PubMed1990

Optimization of non-radioactive in situ hybridization: image analysis of varying pretreatment, hybridization and probe labelling conditions.

Using detection of proopiomelanocortin (POMC) mRNA in rat pituitary as a model, varying conditions of tissue pretreatment, hybridization and probe labelling have been tested. Results were evaluated both by visual assessment and by image analysis of coded specimens. Good correlations between visual gradation, optical densities and cell area percentages were obtained. However, determinations of optical densities (or pixel values) provided most detailed information. The data obtained emphasize the interdependence of fixation and permeabilization conditions and clearly show that the stronger the primary fixation, the more efficient the permeabilization by proteinase K must be. The hybridization temperature is also of importance and temperatures between 40-45 degrees C produced the best signal to noise ratio. The POMC-directed 24-mer probe had a theoretical melting point (Tm) of 49.4 degrees C (in the absence of formamide) and four individual experimental determinations of Tm produced a mean value of 48.9 degrees C. Detection of the biotinylated probe was best accomplished with monoclonal antibiotin antibodies and the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. Short washes at high-stringency (0.1 x SSC, 45 degrees C) produced an optimal signal to noise ratio. Inclusion of 50% formamide in the hybridization buffer produced an enhanced signal to noise ratio, in spite of a higher background staining. The probe employed for most studies was a synthetic 24-mer oligodeoxynucleotide, complementary to the MSH[4-11]-coding region of POMC mRNA. It was labelled with biotinylated dUTP and unlabelled dCTP using terminal transferase. Chromatographical analyses revealed the labelled probe to be heterogeneous in tail length.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗