Measuring the translational diffusion coefficients of small DNA molecules by capillary electrophoresis

The apparent translational diffusion coefficients of four 20 base pair (bp) DNA oligonucleotides with different sequences have been measured by capillary electrophoresis, using the stopped migration method. The diffusion coefficients of the four oligomers were equal within experimental error, and averaged (120 +/- 10) x 10(-8) cm(2) s(-1) in 40 mM Tris-acetate-EDTA buffer at 25 degrees C. Since this value is nearly identical to the translational diffusion coefficient determined for a different 20-bp oligomer using other methods, the stopped migration method can accurately measure the diffusion coefficients of small DNA oligomers. The apparent diffusion coefficient of a 118-bp DNA restriction fragment was also measured by the stopped migration method. However, the observed value was approximately 25% larger than expected from other measurements, possibly because the diffusion coefficients of larger DNA molecules are somewhat dependent on the ionic strength of the solution.     

Physiochemical properties of rat liver mitochondrial ribosomes

In the present study, the physiochemical properties of rat liver mitochondrial ribosomes were examined and compared with Escherichia coli ribosomes. The sedimentation and translational diffusion coefficients as well as the molecular weight and buoyant density of rat mitochondrial ribosomes were determined. Sedimentation coefficients were established using the time-derivative algorithm (Philo, J. S. (2000) Anal. Biochem. 279, 151-163). The sedimentation coefficients of the intact monosome, large subunit, and small subunit were 55, 39, and 28 S, respectively. Mitochondrial ribosomes had a particle composition of 75% protein and 25% RNA. The partial specific volume was 0.688 ml/g, as determined from the protein and RNA composition. The buoyant density of formaldehyde-fixed ribosomes in cesium chloride was 1.41 g/cm(3). The molecular masses of mitochondrial and E. coli ribosomes determined by static light-scattering experiments were 3.57 +/- 0.14 MDa and 2.49 +/- 0.06 MDa, respectively. The diffusion coefficient obtained from dynamic light-scattering measurements was 1.10 +/- 0.01 x 10(-7) cm(2) s(-1) for mitochondrial ribosomes and 1.72 +/- 0.03 x 10(-7) cm(2) s(-1) for the 70 S E. coli monosome. The hydration factor determined from these hydrodynamic parameters were 4.6 g of water/g of ribosome and 1.3 g/g for mitochondrial and E. coli ribosomes, respectively. A calculated hydration factor of 3.3 g/g for mitochondrial ribosomes was also obtained utilizing a calculated molecular mass and the Svedberg equation. These measurements of solvation suggest that ribosomes are highly hydrated structures. They are also in agreement with current models depicting ribosomes as porous structures containing numerous gaps and tunnels.     

Diffusion and electrophoretic mobility of single-stranded RNA from molecular dynamics simulations

Hydrodynamic properties of small single-stranded RNA homopolymers with three and six nucleotides in free solution are determined from molecular dynamics simulations in explicit solvent. We find that the electrophoretic mobility increases with increasing RNA length, consistent with experiment. Diffusion coefficients of RNA, corrected for finite-size effects and solvent viscosity, agree well with those estimated from experiments and hydrodynamic calculations. The diffusion coefficients and electrophoretic mobilities satisfy a Nernst-Einstein relation in which the effective charge of RNA is reduced by the charge of transiently bound counterions. Fluctuations in the counterion atmosphere are shown to enhance the diffusive spread of RNA molecules drifting along the direction of the external electric field. As a consequence, apparent diffusion coefficients measured by capillary zone electrophoresis can be significantly larger than the actual values at certain experimental conditions.     

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.     

Use of EDTA derivatization to characterize interactions between oligodeoxyribonucleoside methylphosphonates and nucleic acids

EDTA-derivatized oligonucleoside methylphosphonates were prepared and used to characterize hybridization between the oligomers and single-stranded DNA or RNA. The melting temperatures of duplexes formed between an oligodeoxyribonucleotide 35-mer and complementary methylphosphonate 12-mers were 4-12 degrees C higher than those of duplexes formed by oligodeoxyribonucleotide 12-mers as determined by spectrophotometric measurements. Derivatization of the methylphosphonate oligomers with EDTA reduced the melting temperature by 5 degrees C. Methylphosphonate oligomer-nucleic acid complexes were stabilized by base stacking interactions between the terminal bases of the two oligomers binding to adjacent binding sites on the target. In the presence of Fe2+ and DTT, the EDTA-derivatized oligomers produce hydroxyl radicals that cause degradation of the sugar-phosphate backbone of both targeted DNA and RNA. Degradation occurs specifically in the region of the oligomer binding site and is approximately 20-fold more efficient for single-stranded DNA than for RNA. In comparison to the presence of one oligomer, the extent of target degradation was increased considerably by additions of two oligomers that bind at adjacent sites on the target. For example, the extent of degradation of a single-stranded DNA 35-mer caused by two contiguously binding oligomers, one of which was derivatized by EDTA, was approximately 2 times greater than that caused by the EDTA-derivatized oligomer alone. Although EDTA-derivatized oligomers are stable for long periods of time in aqueous solution, they undergo rapid autodegradation in the presence of Fe2+ and DTT with half-lives of approximately 30 min. This autodegradation reaction renders the EDTA-derivatized oligomers unable to cause degradation of their complementary target nucleic acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Deoxyribonueleate solutions: Sedimentation in a density gradient,partial specific volumes,density and refractive index increments,and preferential interactions

Density increments (?ρ/?c₂)°_μ in solutions of NaDNA in NaCl and CsDNA in CsCl were determined over a wide range of salt concentrations; calf thymus DNA, fragmented by sonic irradiation to a molecular weight of 4–6 × 10⁵ was used. The partial specific volume v?₂° of NaDNA at 25°C was found to ho 0.500 ml/g in water, and that of CsDNA 0.440 ml/g. Both values increase with increasing NaCl and CsCl concentration. Refractive index increments under various experimental conditions were also determined. The relevance of the density increments (at constant, chemical potential of diffusible solutes) to equilibrium sedimentation in a density gradient and the evaluation of molecular weights is discussed. Distribution coefficients of diffusible components, sometimes referred to as preferential solvation or net hydration, were derived from the density increments and partial volumes and compared with direct experimental results, whenever available, from membrane distribution and isopiestic distillation. The thermo-dynamic significance of the distribution coefficients as well as possible interpretations in terms of specific molecular mechanisms are considered.     

Revised UV extinction coefficients for nucleoside-5'-monophosphates and unpaired DNA and RNA

Ultraviolet absorption provides the nearly universal basis for determining concentrations of nucleic acids. Values for the UV extinction coefficients of DNA and RNA rely on the mononucleotide values determined 30-50 years ago. We show that nearly all of the previously published extinction coefficients for the nucleoside-5'-monophosphates are too large, and in error by as much as 7%. Concentrations based on complete hydrolysis and the older set of values are too low by approximately 4% for typical RNA and 2-3% for typical DNA samples. We also analyzed data in the literature for the extinction coefficients of unpaired DNA oligomers. Robust prediction of concentrations can be made using 38 microg/A260 unit for single-stranded DNA (ssDNA) having non-repetitive sequences and 40-80% GC. This is superior to currently used predictions that account for nearest-neighbor frequency or base composition. The latter result in concentrations that are 10-30% too low for typical ssDNA used as primers for PCR and other similar techniques. Methods are described here to accurately measure concentrations of nucleotides by nuclear magnetic resonance. NMR can be used to accurately determine concentrations (and extinction coefficients) of biomolecules within 1%.     

Interactions of oligonucleotide analogs containing methylphosphonate internucleotide linkages and 2'-O-methylribonucleosides.

The interactions of oligonucleotide analogs, 12-mers, which contain deoxyribo- or 2'-O-methylribose sugars and methylphosphonate internucleotide linkages with complementary 12-mer DNA and RNA targets and the effect of chirality of the methylphosphonate linkage on oligomer-target interactions was studied. Oligomers containing a single Rp or Sp methylphosphonate linkage (type 1) or oligomers containing a single phosphodiester linkage at the 5'-end followed by 10 contiguous methylphosphonate linkages of random chirality (type 2) were prepared. The deoxyribo- and 2'-O-methylribo- type 1 12-mers formed stable duplexes with both the RNA and DNA as determined by UV melting experiments. The melting temperatures, Tms, of the 2'-O-methylribo-12-mer/RNA duplexes (49-53 degrees C) were higher than those of the deoxyribo-12mer/RNA duplexes (31-36 degrees C). The Tms of the duplexes formed by the Rp isomers of these oligomers were approximately 3-5 degrees C higher than those formed by the corresponding Sp isomers. The deoxyribo type 2 12-mer formed a stable duplex, Tm 34 degrees C, with the DNA target and a much less stable duplex with the RNA target, Tm < 5 degrees C. In contrast, the 2'-O-methylribo type 2 12-mer formed a stable duplex with the RNA target, Tm 20 degrees C, and a duplex of lower stability with the DNA target, Tm < 5 degrees C. These results show that the previously observed greater stability of oligo-2'-O-methylribonucleotide/RNA duplexes versus oligodeoxyribonucleotide/RNA duplexes extends to oligomers containing methylphosphonate linkages and that the configuration of the methylphosphonate linkage strongly influences the stability of the duplexes.     

Biochemical, biophysical, and biological properties of densonucleosis virus (paravovirus). II. Two types of infectious virions.

Among the infectious densonucleosis virus (DNV) particles, two types of virions, DNV-I and DNV-II, have been identified. The density of DNV-I was about 1.40 g/ml and depended on the pH, whereas the density of DNV-II was independent of the pH and was 1.44 g/ml. The sedimentation rates of these particles were 111S and 89S, respectively. The specific extinction coefficients and the absorption spectra, corrected for light scattering, were also different for these two types. The electrophoretic mobility of both types of virions was identical under the experimental conditions used. Both types of particles contained single-stranded DNA with similar physicochemical properties. The difference between these types of virions seemed to be due to a different protein content, DNV-II containing about 600,000 daltons less protein, and to a different quaternary structure as indicated by the change in the density of DNV-II, in the presence of divalent cations, without any change in the density of DNV-I.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Determination of amitraz in canine plasma by solid-phase microextraction-gas chromatography with thermionic specific detection

A simple and rapid analytical method is presented for the determination of amitraz in canine plasma samples using solid-phase microextraction (SPME) and gas chromatography with thermionic specific detection (GC-TSD). The best conditions for the SPME procedure were: direct extraction on a polydimethlysiloxane (PDMS) fiber with 100-microm film thickness; 400 µl of sample plasma matrix modified with 4 ml sodium borate solution (0.01 mol l(-1), pH 6.5); extraction temperature 70 degrees C, with stirring at 2500 rpm for 45 min. The method was linear between 20 and 400 ng ml(-1) with regression coefficients corresponding to 0.998 and coefficient of the variation of the points of the calibration curve lower than 15%. The lowest limit of quantification (LOQ) for amitraz in plasma was 20 ng ml(-1). This LOQ was determined as the lowest concentration on the calibration curve in which the coefficient of variation was lower than 15%. The proposed method was applied to determine amitraz concentrations in canine plasma to look for toxicity after treatment with amitraz in a dipping bath.     

Trinitrophenylation of nucleic acids and their constituents.

1. Under relatively mild conditions, nucleic acids and their constituents were trinitrophenylated with 2,4,6-trinitrobenzenesulfonate (TNBS) in aqueous solution (pH 8-11), yielding reddish-orange trinitrophenyl (TNP) derivatives. Guanine residues were trinitrophenylated on the base residues at the 2-amino group (N2-TNP derivatives), and in addition, 2'- and 3'-hydroxyl groups of the ribose moieties of nucleosides or nucleotides were trinitrophenylated to form Meisenheimer complexes. 2. The preparation of TNP derivatives (N2-TNP-guanine, -guanosine, N2, O-bis-TNP-guanosine, O-TNP-guanosine, -adenosine, -cytidine , and -uridine), their rates of formation, absorption spectra (UV, visible, and infrared), molar extinction coefficients, Rf value, electrophoretic mobilities, and stability in acid or alkaline solution, are presented. 3. Trinitrophenylation of several kinds of nucleic acid was investigated. Calf thymus DNA and yeast transfer RNA showed a resistance to trinitrophenylation compared to guanosine 3'(2')-phosphate, yeast RNA or denatured calf thymus DNA. TNP-RNA showed resistance to the action of ribonucleases T1 and T2 [EC 3.1.4.8 and 3.1.4.23]. 4. Trinitrophenylation reactions using 2,4,6-trinitrochlorobenzene and 2,4,6-trinitrofluorobenzene were compared with that using TNBS as regards specificity and reaction rate.     

Affinity Purification of DNA and RNA from Environmental Samples with Peptide Nucleic Acid Clamps

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO₄, 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO₄, 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of ≥100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10⁻²¹ M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.     

Physical properties of Artemia salina ribosomes.

Eukaryotic ribosomes were isolated from the cryptobiotic embryos and from the further-developed free-swimming nauplii of the brine shrimp Artemia salina. Analytical boundary sedimentation and photon correlation spectroscopy yielded, respectively, the standard sedimentation and diffusion coefficients at infinite dilution, s degrees 20,w = 81 +/- 1 S and D degrees 20,w = (1.41 +/- 0.02) x 10(-7) cm2/s, for the unfixed and formaldehyde-fixed ribosomes from different developmental stages and for ribosomes attached to a messenger RNA fragment. Also, the density increment was determined, from which the partial specific volume was derived (0.63 +/- 0.01 cm3/g). Combination of the different measured parameters gives accurate values for the molecular weight (3.8 +/- 0.1) x 106 and for size and solvation parameters. These results are compared with their counterparts for the smaller ribosomes from the prokaryote Escherichia coli.     

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.     

Electrophoretic mobility is a reporter of hairpin structure in single-stranded DNA oligomers

The electrophoretic mobilities of 24 single-stranded DNA oligomers, each containing 26 nucleotide residues, have been measured in polyacrylamide gels and in free solution. The mobilities observed at 20 degrees C differed by approximately 20% in polyacrylamide gels and by approximately 10% in free solution, even though the oligomers contained the same number of bases. Increasing the temperature or adding urea to the solution equalized the mobilities of the oligomers, suggesting that the variable mobilities observed at 20 degrees C are due to the formation of stable secondary structures, most likely hairpins. Thermal melting profiles were measured for eight oligomers in 40 mM Tris acetate buffer. The observed melting temperatures of most oligomers correlated roughly with the mobilities observed at 20 degrees C; however, one oligomer was much more stable than the others. The melting temperatures of four of the oligomers were close to the values predicted by DINAMelt [Markham, N. R., and Zuker, M. (2005) Nucleic Acids Res. 33, W577-W581]; melting temperatures of the other oligomers differed significantly from the predicted values. Thermal melting profiles were also measured for two oligomers as a function of the Tris acetate buffer concentration. The salt concentration dependence of the melting temperatures suggests that 0.15 Tris+ ion per phosphate is released upon denaturation. Because the apparent number of Tris+ ions released is greater than that observed by others for the release of Na+ ions from similar hairpins, the results suggest that DNA hairpins (and, presumably, duplexes) bind more Tris+ ions than Na+ ions in solution.     

Syntheses of alternating oligo-2'-O-methylribonucleoside methylphosphonates and their interactions with HIV TAR RNA

Oligonucleotide analogues 15-20 nucleotides in length have been prepared, whose sequences are complementary to nucleotides in the upper hairpin of HIV TAR RNA. These alternating oligonucleoside methylphosphonates, mr-AOMPs, contain 2'-O-methylribonucleosides and alternating methylphosphonate and phosphodiester internucleotide linkages. The methylphosphonate and phosphodiester linkages of these oligomers are highly resistant to hydrolysis by exonuclease activity found in mammalian serum and to endonucleases, such as S1 nuclease. The oligomers were prepared using automated phosphoramidite chemistry and terminate with a 5'-phosphate group, which provides an affinity handle for purification by strong anion exchange HPLC. A 15-mer mr-AOMP, 1676, that is complementary to the 5'-side of the TAR RNA hairpin, including the 3-base bulge and 6-base loop region, forms a 1:1 duplex with a complementary RNA 18-mer, mini-TAR RNA. The T(m) of this duplex is 71 degrees C, which is similar to that of the duplex formed by the corresponding all phosphodiester 15-mer. Introduction of two mismatched bases reduces the T(m) by 17 degrees C. The apparent dissociation constant, K(d), for the 1676/mini-TAR RNA duplex as determined by an electrophoretic mobility shift assay at 37 degrees C is 0.3 nM. Oligomer 1676 also binds tightly to the full length TAR RNA target under physiological conditions (K(d) = 20 nM), whereas no binding was observed by the mismatched oligomer. A 19-mer that is complementary to the entire upper hairpin also binds to TAR RNA with a K(d) that is similar to that of 1676, a result that suggests only part of the oligomer binds. When two of the methylphosphonate linkages in the region complementary to the 6-base loop are replaced with phosphodiester linkages, the K(d) is reduced by approximately a factor of 10. This result suggests that interactions between TAR RNA and the oligomer occur initially with nucleotides in the 6-base loop, and that these interactions are sensitive to presence and possibly the chirality of the methylphosphonate linkages in the oligomer. The high affinities of mr-AOMPs for TAR RNA and their resistance to nuclease hydrolysis suggests their potential utility as antisense agents in cell culture.     

Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.     

Stability of HCV, HIV-1 and HBV nucleic acids in plasma samples under long-term storage.

The implementation of nucleic acid amplification technology (NAT) for detection of HCV, HIV-1 and HBV has undoubtedly contributed to the viral safety of blood, reducing the window period. One important matter related to the stability of RNA/DNA is the effect of the storage conditions on samples. In a previous work, we studied the stability of HCV RNA in plasma samples after storage at different temperatures. This work is an update on the follow-up of a sample containing 100 IU/ml HCV RNA for 5 years at -20 degrees C, showing no decrease in the initial titre. The nucleic acid stability of other viruses, such as HIV-1 and HBV, has also been studied. At -20 degrees C, samples containing HIV-1 were followed up for approximately 3 years and the results obtained show no decay in HIV-1 RNA detectability. Regardless of the HIV-1 RNA concentration, samples stored at 5 degrees C maintain their titre for at least 14 days. At 25 degrees C, the HIV-1 RNA half-life was determined at nearly 7 days. The HBV DNA, at 5 degrees C and 25 degrees C, is stable for at least 28 days, regardless of the initial titre.     

Re-examining the oligomerization state of macrophage migration inhibitory factor (MIF) in solution

The state of oligomerization of macrophage migration inhibitory factor (MIF, also known as glycosylation inhibiting factor, GIF) in solution has been variously reported as monomer, dimer, trimer, or mixtures of all three. Several crystal structures show MIF to be a trimer. Sedimentation velocity shows a recombinant human MIF sample is quite homogeneous, with 98% as a species with s(20,w)=3.07 S and D(20,w)=8.29 x 10(-7) cm(2)/s. Using the partial specific volume calculated from the amino acid composition these values imply a mass of 33.56 kDa, well above that of dimer, but also 9% below the trimer mass of 37.035 kDa. Sedimentation equilibrium data at loading concentrations from 0.01 to 1 mg/ml show unequivocally that the self-association is extremely tight. However, the apparent mass is 33.53 kDa [95% confidence 33.25-33.82], again 9% below that expected for 100% trimer. To examine the possibility this protein has an unusual partial specific volume, sedimentation equilibrium was also done in H(2)O/D(2)O mixtures, giving 0.765+/-0.017 ml/g rather than the calculated 0.735 ml/g. With this revised partial specific volume, the equilibrium and velocity data each give M=37.9+/-2.8 kDa, fully consistent with a strongly-associated trimeric quaternary structure.