Charge effect in the interaction of doxorubicin and derivatives with polydeoxynucleotides.

The equilibrium interaction of doxorubicin and its N-acetyl derivative with a series of purine-pyrimidine alternating polydeoxynucleotides has been studied through spectrofluorometry to assess the relevance of the electrostatic contribution to DNA intercalation. The results have shown that: (a) the suppression of the positive charge on the aminosugar has: (I) a profound negative effect on the free energy of intercalation, as expected, and (II) a negligible influence on the base specificity, which supports the notion of an essentially electrostatic effect of N-acetylation on intercalation; (b) a reasonably good accord with the demands of a polyelectrolytic model, due to Friedman and Manning, is found.     

The subunit structure of the arom multienzyme complex of Neurospora crassa. A possible pentafunctional polypeptide chain.

A new procedure for the purification of the arom multienzyme complex from Neurospora crassa is presented. Important factors are the inactivation of proteinases by phenylmethanesulphonyl fluoride and the use of cellulose phosphate as an affinity adsorbent. A homogeneous enzyme, with a specific shikimate dehydrogenase activity of 70 units/mg of protein, is obtained in 25% yield. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, combined with cross-linking studies using dimethyl suberimidate, suggest that the complex is composed of two subunits of molecular weight 165000. Glycerol-density-gradient centrifugation indicates a molecular weight for the intact complex of about 270000. Evidence for the effects of proteolysis, both during the preparation and on storage of the purified complex, is presented, and previous reports in the literature of the occurrence of multiple subunits are discussed in this light.     

Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.     

Normal T cell development is possible without ''functional'' gamma chain genes.

The T cell-specific gamma gene family is organized into four V, J and C gene segments containing clusters (gamma 1, gamma 2, gamma 3, gamma 4) in germline DNA. We found that the V, J and C elements of gamma 2 are physically linked on a stretch of 6 kb of DNA while those of gamma 3 are found within a 15-kb region. Rearrangements take place only within the clusters, explaining the rigid rearrangement patterns seen in T lymphocytes. New V gamma, J gamma and C gamma gene segments were discovered and characterized allowing the better understanding of the potential germline diversity of the gamma gene family. No correlation with T cell function, i.e. cytolytic or helper, and the type of the productive gamma rearrangement could be established. In contrast we found that functional T cell clones have been able to mature without any functional gamma chain genes.     

A simple method for the preparation of single-stranded polydeoxynucleotides of desired length.

A method for the preparation of homogeneous, single-stranded polydeoxynucleotides of desired length up to 800 bases is described. The procedure entails 1) generation of double-stranded DNA of desired length by PCR using a pair of primers of which one is biotinylated and the other is either unlabeled or fluorescently labeled, 2) isolation of PCR products by agarose slab gel electrophoresis, 3) recovery of desired product by electroelution, 4) binding of the product to streptavidin-coated magnetic beads and is followed by 5) duplex denaturation and removal of the unbound single strand that is either unlabeled or fluorescently labeled. Final product characteristics were determined by capillary gel electrophoresis with fluorescence detection. Up to microgram quantities of homogeneous single-stranded DNA of a desired length were obtained. These can be used as single-stranded size standards in capillary gel electrophoresis experiments as well as in other techniques requiring such standards.     

The relative importance of Escherichia coli exonuclease III and endonuclease IV for the hydrolysis of 3''-phosphoglycolate ends in polydeoxynucleotides.

In vitro, in the presence of Mg++, the 3'-phosphoglycolatase activity of endonuclease IV is about 4-times smaller than that of exonuclease III for the same AP endonuclease activity. It thus seems that endonuclease IV has only a minor role in the repair of strand breaks limited by 3'-phosphoglycolate ends in Escherichia coli even after the amount of enzyme has been increased by induction with O2 -generating agents.     

Formation of alpha-deoxyadenosine in polydeoxynucleotides exposed to ionizing radiation under anoxic conditions

When poly(dA), poly(dA-dT), and salmon testis DNA were gamma-irradiated under nitrogen, the major deoxyadenosine damage product (excluding liberated adenine) was identified as the alpha-anomer of deoxyadenosine. The yields of alpha-deoxyadenosine from poly(dA), poly(dA-dT), and salmon testis DNA irradiated with a dose of 500 Gy under anoxic conditions were 1.5, 1.3, and 1.3%, respectively. No alpha-deoxyadenosine was detected after irradiation under oxic conditions. The presence of nucleotides with the alpha-configuration at the anomeric carbon atom in the DNA chain may have a significant effect on its tertiary structure and possibly modify its biological activity.     

Free radical yields in A:T polydeoxynucleotides, oligodeoxynucleotides, and monodeoxynucleotides at 4 K.

Dose-response curves for free radical trapping in monomers, dimers, and polymers of dAMP and TMP have been measured. Powder samples pressed into pellets were X-irradiated and observed at 4 K using EPR at Q-band microwave frequencies. The initial free-radical yield (G value) and the destruction rate (k value) are reported for 22 samples. The absolute magnitude and relative changes in G are informative. For example, the duplex of homopolymers, poly(dA):poly(T)(Na), gives a large G of 6 while the closely related duplex of alternating ATs, poly(dA-T)(Na), gives a G value of 3. At G of around 6, the range of the bound electrons (e-) and electron loss centers (holes) is believed to be very limited. Two factors are suggested as important to limiting the range, proton transfer between strands and the low probability of radical transfer between strands. The lower G values are viewed as being due to relatively small increases in the range of e- and/or holes.     

Conformation of double-stranded polydeoxynucleotides in solution by proton two-dimensional nuclear Overhauser enhancement spectroscopy

Proton 2D-NOE spectroscopy has been used to investigate the three-dimensional conformations of several sonicated polydeoxynucleotides in solution. The observed pattern of cross peaks indicate that poly(dA-dT) · poly(dA-dT) in all salt concentrations studied (up to 6.6M CsF), and poly(dG-m⁵dC) · poly(dG-m⁵dC) in low salt (0.1M NaCl) are righthanded B-structures. Poly(dG-m⁵dC) · poly(dG-m⁵dC) in Mg²⁺ (3 mM) solution exhibits a pattern characteristic of the left-handed Z-form. These results for poly(dA-dT) · poly(dA-dT) are in contrast to suggestions that this copolymer exists as a left-handed form, either in low or high salt. We present pure absorption-mode 2D-NOE spectra that enable us to compare several distances and define the conformations of these polydeoxynucleotides in solution.     

Poly[d(A.T)] and other synthetic polydeoxynucleotides containing oligoadenosine tracts form nucleosomes easily.

Synthetic double-stranded polydeoxynucleotides of the general form poly[d(AnT).d(ATn)], with n ranging from 3 to 11, have been synthesized. The conformation of the polymers was investigated by circular dichroism spectroscopy and the polymers were examined for their ability to form nucleosomes. Although spectra show that a circular dichroism band characteristic of poly[d(A.T)] appears in the polymer family for n greater than 7, we demonstrate that even polynucleotides with the longest tracts of contiguous adenosine bases (n = 11) are able to form nucleosomes when reconstituted using a histone exchange procedure. Thus resistance to nucleosome formation does not coincide with the appearance of features similar to that of poly[d(A.T)] over the bulk of the nucleosomal DNA. Furthermore, we show that an approximately 150 base-pair poly[d(A.T)] itself, long thought to be refractory to nucleosome formation, can assemble into such a protein-DNA complex when reconstituted by a low-salt exchange procedure. Competitive assays show that the homopolymer reconstitutes about as well as heterogeneous sequences DNA. Our work, therefore, suggests that highly adenosine-rich sequences in vivo apparently have a function that operates at a level other than that of nucleosome structure.     

Alternative respiratory chain enzymes: Therapeutic potential and possible pitfalls

The alternative respiratory chain (aRC), comprising the alternative NADH dehydrogenases (NDX) and quinone oxidases (AOX), is found in microbes, fungi and plants, where it buffers stresses arising from restrictions on electron flow in the oxidative phosphorylation system. The aRC enzymes are also found in species belonging to most metazoan phyla, including some chordates and arthropods species, although not in vertebrates or in Drosophila. We postulated that the aRC enzymes might be deployed to alleviate pathological stresses arising from mitochondrial dysfunction in a wide variety of disease states. However, before such therapies can be contemplated, it is essential to understand the effects of aRC enzymes on cell metabolism and organismal physiology. Here we report and discuss new findings that shed light on the functions of the aRC enzymes in animals, and the unexpected benefits and detriments that they confer on model organisms. In Ciona intestinalis, the aRC is induced by hypoxia and by sulfide, but is unresponsive to other environmental stressors. When expressed in Drosophila, AOX results in impaired survival under restricted nutrition, in addition to the previously reported male reproductive anomalies. In contrast, it confers cold resistance to developing and adult flies, and counteracts cell signaling defects that underlie developmental dysmorphologies. The aRC enzymes may also influence lifespan and stress resistance more generally, by eliciting or interfering with hormetic mechanisms. In sum, their judicious use may lead to major benefits in medicine, but this will require a thorough characterization of their properties and physiological effects.