Structural perturbation in supercoiled DNA: hypersensitivity to modification by a single-strand-selective chemical reagent conferred by inverted repeat sequences.

Bromoacetaldehyde, a reagent which modifies unpaired adenine residues, selectively modifies supercoiled DNA in the region of inverted repeats which are known targets for single-strand-specific nucleases. The reaction is dependent upon the topological state of the molecule, and the absolute importance of the inverted repeat has been demonstrated. Finer mapping of the distribution of the modification pattern reveals significant and interesting differences from the S1 nuclease target positions. Bromoacetaldehyde modification is distributed over a wider region covering the whole inverted repeat, with greatest extent of reaction in the regions which flank the inverted repeat. It is suggested that an altered conformation may be propagated into these sequences. These results further support the contention that inverted repeats adopt an altered conformation when negatively supercoiled, for which the principal suggestion remains the cruciform structure.     

The structure and properties of histone F2a comprising the heterologous group F2a1 and F2a2 studied by 13C nuclear magnetic resonance

¹³C n.m.r. spectra have been obtained for aqueous solutions of histones F2a₁ and F2a₂, for the group F2a, for the appropriate amino acid mixturesand for the corresponding hydrolysates. These, when compared with computer simulated spectra give good agreement for secondary structure with that calculated from the known primary structure of the proteins. Evidence based on the spectra obtained at various salt concentrations leads to the conclusion that F2a is not a simple mixture but an interacting heterologous group of histones F2a₁ and F2a₂.     

Plasma levels of gonadotropin and 17α,20β-dihydroxy-4-pregnen-3-one in relation to spawning behaviour of rainbow trout, Oncorhynchus mykiss(Walbaum)

Manipulation of the opportunity to spawn was used to investigate the relationship between endocrine events, egg viability and spawning behaviour in female rainbow trout. Females were prevented from spawning by isolating them from males and gravel for up to 21 days after ovula- tion. Blood samples were taken before pairing with a male, at the onset of nesting activity, and at the completion of spawning. Plasma hormone levels of gonadotropin (GtH) and 17α,20β-dihydroxy-4-pregnen-3-one (17,2OP) were measured by specific radioimmunoassays. There were no qualitative or quantitative differences in the spawning behaviour of females paired on the day of ovulation or 7. 14, or 21 days after ovulation. There was a general decrease in the viability of eggs with increasing retention times. In females paired on the day of ovulation, or after 7 or 14 days, GtH levels increased with the onset of nesting behaviour and declined as fish reached the post-spawning condition. By day 21, GtH levels before pairing were significantly higher than prepairing levels in the other three treatment groups, and did not increase at the onset of nesting, or decrease in post-spawning fish. Plasma 17,20P remained high in prepairing and nesting samples of all four groups and declined to low levels in fish in post-spawning condition. In females paired on the day of ovulation there was a significant increase in 17,20P from the prepairing to the nesting stage. These results suggest that 17,20P plays a key role in the synchronization of behavioural and maturational events at the time of spawning.     

Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity

We have devised a procedure to generate any single base mismatch in a constant sequence context, and have studied these from two points of view. (1) We have examined electrophoretic mobility of 458 base-pair fragments containing approximately centrally located single mismatches, in polyacrylamide gels, compared to fully matched DNA fragments. We found that no single mismatch caused a significant perturbation of gel mobility, and we conclude that all the mismatches may be accommodated within a helical geometry such that there is no alteration of the path of the helix axis in a straight DNA molecule. (2) We have studied all the single mismatches with respect to reactivity to a number of chemical probes. We found that: (a) No mispaired adenine bases are reactive to diethyl pyrocarbonate and are therefore not simply unpaired such that N-7 is exposed. (b) A number of mispaired thymine bases are reactive to osmium tetroxide, and cytosine bases to hydroxylamine. (c) Where crystal or nuclear magnetic resonance structures are available, the reactivity correlates with exposure of the pyrimidine 5,6 double bonds to attack in the major groove as a result of wobble base-pair formation. This is particularly clear for G.T and I.T base-pairs. (d) Reactivity of bases in mismatched pairs can be dependent on sequence context. (e) Reactivity of the C.C mismatch to hydroxylamine is suppressed at low pH, suggesting that a rearrangement of base-pairing occurs on protonation. The results overall are consistent with the formation of stacked intrahelical base-pairs wherever possible, resulting in no global distortion of the DNA structure, but specific enhancement of chemical reactivity in some cases.     

The role of metal ions in the conformation of the four-way DNA junction.

Metal ions fold DNA junctions into a compact conformation that confers protection of all thymine bases to modification by osmium tetroxide. In the absence of the cation the arms of the junction are fully extended in an approximately square-planar configuration. Group IIa cations are effective in achieving a folded conformation of the junction at 80-100 microM, and there is an excellent agreement between the ionic concentrations that fold the junctions as deduced from gel electrophoretic experiments, and those that prevent osmium tetroxide reaction at the junction. Hexamminecobalt(III) achieves full folding at 2 microM, while spermine and spermidine are effective at 25 microM. Some transition metal ions such as Ni(II) may replace the group IIA cations. Monovalent ions of group IA are only partially effective in folding the junctions. Very much higher concentrations are necessary, gel electrophoretic mobilities suggest that a less symmetrical conformation is adopted and thymine bases at the junction remain reactive to osmium tetroxide. Charge-charge interactions at the centre of the junction are structurally extremely important. Substitution of junction phosphate groups by uncharged methyl phosphonates severely perturbs the structure of the junction. If just two phosphates are substituted, diametrically facing across the junction, the structure always folds in order to place the electrically neutral phosphate on the exchanging strands. We suggest that folding of the junction into the stacked X-structure generates electronegative clefts that can selectively bind metal ions, depending on the chemistry, size and charge of the ion. Moreover, occupation of these cavities is essential for junction folding, in order to reduce electrostatic repulsion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoaffinity Purification of Epitope-Tagged Human β2-Adrenergic Receptor to Homogeneity

To obtain large quantities of pure human β₂-adrenergic receptor (β₂-AR) needed for structural studies, an efficient method for β₂-AR purification was developed using a recombinant receptor with an eight amino acid epitope at its C-terminus. This epitope is recognized by KT3-monoclonal antibody. The epitope tagged β₂-AR was expressed in Sf9 cells with a specific activity of 5–20 pmol/mg of membrane protein. The epitope-tagged and wild-type receptors had identical ligand binding properties. The tagged receptor was solubilized using dodecyl-β-maltoside with a quantitative yield. Solubilized epitope-tagged receptors were partially purified by KT3-mAb immunoaffinity in 60–70% yield. Further purification of the receptors on an alprenolol-affinity column resulted in a homogenous preparation with an overall yield of >30%. The purified receptor was concentrated to >1 mg/ml without loss of ligand binding activity.     

Large-scale opening of A + T rich regions within supercoiled DNA molecules is suppressed by salt.

Large-scale cooperative helix opening has been previously observed in A + T rich sequences contained in supercoiled DNA molecules at elevated temperatures. Since it is well known that helix melting of linear DNA is suppressed by addition of salt, we have investigated the effects of added salts on opening transitions in negatively supercoiled DNA circles. We have found that localised large-scale stable melting in supercoiled DNA is strongly suppressed by modest elevation of salt concentration, in the range 10 to 30 mM sodium. This has been shown in a number of independent ways: 1. The temperature required to promote cruciform extrusion by the pathway that proceeds via the coordinate large-scale opening of an A + T rich region surrounding the inverted repeat (the C-type pathway, first observed in the extrusion of the ColE1 inverted repeat) is elevated by addition of salt. The temperature required for extrusion was increased by about 4 deg for an addition of 10 mM NaCl. 2. A + T rich regions in supercoiled DNA exhibit hyperreactivity towards osmium tetroxide as the temperature is raised; this reactivity is strongly suppressed by the addition of salt. At low salt concentrations of added NaCl (10 mM) we observe that there is an approximate equivalence between reducing the salt concentration, and the elevation of temperature. Above 30 mM NaCl the reactivity of the ColE1 sequences is completely supressed at normal temperatures. 3. Stable helix opening transitions in A + T rich sequences may be observed with elevated temperature, using two-dimensional gel electrophoresis; these transitions become progressively harder to demonstrate with the addition of salt. With the addition of low concentrations of salt, the onset of opening transitions shifts to higher superhelix density, and by 30 mM NaCl or more, no transitions are visible up to a temperature of 50 degrees C. Statistical mechanical simulation of the data indicate that the cooperativity free energy for the transition is unaltered by addition of salt, but that the free energy cost for opening each basepair is increased. These results demonstrate that addition of even relatively low concentrations of salt strongly suppress the large-scale helix opening of A + T rich regions, even at high levels of negative supercoiling. While the opening at low salt concentrations may reveal a propensity for such transitions, spontaneous opening is very unlikely under physiological conditions of salt, temperature and superhelicity, and we conclude that proteins will therefore be required to facilitate opening transitions in cellular DNA.     

Structures of bulged three-way DNA junctions.

We have studied a series of three-way DNA junctions containing unpaired bases on one strand at the branch-point of the junctions. The global conformation of the arms of the junctions has been analysed by means of polyacrylamide gel electrophoresis, as a function of conditions. We find that in the absence of added metal ions, all the results for all the junctions can be accounted for by extended structures, with the largest angle being that between the arms defined by the strand containing the extra bases. Upon addition of magnesium (II) or hexamine cobalt (III) ions, the electrophoretic patterns change markedly, indicative of ion-dependent folding transitions for some of the junctions. For the junction lacking the unpaired bases, the three inter-arm angles appear to be quite similar, suggesting an extended structure. However, the addition of unpaired bases permits the three-way junction to adopt a significantly different structure, in which one angle becomes smaller than the other two. These species also exhibit marked protection against osmium addition to thymine bases at the point of strand exchange. These results are consistent with a model in which two of the helical arms undergo coaxial stacking in the presence of magnesium ions, with the third arm defining an angle that depends upon the number of unpaired bases.     

LYCOSPORA FROM CARBONIFEROUS LEPIDOSTROBUS COMPRESSIONS

Spores were extracted from Carboniferous Lepidostrobus compressions in order to associate in situ microspores with dispersed species of Lycospora. Two hundred twenty-six cones were examined, of which 61 contained spores. Fertile cones came from the Westphalian D of England, Namurian B through Westphalian D of the Appalachian and Illinois basins, and the Westphalian D of the Western Interior. Cones were separated into species based on microspore and cone morphology. Lycospora trigonoreticulata was produced by Lepidostrobus princeps from Westphalian C-D rocks from Missouri, the Illinois Basin, and the Appalachian Basin. Lycospora rotunda was produced by Lepidostrobus sp. A from Westphalian A rocks of Alabama. Two cone species produced Lycospora torquifer: Lepidostrobus praelongus from the Westphalian D of Pennsylvania and Lepidostrobus variabilis from the Westphalian A and C of the Illinois and Appalachian basins. Lycospora punctata was produced by Lepidostrobus cf. squarrosus from the Westphalian D of England, the Appalachian Basin, and Illinois Basin. Lycospora noctuina was produced by Lepidostrobus haslingdenensis from the Namurian B/C of Illinois. Microspore species are differentiated primarily on the basis of size, cingulum structure and width, and ornamentation. Cone species differ in width and distal lamina size, shape, and attitude. Lycospora species isolated from clastic species of Lepidostrobus differ completely from those of coal-swamp species, confirming that lycopod trees from clastic environments represent biologically different species from those centered in coal swamps.