Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves

Summary Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y ₀), and the rate constants for depurination or denaturation (k ₁) and depolymerization (k ₂). The values for k ₁ (1/k ₁ is correlated with the degree of chromatin condensation) and k ₂ (which reflects the degree of DNA instability) steadily increased with age. The values for y ₀, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum untill 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk ₁ and lnk ₂ were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k ₂ decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.In honour of Prof. P. van Duijn     

The organization and repair of DNA in the mammalian chromosome. III. Determination of the molecular weight of a mammalian native DNA

The necessary conditions for a unique solution of the sedimentation vs DNA molecular weight equations are considered and applied to the native DNA of the L5178Y mouse leukemia cell. A brief review and critique of the literature of sedimentation anomalies is given to demonstrate that such anomalies are not present in the data reported here. It is shown that the chromosomal DNA of L5178Y cells comes in uniform packages of 1.0 (0.5–2.0) × 10¹⁰ daltons. All pieces are of an identical size which corresponds to the DNA content of about 1/13 the average chromatid. Both the size estimate and the number of such molecules/cell are confirmed by viscoelastometry. This DNA is shown to be free of radioactively demonstrable protein and/or lipid contaminants and of the same isopycnic density as T₄ DNA. Variance analysis is applied to determine the precision of all measurements and to pinpoint major sources of error. A relationship between [η] and M is derived for native DNA in 1.0M NaCl. A necessary conclusion from these data is that mammalian chromosome models requiring degrees of polynemy greater than 16-neme (in G₁) are incorrect (to the extent that the L5178Y cell is typical of mammalian cells).     

Stretching and overstretching of DNA in pulsed field gel electrophoresis. I. A quantitative study from the steady state birefringence decay

Using a sensitive birefringence instrument, the birefringence arising from the orientation of the DNA chain during electrophoretic transport has been recorded. This birefringence is shown to proceed both from the alignment (stretching) of the molecule in the direction of the electric field and from the extension of the length of its primitive path (overstretching). The contribution of these two processes can be separated in the decay of the birefringence after the end of the application of the electric field. The fast relaxation of the overstretching occurs first and is demonstrated to be the main contribution to the birefringence. The orientation factor of the remaining stretched state and its decay can be quantitatively understood using the biased reptation model. It provides, in addition, a high value for the tube diameter or gel pore size a (4500 ± 450 Å for a 0.7% agarose gel with a c^?0.6_g dependence in the agarose concentration c_g) and a low value for the effective charge per base pair (0.2e as compared to 0.5e using the condensation hypothesis). The contribution of overstretching to the birefringence is also quantitatively interpreted in term of the change in the mean length l of DNA inside a pore size a. The dynamics of decay of this overstretching is well represented by a stretched exponential with a stretching exponent α = 0.44. The mean decay time decreases slightly with increasing fields and scales with the overall DNA length close to N²₀. © 1993 John Wiley & Sons, Inc.     

On the hydration of DNA. I. Preferential hydration and stability of DNA in concentrated trifluoroacetate solution

The hydration of DNA is an important factor in the stability of its secondary structure. Methods for measuring the hydration of DNA in solution and the results of various techniques are compared and discussed critically. The buoyant density of native and denatured T-7 bacteriophage DNA in potassium trifluoroacetate (KTFA) solution has been measured as a function of temperature between 5 and 50°C. The buoyant density of native DNA increased linearly with temperature, with a dependence of (2.3 ± 0.5) × 10^?4 g/cc-°C. DNA which has been heat denatured and quenched at 0°C in the salt solution shows a similar dependence of buoyant density on temperature at temperatures far below the T_m, and above the T_m. However, there is an inflection region in the buoyant density versus T curve over a wide range of temperatures below the T_m. Optical density versus temperature studies showed that this is due to the. inhibition by KTFA of recovery of secondary structure on quenching. If the partial specific volume is assumed to be the same for native and denatured DNA, the loss of water of hydration on denaturation is calculated to be about 20% in KTFA at a water activity of 0.7 at 25°C. By treating the denaturation of DNA as a phase transition, an equation has immmi derived relating the destabilizing effect of trifluoroacetate to the loss of hydration on denaturation. The hydration of native DNA is abnormally high in the presence of this anion, and the loss of hydration on denaturation is greater than in CsCl. In addition, trifluoroacetate appears to decrease the ΔHof denaturation.     

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T₄ single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5′-AGGAAGGAGGAAAGAG-3′. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5′-TCCTTCCTCCTTTCTC-3′ were also prepared and studied. At 22°C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 μM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 μM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts ～20°C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (～ 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand. From the model analysis a straightforward derivation of f_T(T), the fraction of triplex as a function of temperature, was obtained. Analysis of f_T vs temperature curves, in effect melting curves of the triplexes, provided evaluation of thermodynamic parameters of the melting transition. For the triplex formed with the dumbbell substrate, the total transition enthalpy is ΔH_T = 118.4 ± 12.8 kcal/mol (7.4 ± 0.8 kcal/mol per triplet unit) and the total transition entropy is ΔS_T = 344 ± 36.8 cal/K · mol (eu) (21.5 ± 2.3 eu per triple unit). The transition curves of the triplex formed with the linear duplex substrate displayed two distinct regions. A broad pretransition region from f_T = 0 to 0.55 and a higher, sharper transition above f_T = 0.55. The transition parameters derived from the lower temperature region of the curve are ΔH′_T = 44.8 ± 9.6 kcal/mol and ΔS′_T = 112 ± 33.6 eu (or ΔH′ = 2.8 ± 0.6 kcal/mol and ΔS′ = 7.0 ± 2.1 eu per triplet). These values are probably too small to correspond to actual melting of the triplex but instead likely reveal effects of end fraying of the duplex substrate on triplex stability. Transition parameters of the upper transition are ΔH′_T = 128.0 ± 2.3 kcal/mol and ΔS′_T = 379.2 ± 6.4 eu (ΔH′ = 8.0 ± 0.2 kcal/mol and ΔS′ = 23.7 ± 0.4 eu per triplet) in good agreement (within experimental error) with the transition parameters of the triplex formed with the dumbbell substrate. Supposing this upper transition reflects actual dissociation of the third strand from the linear duplex substrate this triplex is comparable in thermodynamic stability to the triplex formed with a dumbbell substrate. Even so, the biphasic melting character of the linear triplex obscures the whole analysis, casting doubt on its absolute reliability. Apparently triplexes formed with a dumbbell substrate offer technical advantages over triplexes formed from linear or hairpin duplex substrates for studies of DNA triplex stability. © 1993 John Wiley & Sons, Inc.     

DNA orientation in shear flow

The linear dichroism (LD) has been measured for DNA molecules 239–164,000 base pairs long oriented in shear flow over a large range of velocity gradients (30–3,000 s ^?1) and ionic strengths (2–250 mM). At very low gradients, the degree of DNA orientation increases quadratically with the applied shear as predicted by the Zimm theory [J. Zimm, (1956) Chemical Physics, Vol. 24, p. 269]. At higher gradients, the orientation of fragments ≥ 7 kilobase pairs (kbp) increases linearly with increasing shear, whereas the orientation of fragments ≥ 15 kbp shows a more complicated dependence. In general, the orientation decreases with increasing ionic strength throughout the studied ionic strength interval, owing to a decrease in the persistence length of the DNA. The effect is most dramatic at ionic strengths below 10 mM, and is more pronounced for longer DNA fragments. For fragments ≥ 15 kbp and velocity gradients ≥ 100 s^?1, the orientation can be adequately described by the empirical relation: LD^r= –(k₁-G)/(k₂ + G), where k₁is a linear function of the square root of the ionic strength and k₂ depends on the DNA contour length. Since the DNA persistence length can be represented as a linear function of the reciprocal square root of the ionic strength [D. Porschke, (1991) Biophysical Chemistry, Vol. 40, p. 169], extrapolation of the empirical relation provides information about the stiffness of the DNA fibers. © 1993 John Wiley & Sons, Inc.     

Intermediates in the strand-separation transition of T7 DNA.

Sedimentation velocity runs as a function of temperature in the region of the alkaline helix-coil transition have enabled us to demonstrate the existence of stable two-stranded intermediates in the strand-separation process for T7 DNA. The strand-separation transition under these conditions has an intrinsic breadth of ～1°C, and within this temperature range (T_m + 2°C < T < T_m + 3°C) the intermediate forms are progressively converted (as a function of temperature) to single-stranded DNA. Parallel characterizations of the strand-separation transition by viscosity and absorbance–renaturation studies in the alkaline solvent are entirely consistent with the sedimentation experiments. Comparison of the experimental mean sedimentation coefficient of the intermediate forms with theoretical predictions for branched structures suggests that in these molecules the two strands are connected at a single point, not centrally located with respect to the ends of the molecule.     

Dynamics of molecular organization in agarose sulphate

Nongelling solutions of structurally regular chain segments of agarose sulphate show disorder–order and order–disorder transitions (as monitored by the temperature dependence of optical rotation) that are closely similar to the conformational changes that accompany the sol–gel and gel–sol transitions of the unsegmented polymer. The transition midpoint temperature (T_m) for formation of the ordered structure on cooling is ～25 K lower than T_m for melting. Salt-induced conformational ordering, monitored by polarimetric stopped-flow, occurs on a millisecond time scale, and follows the dynamics expected for the process 2 coil ? helix. The equilibrium constant for helix growth (s) was calculated as a function of temperature from the calorimetric enthalpy change for helix formation (ΔH_cal = ?3.0 ± 0.3 kJ per mole of disaccharide pairs in the ordered state), measured by differential scanning calorimetry. The temperature dependence of the nucleation rate constant (k_nuc), calculated from the observed second-order rate constant (k_obs) by the relationship k_obs = k_nuc(1 ? 1/s) gave the following activation parameters for nucleation of the ordered structure of agarose sulphate (1 mg mL^?1; 0.5M Me₄NCl or KCl): ΔH* = 112 ± 5 kJ mol^?1; ΔS* = 262 ± 20 J mol^?1 K^?1; ΔG*₂₉₈ = 34 ± 6 kJ mol^?1; (k_nuc)₂₉₈ = (7.5 ± 0.5) × 10⁶ dm³ mol^?1 s^?1. The endpoint of the fast relaxation process corresponds to the metastable optical rotation values observed on cooling from the fully disordered form. Subsequent slow relaxation to the true equilibrium values (i.e., coincident with those observed on heating from the fully ordered state) was monitored by conventional optical rotation measurements over several weeks and follows second-order kinetics, with rate constants of (2.25 ± 0.07) × 10^?4 and (3.10 ± 0.10) × 10^?4 dm³ mol^?1 s^?1 at 293.7 and 296.2 K, respectively. This relaxation is attributed to the sequential aggregation processes helix + helix → dimer, helix + dimer → trimer, etc., with depletion of isolated helix driving the much faster coil–helix equilibrium to completion. Light-scattering measurements above and below the temperature range of the conformational transitions indicate an average aggregate size of 2–3 helices.     

Temporal and latitudinal comparisons of reproductive parameters in a heavily exploited shark,the bonnethead,Sphyrna tiburo (L. 1758), in the southern Gulf of Mexico

In the southern Gulf of Mexico, the bonnethead shark, Sphyrna tiburo, is one of the most frequently captured species in landings of small-scale fisheries. Based on the analysis of two fishery-dependent sampling periods (1993–1994 and 2007–2014), this study aimed to determine reproductive parameters and identify temporal differences between the two time periods. In the first sampling period, 776 males and 352 females with a size range of 28.0–120.0 cm total stretched length (L_T) were analysed, and in the second sampling period, 387 males and 432 females with a size range of 28.0–122.0 cm L_T were analysed. The size at 50% maturity in the second sampling period was significantly different between sexes, 82.6 cm L_T for females and 73.8 cm L_T for males (no estimation was possible for the first sampling period). The size at 50% maternity was not different between sampling periods, 97.3 cm L_T for the first sampling period and 99.0 cm L_T for the second sampling period. Litter size varied from 3 to 19 embryos and the average was not statistically different in both periods, 10.1 (S.D. = 3.8) for the first sampling period and 11.3 (S.D. = 3.5) for the second sampling period. The female reproductive cycle is asynchronous, and it seems to be annual, with a gestation period of 5–6 months, and a consecutive ovarian cycle and gestation period. Temporal (between sampling periods) and latitudinal (southern Gulf versus northern regions) variations occur in the synchronicity of the reproductive cycle, temporal variation in the relationship between maternal length and litter size, and latitudinal variation in average size of mature sharks.     

Light scattering studies of supercoiled and nicked DNA

Static and dynamic light scattering measurements were made of solutions of pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms. The static structure factor and the spectrum of decay modes in the autocorrelation function were examined in order to determine the salient differences between the behaviors of nicked DNA and supercoiled DNA. The concentrations studied are within the dilute regime, which is to say that the structure and dynamics of an isolated DNA molecule were probed. Static light scattering measurements yielded estimates for the molecular weight M, second virial coefficient A₂, and radius of gyration R_G. For the nicked DNA, M = (2.8 ± 0.4) × 10⁶g/mol, A₂ = (0.9 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 90 ± 3 nm were obtained. For the supercoiled DNA, M = (2.5 ± 0.4) × 10⁶ g/mol, A₂ = (1.2 ± 0.2) × 10⁻³ mol cm³/g², and R_G = 82 ± 2.5 nm were obtained. The static structure factors for the nicked and supercoiled DNA were found to superpose when they were scaled by the radius of gyration. The intrinsic stiffness of DNA was evident in the static light scattering data. Homodyne intensity autocorrelation functions were collected for both DNAs at several angles, or scattering vectors. At the smallest scattering vectors the probe size was comparable to the longest intramolecular distance, while at the largest scattering vectors the probe size was smaller than the persistence length of the DNA. Values of the self-diffusion coefficients D were obtained from the low-angle data. For the nicked DNA, D = (2.9 ± 0.3) × 10⁻⁸ cm²/s, and for the supercoiled DNA, D = (4.11 ± 0.21) × 10⁻⁸ cm²/s. The contribution to the correlation function from the internal dynamics of the DNA was seen to result in a strictly bimodal decay function. The rates of the faster mode Γ_int, reached plateau values at low angles. For the nicked DNA, Γ_int = 2500 ± 500 s⁻¹, and for the supercoiled DNA, Γ_int = 5000 ± 500 s⁻¹. These rates correspond to the slowest internal relaxation modes of the DNAs. The dependence of the relaxation rates on scattering vector was monitored with the aid of cumulants analysis and compared with theoretical predictions for the semiflexible ring molecule. The internal mode rates and the dependence of the cumulants moments reflected the difference between the nicked DNA and the supercoiled DNA dynamical behavior. The supercoiled DNA behavior seen here indicates that conformational dynamics might play a larger role in DNA behavior than is suggested by the notion of a branched interwound structure. © 1996 John Wiley & Sons, Inc.     

Dynamic light-scattering studies of internal motions in DNA. III. Evidence for titratable joints associated with bound polycations

Dynamic light-scattering techniques are employed to study the internal Brownian motions of a commercial calf thymus DNA, clean and contaminated ?29 DNAs, and a clean ?29 DNA with bound spermidine as a function of pH. The Rouse-Zimm model parameters of both calf thymus and contaminated ?29 DNAs differ substantially from those of clean ?29 DNA in the neutral-pH region. However, this difference is largely removed by adding 0.01M EDTA (which has no effect on clean ?29 DNA) to the calf thymus DNA sample. These findings imply the existence in that preparation of polycation contaminants, presumably basic proteins, that can substantially alter the local mechanical properties of the DNA near their binding sites. The internal motion parameters k_BT/f and b of both calf thymus and contaminated ?29 DNAs are found to exhibit pronounced characteristic variations between pH 8.5 and 10.5, over which range there is essentially no detectable titration to a resolution of about 1% of the base pairs. These variations, which are not observed for clean ?29 DNA, are qualitatively similar to those previously reported for a ?29 DNA with 21 single-strand breaks per chain. This indicates the formation of titratable joints associated with bound polycation contaminants. These basic ligands presumably facilitate local denaturation by stabilizing the titration of one or more protons on base-ring nitrogens near their binding sites. Spermidine binding up to 85–87% of neutralization of the total DNA charge has only a relatively minor effect on the internal motion parameters at neutral pH in 0.01M NaCl. However on raising the pH to 10.2, the internal motion parameter k_BT/f undergoes a marked decrease similar to that observed for both calf thymus and contaminated ?29 DNAs and also ?29 DNA with single-strand breaks. This indicates that spermidine, too, is capable of inducing titratable joints. Evidence is presented that the titratable joints associated with bound polycations on the calf thymus DNA may serve primarily as torsion joints, as was found previously for the titratable joints associated with single-strand breaks.     

On the application of polyelectrolyte limiting laws to the helix–coil transition of DNA. V. Ionic effects on renaturation kinetics

The bimolecular rate constant k₂ for the association of complementary polynucleotide strands has been observed to increase strongly with increasing ionic strength—in fact, proportional to its third or fourth power. This effect is here interpreted quantitatively by means of polyelectrolyte theory starting with the Wetmur–Davidson postulate of a pre-equilibrium between separated strands and aligned segments close to one another but unbonded. The correct form, a power dependence of k₂ on ionic strength, is predicted. Comparison of the theoretical exponent with data allows the conclusion that each of the two single-stranded segments in the aligned but unbonded configuration consists of about 13–16 nucletides (not to be confused with the much smaller number of bonded base pairs in the nucleus), and that this number, denoted by Q, is possibly correlated either with a minimum length for duplex stability or with the persistence length of a single polynucleotide strand. It is suggested that experimental determination of the dependence of Q on (G+C)-content may distinguish between these possibilities. It is also suggested that addition of sufficient amounts of divalent metal ions such as Mg²⁺, Ca²⁺, or Co²⁺ may reverse the dependence of k₂ on ionic strength; under these conditions, k₂ is predicted to decrease with about the first power of ionic strength. At fixed ionic strength, k₂ should increase with increasing concentration of divalent metal ion, and, in fact, the published observation that the formation of poly(A)·2 poly(U) from poly(A)·poly(U) and poly(U) is second order in Mg²⁺ concentration is here correctly predicted from a priori molecular considerations. Finally, published association rate data for oligonucleotides are discussed in the present theoretical context.     

DNA–DNA sequence homology recognition: physical mechanisms and open questions

First, we summarize recent experimental facts on homologous DNA pairing in vitro and discuss possible mechanisms of DNA–DNA sequence recognition. Then, we overview the mechanism of DNA–DNA recognition based on complementarity of DNA charge patterns. The theory predicts the recognition energy up to 10 k_BT for close parallel homologous DNA fragments of gene‐relevant lengths. We argue why this estimate cannot be directly applied to pairing of homologous DNA loci in experiments on yeast chromosomes. Namely, DNA–DNA distances assessed from experiments are much larger than those typically used in theory. Finally, we suggest some experiments that could help to judge whether short‐range electrostatic forces indeed govern DNA pairing. This viewpoint paper introduces recently developed theoretical concepts to molecular biologists, with a hope to generate a junction between theory and future experiments on DNA recognition. Copyright © 2010 John Wiley & Sons, Ltd.     

Slow relaxational processes in the melting of linear biopolymers: A theory and its application to nucleic acids

We treat the problem of the mean time of complete separation of complementary chains of a duplex containing N base pairs. A combination of analytical and computer methods is used to obtain the exact solution in the form of a compact expression. This expression is used to analyze the limits of application of the equilibrium theory of helix–coil transition in oligo- and polynucleotides. It also allows the melting behavior of a biopolymer to be predicted when its melting is nonequilibrium. In the case of oligonucleotides for which the equilibrium melting takes place at a high value of the stability constant s, the general expression turns into the equation of Craig, Crothers, and Doty, used by them to determine the rate constant k_f of the growth of a helical region from temperature-jump experiments. For the case of fragmented DNA with N ～ 10², the melting process is shown to be completely nonequilibrium, and as a result, the observed melting temperature should be higher than that for the equilibrium. A simple equation is obtained that makes possible calculation of the real, “kinetic” melting temperature T_k. As N increases, the observed melting temperature should approach the equilibrium value, T_m. This analysis has explained quantitatively the peculiar chain-length dependence of the experimentally observed shift in the DNA melting temperature during fragmentation. A thorough analysis is given of the nonequilibrium effects in the melting process of long DNA molecules (N ? 10³). The main conclusion is that even in the presence of profound hysteresis phenomena, the melting profile observed on heating may differ only slightly from the equilibrium profile.     

Very low-angle light scattering. A characterization method for high-molecular-weight DNA

A very low-angle light-scattering photometer is described with respect to optical features, scattering cell, correction factors, and absolute calibration in the angular range 2°–35°. An improved microfiltration apparatus was used to obtain essentially dust-free aqueous solutions for very low-angle light scattering. The instrument was calibrated with silicotungstic acid, an absolute molecular-weight standard, and the calibration was confirmed with the use of several secondary standards. Very low-angle light-scattering measurements were made to determine the weight-average molecular weight M?_r and z-average radius of gyration R_g,z of a commerical preparation of calf-thymus DNA. Microfiltration of the solutions allowed measurements down to 6°. The value M?_r = 20.0 × 10⁶ obtained by extrapolating 6°–9° data to 0° is more than three times that from 30°–75° data (6.38 × 10⁶) but ～20% smaller than that from 10–35° data (23.7 × 10⁶). The experimental errors in M?_r and R_g,z are estimated to be ±8% and ±14%, respectively. Combined 6°–75° data from two photometers fit well a theoretical scattering curve for a model wormlike coil of the same M?_r as the DNA sample.     

Description of two nitrogen-fixing bacteria,Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., isolated from paddy soils

Two strictly anaerobic nitrogen-fixing strains, designated RG17^T and RG53^T, were isolated from paddy soils in China. Strains RG17^T and RG53^T showed the highest 16S rRNA gene sequence similarities to the type strain Geomonas paludis (97.9–98.4%). Phylogenetic tree based on 16S rRNA gene sequences showed that two strains clustered with members of the genus Geomonas. Growth of strain RG17^T was observed at 20–42 °C, pH 5.5–8.5 and 0–0.3% (w/v) NaCl while strain RG53^T growth was observed at 20–42 °C, pH 5.5–9.5 and 0–0.7% (w/v) NaCl. Strains RG17^T and RG53^T contained MK-8 as main menaquinone and C_15:1 ω6c, iso-C_15:0, and Summed Feature 3 as the major fatty acids. The genomic DNA G?+?C content of strains RG17^T and RG53^T were 61.6 and 60.7%, respectively. The digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between the isolated strains and the closely related Geomonas species were lower than the cut-off value (dDDH 70% and ANI 95–96%) for prokaryotic species delineation. Both strains possessed nif genes nifHDK and nitrogenase activities. Based on the above results, the two strains represent two novel species of the genus Geomonas, for which the names Geomonas fuzhouensis sp. nov. and Geomonas agri sp. nov., are proposed. The type strains are RG17^T (=?GDMCC 1.2687^T?=?KTCC 25332^T) and RG53^T (=?GDMCC 1.2630^T?=?KCTC 25331^T), respectively.

     

Reproductive aspects of the Atlantic angel shark Squatina dumeril in the southern Caribbean Sea

The maturity and reproduction of the Atlantic angel shark Squatina dumeril were assessed using 77 females (29·2–110·4 cm total length; L_T) and 269 males (58·7–108·2 cm L_T) harvested by artisanal gillnetters off Venezuela. The biased sex ratio implied segregation or sex‐specific gear selectivity. Based on the development of the reproductive tract, 50% L_T at sexual maturity (L_T50, mean ± s.e .) for females and males were estimated at 86·14 ± 0·64 and 81·55 ± 0·12 cm, respectively. Uterine fecundity ranged between one and six and with a maximum embryo size of 25·7 cm L_T. Gravid females were observed from August to December, including those close to parturition and while the gestation period was not confirmed, the size of ovarian follicles among some specimens implied protraction. The low fecundity of the species supports close monitoring of catches.     

Life‐history traits of the long‐nosed skate Dipturus oxyrinchus

This work investigates life‐history traits of the long‐nosed skate Dipturus oxyrinchus, which is a common by‐catch in Sardinian waters. The reproductive variables were analysed from 979 specimens sampled during scientific and commercial hauls. Females (10·4–117·5 cm total length, L_T) attained larger sizes than males (14·5–99·5 cm L_T). To evaluate age and growth, a sub‐sample of 130 individuals (76 females and 54 males) were used. The age was estimated by annuli counts of sectioned vertebral centra. Four models were used for the length‐at‐age data: the von Bertalanffy, the exponential, the Gompertz and the logistic functions. According to the Akaike's information criterion, the Gompertz model seemed to provide the best fitting curve (L_∞ mean ± s.e. : 127·55 ± 4·90 cm, k: 0·14 ± 0·09, IP: 3·97 ± 0·90 years). The oldest female and male were aged 17 (115·5 cm L_T) and 15 years (96·0 cm L_T), respectively. Lengths at maturity were 103·5 cm for females and 91·0 cm for males, corresponding to 90% of the maximum observed length in both sexes. The monthly distribution of maturity stages highlighted an extended reproductive cycle, with spawning females and active males being present almost throughout the year, as confirmed by the gonado‐somatic index. Ovarian fecundity reached a maximum of 26 yolked follicles with a mean ± s.e. size of 19·7 ± 6·5 mm.     

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base-pair sequence 5′G-T-A-T-C-C-C-T-C-T-G-G-A-T-A-C3′ linked on the ends by dodecyl chains

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C¹²H²⁴ (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T₄ loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na⁺ and revealed, regardless of [Na ⁺], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T₄ loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T₄ end loops are essentially two state in 25 and 55 mM Na⁺. In contrast, significant deviations from two-state behavior were observed in 85 m MNa⁺. From our previously published melting data of DNA dumbbells with T_n end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881–892] and the dumbbell with T₄ end loops of this study, a plot of d(T_m)/d ln [Na⁺] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (T_m)/d ln [Na⁺] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.     

DNA, rRNA and RNA partition isotherms rapidly labeled in a saline solvent system

Partition isotherms of DNA, rRNA, and rapidly labeled RNA in a salt solvent system are determined. In the salt solvent system PMB (potassium phosphate buffer 1.50 M pH 7.0:2-methoxyethanol:2-butoxyethanol; 3:1:variable volume), equilibrated at the temperature T, the partition isotherms of animal ribosomal RNA and DNA form a series of straight lines according to the basic relation log k = B ? A_T(BuO), where k is the partition coefficient, B and A_T are parameters depending upon the base composition, the molecular weight, the helix content, and conformation of nucleic acids, and (BuO) refers to the 2-butoxyethanol amount in volume percent. This relation is also valid for other nucleic acid compounds (bases, nucleosides, nucleotides, oligoribonucleotides, and transfer RNA). For RNA fractions distributed in different Kirby salt solvent systems, log k is proportional to the relative levels of adenine and guanine expressed as the A/(A+G) ratio. Partition isotherms of high molecular weight RNA from mouse plasmocytoma, labeled during a few hours, are not straight lines. This behavior indicates a heterogeneous RNA population. By plotting the specific activity of this mixture (rRNA and rapidly labeled RNA) determined in the top phase of our salt solvent system as a function of the 2-but-oxyethanol content, we obtained a sigmoidal curve, which characterizes the nature of the RNA population investigated. Formulas are given for the calculation of the specific activities of rRNA and rapidly labeled RNA and for the calculation of their relative ratio.