EVOLUTIONARY IMPLICATIONS OF DNA DIVERGENCE IN THE DROSOPHILA OBSCURA GROUP

Using DNA–DNA hybridization, we have determined the degree of single-copy DNA (scDNA) divergence among eight species of the Drosophila obscura group. These include Old World and New World species as well as members of two subgroups. Contrary to classical systematics, members of the affinis subgroup are more closely related to American members of the obscura subgroup than are Old World species. The Old World species are not a monophyletic group. The degree of scDNA divergence among species is not necessarily correlated with morphology, chromosomal divergence, or ability to form hybrids. A unique pattern of hybrid formation was found: species separated by a ΔT_m of 6.5°C can form hybrids whereas species separated by a ΔT_m of 2.5°C cannot. As with other groups of Drosophila, the obscura group has discrete parts of the genome evolving at very different rates. The slow evolving fraction of the nuclear genome is evolving at about the same rate as mitochondrial DNA. The additional scDNA divergence accompanying the step from partial reproductive isolation (between North American pseudoobscura and the isolated Bogotà population) to full isolation is very small. The resolution of the technique was challenged by five closely related taxa with a maximum ΔT_m of 2.5°C separating them; the taxa were unambiguously resolved and the “correct” phylogeny recovered. Finally, there is some indication that scDNA in the obscura group may be evolving considerably slower than in the melanogaster subgroup.     

Helix–coil transition and conformational studies of protamine–DNA complexes

Protamine–DNA complexes prepared by the method of direct and slow mixing in 2.5 × 10^?4M EDTA, pH 8.0, have been studied by thermal denaturation and circular dichroism. The complexes show biphasic melting with T_m at about 50 °C corresponding to the melting of free DNA regions and T_m′ at about 92 °C corresponding to the melting of protamine-bound regions. In protamine-bound regions there are 1.38 amino acid residues per nucleotide, indicating a nearly completely charge neutralization. T_m is increased but T_m′ is not when the ionic strength of the buffer is raised. This also supports a full charge neutralization in protamine-bound regions. The circular dichroism of the complexes can be decomposed into two components, Δ_ε0 of free DNA regions in B-form conformation and Δ_εb of protamine-bound regions in a characteristic conformation neither that of B- nor C-form but somewhere between them.     

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.     

Effects of deuterium on the thermal stability of the poly A-poly U helix

The effect of deuterium on the thermal stability of the polynecleotide double helix formed by the homopolymers polyadenylie acid (poly A)and polyuridylic acid (poly U)has been invertigated by measuring the best capacity as a function of temperature in an automatic scanning adiabatic calorimeter. Hydrogen-bounded and deuterium-bonded helical conformations of the polynecleotides used have been melted in H₂O and D₂O₂ respectively, as solvent. Within the limits of experimental error, there is no dfference in the measured enthallpy change accompanying the helix-random coil transition. The enthalpy change ΔH is 6.6 Kcal/MBP ub any case. The half-conversion temperatures T_m differ by two degrees. T_m for poly (AU) in H₂O is 45.8°C, T_m for poly (AU) in D₂is 47.7.°C.     

Microcalorimetric Investigation of DNA,poly(dA)poly(dT) and poly[d(A-C)]poly[d(G-T)] Melting in the Presence of Water Soluble (Meso tetra (4 N oxyethylpyridyl) Porphyrin) and its Zn Complex

Abstract

Thermodynamic parameters of melting process (δH_m, T_m, δT_m) of calf thymus DNA, poly(dA)poly(dT) and poly(d(A-C))·poly(d(G-T)) were determined in the presence of various concentrations of TOEPyP(4) and its Zn complex. The investigated porphyrins caused serious stabilization of calf thymus DNA and poly poly(dA)poly(dT), but not poly(d(A-C))poly(d(G-T)). It was shown that TOEpyp(4) revealed GC specificity, it increased T_m of satellite fraction by 24°C, but ZnTOEpyp(4), on the contrary, predominately bound with AT-rich sites and increased DNA main stage T_m by 18°C, and T_m of poly(dA)poly(dT) increased by 40 °C, in comparison with the same polymers without porphyrin. ZnTOEpyp(4) binds with DNA and poly(dA)poly(dT) in two modes—strong and weak ones. In the range of r from 0.005 to 0.08 both modes were fulfilled, and in the range of r from 0.165 to 0.25 only one mode—strong binding—took place. The weak binding is characterized with shifting of T_m by some grades, and for the strong binding T_m shifts by ～ 30–40°C. Invariability of ΔH_m of DNA and poly(dA)poly(dT), and sharp increase of T_m in the range of r from 0.08 to 0.25 for thymus DNA and 0.01–0.2 for poly(dA)poly(dT) we interpret as entropic character of these complexes melting. It was suggested that this entropic character of melting is connected with forcing out of H₂O molecules from AT sites by ZnTOEpyp(4) and with formation of outside stacking at the sites of binding. Four-fold decrease of calf thymus DNA melting range width ΔT_m caused by increase of added ZnTO- Epyp(4) concentration is explained by rapprochement of AT and GC pairs thermal stability, and it is in agreement with a well-known dependence, according to which ΔT～T_GC-T_AT for DNA obtained from higher organisms (L. V. Berestetskaya, M. D. Frank-Kamenetskii, and Yu. S. Lazurkin. Biopolymers 13, 193–205 (1974)). Poly (d(A-C))poly(d(G-T)) in the presence of ZnTOEpyp(4) gives only one mode of weak binding. The conclusion is that binding of ZnTOEpyp(4) with DNA depends on its nucleotide sequence.     

Correlation of Tm and sequence of DNA duplexes with delta H computed by an improved empirical potential method

T_m values for 20 DNA duplexes with different repeating base sequences provided the data base for developing a rational and relatively simple methodology for computing apparent enthalpies for the helix → coil transitions of DNA helices, ΔH _calc. The computational variables and their range of acceptable values were selected on the basis of physically plausible arguments. Over 350,000 different combinations of the variables were tested for degree of fit. It was therby possible to find a combination giving a high degree of linear fit between T_m and ΔH _calc (correlation coefficient, 0.99), with T_m values deviating (on average) from the regression line by only ±2.17°C. Most of this uncertainty is attributed to experimental limitations, although computational approximations also contribute. With ΔH _calc for the melting of each of the unique complementary dinucleotide fragments computed by the method developed, it is possible to estimate T_m and (relative) ΔH _calc reliable for the melting of any particular DNA [with base pairs G(I)·C and A·T] given only its base sequence. The ΔH_calc values for the complementary dinucleotide fragments, together with statistical considerations, make it apparent that T_m of DNAs with repeating base sequence show only an approximate linear dependence on G·C content because A·T and G·G pairs do not contribute to helix stability independently of the base-pair sequence in which they occur. In fact, the nearestneighbor stacking interactions are so significant that certain complementary dinucleotide fragment sequences with 0,50, and 100% G·C content have the same stability.     

DNA Hybridization as a Guide to Phylogenies: a Critical Analysis

Abstract— This article evaluates the use of DNA hybridization for estimating the extent of divergence among the single-copy fractions of vertebrate genomes. It focuses, in particular, on the nature and informational content of the melting profiles as a guide to phylogenetic relationships. While concluding that the DNA hybridization approach remains the best and most cost-effective guide to such relationships over its useful range, it demonstrates serious flaws in certain recent attempts to apply the method to specific cases among primates and birds. The major points are:

• 1 The T₅₀H statistic is flawed as a measure of mean sequence divergence, and also, therefore, as a measure of phylogenetic distance.
• 2 The T_mode statistic overcomes many of the problems inherent in interpreting thermal stabilities of DNA heteroduplexes for phylogenetic purposes.
• 3 The phylogenetic significance of ΔT_modes of > 15d? or so cannot be accurately assessed.
• 4 The putative slowdown in the rate of nuclear DNA sequence change among the lemurs is not justified by the data.
• 5 The claims of Sibley and Ahlquist to have resolved the human/chimpanzee/gorilla trichotomy are not supported by their data.
• 6 There are major problems in the published Sibley and Ahlquist avian phylogenies; in particular, with those containing evolutionary “staircases” of nodes separated by less than 1d? from one another.
• 7 There would appear to be a lineage misplacement involving a ΔT of at least 4d? in a recent publication on avian phylogeny.
• 8 Certain of the published ΔT₅₀ H values seem not to be representative of the actual data on which they are based.
• 9 Most important, it is recommended that no phytogenies based on DNA hybridization comparisons should be presented without being accompanied by the data relevant to each claim of a resolved lineage.

     

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.     

Univalent ions in phospholipid model membranes: Thermodynamic and hydration aspects

By means of differential scanning calorimetry, effects of systematic series of Group I and VII ions on the phase state of model multibilayer dimyristoylphosphatidylcholine (di(14:0)PC) membranes have been studied at a lipid/ion molar ratio of 3/1. The sign-changing correlations between the ionic radii of cations and temperature shifts of di(14:0)PC phase transition were obtained. For cosmotropic Li⁺ and Na⁺, the observed shifts were positive (LiCl: ΔT _m = 0.6°C; ΔT _p = 1.9°C), whereas chaotropic K⁺ and Rb⁺ presence resulted in negative shifts (RbCl: ΔT _m = ?0.3°C; ΔT _p = ?2.5°C). The anions (Cl^?, Br^?, I^?) showed a similar effect increasing with the ion chaotropicity. An essentially weaker effect of Cs⁺ as compared to other alkali metal ions (CsCl: ΔT _m ≈ 0°C; ΔT _p = ?0.1°C) can be one of the reasons of its accumulation in living organisms. Generalization of all available data allowed us to specify some important factors of lipid-ion interactions that should be taken into account in further investigations in this field.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

GEOLOGICALLY DATED SEA BARRIERS CALIBRATE A PROTEIN CLOCK FOR AEGEAN WATER FROGS

Reliable estimates of phylogenetic relationships and divergence times are a crucial requirement for many evolutionary studies, but are usually difficult because fossils are scarce and their interpretation is often uncertain. Frogs are fresh water animals that generally are unable to cross salt water barriers (their skin is readily permeable to both salt and water). The geologically determined ages of salt water barriers that isolate related frog populations thus provide an independent measure of the minimum date of genetic divergence between pairs of such populations. For the genetically well-studied western Palearctic water frogs (Rana esculenta group), the Aegean region provides an ideal area for determining the relationship between genetic divergence and time of spatial isolation, using a nested set of geologically determined isolation times (12,000 yr, 200,000 yr, 1.8 Myr, 2–3 Myr, and 5.2 Myr). Using 31 electrophoretic loci for 33 pairs of neighboring frog populations, a linear relationship between geologically determined isolation time and Hillis' modified Nei genetic distance was found: D^*_Nei = (0.04 ± 0.01) + (0.10 ± 0.01) isolation time [Myr] corresponding to an average divergence rate (“molecular clock” pace) of 0.10 D^*_Nei/Myr (0.10 D_Nei/Myr). This rate is in the range of previous estimates reported for protein electrophoretic data; the value is conservative because relatively few of the loci used are “fast evolvers” (13%; sAAT, ALB, EST-5, MPI). Removing these fast evolvers from the analysis results in 0.08 D^*_Nei/Myr (0.08 D_Nei/Myr). The confidence limits for estimation of the divergence time given the genetic distance are large, but unusually narrow for this kind of study; they permit us to estimate divergence times during the Pliocene and Miocene. Few previous studies, including sequence analyses, have provided reasonable estimates of divergence time for the Pliocene. A test using the outgroup taxa Rana perezi and Rana saharica (also isolated for 5.2 Myr by the Strait of Gibraltar) fits the calibration well: observed genetic Nei distance D^*_Nei = 0.55, expected D^*_Nei = 0.56. The calculated divergence times, based on this absolute molecular clock, suggest a series of speciation events after the Messinian (5.2 Myr), possibly triggered by the rapid ecological changes accompanying the desiccation and refilling of the Mediterranean Basin.     

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.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

Oligonucleotides containing a peptide internucleotide linkage. A novel class of modified oligonucleotides

Oligonucleotide analogues containing one or a few glycine, L-, and D-alanine residues instead of phosphodiester internucleotide linkages were synthesized (C3′-NH-C(O)-CH(X)-NH-C(O)-C4′, where X = H, (S)-CH₃, and (R)-CH₃. The stability of the duplexes of modified oligonucleotides with their wild-type complements was studied. The incorporation of glycine and L-alanine residues into internucleotide linkages was shown to noticeably decrease the stability of modified duplexes as compared to that of native ones (ΔT _m∼−2°C per modification), whereas analogues containing D-alanine linkers form duplexes with increased stability (ΔT _m∼+2°C per modification).     

Use of principal component analysis in interpretation of deoxyribonucleic acid relatedness

Principal component analysis (PCA) of published DNA-relatedness data showed the usefulness of this method in displaying relationships among closely related bacteria. Very similar ordinations were obtained when relative binding ratios (RBR) at 60°C or 75°C or ΔT _m values were used to form the data matrix. A curvilinear relationship and a (quasi) linear relationship were found, respectively, between 75°C and 60°C RBR and ΔT _m and 60°C RBR. These statistical relationships explain the similarity of PCA results using either measurement (60°C RBR, 75°C RBR, or ΔT _m). Use of PCA is suggested to delineate groups within a complex set of DNA-relatedness data. The level of ΔT _m within groups and between groups should help decide whether these groups are genospecies.     

Macroevolution of thermal tolerance in intertidal crabs from Neotropical provinces: A phylogenetic comparative evaluation of critical limits

Thermal tolerance underpins most biogeographical patterns in ectothermic animals. Macroevolutionary patterns of thermal limits have been historically evaluated, but a role for the phylogenetic component in physiological variation has been neglected. Three marine zoogeographical provinces are recognized throughout the Neotropical region based on mean seawater temperature (T_m): the Brazilian (T_m = 26 °C), Argentinian (T_m = 15 °C), and Magellanic (T_m = 9 °C) provinces. Microhabitat temperature (MHT) was measured, and the upper (UL₅₀) and lower (LL₅₀) critical thermal limits were established for 12 eubrachyuran crab species from intertidal zones within these three provinces. A molecular phylogenetic analysis was performed by maximum likelihood using the 16S mitochondrial gene, also considering other representative species to enable comparative evaluations. We tested for: (1) phylogenetic pattern of MHT, UL₅₀, and LL₅₀; (2) effect of zoogeographical province on the evolution of both limits; and (3) evolutionary correlation between MHT and thermal limits. MHT and UL₅₀ showed strong phylogenetic signal at the species level while LL₅₀ was unrelated to phylogeny, suggesting a more plastic evolution. Province seems to have affected the evolution of thermal tolerance, and only UL₅₀ was dependent on MHT. UL₅₀ was similar between the two northern provinces compared to the southernmost while LL₅₀ differed markedly among provinces. Apparently, critical limits are subject to different environmental pressures and thus manifest unique evolutionary histories. An asymmetrical macroevolutionary scenario for eubrachyuran thermal tolerance seems likely, as the critical thermal limits are differentially inherited and environmentally driven.     

Chain length and oligonucleotide stability at high pressure

The effect of hydrostatic pressure on the helix-coil transition temperature (T_m) was measured for the DNA oligomers (dA)_n(dT)_n, where n = 11, 15, and 19, in 50 mM NaCl. The data were analyzed in light of previously published data for the polymer, poly(dA)·poly(dT) under the same conditions. As has been observed for DNA polymers, increasing the hydrostatic pressure led to an increase in the T_m of the oligomers; however, the effect of pressure diminished with decreasing chain length. The value of dT_m/dP decreased linearly with the inverse of the chain length varying from 3.15 × 10⁻²°C MPa⁻¹ for the polymer to 0.7 × 10⁻²°C MPa⁻¹ for the 11-mer. The two-state or van't Hoff enthalpy (ΔH_vH) of the helix-coil transition was obtained by analysis of the half-width of the thermal transition. As expected, ΔH_vH decreases with decreasing chain length. In contrast to the behavior of the polymer, poly(dA)·poly(dT), and (dA)₁₉(dT)₁₉, the ΔH_vH of the two shorter duplex oligonucleotides displayed a small pressure dependence dΔH_vH/dP≃−0.4 kJ MPa⁻¹ in both cases. The changes observed in the T_m and ΔH_vH were not sufficient to explain the magnitude of the chain-length dependence of the pressure effect. To interpret the large chain-length dependence of dT_m/dP, we propose that the terminal base pairs contribute a negative volume change to the helix-coil transition. Base pairs distant from the ends exhibit behavior characterized by the polymer where end effects are assumed to be negligible, i.e., a positive volume change for the helix-coil transition. The negative volume change of separating terminal bases may originate from the imperfect interactions these base pairs form with water due to the existence of several energetically equivalent conformations. This is reminiscent of one of the mechanisms proposed to be important in the pressure-induced dissociation of multimeric proteins into their constituent subunits. © 1996 John Wiley & Sons, Inc.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Phenotypic divergence of thermotolerance: Molecular basis and cold adaptive evolution related to intrinsic DNA flexibility of glacier-inhabiting Cryobacterium strains

The link between guanine–cytosine (GC) content and thermal adaptation is controversial. Here, we compared maximum growth temperature (T_MGT) and genomics of 78 Cryobacterium strains to avoid unreliable conclusions resulting from distantly phylogenetic groups. Phylogenomic analysis revealed this taxon had much higher diversification than we knew. Interestingly, these strains showed thermotolerance divergence with phylogenetic cohesion. A significant difference was found between T_MGT ≤ 20°C strains and T_MGT > 20°C strains in genomic GC content which mainly caused by variation of GC3. T_MGT ≤ 20°C strains tended to use synonymous codons ended with A/U, but T_MGT > 20°C strains tended to use G/C. Lower GC content at synonymous sites (≈GC3) of T_MGT ≤ 20°C strains could provide lower intrinsic DNA flexibility which strongly associated with optimal molecular dynamics, and then guarantee DNA function at lower growth temperatures. This analysis of codon bias revealed close relationships for thermal adaptation, GC content at synonymous sites (≈GC3), intrinsic DNA flexibility and optimal DNA dynamics. Natural selection was main force driving this codon bias; strains with lower T_MGT endured stronger natural selection. Therefore, this study provided molecular basis for bacterial adaptive evolution from moderate temperature to low temperature.