Calorimetric measurements of the destabilisation of a ribosomal RNA hairpin by dimethylation of two adjacent adenosines.

Fragments of 16S ribosomal RNA from E. coli and B. stearothermophilus, respectively comprising the 49 and 52 3' terminal nucleotides have been studied thermodynamically using high sensitivity differential scanning calorimetry. The fragments were isolated after cleavage of 16S rRNA in the ribosome by the bacteriocin cloacin DF13. Comparison of the thermal properties of the E. coli fragments with those derived from a kasugamycin resistant mutant, which specifically lacks dimethylation of two adjacent adenosines was employed to study the effect of the methylgroups on the thermal stability. Both E. coli species exhibit similar complex melting patterns with several transitions. Overall molar transition enthalpies are equal and do not depend significantly on buffer conditions (120 kcal/mol at 15 mM Na+ to 136 kcal/mol at 215 mM Na+). However, the transition with the highest Tm, corresponding to unfolding of a nine basepair central helix is lowered by the dimethylation of the adenines in the four-membered loop. This decrease amounts to 4 degrees C at 15 mM Na+ and 2 degrees C at 215 mM Na+. The corresponding nine basepair long hairpin in the Bacillus fragment melts at a temperature of 70 degrees C at 15 mM Na+. This Tmax is much higher than expected on the basis of the sequence in the hairpin.     

Preparation of milligram amounts of 21 deoxyribonucleic acid restriction fragments

Twenty-one DNA restriction fragments ranging in size from 12 to 880 base pairs (bp) were purified to homogeneity in milligram amounts. The developments which facilitated this work were (a) procedures for the rapid preparation of gram quantities of pure recombinant plasmid DNAs, (b) selective poly(ethylene glycol) (PEG) precipitation of DNAs according to broad classes of lengths, and (c) large-scale high-pressure liquid chromatography on RPC-5 for the purification of fragments to homogeneity. The 95- and 301-bp sequences from the lactose control region of Escherichia coli were cloned into the single EcoRI site of pVH51 in up to four copies per plasmid. These tandem inserts are separated by EcoRI sites and have a head to tail orientation in all cases. A total of 50 and 90 mg of th 95- and 301-bp fragments, respectively, were prepared from 300-L fermentations of E. coli cells transformed with these plasmids. A rapid and improved method, which can easily be scaled up, for the purification of plasmids and DNA restriction fragments was developed. Also, the linear pVH51 vector DNA was digested with HaeIII to yield fragments ranging in size from 12 to 880 bp. The five smaller fragments (from 12 to 180 bp) were purified quantitatively by a selective PEG precipitation enrichment step followed by RPC-5 column fractionation. The larger fragments (245-880 bp) were prepared in milligram amounts. Ten subfragments from the 301-bp lac fragment were prepared by HpaII, HinfI, or HaeIII/AluI digestions followed by separation of the reaction products on RPC-5.     

High resolution experimental and theoretical thermal denaturation studies on small overlapping restriction fragments containing the Escherichia coli lactose genetic control region

The distribution of thermal stability in the Escherichia coli lac control region is evaluated from the melting behavior of 5 short (80-219 base pairs (bp)) sequenced DNA restriction fragments containing various parts of this sequence. The thermal denaturation of these fragments was measured at 3 salt concentrations. The previous notion that the melting curves for small fragments are sharp and asymmetric in 0.01 M Na+ and broadened and less asymmetric at 0.105 and 0.505 M Na+ is confirmed and the possible explanations are discussed. The existence of two thermodynamic boundaries in this region is also confirmed. The exact location of the boundary upstream of the cyclic AMP receptor protein (CAP) binding site is accurately determined from melting experiments at 260 and 282 nm. The secondary boundary located between the promoter and operator sequence is apparent at the two higher salt concentrations and begins to disappear at the lower salt concentration. The physical interpretation of the melting experiments is compared to the results of theoretical predictions derived from the known sequence of the fragments.     

Melting of oligodeoxynucleotides with various structures

Effects of DNA fragments end structures on their melting profiles were studied experimentally and theoretically. We examined melting of hairpins and dumbbells obtained from 62-bp-long linear DNA duplex which is a perfect palindromic sequence. To fit theoretical melting profile to experimental ones additional theoretical parameters were incorporated into the standard statistical mechanical helix-coil transition theory. From comparison theoretical and experimental melting profiles theoretical parameters connected with end-structure effects were evaluated. Analysis revealed the stabilization effect of the hairpin loops and helix ends with respect to DNA duplex melting. Both type of ends make melting these oligodeoxynucleotides more cooperative than predicted by the standard helix-coil transition theory. At low ionic strength ([Na+] less than 0.04 M) this effect becomes so pronounced that melting of the DNA duplexes 30-40 bp-long conforms to the two state model. From the analysis experimental data obtained for dumbbell structures loop-weighting factor for single-stranded loop consisting of 132 nucleotides was determined. This parameter decreases 10 times with the ionic strength decreasing by an order of magnitude from 0.2 to 0.02 M Na+.     

Physical properties of moloney murine leukemia virus high-molecular-weight RNA: a two subunit structure.

The high-molecular-weight RNA of Moloney murine leukemia virus (MuLV) was analyzed by sedimentation equilibrium ultracentrifugation. Molecular weights of 7.2 x 10(6) and 3.4 x 10(6) were found for the native and subunit forms, respectively, indicating that the native structure is a dimer. S20,w and frictional coefficients were determined for MuLV RNA by analytical velocity centrifugation as a function of ionic strength. The apparent S20,w of native MuLV RNA was 47.3, 57.4, and 66.5 in 0.01, 0.1, and 0.20 M Na+, respectively; the corresponding frictional coefficients were 5.44, 4.48, and 3.87. Native RNA was estimated by circular dichroism to be 85% helical, whereas denatured RNA was 54% helical. Thermal denaturation profiles were obtained from uv absorbance scans. Melting temperatures of 57 and 68 C were obtained for high-molecular-weight RNA in 0.01 M Na+ and 0.122 M Na+, 1mM Mg2+, respectively. van't Hoff plots of the thermal denaturation data gave enthalpies for the helix-coil transition of 21,600 cal (ca. 90,500 J) per mol of cooperatively melting unit in high salt and 19,600 cal (ca. 82,100 J) per mol in low salt, consistent with both base stacking and pairing. The melting of Mu LV RNA occurred over a broad temprange and van't Hoff plots were linear over most of the melting range, indicating a noncooperative process of helix stabilization.     

Time-resolved synchrotron radiation X-ray solution scattering study of DNA melting

Time resolved x-ray solution scattering measurements were made during thermal denaturation of DNA from various sources in the temperature range of 20-90 degrees C. Preliminary results on the influence of fragment length, ionic strength, and origin of the DNA on the time course of the scattering are described. Interpretation is based on model calculations of the scattering patterns. The results indicate that, for long DNA fragments at very low ionic strength, the melting process is a continuous phenomenon over the whole temperature range. It is accompanied by a progressive decrease of the radius of gyration of the cross section and of the mass per unit length. For short fragments of 146 base pair nucleosomal core DNA, stiffening of the DNA appears to precede a sharp melting transition.     

Conformational and thermodynamic effects of naturally occurring base methylations in a ribosomal RNA hairpin of Bacillus stearothermophilus.

The 3'-terminal colicin fragments of 16S ribosomal RNA were isolated from Bacillus stearothermophilus and from its kasugamycin-resistant (ksgA) derivative lacking N6-dimethylation of the two adjacent adenosines in a hairpin loop. The fragment from the ksgA strain still contains a naturally occurring N2-methylguanosine in the loop. An RNA molecule resembling the B. stearothermophilus colicin fragment but without modified nucleosides was synthesized in vitro using a DNA template and bacteriophage T7 RNA polymerase. Proton-NMR spectra of the RNAs were recorded at 500 MHz. The imino-proton resonances of base-paired G and U residues could be assigned on the basis of previous NMR studies of the colicin fragment of Escherichia coli and by a combination of methylation-induced shifts and thermal melting of base pairs. The assignments were partly confirmed by NOE measurements. Adenosine dimethylation in the loop has a distinct conformational effect on the base pairs adjoining the loop. The thermal denaturation melting curve of the enzymatically synthesized RNA fragment was also determined and the transition midpoint (tm) was found to be 73 degrees C at 15 mM Na+. A comparison with previously determined thermodynamic parameters for various colicin fragments demonstrates that base methylations in the loop lead to a relatively strong destabilization of the hairpin helix. In terms of free energy the positive contribution of the methylations are in the order of the deletion of one base pair from the stem. Other data show that recently published free-energy parameters do not apply for certain RNA hairpins.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

Cloning DNA restriction endonuclease fragments with protruding single-stranded ends

A new method of in vitro recombination was employed to construct plasmids containing lac promoter fragments 64 bp and 144 bp long. The 64 bp HpaII-HhaI fragment contains the binding site for the catabolite activator protein (CAP). The HpaII-HaeIII 144 bp fragment includes the binding sites for RNA polymerase, the lac repressor and CAP. The method utilizes the ability of T4 DNA polymerase to make flush-ended DNA either by filling in a recessed 3'-end or by exonucleolytic removal of a protruding 3'-end. The treated fragments were then blunt-end ligated to the filled-in EcoRI cloning sites of the plasmids pVH51 and pBR322 using T4 ligase. In this process, the EcoRI sites were regenerated on the fragment ends thus facilitating the subsequent isolation of the fragments from their cloning vectors.     

Calcium-sensitive thermal transitions and domain structure of human complement subcomponent C1r

Fluorescent probes and other methods have been used to investigate the thermal stability of activated C1r and functionally intact fragments isolated from tryptic digests of the protein. This enzyme exhibits two irreversible transitions that differ with respect to their sensitivity to metal ions. The high-temperature transition occurs with a midpoint near 53 degrees C in 0.02 M tris(hydroxymethyl)aminomethane buffer and 0.15 M NaCl, pH 7.4. It is relatively insensitive to Ca2+ and ionic strength and is accompanied by a loss of catalytic activity. The low-temperature transition is most easily observed in the presence of ethylenediaminetetraacetic acid and is completely abolished by 100 microM Ca2+. Its midpoint varies between 26 degrees C at low ionic strength and 40 degrees C in the presence of 0.5 M NaCl. The low-temperature transition results in extensive polymerization of the protein without loss of the esterolytic activity or the ability to react with C1 inhibitor; however, the ability to reconstitute hemolytically active C1 or even bind to C1s in the presence of Ca2+ is destroyed. A highly purified N-terminal fragment generated by tryptic digestion of C1r in the presence of Ca2+ retained its ability to interact with C1s, disrupting the formation of C1s dimers in the presence of Ca2+. In the absence of Ca2+, this fragment displays only a low-temperature transition that is very similar to the one observed with the whole protein and that destroys its ability to bind to C1s. Addition of Ca2+ stabilizes this fragment, shifting the midpoint of its melting transition upward by more than 20 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The thermotropic properties of human plasma fibronectin

Thermal denaturation profiles for human plasma fibronectin under a variety of conditions have been determined. Although a single melting curve for this protein, with a thermal transition midpoint of 58.4 +/- 1.0 degree C and a calorimetric enthalpy change (delta Hc) of 1040 +/- 100 kcal/mol, is obtained in dilute neutral salt solutions, it is estimated that a total of seven to eight independent two-state thermal transitions are present in this endotherm. These values are not significantly altered by the presence of Ca2+, up to levels of at least 20 mM. Upon variation of the pH, no distinct thermal transitions are noted at values below pH 5.0 and above pH 10.0. Between pH 7.0 and 10.0, virtually no alterations in the thermotropic properties of fibronectin are observed, indicating that the individual domains of this protein, which contribute to the thermogram, are preserved in this pH range. Upon alteration of the ionic strength of the buffer, from 0.05 to 0.4 M KCl, small changes are observed in the thermal transition profiles of fibronectin, indicative of conformational changes in the protein resulting in a larger number of cooperative units undergoing the temperature-induced unfolding reaction.     

Correlation of thermodynamic and genetic properties in the Tn10 encoded TET gene control region.

The thermal stability of the Tn10 encoded tetracycline resistance (TET) gene control region is investigated by melting studies using purified DNA restriction fragments containing various amounts of flanking sequences. In order to study the thermodynamic properties of this control region under conditions, where enough flanking DNA is present to mimic the situation in the chromosome, the five step melting process of a 1450-bp DNA fragment is analyzed. Because most of the sequence of this DNA is not known, the assignment of the melting transitions to segments of the DNA is done by an experimental method. This employs the preparation of subfragments from the 1450-bp DNA and comparison of their denaturation profiles with the one of the intact sequence. This approach results in the complete assignment of the five denaturation steps. Rather than from the ends, the unwinding starts from the TET gene control region in the middle of the 1450-bp sequence. A clear correlation between the thermodynamic and genetic properties of this DNA is observed. The regulatory sequence forms a small cooperative unit with the lowest stability in the entire fragment. The thermal denaturation of the TET repressor. TET operator complex reveals, that the TET repressor specifically recognizes the double stranded TET operator DNA and stabilizes this structure by 2.4 degrees C. This results is also discussed as an example of the possible action of denaturing or stabilizing proteins on this genetic control region.     

Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.     

Construction and mapping of recombinant plasmids used for the preparation of DNA fragments containing the Escherichia coli lactose operator and promoter.

Three DNA restriction fragments of established sequence containing the Escherichia coli lac genetic controlling regions were cloned. In each case a recombinant plasmid was constructed which was suitable for the subsequent large scale purification of the lac fragment. A 789-base pair HindII fragment, containing the lac operator, promoter, and cyclic AMP receptor protein binding site, was ligated into the single HindII site of the amplifiable plasmid minicolicin E1 DNA (pVH51). A 203-base pair Hae III fragment containing the same genetic sites was ligated into the single Eco RI site of pVH51 which had been "filled in" by the Micrococcus luteus DNA polymerase. Thus, the lac fragment was inserted between two Eco RI sites. Plasmids containing multiple copies of this Eco RI fragment were then constructed. A 95-base pair Alu I fragment containing the lac promoter and operator was cloned similarly. Also, the 203-base pair fragment was cloned into the Eco RI site of pVH51 using a 300-base pair linker fragment (isolated by RPC-5 column chromatography) which permitted retention of its Hae III ends. Mapping studies on pVH51 DNA with a number of DNA restriction endonucleases, including Alu I, Taq I, and Hpa II, are described.     

Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+.

Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2-8.7 kcal/mol bp and 75.6-188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit (<nmelt>) varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I1335/I1374. At room temperature, DNA-metal interactions result in a pH drop of 1.2-2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations; 1) The cations initially interact with the DNA at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. 3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross-links break, resulting in single-stranded DNA complexed with metal ions.     

High resolution thermal denaturation of DNA: thermalites of bacteriophage DNA.

High resolution thermal denaturation profiles are presented for the DNAs of bacteriophages lambda and T7. It is concluded that the temperature increment in data gathering and the method of calculating results meet the requirements for quantitative recording of the large amount of information found in the thermal transitions of both DNAs. The high resolution derivative denaturation profiles of these bacteriophage DNAs demonstrate that individual subtransitions (thermalites) of natural DNA are Gaussian in form and have narrow transition widths. Curve resolution performed on these profiles indicates that the mean thermalite width (2 sigma) is 0.33 degrees C and that this breadth is relatively invariant. Transition widths are not influenced by the position of thermalites in the profile or by cation concentration in the range from 5 to 30 mM Na+. However, the relative position of thermalites within a denaturation profile is a function of the solution ionic strength. The distribution of lengths of the DNA sequences which these thermalites represent is broad, with a number average length of 900 base pairs. Although we find an approximate similarity between the number of thermalites in the denaturation profile of T7 DNA and the number of looping regions in the electron microscopic partial denaturation map of Gomez and Lang ((1972), J. Mol. Biol. 70, 239-251) we conclude that free solution thermal denaturation experiments can be compared only superficially to the mapping results.     

Thermal denaturation of engineered tet repressor proteins and their complexes with tet operator and tetracycline studied by temperature gradient gel electrophoresis

The effects of Trp to Phe exchanges in the Tet repressor on the thermal stability of the proteins and their complexes with operator DNA and inducer have been studied by temperature gradient polyacrylamide gel electrophoresis. The denaturation temperatures obtained by this method are compared with the results from temperature-dependent fluorescence and binding activities of the proteins. It is established that exchanging the interior Trp75 to Phe reduces the thermal stability of the Tet repressor by 8 degrees C while exchanging the exterior Trp43 to Phe has no effect on the stability of the protein. Binding of the inducer tetracycline increases the thermal stability of wild-type and Trp43 to Phe mutant Tet repressors by 5 degrees C, while the ones with the Trp75 to Phe mutation are stabilized by 10 degrees C. The stabilizing effect of operator binding is 20 degrees C in the Trp75 to Phe mutant and only 9 degrees C in the ones with the Trp43 to Phe exchange. In addition to the denaturation temperatures, the gel mobility shifts observed in temperature gradient gel electrophoresis reveal also information about the intermediates of the denaturation reaction. The free proteins and their complexes with the inducer tetracycline exhibit monophasic transitions upon denaturation. The operator complexes of wild-type and Trp75 to Phe mutant repressors denature in more complex reactions. At low temperature they exhibit a stoichiometry of two repressor dimers per tandem tet operator DNA. Upon elevating the temperature they form complexes with only one repressor dimer per DNA fragment. When the temperature is further increased the double-stranded DNA begins to melt from one end resulting in a complex with partially single-stranded DNA which exists only in a narrow temperature range. Finally, the denatured protein and single-stranded DNA are formed at high temperature. The associated mobility shifts are analyzed by changing the ionic strength and characterizing multiphasic melting of a pure DNA fragment by temperature gradient gel electrophoresis.     

Salt effects on the denaturation of DNA. IV. A calorimetric study of the helix-coil conversion of the alternating copolymer poly[d(A-T)].

The enthalpy deltaH, entropy deltaS, and the temperature Tm of the conformational transition of poly[d (A-T)] from the ordered to the randomly oriented state have been determined at pH 6.8 with the help of an adiabatic differential scanning calorimeter in Na2SO4 solutions of increasing ionic strength. Spectrophotometric denaturation experiments supplemented the calorimetric measurements. All thermodynamic parameters were found to vary strongly with salt concentration: both deltaH and Tm increase linearly with the logarithm of the mean molal activity alpha plus or minus of Na2SO4. However, whereas the dependence of Tm on salt activity remains linear over the entire salt concentration range employed deltaH decreases abruptly in the most concentrated Na2SO4 solutions. The entropy of melting changes with salt concentration in a pattern similar to that displayed by deltaH. The data on deltaH as well as data derived from the maximum slopes of the calorimetric heat denaturation curves were used to calculate the cooperative length Lh, the stacking free energy epsilon, and the cooperativity parameter sigma of poly[d(A-T)] as a function of ionic strength. Lh decreases with increasing salt concentration whereas sigma increases. Epsilon assumes more positive values with increasing salt molality. These changes then are in agreement with the generally held belief that an increase in salt concentration leads to an increase in the "loop" content of the copolymer.