SynopsisProton and phosphorus-31 nuclear spin-lattice relaxation times 2' 1 and spin-spin relaxation times T2 have been measured on the single-stranded polyriboadenylic acid [poly(A)]-Mn2+ system in a neutral D2O solution in the temperature range 10"-90"C a t 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T I have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn2+ to H2, Hs, HI,, and the phosphorus nucleus to be 4.7,4.1, 5.2, and 3.0 A, respectively. The electron spin of Mn2+ penetrates into the phosphorus nucleus, giving 31P hyperfine coupling of more than lo6 Hz. Evidence of penetration of the electron spin into H g and H2 is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn2+ ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn2+ ion added is bound directly to two phosphate groups with a Mn2+-phosphorus distance of 3.3 A, while a part of the Mn2+ ions are simultaneously bound to the adenine ring.It is estimated that 39 f 13% and 13 f 5% of Mn2+ are coordinated by N7 and N3 (or NI), respectively. The motional freedom of poly(A) in the environment of the Mn2+ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn2+-free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn2+-free poly(A), and are found to be 8.3,8.5,6.1, and 8.7 kcal/mol for Hs, H2, HI,, and phosphorus, respectively.T:! of phosphorus is determined by the dissociation rate (k-1) of Mn2+ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn2+ from the adenine ring are also estimated from proton 2'2 values below 50°C.

We investigated to determine whether dried bonito broth flavor induces a reinforcing effect using the conditioned place preference (CPP) test. Only dried bonito broth did not induce CPP. Sucrose induced CPP in 20% solution. A 21.86% dextrin solution, with the same calorie content as the 20% sucrose solution, did not induce CPP, but a dextrin solution flavored with dried bonito broth (BD) induced CPP. An AD solution containing the same concentrations of dextrin, NaCl, IMP, GMP, and amino acids as found in BD tended to increase the time spent in the conditioned box but did not significantly. Aromatic compounds, such as citral, vanillin, and menthol flavored AD solutions did not induce CPP, whereas an AD solution supplemented with dried bonito flavoring agent induced CPP. In mice with transected olfactory nerves, CPP was not induced by voluntary intake of BD. These results suggest that the aromatic profile of the dried bonito broth plays an important role in BD-induced CPP.

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Phosphorus-31 nuclear spin-lattice relaxation times and nuclear Overhauser enhancement upon nonselective proton saturation were measured on polyriboadenylic acid, polyribocytidylic acid, and polyribouridylic acid. A method is described to separate dipolar contribution from the contribution of chemical shift anisotropy for the phosphorus relaxation by the combined use of T1 and NOE data. Chemical shift anisotropies (δ⁄⁄−δ⊥) of phosphorus in these polynucleotides in solution are estimated to be in the range 120–165 ppm. The molecular motion of the phosphorus moiety becomes faster in the order, poly(A)<poly(C)<poly(U), the thermal activation processes being characterized by single activation energies, i.e., 5.1, 5.3–6.0, and 7.2–8.1 kcal/mol for poly(U), poly(C), and poly(A), respectively.

We investigated how gustatory and olfactory information contributes to the preference for dried bonito broth in mice. In the two-bottle preference test, intact mice consumed dried bonito broth in preference to water or an amino acid-nucleotide (AN) solution containing the same concentration of amino acids and nucleotides as that in dried bonito broth. It was observed that mice with transected bilateral chorda tympani (CT) nerves, those with transected bilateral glossopharyngeal (GL) nerves, and those that were intranasally administered with zinc sulfate preferred dried bonito broth to water. Zinc sulfate was used to produce a temporary loss of olfaction. In the two-bottle preference test with dried bonito broth and an AN solution, the preference for the former was reduced in mice with transected bilateral GL nerves and in those with an olfactory blockade, but not in mice with transected bilateral CT nerves. These results suggest that dried bonito broth was preferred over the AN solution, and that simultaneous inputs from olfaction and the GL nerve contributed to this preference.

Recent 3'P NMR studies of tRNA have disclosed that 3'P chemical shift sensitively reflects the higher order structure of tRNA, e.g., double-stranded or singlestranded [l-4] . The origin of such differential chemical shift was discussed extensively in [5 ] where empirical correlation between 31P chemical shift and phosphodiester conformation was presented, in particular the P-O torsional angles (o,~'), by using calculated electronic density on the phosphorus atom. Evidence is accumuiating to show that such relation indeeds holds at least qu~tatively in some model systems, including polynucleotides [6] and short oligonucleotide duplex [7]. It is generally found that 31P resonance is shifted downfield when the polynucleotides transform from helix (with the P-O configuration locked to @zushe, gaushe) position) to coil (where the P-O configuration can also take nongaushe positions).In the present paper, we first show that such downfield shift upon helix-coil transition is also observable with an actual DNA sample. Our major purpose of the present paper, however, is to give evidence that there is substanti~ heterogeneity in the DNA backbone structure in the single-stranded form and that this heterogeneity depends on the base-sequence, based on the 31P NMR spectra of DNA and apurinic acid. ExperimentalSalmon testes DNA was purchased from Sigma Chemical Company. Apurinic acid was prepared from salmon testes DNA as in [S] . DNA and apurinic acid 16 were diaiyzed before NMR measurements, DNA and apurinic acid was 1 S mM and 0.1 M, respectively, and final NaCl was 10V5 M and 0.1 M for DNA and apurinic acid solution, respectively. All the NMR measurements described below was carried out in DzO solution. The pH of the solution was adjusted by add. ing NaOH to the solution.3*P NMR spectra of DNA were measured at 145.7 MHz with a Bruker HX360 in a Fourier-transform mode. 31P NMR spectraof apurinic acid were recorded on a JEOL PS-100 Fourier transform unit, All the spectra of apurinic acid were obta~ed under proton noise decoupl~g. Chemical shifts were referenced to an external standard of H3P04 (85%) in a cylindrical capillary. Results and discussionFigure la shows 31P NMR spectrum of salmon testes DNA measured at pH 3.4. The resonance lmeshape is not symmetrical and apparently consists of several resonance lines. Since double-stranded DNA is not stable at low ionic strength [9] a large part of the molecule must be converted to single stranded form coexisting with the rest in double-stranded form under our experimental condition (ionic strength < lo4 M).When the solution is increased to pH 11.2, the largest peak in the higher field (1 .15 ppm) decreases and the resonance intensity in the lower field (less than 1 .O ppm) increases ( fig.lb). The change in the spectrum is more clearly seen in the difference spectrum shown in fig. 1 c. Since at pH 11.2, guanme and thymid~e bases are deprotonated and all hydrogen

SynopsisAn NH proton signal was observed in a neutral DzO solution (pH 5.8 f 0.2) of poly(G) (M, > 150,000) that had been lyophilized from HzO. This NH proton is slowly exchanged with deuterium with a rate constant of 5 X sec-l at 29.5"C. This proves that poly(G) exists in a relatively rigid multistranded form after lyophilization in neutral aqueous solution. The number of NH protons observed is approximately 0.5 per nucleotide, indicating that this multistrand would not be a complete four-stranded form as suggested by x-ray works [Zimmerman et al. (1975) J . Mol. Biol. 92, 181-192; and Arnot et al. (1974) Biochern. J. 141, 537-5431. The large difference between phosphorus-31 spin-spin relaxation time (2'2) measured with the spin-echo method and that calculated from the line width suggests that there is a distribution of rotational angles about the phosphodiester bond. The rate of conversion among different conformational states is slow in the nmr time scale and is estimated to be less than 300 sec-' a t 28°C.For both phosphorus and Hg proton, spin-lattice relaxation time (2' 1) and Tz show opposite temperature dependence and the ratio of 2' 1 to T2 gives the correlation time T~ of 5 X sec and 7 X lo-* sec (at 27OC) for H8 and phosphorus, respectively. These correlation times are considered to represent the slow rotational diffusion of the multistranded form of polr(G).