Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

Electronic circular dichroism of monomethyl [O, O, O]-phosphate and [O, O, O]-thiophosphate revisited

Phosphoryl-transfer reactions have long been of interest due to their importance in maintaining numerous cellular functions. A phosphoryl-transfer reaction results in two possible stereochemical outcomes: either retention or inversion of configuration at the transferred phosphorus atom. When the product is phosphate, isotopically-labeled [¹⁶O, ¹⁷O, ¹⁸O]-phosphate derivatives can be used to distinguish these outcomes; one oxygen must be replaced by sulfur or esterified to achieve isotopic chirality. Conventionally, stereochemical analysis of isotopically chiral phosphate has been based on ³¹P NMR spectroscopy and involves complex chemical or enzymatic transformations. An attractive alternative would be direct determination of the enantiomeric excess using chiroptical spectroscopy. (S)-Methyl-[¹⁶O, ¹⁷O, ¹⁸O]-phosphate (MePi^∗), 7 and enantiomeric [¹⁶O, ¹⁷O, ¹⁸O]-thiophosphate (TPi^∗), 10, were previously reported to exhibit weak electronic circular dichroism (ECD), although with 10 the result was considered to be uncertain. We have now re-examined the possibility that excesses of 7 and 10 enantiomers can be detected by ECD spectrometry, using both experimental and theoretical approaches. 7 and both the (R) and (S) enantiomers of 10 (10a, 10b) were synthesized by the ‘Oxford route’ and characterized by ¹H, ³¹P and ¹⁷O NMR, and by MS analysis. Weak ECD could be found for 7, with suboptimal S/N. No significant ECD could be detected for the 10 enantiomers.Time-dependent DFT (TDDFT) calculations of the electronic excitation energies and rotational strengths of the same three enantiomers were carried out using the functional B3LYP and the basis set 6-311G^∗∗. The isotopically-perturbed geometries were predicted using the anharmonic vibrational frequency calculational code in GAUSSIAN 03. In the case of 10, calculations were also carried out for the hexahydrated complex to investigate the influence of the aqueous solvent. The predicted excitation wavelengths are greater than the observed wavelengths, a not unusual result of TDDFT calculations. The predicted anisotropy ratios are 2.9 × 10⁻⁵ for 7, −5.3 × 10⁻⁶ for 10a/b, and 1.7 × 10⁻⁶ for 10a/b⋅(H₂O)₆. For 7 the predicted anisotropy ratio approximates that observed in this work, 4.5 × 10⁻⁵ at 208 nm. For 10a/b, the upper limits of the experimental anisotropy ratios (<5 × 10⁻⁶ at 225 nm, pH 9; <5 × 10⁻⁶ at 236 nm, pH 12) are comparable to the predicted magnitude of the value for 10a/b. The lower predicted value for 10a/b · (H₂O)₆ suggests that the aqueous environment affects the ECD significantly. Altogether, the TDDFT calculations together with a stereochemical analysis based on NMR and the MS data support the conclusion that the experimental ECD results for MePi^∗ and TPi^∗ may be reliable in order of magnitude.