Recent studies of genome-wide nucleosomal organization suggest that the DNA 
sequence is one of the major determinants of nucleosome positioning. Although 
the search for underlying patterns encoded in nucleosomal DNA has been going on 
for about 30 years, our knowledge of these patterns still remains limited. Based 
on our evaluations of DNA deformation energy, we developed new scoring functions 
to predict nucleosome positioning. There are three principal differences between 
our approach and earlier studies: (i) we assume that the length of nucleosomal 
DNA varies from 146 to 147 bp; (ii) we consider the anisotropic flexibility of 
pyrimidine-purine (YR) dimeric steps in the context of their neighbors (e.g., 
YYRR versus RYRY); (iii) we postulate that alternating AT-rich and GC-rich 
motifs reflect sequence-dependent interactions between histone arginines and DNA 
in the minor groove. Using these functions, we analyzed 20 nucleosome positions 
mapped in vitro at single nucleotide resolution (including clones 601, 603, 605, 
the pGUB plasmid, chicken beta-globin and three 5S rDNA genes). We predicted 15 
of the 20 positions with 1-bp precision, and two positions with 2-bp precision. 
The predicted position of the '601' nucleosome (i.e., the optimum of the 
computed score) deviates from the experimentally determined unique position by 
no more than 1 bp - an accuracy exceeding that of earlier predictions. Our 
analysis reveals a clear heterogeneity of the nucleosomal sequences which can be 
divided into two groups based on the positioning 'rules' they follow. The 
sequences of one group are enriched by highly deformable YR/YYRR motifs at the 
minor-groove bending sites SHL+/- 3.5 and +/- 5.5, which is similar to the 
alpha-satellite sequence used in most crystallized nucleosomes. Apparently, the 
positioning of these nucleosomes is determined by the interactions between 
histones H2A/H2B and the terminal parts of nucleosomal DNA. In the other group 
(that includes the '601' clone) the same YR/YYRR motifs occur predominantly at 
the sites SHL +/- 1.5. The interaction between the H3/H4 tetramer and the 
central part of the nucleosomal DNA is likely to be responsible for the 
positioning of nucleosomes of this group, and the DNA trajectory in these 
nucleosomes may differ in detail from the published structures. Thus, from the 
stereochemical perspective, the in vitro nucleosomes studied here follow either 
an X-ray-like pattern (with strong deformations in the terminal parts of 
nucleosomal DNA), or an alternative pattern (with the deformations occurring 
predominantly in the central part of the nucleosomal DNA). The results presented 
here may be useful for genome-wide classification of nucleosomes, linking 
together structural and thermodynamic characteristics of nucleosomes with the 
underlying DNA sequence patterns guiding their positions.