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A method for the determination of the primary structure of spliced mRNA junction and leader sequences is described. By analogy to the DNA sequencing procedure of Sanger et al. [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci USA 74, 5463--5467], we use 2",3'-dideoxynucleoside triphosphates as chain-terminating inhibitors of the reverse transcriptase (RNA-dependent DNA polymerase) reaction. By using specific DNA restriction fragments as primers in combination with this technique, we have determined the sequence of the spliced junction between the body and the leader sequence of the 16S late mRNA of simian virus 40. The method described should be of general utility in mapping spliced mRNA regions for which the corresponding protein sequence (if any) is unknown.

We have examined the 270 MHz nuclear magnetic resonance spectrum and relaxation kinetic behavior of tRNA2Glu (E. coli) in the absence of Mg++, a condition which produces an inactive form of this tRNA. The results show that the denatured form has about five fewer proton resonances in the region from -12 to -15 ppm. Relaxation kinetic measurements reveal that the denatured conformer contains three separately melting helices. The results support a model in which the tertiary structure and dihydrouridine helix characteristic of the native form are unfolded in the denatured state, and are replaced by an altered tertiary structure. The acceptor stem, anticodon, and TpsiC helices are intact in this model for the denatured conformation. The optical changes that accompany melting of the denatured tertiary structure are faster than 10 musec.

When histones in chromatin core particles were crosslinked with dimethylsuberimidate, the resulting particles had properties closely similar to those of native core particles. A crosslinked octameric histone complex was isolated from these particles under nondenaturing conditions. Upon reaction with DNA, this octameric protein folded the DNA into a structure closely resembling that of native core particles as verified by various techniques; protein denaturants were necessary for reassociation. The histone octamer is useful as a model of the nucleosome protein core and for studying histone-DNA interactions that occur in nucleosomes.