Electron-microscopic demonstration of terminal and internal initiation sites for cDNA synthesis on vitellogenin mRNA.

cDNA synthesized on purified vitellogenin mRNA from Xenopus liver was hybridized to the template in formamide/urea at 22 degrees C to avoid degradation of the RNA. The hybrids formed were visualized by spreading for electron microscopy. Contour length measurements proved that most of the RNA molecules in the hybrids were still intact showing the expected molecular weight of 2.3 x 10(6). The hybridized cDNA corresponded on the average to 12% of the RNA length. In about 80% of the molecules the cDNA was located at one end. Since cDNA synthesis was primed by oligo(dT), the terminal duplex region marks the 3' end of the vitellogenin mRNA molecule. Internal duplex regions were mainly located at a specific position starting about 2800 nucleotides from the 3' end. Since the cDNA hybridizing at the internal position could specifically be synthesized on a vitellogenin RNA fragment isolated on poly(U)-Sepharose as an oligo(A)-containing RNA, we conclude that cDNA synthesis is not only initiated by the poly(A) of the 3' end, but also by a specific internal sequence.