)NA/restriction fragment extension/

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. Nat). Acad. Sci. USA 74, 5463-5467), we use 2',3'-dideoxynu- cleoside triphosphates as chain-terminating inhibitors of the reverse transcriptase (RNA-dependent DNA polymerase) reac- tion. 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 se- quence (if any) is unknown. Recent discoveries that many eukaryotic and viral mRNAs result from the excision of segments from primary transcripts and subsequent splicing (for reviews see ref. 1-3) suggest that a detailed knowledge of mRNA structure is necessary for an understanding of gene regulation. A number of investigators have analyzed the primary structure of mRNA by sequence determination of the complementary DNA (cDNA) (4-8). In those studies, the cDNA synthesis has been primed with syn- thetic oligonucleotides and the cDNAs have been sequenced either by Maxam and Gilbert's chemical method (7-9) or by Sanger and Coulson's plus and minus method (4-6, 10). We have approached the problem of sequencing desired regions of selected mRNAs by using given DNA restriction fragments, which anneal to the mRNA at specific regions, as primers for the reverse transcriptase (RNA-dependent DNA polymerase) reaction. Addition of dideoxynucleoside triphosphates as chain terminators (11) during the reverse transcriptase reaction allows rapid sequence determination of the cDNA. Using this method, we have studied the major late cytoplasmic 16S mRNA of simian virus 40 (SV40), the message that codes for the structural coat protein VP1 (12). It had been previously shown that VP1 mRNA is transcribed from two noncontiguous regions in the genome; the 5' end of the mRNA is from position 0.72 to 0.76, while the body of the mRNA is transcribed from the region between 0.938 and 0.17 on the viral genome (8, 13--16) (Fig. 1A). We have confirmed the results showing that the 5' leader sequence is about 200 nucleotides long (8). We find that the junction of the leader with the main body of the 16S mRNA precedes the initiation codon by 44 nucleotides, and we suggest that a subset of mRNAs with shorter leader sequences may exist.