Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing

Abstract

Three synthetic oligonucleotides were prepared to be complementary to known regions of the mouse immunoglublin light chain mRNA, and their ability to prime the transcription of complementary DNA (cDNA) was studied. The sequence of the cDNA was determined by adapting for mRNA the DNA sequencing method of Sanger, Nicklen and Coulson (1977) which uses 2'3' dideoxy ribonucleotides. A continuous sequence of 532 nucleotides was obtained, 321 corresponding to the whole of the constant region of the mRNA and the remaining 211 being the complete 3' noncoding region of the mRNA. The termination codon U-A-G occurs at the expected position in the mRNA corresponding to the triplet following the C terminal cystine. The nucleotide sequence is partially corroborated by the sequence of fragments obtained previously from 32P-mRNA fingerprints and endonuclease IV digests of 32P-cDNA, and is in agreement with the amino acid sequence of the constant region, except for a rearrangement of four amino acids (between amino acid positions 163 and 166). A revision of the amino acid sequence confirms the nucleic acid sequence.