Specific binding of a synthetic oligodeoxyribonucleotide to yeast cytochrome c mRNA

THE investigation of specific messenger RNAs in terms of sequence analysis and of the control of their synthesis, processing, translation and degradation is one of the major interests of modern molecular biology. These studies have been possible so far in only the few cases where a cell type is known to synthesise predominantly one species of protein. Since most mRNAs are chemically similar, differing only in size and sequence, it has been difficult to purify them by conventional methods. Given the amino acid sequence of a protein, one can use the genetic code to deduce a sequence of nucleotides in a specific region of the mRNA and then synthesise an oligodeoxyribonucleotide complementary to that region 1–4 . We have chosen to investigate the yeast iso-1 cytochrome c mRNA as a model system for the use of synthetic oligonucleotides as specific probes of eucaryotic mRNA structure and function. The genetics of yeast iso-1 cytochrome c has been studied in great detail by Sherman, Stewart and co-workers 5,6 , who have determined the sequence of 44 nucleotides of the mRNA from the analysis of altered cytochromes produced by a series of double frameshift mutants. In addition, a number of well defined deletion mutants are available within this region. The pentadecadeoxyribonucleotide (15-mer) that we decided to synthesise, 5′–d(A–G–C–A–C–C–T–T–T–C–T–T–A–G–C)3′ is complementary to nucleotides 25–39 of the cytochrome c mRNA (Fig. 1). This sequence was chosen because it is relatively easy to synthesise, should form a very stable hybrid with mRNA, and is close to the 5′ end of the coding region of the mRNA. A known deletion mutation covers this region, yet allows the production of a functional cytochrome c in normal amounts 5,6 , and thus provides an excellent control for hybridisation experiments. We have been able to show that the synthetic 15-mer does indeed bind specifically to the expected region of the mRNA, and therefore should be useful as a specific probe for the study of this mRNA.