We have determined whether two 46-base pair (bp) GC-rich clusters present in the coding region of the yeast mitochondrial var 1 gene are transcribed and retained in the putative mRNA for the var 1 protein. One GC cluster is found in all var 1 alleles, while the other is optional; both are identical in sequence and, when present together in var 1, are arranged in opposite orientation 158 bp apart. Because of their dyad symmetry, these GC clusters would form stem and loop structures if present in RNA. We have exploited this potential for secondary structure to show that these GC clusters are transcribed. Using a 46-bp RsaI-MboI var 1 restriction fragment as primer and 16 S RNA (the putative var 1 mRNA (Farrelly, F., Zassenhaus, H. P., and Butow, R. A. (1982) J. Biol. Chem. 257, 6581-6587) purified from strains whose var 1 allele contains one or both of these GC clusters as template for AMV reverse transcriptase, we find the accumulation of a major class of cDNAs whose size is consistent with an impediment of reverse transcription at the predicted positions of the GC clusters in the RNA template. Moreover, depending upon the RNA template, we find that the small fraction of full length cDNA molecules made in the reaction contain one or two SstII sites. Because of fold-back, these sites are predicted to be present in the otherwise single-stranded cDNAs if the GC clusters are transcribed.
Base Sequence DNA