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The DNA sequence that encodes 23S rRNA domain V of Bacillus subtilis, nucleotides 2036 to 2672 (C. J. Green, G. C. Stewart, M. A. Hollis, B. S. Vold, and K. F. Bott, Gene 37:261-266, 1985), was cloned and used as a template from which to transcribe defined domain V RNA in vitro. The RNA transcripts served as a substrate in vitro for specific methylation of B. subtilis adenine 2085 (adenine 2058 in Escherichia coli 23S rRNA) by the ErmSF methyltransferase, an enzyme that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics on Streptomyces fradiae NRRL 2702, the host from which it was cloned. Thus, neither RNA sequences belonging to domains other than V nor the association of 23S rRNA with ribosomal proteins is needed for the specific methylation of adenine that confers resistance to the macrolide-lincosamide-streptogramin B group of antibiotics.