Fromtwo types of class V act mutants of Streptomyces coelicolor two monomericprecursors of actinorhodin have been isolated and their structures determined. One is the known antibiotic kalafungin (2) and the other a new compound(6). Their relationship to actinorhodin biosynthesis is discussed.Streptomyces coelicolor A3(2) is the genetically most characterized actinomycete, with a well developed circular chromosomelinkage map1}. A fragment of this chromosomebetween the markers gua A and his D contains a cluster of structural genes for the biosynthesis of the isochromanequinone antibiotic, actinorhodin (1) (Scheme 1)2). Rudd and Hopwoodisolated 75 mutants which are blocked in the biosynthesis of this antibiotic, and were able to group them into seven classes. The classification was based on the mutants' ability to secrete intermediates which other mutants, blocked earlier in the biosynthetic pathway, could convert into actinorhodin. Production of this antibiotic could easily be recognized by fuming the agar plates over ammonia, since actinorhodin is a red-blue acidbase indicating compound. If a donor strain secreted a precursor which a converter strain could use to produce actinorhodin, then a blue zone would appear on the edge of the converting strain colonies closest to the donor. The classes could thus be ordered as to their place in the actinorhodin biosynthetic pathway.Class V mutants secreted (a) diffusible intermediate(s) into the medium which all other classes (except a putative regulatory class, II) could convert into actinorhodin. Membersof this class could not convert compoundssecreted by any other mutants into actinorhodin, and therefore must be blocked at the latest stage in the biosynthesis.The steps of isochromanequinone antibiotic biosynthesis following polyketide chain formation have recently been of interest since the act genes from class V have been cloned and inserted into another species, Streptomyces sp. AM-71613). This latter species normally produces the yellow-brown acid-base indicating antibiotic, medermycin but after transfer of the class V act genes, a new, hybrid orange-purple acid-base indicating pigment named mederrhodin A was produced. Another new hybrid structure, dihydrogranatirhodin was produced by a transformant of the dihydrogranticin-producing S. coelicolor A3(2). This compoundhas the granaticin stereochemistry at one chiral center and the actinorhodin stereochemistry at the other. With this ability to clone antibiotic genes and transfer them between species, the design and syn-

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Direct study of the methylation of ribonucleic acid with methyl methanesulfonate by carbon-13 nuclear magnetic resonance spectroscopy has demonstrated the usefulness of this method in studying the chemical modification of biomacro-molecules and the interaction between nucleic acids and biologically active agents. This direct stable isotope method eliminated all tedious and questionable degradation processes for determining the reactive sites and the product distributions. Six methylated products, 7-methylguanosine, 1-methyladenosine, 3-methylcytidine, 1-methylguanosine, 3-methyluridine, and methyl phosphodiester, were identified by comparison with many model compounds and careful examination of spin-spin coupling and spin-lattice relaxation time. An extensive study of the interaction of phosphate buffer with methyl methansulfonate accounted for the sharp difference in the 13C spectra of the methylated RNA isolated from the reactions controlled by a pH-stat and phosphate buffer, respectively. The 13C-enriched agent significantly enhances the specificity and sensitivity of the method and provides better quantitative results.