| dc.description | From their growth requirements, the metabolic intermediates accumulated, and the enzymes present, ten auxotrophs of Streptomyces venezuelae and two of Streptomyces lividans were characterized and their trp mutations determined. All classes of trp mutations except trpF, trpE, and trpG were present. When considered with the relative location of trp mutations obtained in this and earlier studies, the results showed that in S. venezuelae trpC, trpB, and trpA are clustered near hisA and hisB while trpD is at another location near nicB. Fragments of S. venezuelae DNA encoding trpC and trpD were cloned by complementation of relevant S. lividans auxotrophs. A trpE-encoding DNA fragment was cloned by complementation of an E. coli trpE auxotroph. Growth studies and enzyme assays indicated that trpG was clustered with trpE on a 2.4-kb DNA fragment. By developing a technique for integration of a vector carrying a resistance marker and homologous DNA of the gene under study, and in conjunction with "classical" genetic analysis, trpEG was mapped close to but separate from the trpCBA cluster. The arrangement of trp genes found in S. venezuelae is unique among bacteria. An attempt was made to introduce a trpE or trpG mutation by in vitro modification of the cloned 2.4-kb DNA fragment containing trpEG and introduction of the modified fragment into the host chromosome by allele replacement. Although DNA replacement occurred, it did not result in tryptophan auxotrophy. | en_US |
