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THE ACTIXOMYCETES, Vol. I 



colonies and sporulated on synthetic agar 

 with ammonium sulfate and glucose as the 

 only nitrogen and carbon sources. Nutri- 

 tionally deficient mutants were developed 

 by ultraviolet irradiation of the parental 

 prototrophic culture, concentrating the mu- 

 tants by the filtration technique, and finally 

 employing selective media and the replica- 

 plate principle to detect and identify the 

 mutants. Auxotrophic strains were isolated 

 which required single or multiple supple- 

 ments of various amino acids or alternati\'e 

 requirements of two, three, and even four 

 amino acids, often as the result of a single 

 mutation. Several antibiotic-resistant mu- 

 tants were also isolated with the help of the 

 gradient-plate technique (Szybalski, 1958). 

 The first type of genetic interaction, 

 widely observed in these studies, was the 

 formation of heterokaryotic mycelium con- 

 taining both types of parental nuclei in a 

 common cytoplasm. This phenomenon was 

 easily demonstrated by plating a mixture of 

 two types of nutritionally marked conidia 

 on a selective medium. A cross was per- 

 formed between two strains of S. fradiae, 

 one streptomycin-resistant and requiring 

 methionine and leucine, and the other re- 



quiring histidine and arginine but strepto- 

 mycin-sensitive (Fig. 53). A mixture, con- 

 sisting of approximately lO'"* spores from each 

 parent, was incubated for 2 to 6 days on 

 minimal agar. This mixture yielded several 

 hundi'ed prototrophic "recombinant" colo- 

 nies, whereas the plates seeded with spore 

 suspensions of only one of the parents 

 showed no growth. The formation of hetero- 

 karyons, nutritionally balanced, i.e. able to 

 grow in the absence of all the nutritional 

 reciuirements exhibited by any one of the 

 parents, was demonstrated for S. griseus, S. 

 fradiae, S. venezuelae, and S. albus. Only *S'. 

 coelicolor produced nutritionally unbalanced 

 heterokaryons, which formed tufts of growth 

 between proximal parental colonies grown 

 on the synthetic medium enriched with a 

 small amount of an amino acid mixture. 

 These inibalanced types did not grow on an 

 unsupplemented medium. 



Heterokaryon formation was observed 

 only between mutants derived from the same 

 parental culture. The interspecific crosses 

 and a limited number of intraspecific crosses 

 between different strains designated as S. 

 griseus were unsuccessful. 



