Transformation of Bacillus subtilis using hybrid DNA molecules constructed by annealing resolved complementary strands.

Abstract

HE question of whether one or both strands of the transforming deoxyriboTnucleic acid (DNA) molecule is inserted into the host chromosome of Bacillus subtilis has been the subject of several recent reports. BODMER and GANESAN (1964) presented physical evidence for the incorporation of one strand. AYAD and BARKER (1969) in contrast, presented physical evidence suggesting that both strands of the duplex are incorporated. Evidence bearing on this question using D. pneumoniae (Fox and ALLEN 1964) and H. influenzae (NOTANI and GOODGAL 1966) favored the incorporation of only one strand of the donor duplex. BRESLER et al. (1964) and VESTRI, FELICETTI and LOSTIA (1966) have reported genetic evidence in B. subtilis transformation for the use of both strands with low frequency. With the advent of methods for separating the complementary strands of B. subtilis DNA (ROGER, RECKMANN and HOTCHKISS 1966; RUDNER, KARKAS and CHARGAFF 1968), it became possible to prepare pure hybrid duplexes each strand of which contained a unique genetic marker. The use of such preparations permits a more critical evaluation of the role of the duplex in transformation. The transformants which arise from the use of these duplexes are easily analyzed and permit one to examine the behavior of hybrid duplex free of its mirror image. ( GABOR and HOTCHKISS 1966,1969; PETERSON and GUILD 1968).