Asymmetric distribution of the transcribing regions on the complementary strands of coliphage lambda DNA.

Abstract

Hybridization of the various \-specific mRNA''s with the separate strands of A DNA provides a new technique for determining the distribution of the transcribing regions. Several technical refinements include self-annealing of the separated DNA strands which results in preparations of individual strands displaying a purity of over 99% in hybridization tests, and a highly selective 2-step hybridization procedure with prehybridization including RNAse treatment followed by inactivation of RNase by iodoacetate. With the latter method, the levels of X-specific mRNA in nonlysogenic E. coli (0.001-0.002%), in noninduced lysogens (0.03-0.06%) and early (0.5- 2.0%) or late (6-12%) in induced nondefective lysogens or in infected cells were compared with the simplified ratio of these figures being represented as 1 (nonlysogenic): 50 (noninduced): 500 ("early" induced): 5000 ("late" induced). In the noninduced state the majority mRNA is transcribed from the W strand probably being the product of gene cI. Upon induction or infection, 2 regions adjoining the cI gene start to be transcribed: the predominant product (90%) is copied from strand W in the same direction as gene q through genes N-to-a, whereas the minority mRNA (10%) is copied in the opposite direction from strand C through the x-0 operon. Both of these transcription and translation products seem to be required to activate the further transcription of the X. genome since the early transcription pattern could be frozen either by inhibition of protein synthesis (100 [mu]g CM/ml) or by the nonsense or polar mutations in genes N or x. Within 10-20 min. after infection, the transcription changes to the "late" pattern with over 85% of the mRNA being transcribed from the C strand pro-gressively more and more from its left arm. During the development of X , the transcription of strand W decreases 10-30%; whereas the transcription of strand C increases 30-to 70-fold. Transcription in the induced susO and P lysogens unable to synthesize DNA does not proceed far beyond the early stage. The hybridization pattern ob-tained with the Adg-J mutant, which transcribes both DNA strands confirms that a divergency switch in the direction of mRNA synthesis is on the right arm of X DNA. The segments of the individual DNA strands characterized as transcribing regions contain all the dC-rich clusters which may initiate the DNA-to-RNA transcription process. The asymmetry in the distribution of the poly IG-binding dC clusters, seems to be directly related to the asymmetric transcription pattern of mRNA and the changes in the orientation of this transcription.