ALBERT

Notes: Summary Twenty-two amber mutants of the thermoinducible mutator phage Mu-c4ts were isolated. These mutants fall into 11 complementation groups. The data obtained by crossing these amber mutants suggest that bacteriophage Mu-1 has a linear vegetative linkage map. In a recombination deficient host of the RecA type the recombination frequencies are extremely low, indicating that Mu-1, in contrast to many other E. coli phages, is dependent on the recombination system of its host. With λ as a helper phage, recombination between Mu phages in a RecA host is restored to about 1/3 of the frequency in a Rec+ host. Although Mu-1 is able to integrate efficiently into the chromosome of a RecA strain, it seems that its integration system does not contribute to vegetative recombination. The survival of UV-irradiated Mu-1 was measured on different radiation sensitive mutants of E. coli. The survival on a UvrB strain was very low as compared to the wild-type; the survival on a RecA strain was almost the same as on the wild-type.

Notes: Summary It has previously been shown that the transcription of Mu is asymmetric and takes place on the heavy DNA strand (Bade, 1972; Wijffelman et al., 1974). The direction of transcription of Mu has now been determined by RNA-DNA hybridizations between purified Mu-RNA and the separated strands of λ-Mu hybrid phages. The direction of transcription is from the c-gene (immunity gene) end of the heavy strand to the β-end (immunity distal end) (Fig. 1). Thermo-inducible, defective Mu lysogens, in which the prophage is deleted from the β-end, have a normal early transcription pattern, but the increase of RNA at later times is absent. A defective lysogen, which contains only the immunity gene c and the genes A and B, still has an early transcription pattern similar to that of the wild-type. Therefore, we conclude that the early RNA is transcribed from that region of the Mu genome. The early Mu-RNA synthesis is negatively regulated with a minimum rate of transcription at 9 minutes after induction. Before the onset of the late RNA synthesis, at about 22 minutes there is a rather long period in which the rate of Mu-RNA synthesis slowly increases. Using DNA strands of λ-Mu hybrids which contain only that part of the Mu-DNA on which the early RNA synthesis takes place, we have determined that during the first half in the intermediate phase only early genes are transcribed. The amount of Mu-RNA synthesized by a Mu prophage carrying the X-mutation, which influences the excision of Mu, is greatly reduced. Negative regulation of early transcription occurs normally in this mutant.

Notes: Summary The transcription pattern of bacteriophage Mu has been studied with the use of Mu-1 cts62, a thermo-inducible derivative of wild-type Mu. The rate of transcription at various times after induction was measured by pulse-labeling the RNA during synthesis and determining the fraction of Mu-specific RNA by hybridization with the separated strands of Mu-DNA. Transcription was found to take place predominantly from the heavy strand of Mu-DNA, as was found previously by Bade (1972). A study of the kinetics of this process revealed four phases. Initially after the induction the rate of transcription increases and reaches a maximum after four minutes. In the second phase during five minutes the rate falls down. During the third phase, up to 25 minutes after induction, the rate of transcription rises slowly, followed by a very rapid increase in the final phase, at the end of the lytic cycle. Phage Mu can be integrated in the host chromosome in two opposite orientations. The strand specificity, rate and time-course of transcription appeared not to be influenced by the orientation. The presence of chloramphenicol during the induction of the phage does not have an effect on the initial phase of transcription, but it prevents the decrease in the second phase. This suggests that in the early phase a Mu-specific protein is synthesized which acts as a negative regulator of trancription. In non-permissive strains, lysogenic for a phage with an amber mutation in gene A or B, the transcription during the first and the second phase is the same as with wild-type phage; in the third phase, however, there is much less transcription than with wild type phage, whereas in the final phase the increase of the transcription rate is completely absent. Control experiments showed that DNA synthesis does not take place when a non-permissive strain is infected with a phage with an amber mutation in gene A or B. Therefore we conclude that the products of the genes A and B are required, directly or indirectly, for the autonomous replication of phage DNA. Since these amber mutants are also impaired in the integration process, we conclude that the genes A and B code for regulator proteins with a crucial role in the development of bacteriophage Mu.