Kjell Bøvre

Patterns of convergent and overlapping transcription within the b2 region of coliphage λ

Abstract The central b2 region in the conventional vegetative map of the coliphage λ genome was found to be a unique DNA segment in which two opposingly oriented mRNA syntheses, one originating on the r strand of the left arm and the other on the l strand of the right arm, converge and partially overlap (Figs. 1 and 2). Thus, a segment located to the left of the center of the b2 region, for which no functional genes have been found, appears to act as a buffer zone between the two converging transcriptions, some complementary RNA sequences being produced in the region of overlapping syntheses. The l strand of the b2 region is transcribed early and late after infection or induction. However, there is little or no such transcription upon induction of singly lysogenized bacteria when the excision and circularization of λ are blocked. It appears, therefore, that the l strand transcription of the b2 region does depend on coupling between b2 and the early N- a ′ genes; this link is characteristic of the DNA extracted from phage particles and of the circularized genomes, but is disrupted in the singly lysogenic form of λ, which is cyclicly permuted (Fig. 1). The r strand within the left segment of the b2 region becomes transcribed only late after infection or induction, as if it were coupled to the A-J genes, which are expressed late and transcribed from strand r . When protein synthesis is inhibited by chloramphenicol or when gene N is nonfunctional, the b2 region is not transcribed from either strand of DNA. The possible roles of the b2 region, which constitutes a model system for studying convergent transcriptions, are discussed.

Controls of rightward transcription in coliphage λ

Abstract Hybridization between [ 3 H]RNA, produced after thermal induction of λ lysogens, and the r strands of λ and the appropriate λ−φ80 hybrid phage DNAs was employed to study the rightward transcription of the well-defined λ regions designated A-J-b2, x-P-Q , and Q-S-R , including segments A-J plus half of the b2 region, the region from x to the left boundary of Q , and genes Q to R , respectively. Control of the x-P-Q region, which is transcribed early after induction and remains low, is independent of the late controlsof the Q-S-R and A-J-b2 regions, the transcription of which positively depends on the Q product and λ DNA replication. Mutation in the p L promoter ( sex − ) reduces the total leftward transcription by more than tenfold, and indirectly reduces the rightward transcription, as in the λ N − mutants. Mutation in the p R promoter ( x 13 ) abolishes the rightward transcription and indirectly results in uncontrolled leftward transcription. Mutation zd results in a strong reduction of the rightward transcription in all three regions, whereas the Q − mutation abolishes the late augmentation of transcription only in the Q-S-R and A-J-b2 regions.

Techniques of RNA-DNA Hybridization in Solution for the Study of Viral Transcription

Publisher Summary This chapter discusses the techniques of RNA-DNA hybridization in solution for the study of viral transcription. The first technique of RNA-DNA hybridization designated as hybridization in solution technique is based on the original method of A. P. Nygaard and B. D. Hall and involves hybridization between denatured DNA and 3 H - or 32 P-labeled RNA in an appropriate fluid medium. The second technique, designated as filter hybridization, is based on the D. Gillespie and S. Spiegelman’ s modification of the above technique. It involves hybridization between a solution of labeled RNA and denatured DNA already fixed on nitrocellulose filters. The chapter discusses the technique that combines the advantages of both hybridizaxadtion methods. The use of separated DNA strands isolated from viral mutants carrying various genetic deletions or substitutions permits a detailed study of transcription from the various segments of the viral genome. This is based on the fact that those segments of the DNA strand that are transcribed also hybridize with their complementary RNA product. Multistep hybridization in solution is a flexible means of increasing the discriminatory power of RNA-DNA hybridization and of controlling the specificity of the ordinary one-step procedure.

Regulation of leftward transcription in the J-b2-att region of coliphage lambda

SummaryThe leftward transcription of the central region of the mature coliphage λ genome, which extends from gene J to the attachment point att and which includes the b2 region, was studied. Upon thermal induction of λcI, 857 lysogens, the rate of the leftward transcription of the central region reaches a sharp maximum (1–2% of the total, i.e., λ + Escherichia coli, transcription) at about 5 min at 42° C, and then rapidly declines. This rapid decline was not observed in induced λx− (pR−; phenotypically tof−) lysogens. Upon induction of a lysogen carrying a single, nonexcisable int− prophage, the l strand of the central region is not transcribed, whereas in an induced int+ or a double int− or int+ lysogen the l strand of the central region is transcribed both early and late in λ development. It is concluded that the leftward transcription of the central region is not autonomously initiated, but depends on the coupling between b2 and the att-N region containing the autonomous PL promoter. The leftward transcription of the central region is not restricted to b2, but can also proceed to the left of it. The results confirm the previous conclusion, based on a study of the late rightward transcription of the b2 segment (Bøvre and Szybalski, 1969), that convergent transcriptions overlap in the J-b2-att region, which comprises about 20% of the λ genome, leading to the synthesis of mutually complementary RNA sequences.

Control of Leftward Transcription in the Immunity Region of Escherichia coli Phage Lambda

SummaryIt was shown by various RNA-DNA hybridization techniques that the l-strand transcription of the λ immunity region, including genes cI and rex (see i434, Fig. 1), of Escherichia coli phage λ rapidly decreases after induction of lysogenic cells. This decrease appears to depend on protein synthesis, since the cI transcription was not reduced after heat induction of a λcI,857 lysogen in the presence of chloramphenicol. Upon infection of a sensitive E. coli host with λbio256, an immediate, but very low and transient synthesis of l-strand specific mRNA from the immunity region was observed.