The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Summary Intermediates of DNA replication in the second half of the latent period after phage infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative froms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the genome. Replicating circles with a linear DNA tail longer than one unit length of genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.     

Stability of coliphage lambda DNA replication initiator, the lambda O protein.

The initiator of coliphage lambda DNA replication, lambda O protein, may be detected among other 35S-labeled phage and bacterial proteins by a method based on immunoprecipitation. This method makes it possible to study lambda O proteolytic degradation in lambda plasmid-harboring or lambda phage-infected cells; it avoids ultraviolet (u.v.)-irradiation of bacteria, used for depression of host protein synthesis, prior to lambda phage infection. We confirm the rapid decay of lambda O protein (half-time of 80 s), but we demonstrate the existence of a stable lambda O fraction. In the standard five minute pulse-chase experiments, 20% of synthesized lambda O is stable. The extension of the [35S]methionine pulse, possible in lambda plasmid-harboring cells, leads to a linear increase of this fraction, as if a part of the synthesized lambda O was constantly made resistant to proteolysis. Less than 5% of lambda O protein synthesized during one minute is transformed into a stable form. We presume that the stable lambda O is identical with lambda O present in the normal replication complex and thus protected from proteases. We cannot find any stable lambda O in Escherichia coli recA+ cells that were irradiated with u.v. light prior to lambda phage infection, but their recA- counterparts behave normally, suggesting that recA function interferes in the assembly of a normal replication complex in u.v.-irradiated bacteria. The stable lambda O found in lambda plasmid-harboring, amino acid-starved relA cells is responsible for the lambda O-dependent lambda plasmid replication that occurs in this system in the absence of lambda O synthesis. The existence of stable lambda O raises doubt concerning its role as the limiting initiator protein in the control of replication. Another significance of lambda O rapid degradation is proposed.     

Mutations in coliphage p1 affecting host cell lysis

A total of 103 amber mutants of coliphage P1 were tested for lysis of nonpermissive cells. Of these, 83 caused cell lysis at the normal lysis time and have defects in particle morphogenesis. Five amber mutants, with mutations in the same gene (gene 2), caused premature lysis and may have a defect in a lysis regulator. Fifteen amber mutants were unable to cause cell lysis. Artificially lysed cells infected with five of these mutants produced viable phage particles, and phage particles were seen in thin sections of unlysed, infected cells. However, phage production by these mutants was not continued after the normal lysis time. We conclude that the defect of these five mutants is in a lysis function. The five mutations were found to be in the same gene (designated gene 17). The remaining 10 amber mutants, whose mutations were found to be in the same gene (gene 10), were also unable to cause cell lysis. They differed from those in gene 17 in that no viable phage particles were produced from artificially lysed cells, and no phage particles were seen in thin sections of unlysed, infected cells. We conclude that the gene 10 mutants cannot synthesize late proteins, and it is possible that gene 10 may code for a regulator of late gene expression for P1.     

Mutations affecting two distinct functions of the RNA component of RNase P.

The effect of structural changes on the functions of the RNA component (M1 RNA) of ribonuclease P (RNase P) of Escherichia coli has been studied using the thermosensitive mutants of the rnpB gene. One of the mutants, ts709, has two G--A substitutions at positions 89 and 365 from the 5' end of M1 RNA. Of these substitutions, the one at position 89 from the 5' end is responsible for the phenotype of this mutant. Although the RNase P activity of ts709 is thermosensitive, the mutant M1 RNA has the same catalytic activity as the wild-type RNA. M1 RNA of another mutant, ts2418, has a G--A substitution at position 329. This mutant RNA has extremely low catalytic activity. The upstream mutational site of ts709 appears to play a role in the association with the protein subunit, whereas the mutational site of ts2418 is related to the catalytic function of M1 RNA.     

Secondary-site attachment of coliphage lambda near the thr operon

Phage λ has been integrated at a secondary attachment site near the thr oporon of Escherichia coli. The integration caused a pleiotropic effect since both thrA and thrB enzyme activities were lost. Lysogenic, thr⁺ revertants regained thrA and thrB activities although the enzymes were synthetized constitutively at low levels. Four classes of prophage deletion strains were isolated with deletions extending into trpR and the thr operon. Genetic and biochemical analyses indicated a gene order: trpR .. λ .. thrA .. B .. C. Defective transducing phage carrying thr A, B,C were also isolated.     

N-independent leftward transcription in coliphage lambda: deletions, insertions and new promoters bypassing termination functions.

Lambda mutants capable of N-independent red-gam gene expression were isolated by selecting Fec+ plaque-forming derivatives of lambda N+ nutL- (Fec-) strains. In addition to true nutL+ reversions, three classes of second-site mutations were identified: (1) ninL deletions that remove a region containing either tL1 or both tL1 and tL2 termination signals, or only a small region (defining the rut site) just upstream from tL1, (2) new constitutive promoters that map just upstream from the tL2 termination site and which are created either by point mutations (hip) or by short insertion sequences (isp), (3) small internal deletions in gene cro. The positions and individual effects of these mutations, some of which only partially abolish termination function, provide evidence for a complex multipartite structure of the termination signals.     

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Summary Genetically marked and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the protein coat were selected on WR4027 (), a -immune, P22-resistant derivative of WR4028. In these immP22 hybrids, at least the c through P genes of were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (, immP22). One such hybrid, immP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that immP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for immP22dis. In addition, immP22dis contains the P22 a1 locus responsible for somatic 0–1 antigen conversion in Salmonella. Although the immP22dis phage particle has the head and tail, the phage genome also carries P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the immP22dis prophage near the proA locus on the bacterial chromosome.     

Genetic studies of hybrids between coliphage lambda and salmonella phage P22: genetic analysis of the P22-lambda hybrid class.

Summary P22- hybrids which retain the protein coat of P22 have been isolated and characterized into two types. Type 1 hybrids which have the c through O-P genes of are unable to grow lytically on Salmonella typhimurium. On the other hand, type 2 hybrids which contain only the c region of , plated on S. typhimurium. Both hybrid types retained the generalized transducing and antigenic conversion capabilities of P22.