Jun-ichi Tomizawa

Transducing fragments in generalized transduction by phage P1: I. Molecular origin of the fragments

Bromouracil-labelled Escherichia coli thy− was infected with a virulent mutant of phage P1kc and incubated in a medium containing thymine until lysis occurred. The analysis of the phage yield by CsCl density-gradient centrifugation showed that the transducing particles carrying various chromosomal genes have a density and band profile similar to those prepared on bromouracil-labelled bacteria in a medium containing bromouracil. When the phage particles were prepared on the bromouracil-labelled bacteria in a medium containing thymine and 32PO4, radioactivity was not found in fractions which contained transducing particles. These reults indicate that most of the transducing particles lack phage genome and carry only fragments of the bacterial chromosome existing at the time of infection. The results show interruption of the replication of bacterial chromosome by phage infection. The replication of λ prophage and F′ lac was also interrupted by infection. On the other hand, the replication of an R factor was not arrested. In contrast to the behaviour of the virulent mutant, the original P1kc phage did not arrest the replication of bacterial chromosome. When the virulent mutant was grown on [3H]thymidine-labelled bacteria in a medium containing 32PO4, infective particles and transducing particles could be differentially labelled with 32P and 3H, respectively. The results of the analysis of the phage particles showed that the transducing particles comprised 0·3% of the total phage particles. Physical properties of DNAs of infective and transducing particles were studied. The density of P1 DNA is 1·706 g cm−3, which corresponds to a guanine–cytosine content of 46%. The molecular weights of DNAs of infective and transducing particles are the same, namely, 6 × 107 daltons. Homology between PI DNA and E. coli DNA is rare.

Plasmid ColE1 incompatibility determined by interaction of RNA I with primer transcript

Mutants of plasmid pNT7 that can coexist with plasmid pMB9 in growing bacteria have been isolated. These mutants show altered incompatibility properties and increased copy numbers. Each mutant has a single base change at or near the center of one of the three palindromes in the region that specifies two RNA species: a larger one (primer transcripts) that provides a primer for DNA replication and a smaller one (RNA I) that is the incompatibility-specific inhibitor of primer formation. In vitro transcription studies show that the single base changes affect both the ability of RNA I to inhibit primer formation and the sensitivity of primer formation to inhibition by RNA I. RNA I hybridizes to the primer transcript, and the rate of hybridization is reduced by the single base changes. Based on analyses of inhibition of in vitro primer formation by RNA I and of in vivo properties of the mutant plasmids, we conclude that incompatibility between two plasmids can be attributed to inhibition of primer formation on one of the plasmids by the RNA I of the other. Inhibition of primer formation by RNA I appears to be the mechanism that determines the copy number of pNT7 and its derivatives.

Control of cole 1 plasmid replication: Enhancement of binding of RNA I to the primer transcript by the rom protein

RNA I prevents a transcript (RNA II) from the ColE1 primer promoter to form a hybrid with the template DNA and thereby inhibits formation of primer for DNA replication. Binding of RNA I to RNA II is responsible for the inhibition. The Rom protein, a plasmid-specified 63 amino acid protein, enhances the inhibitory effect of RNA I on primer formation by enhancing the binding of RNA I to RNA II. In vivo, RNA I controls plasmid copy number and incompatibility and inhibits expression of a galK gene fused to the primer promoter. The rom gene modulates these activities of RNA I. These functions of the rom gene can be explained by alteration of the binding of RNA I to RNA II by the Rom protein.

Studies on radiation-sensitive mutants of E. coli. I. Mutants defective in the repair synthesis.

SummaryTwenty-four UV sensitive strains which can not carry out the host cell reactivation and two which are deficient in recombination were isolated. By analyzing their properties and genetic locations, 21 UV sensitive strains were classified into the uvrA, B, and C groups and 3 strains into a group which has not been described. Two Rec- strains were classified in the recA group. The UV sensitive mutants of the new group are distinctly different from other mutants in their properties and in the sites of mutation. We named this group the uvrD.These uvrD mutants have the following properties. They have an intermediate sensitivity against UV irradiation and a higher sensitivity against γ-ray irradiation than those of other UV sensitive mutants. The UV damages which are repaired in the participation of the uvrD gene are photoreactivable. In the cells UV irradiated λ phage rapidly loses the susceptibility to photoreactivation during the incubation in broth with chloramphenicol. The DNA of the uvrD mutant is rapidly degraded at a small dose of UV light and to a large extent. The uvrD gene locates very close to the metE gene and uvrD- is dominant over uvrD+. The uvrD cells have the capacity to carry out UV reactivation for UV irradiated λ phage, in contrast to other UV sensitive mutants including the Rec- ones which turned out to have no capacity. A double mutant, uvrB uvrD, is about three times as sensitive as the uvrB mutant against UV irradiation and DNA degradation after UV irradiation takes place at much lower rate than the uvrD mutant. These results show the presence of a functional relation between the uvrB and uvrD genes and suggest that the uvrD gene participate in the repair synthesis at a step following that performed by the uvrB gene.

Studies on radiation-sensitive mutants of E. coli

SummaryThe bacterial recA gene participates in the induction by UV irradiation of the clear mutation of phage λ and the Lac- mutation of bacteria. The necessary function is induced by irradiation of Rec+ bacteria and acts upon DNA irradiated with UV light.

The origin of replication of plasmid p15A and comparative studies on the nucleotide sequences around the origin of related plasmids

Replication of Escherichia coli plasmid p15A was examined by use of a cell extract or a mixture of three purified E. coli enzymes: RNA polymerase; RNAase H; and DNA polymerase I. In each system, replication initiates at any of three consecutive nucleotides located at a unique site. Primer transcription starts 508 bp upstream of the replication origin. The region between 294 and 524 bp upstream of the origin determines the incompatibility property. This region specifies an RNA (RNA I) of about 105 nucleotides that is involved in regulation of primer formation. We compare the nucleotide sequences around the origins of related plasmids p15A, ColE1, pBR322, RSF1030 and CloDF13, and discuss the significance of possible RNA secondary structures in primer formation.

Replication of bacteriophage DNA: I. Replication of DNA of lambda phage defective in early functions☆

Abstract DNA synthesis of early sus (suppressor sensitive) mutants of phage λ in su − cells was studied by the density-gradient centrifugation method. The functions of the O and P cistrons are essential for replication of the phage genome while the function of the N cistron is not. DNA molecules of sus N mutants replicate at a slow rate. A majority of the molecules replicate more than once. For normal replication to occur the N function has to be expressed. The functions of the O and P cistrons are expressed in the absence of the N function. Superinfection of a heteroimmune lysogen by a sus P mutant fails to elicit the function required for replication of the superinfecting phage genome.

Control of ColE1 plasmid replication: initial interaction of RNA I and the primer transcript is reversible.

Replication of ColE1-type plasmids is known to be regulated by a plasmid-specific RNA (RNA I), whose binding to the transcript (RNA II) from the primer promoter results in inhibition of formation of the primer for DNA replication. In this paper, it is shown that binding of RNA I to the homologous RNA II is inhibited by an RNA I specified by a plasmid of different compatibility. The inhibition is caused by the reversible interaction of RNA II with the heterologous RNA I, which competes with reversible interaction of the two homologous RNAs at a step preceding their stable binding. As predicted from these results, the copy numbers of both ColE1 and RSF1030 are increased when both plasmids are present in the same cell. The Rom protein enhances the reversible interaction.

Complex formed by complementary RNA stem-loops and its stabilization by a protein: Function of ColE1 Rom protein

A small plasmid-specified RNA (RNA I) inhibits formation of the RNA primer for CoIE1 DNA replication by binding to its precursor (RNA II). Binding is modulated by the plasmid-specified Rom protein. Both in the presence and absence of Rom, binding starts with interaction between loops of RNAs. To understand the mechanism of binding, we examined the interactions of pairs of single stem-loops that are complementary fragments of RNA I and RNA II. We found that these complementary single stem-loops bind to each other at their loops, forming an RNAase V1-sensitive structure. Rom protects the complex from cleavage and from alkylation of phosphate groups by ethyinitrosourea. A single dimer of Rom binds to the complex by recognizing the structure rather than its exact nucleotide sequence. Rom enhances complex formation by decreasing the rate of dissociation of the complex. Structures of RNA complexes formed in the presence and absence of Rom are proposed.

Control of ColE1 plasmid replication: Binding of RNA I to RNA II and inhibition of primer formation

Formation of the primer for ColE1 DNA replication from the primary transcript, RNA II, is regulated by an antisense transcript, RNA I. Exposure of elongating RNA II 100-360 nucleotides long to RNA I inhibits subsequent primer formation. However, primer forms normally for transcripts longer than 360 nucleotides. Therefore, the rate of binding of RNA I to RNA II is crucial to regulation. The binding rate varies among RNA II transcripts of different lengths. Transcripts longer than 200 nucleotides are bound faster than shorter ones. The Rom protein enhances binding of RNA I to these longer transcripts, whereas it suppresses binding to some shorter ones. The insensitivity to RNA I of transcripts longer than 360 nucleotides is not due to the absence of RNA I binding but to this binding having no effect on primer formation.